Methods, kits and systems for determining the status of lung cancer and methods for treating lung cancer based on same

Abstract

The present disclosure includes, among other things, methods, kits, and systems for determining the status of lung cancer. In various embodiments, the present disclosure relates to the use of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation that that are characteristic of the status of lung cancer. In some embodiments, differential modifications and / or differential accessibility are detected and quantified at one or more genomic loci of a biological sample, e.g., in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from a subject with lung cancer. In various embodiments a determined status is useful, e.g., in selecting treatment for and / or treating a lung cancer.

Description

METHODS, KITS AND SYSTEMS FOR DETERMINING THE STATUS OF LUNG CANCER AND METHODS FOR TREATING LUNG CANCER BASED ON SAMECross-Reference to Related Applications

[0001] This application claims the benefit of U. S. Provisional Application No.63 / 590,215, filed on 13 October 2023; U. S. Provisional Application No. 63 / 591,811, filed on 20 October 2023; U. S. Provisional Application No. 63 / 575,700, filed on 6 April 2024; and U. S. Provisional Application No. 63 / 660,209, filed on 14 June 2024; the entire contents of each application is incorporated herein in its entirety by this referenceGOVERNMENT SUPPORT

[0002] This invention was made with government support under P50 CA265826 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] About 80% to 85% of lung cancers are non-small cell lung cancer (NSCLC). The main subtypes of NSCLC are adenocarcinoma (LUAD), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). These subtypes, which start from different types of lung cells, are grouped together as NSCLC because their treatment and prognoses are often similar.

[0004] About 10% to 15% of all lung cancers are small cell lung cancer (SCLC), a type of neuroendocrine tumor with high malignancy and poor prognosis. Besides de novo SCLC, there is transformed SCLC, which has similar characteristics of pathological morphology, molecular characteristics, clinical manifestations and drug sensitivity. However, de novo SCLC and transformed SCLC have different pathogenesis and tumor microenvironment. SCLC transformation is one of the mechanisms of resistance to chemotherapy, immunotherapy, and targeted therapy in NSCLC. It usually occurs in epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (LUAD) after treatment with tyrosine kinase inhibitors (TKIs) (Sequist et al., Sci Transl Med (2011) 3:75ra26). SCLC transformation can also occur in anaplastic lymphoma kinase (ALK)-positive lung cancer after treatment with ALK inhibitors and in wild-type EGFR or ALK NSCLC treated with immunotherapy (Ferrer et al., J ThoracOncol (2019) 14:130-134). Chemotherapy is currently used to treat transformed SCLC, yet it is associated with an unsatisfactory prognosis.

[0005] Current methods for classifying lung cancer, in particular SCLC (de novo SCLC or transformed SCLC) are limited to histology and staging. These methods do not capture the full complexity of the biology involved in lung cancer and therefore only partially characterize the relevant patient population. They also involve invasive tissue biopsies. Invasive biopsies come with risks to the patient and may not yield sufficient tissue for a comprehensive work up. There remains a need in the art for more comprehensive and precise diagnostic methods for determining the status of lung cancer, in particular SCLC (de novo SCLC or transformed SCLC) including methods that are independent of tissue biopsies. Improved diagnostic methods would also better support future clinical trials that seek to identify subpopulations of patients that respond to lung cancer therapies. They would also expand our understanding of the underlying biology of lung cancer. This would in turn help medical practitioners select the most appropriate agents to treat different subtypes of lung cancer, including de novo SCLC or transformed SCLC. Improved diagnostic methods would also help identify and support new treatments in ongoing clinical trials by more accurately identifying the profiles of lung cancers that respond to treatment and through characterizing the mechanisms of resistance to existing therapies (e.g., if a given NSCLC, e.g., LU AD patient developed transformed SCLC due to NSCLC, e.g., LU AD specific treatment).SUMMARY

[0006] The present disclosure is based, at least in part, on the demonstration that the SCLC / LUAD status of a lung cancer in a subject can be determined by detecting and quantifying the presence of histone modifications and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from the subject. The present disclosure also encompasses methods where chromatin accessibility and / or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and / or DNA methylation. The present disclosure is also based, at least in part, on the demonstration that genomic loci that are differentially modified based on different types of histone modifications (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac) and / orDNA methylation can be combined into multimodal classifiers to determine SCLC / LUAD status. These new monomodal and multimodal classifiers provide minimally invasive ways of determining SCLC / LUAD status that are more accurate, objective, and comprehensive than the current tissue-based approaches.

[0007] Liquid biopsies are now widely utilized in clinical oncology to detect cancer recurrence and inform therapeutic decisions. However, most commercially available cfDNA assays only detect tumor genomic alterations and not all disease states have a characteristic genomic alteration that can be used for detection. For example, the lack of genomic alterations exclusive to tSCLC limits the utility of genomic-based cfDNA approaches to detect small cell transformation in patients with EGFRm LUAD. Among other things, the present disclosure provides tools to analyze multiple tumor epigenomic features from patient plasma, including DNA methylation, chromatin accessibility, and histone modifications. Among other things, the present disclosure demonstrates that epigenomic cfDNA profiling can be used to detect small cell transformation in patients with EGFRm LU AD progressing on EGFR TKIs. Diagnosing tSCLC by cfDNA profiling would be immediately clinically actionable, as guidelines recommend that tSCLC be treated with a de novo SCLC regimen of platinum-etoposide chemotherapy which would not otherwise be used in patients with LUAD.

[0008] The present disclosure includes, among other things, technologies for the determination of SCLC / LUAD status and for the detection, monitoring, and / or treatment of lung cancer based on SCLC / LUAD status. In various embodiments, the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and / or treat lung cancer based on SCLC / LUAD status. The present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are characteristic of lung cancer, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and / or treating lung cancer based on SCLC / LUAD status. The present disclosure includes, among other things, histone modification measurements in cfDNA that are characteristic of SCLC cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating an SCLC cancer. The present disclosure includes, among other things, histone modification measurements in cfDNA that are characteristic of LUAD cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating LUAD cancer. In some embodiments, histonemodification measurements in cfDNA can be used to detect or determine resistance of a lung cancer, e.g., LU AD cancer to a therapy or transformation of a lung cancer, e.g., from LU AD to SCLC. In various embodiments, the present disclosure includes exemplary genomic loci that are differentially modified in SCLC vs. LU AD cancer. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more promoters.

[0009] In various embodiments, a genomic locus is differentially modified if it is characterized by increased or decreased histone modification as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). Increased or decreased histone modification can be or include, e.g., increased or decreased histone methylation (hypermethylation or hypomethylation, respectively) of one or more particular methylation marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and / or increased or decreased pan-acetylation (e.g., pan-H3 acetylation). In various embodiments, histone methylation can be or include histone methylation marks selected from H3K4mel, H3K4me2, H3K4me3, or a combination thereof. In various embodiments, histone methylation can be or include H3K4me3. In various embodiments, histone acetylation can be or include histone acetylation marks selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, or a combination thereof. In various embodiments, histone acetylation can be or include H3K27ac.

[0010] In various embodiments, the present disclosure relates to the measurement of DNA methylation in a sample obtained or derived from a subject to detect and / or treat lung cancer based on SCLC / LUAD status. The present disclosure includes, among other things, DNA methylation measurements in cell-free DNA (cfDNA) that are characteristic of cancer, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and / or treating lung cancer based on SCLC / LUAD status. The present disclosure includes, among other things, DNA methylation measurements in cfDNA that are characteristic of SCLC cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating an SCLC cancer. The present disclosure includes, among other things, DNA methylation measurements in cfDNA that are characteristic of LU AD cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treatingLU AD cancer. In some embodiments, DNA methylation measurements in cfDNA can be used to detect or determine resistance of a lung cancer, e.g., LU AD cancer to a therapy or transformation of a lung cancer, e.g., from LU AD to SCLC. In various embodiments, the present disclosure includes exemplary genomic loci that are differentially DNA methylated in SCLC vs. LUAD cancer. In various embodiments, a genomic locus is differentially modified if it is characterized by increased or decreased DNA methylation as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, genomic loci differentially modified in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more promoters.

[0011] The present disclosure further relates, in various embodiments, to the measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine SCLC / LUAD status. The present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of SCLC cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating an SCLC cancer. The present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of LUAD cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating LUAD cancer. In some embodiments, chromatin accessibility measurements in cfDNA can be used to detect or determine resistance of a lung cancer, e.g., LUAD cancer to a therapy or transformation of a lung cancer, e.g., from LUAD to SCLC. In various embodiments, the present disclosure includes genomic loci that are differentially accessible in SCLC vs. LUAD cancer. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more promoters.

[0012] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular' scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0013] In various embodiments, a genomic locus is differentially accessible if it is characterized by increased or decreased chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). Increased or decreased histone modification can be or include, e.g., increased or decreased accessibility as determined by various chromatin accessibility assays known in the art.

[0014] The present disclosure further relates, in various embodiments, to the measurement of transcription factor binding in cell-free DNA (cfDNA) to determine SCLC / LUAD status. The present disclosure includes, among other things, transcription factor binding measurements in cfDNA that are characteristic of SCLC cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating an SCLC cancer. The present disclosure includes, among other things, transcription factor binding measurements in cfDNA that are characteristic of LU AD cancers, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating LU AD cancer. In some embodiments, transcription factor binding measurements in cfDNA can be used to detect or determine resistance of a lung cancer, e.g., LU AD cancer to a therapy or transformation of a lung cancer, e.g., from LU AD to SCLC. In various embodiments, the present disclosure includes genomic loci that are differentially bound by transcription factors in SCLC vs. LU AD cancer. In various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancers. In various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.

[0015] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0016] In various embodiments, a genomic locus is differentially bound by transcription factors if it is characterized by increased or decreased transcription factor binding as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). Increased or decreased transcription factor binding can be or include, e.g., increased or decreasedtranscription factor binding as determined by various transcription factor binding assays known in the art.

[0017] In one aspect, the present disclosure provides a method of determining the SCLC / LUAD status of a lung cancer in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and / or (iv) DNA methylation.

[0018] In some embodiments, the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, and pan-acetylation. In some embodiments, the histone modification assay detects H3K4me3 modifications. In some embodiments, the histone modification assay detects H3K27ac modifications. In some embodiments, the histone modification assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT& RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT& Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0019] In some embodiments, chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.

[0020] In some embodiments, binding of one or more transcription factors is quantified using a transcription factor binding assay. In some embodiments, the transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT& RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT& Tag (Cleavage Under Targets and Tagmentation) sequencing. In some embodiments, binding of one or more transcription factors is quantified using a transcription factor binding assay that detects binding of one or more of p300, mediator complex, cohesin complex, RNA pol II, FOXA1, ESRI, PR, MYC, EN1, FOXM1, KLF4, AP-2, RARa, or RUNX1.

[0021] In some embodiments, DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).

[0022] In some embodiments, the method comprises quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and / or (iv) DNA methylation. In some embodiments, the method comprises quantifying two or more histone modifications, e.g., quantifying H3K4me3 and H3K27ac modifications. In some embodiments, the method comprises quantifying one or more histone modifications and DNA methylation, e.g., quantifying H3K4me3 and / or H3K27ac modifications and DNA methylation. In some embodiments, the method comprises quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.

[0023] In some embodiment, the biological sample is a liquid biopsy sample, e.g., a plasma sample, serum sample, or urine sample. In some embodiments, a method comprises isolating DNA (e.g., cfDNA) from 1, 2, 3, 4, or 5 mL of a liquid biopsy sample (e.g., plasma sample).

[0024] In some embodiments, quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has SCLC.

[0025] In some embodiments, quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has LUAD.

[0026] In some embodiments, a sample is a liquid biopsy sample comprising cfDNA, and a method comprises:(a) quantifying H3K4me3 modifications at one or more genomic loci using an assay that comprises enriching for cfDNA comprising one or more H3K4me3 modifications and sequencing the cfDNA enriched for H3K4me3 modifications (e.g., using a cf'ChlP-seq assay);(b) quantifying H3K27ac modifications at one or more genomic loci using an assay that comprises enriching for cfDNA comprising one or more H3K27ac modifications andsequencing the cfDNA enriched for H3K27ac modifications (e.g., using a cfChlP-seq assay); and / or;(c) quantifying methylated DNA using an assay that comprises enriching for methylated cfDNA and sequencing the enriched cfDNA to determine a count of sequences with one or more methylated nucleotides (e.g., using a MBD-seq assay).

[0027] In some embodiments,(a) cfDNA comprising H3K4me3 modifications is enriched using a method that comprises incubating a sample with an agent (e.g., an antibody) that binds H3K4me3 modifications;(b) cfDNA comprising H3K27ac modifications is enriched using a method that comprises incubating a sample with an agent (e.g., an antibody) that binds H3K27ac modifications; and / or(c) methylated cfDNA is enriched using a method that comprises incubating a sample with an agent (e.g., an antibody or a methyl binding domain) that binds methylated DNA.

[0028] In some embodiments, an agent that binds H3K4me3 modifications, an agent that binds H3K27ac modifications, and / or an agent that binds methylated DNA are attached (e.g., via a covalent or noncovalent bond) to a physical support (e.g., a bead, a magnetic bead, an agarose bead, or a magnetic epoxy bead) prior to incubating with a sample.

[0029] In some embodiments, if a method comprises incubating with two or more of (a) an agent that binds H3K4 modifications, (b) an agent that binds H3K27ac modifications, and (c) an agent that binds methylated DNA, a sample is incubated with the two or more agents (1) in sequence, or (2) in parallel (e.g., wherein the sample is divided into fractions and each fraction is incubated with a different agent).

[0030] In some embodiments, sequencing is performed using a next generation sequencing method.

[0031] In some embodiments, a method comprises attaching (e.g., ligating) adapters to cfDNA obtained from a subject (e.g., attaching after cfDNA has been enriched for cfDNA comprising one or more H3K4me3 modifications, cfDNA comprising one or more H3K27ac modifications, and / or methylated cfDNA).

[0032] In some embodiments, a method comprises amplifying a plurality of converted DNA fragments after attaching adapters to the plurality of DNA fragments.

[0033] In some embodiments, sequence reads are mapped to a reference genome. In some embodiments, non-uniquely mapped and redundant sequence reads are discarded prior to quantifying one or more epigenetic biomarkers. In some embodiments, sequence reads are mapped to a reference genome, wherein the one or more genomic loci correspond to sequence read peaks, wherein a sequence read peak corresponds to a region of the genome that has a higher number of sequence reads that the local background. In some embodiments, peaks in high noise regions are ignored when identifying genomic loci with a higher number of sequence reads than the local background and / or when identifying genomic loci associated with an SCLC / LUAD disease state. In some embodiments, peaks in white blood cell regions are ignored when identifying genomic loci with a higher number of sequence reads than the local background and / or identifying genomic loci associated with an SCLC / LUAD disease state. In some embodiments, peaks in regions likely to be artifactual are removed. In some embodiments, peaks that are less than 50 bp in length are removed.

[0034] In some embodiments, H3K4me3 modifications, H3K27ac modifications, and / or DNA methylation are quantified by summing the number of sequence reads having at least one nucleotide overlap one or more genomic loci. In some embodiments, sequence reads are adjusting on the basis of sequencing depth (e.g., quantile normalizing sequence reads to a common reference distribution) and / or ChIP quality prior to summing. In some embodiments, sequence counts are normalized to aggregate counts in a given sample across a set of regions (e.g., 10,000 regions) previously determined to have DNAse hypersensitivity in most cell types. In some embodiments, an estimate of local background signal is subtracted from the sequence reads at each genomic loci prior to summing.

[0035] In some embodiments, a method comprises comparing a measure of one or more epigenetic biomarkers to a reference. In some embodiments, a reference is a predetermined threshold, a measurement from a liquid biopsy sample, a measurement from liquid biopsy samples obtained from a cohort of subjects, and / or a normalized value. In some embodiments, a predetermined threshold or a normalized value were previously shown to distinguish LU AD and SCLC subjects (e.g., distinguish with an AUROC of greater than 0.5). In some embodiments, a reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have LU AD or SCLC. In some embodiments, a cohort of subjects had previously been determined to have lung cancer (e.g., LU AD or SCLC).

[0036] In some embodiments, a method comprises calculating sequence read density at one or more genomic loci. In some embodiments, calculating sequence read density can be calculated by:(a) summing background adjusted sequence counts at each of one or more genomic loci and dividing by the sum of the kilobases of the one or more genomic loci; or(b) for each genomic loci, dividing background adjusted fragment count by the number of kilobases of the genomic loci, and then summing for each loci.

[0037] In some embodiments, one or more genomic loci include one or more genomic loci with an increased level of the one or more epigenetic biomarkers in (a) sample(s) obtained from a subject with SCLC as compared to sample(s) obtained from a subject with LU AD, and / or (b) sample(s) obtained from a subject with LU AD as compared to sample(s) obtained from a subject with SCLC.

[0038] In some embodiments, a method described herein comprises calculating an SCLC / LUAD ratio score. In some embodiments, an SCLC / LUAD ratio score can be calculated by a method comprising:(a) calculating an SCLC sequence read density by a method comprising summing background adjusted sequence counts at each of one or more genomic loci with an increased level of one or more epigenetic biomarkers in sample(s) obtained from subjects with SCLC as compared to samples obtained from subjects with LU AD;(b) calculating a LU AD sequence read density by a method comprising summing background adjusted sequence counts at each of one or more genomic loci with an increased level of one or more epigenetic biomarkers in sample(s) obtained from subjects with LU AD as compared to samples obtained from subjects with SCLC; and(c) dividing the SCLC sequence read density by the LU AD sequence read density.

[0039] In some embodiments, a method comprises determining an SCLC / LUAD ratio score for one or more epigenetic biomarkers. In some embodiments, a method comprises determining an SCLC / LUAD ratio score for two or more epigenetic biomarkers. In some embodiments, a method comprises determining an SCLC / LUAD ratio score for two or more epigenetic biomarkers and combining the two or more SCLC / LUAD ratio scores. In some embodiments, a method comprises determining an SCLC / LUAD ratio score for each of H3K4me3 modifications, H3K27ac modifications, and methylated DNA, and combining each ofthe ratio scores. In some embodiments, two or more ratio scores can be combined using fitted values determined using a logistic regression.

[0040] In some embodiments, a method comprises comparing one or more quantified epigenetic biomarkers to a reference, wherein an increase or decrease in the one or more epigenetic biomarkers as compared to the reference indicates that the subject has SCLC or LUAD. In some embodiments, the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value. In some embodiments, the reference is a measurement from liquid biopsy samples obtained from a cohort of subjects who have previously been determined to have LU AD or to be cancer free. In some embodiments, the predetermined threshold and the normalized value were previously shown to distinguish LU AD and SCLC subjects (e.g., to provide an AUROC value of at least 0.5). In some embodiments, the cohort of subjects had previously been determined to have lung cancer (e.g., LUAD or SCLC).

[0041] In some embodiments, quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has SCLC cancer (e.g., de novo SCLC cancer or transformed SCLC cancer). In some embodiments, the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have LUAD cancer.

[0042] In some embodiments, a subject has previously been determined to have lung cancer, an increased susceptibility to lung cancer, and / or a method further comprises determining whether the subject has lung cancer. In some embodiments, a subject has an increased susceptibility to SCLC.

[0043] In some embodiments, quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has LUAD cancer. In some embodiments, the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have SCLC cancer.

[0044] In some embodiments, the lung cancer is metastatic lung cancer. In some embodiments, the lung cancer exhibits loss of TP53 and / or RBI (c.g., comprises one or more loss of function mutations). In some embodiments, the lung cancer exhibits TKI resistance.

[0045] In some embodiments, the SCLC is de novo SCLC. In some embodiments, the SCLC is transformed SCLC. In some embodiments, the SCLC is transformed SCLC and the LU AD is EGFRm LUAD.

[0046] In some embodiments, for a method provided a subject has previously been determined to have EGFRm (EGFR mutant) LU AD, the LU AD is EGFRm LU AD, and the SCLC is transformed SCLC (tSCLC).

[0047] In some embodiments, the method comprises quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in Tables 1-3. In some embodiments, the method comprises quantifying H3K4me3 modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the method comprises quantifying H3K27ac modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the method comprises quantifying H3K27ac for at least 1, 2, 3, or 4 genomic loci in Table 4. In some embodiments, the method comprises quantifying H3K4me3 or H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5. In some embodiments, the method comprises quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the method comprises quantifying DNA methylation for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5.

[0048] In some embodiments, the area under the receiver operating characteristic (AUROC) for determining if a subject has SCLC cancer vs. LU AD cancer is greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95).

[0049] In some embodiments, the SCLC cancer is a SCLC cancer based on histological testing (e.g., IHC testing) and the LU AD cancer is a LU AD cancer based on histological testing (e.g., IHC testing). In some embodiments, the subject has previously been determined to have lung cancer. In some embodiments, a sample comprises a detectable amount of cfDNA (e.g., wherein the estimated tumor fraction is >3%, as determined by iChorCNA).

[0050] In some embodiments, a sample is obtained from a subject having lung cancer wherein a biopsy of the lung cancer is not possible and / or feasible.

[0051] In some embodiments, if the subject is determined to have SCLC, the method further comprises subtyping the SCLC. In some embodiments, the SCLC is subtyped by detecting activity (e.g., expression level) of one or more transcription factors. In some embodiments, the SCLC is subtyped by detecting increased activity (e.g., increased expression) of one or more transcription factors (e.g., increased relative to average expression in a subject having SCLC and / or one or more subjects characterized by an alternative SCLC subtype). In some embodiments, the SCLC is subtyped by detecting decreased activity (e.g., decreased expression) of one or more transcription factors (e.g., decreased relative to average expression in a subject having SCLC and / or one or more subjects characterized by an alternative SCLC subtype). In some embodiments, the one or more transcription factors are ASCL1, NEURODI, YAP1, and / or POU2F3. In some embodiments, the one or more transcription factors are ASCL1, NEURODI, YAP1, and POU2F3. In some embodiments, the SCLC subtype is characterized by increased activity of YAP1 (e.g., increased relative to ASCL1, NEURODI, and POU2F3). In some embodiments, the SCLC subtype is characterized by increased activity of ASCL1, NEURODI, or POU2F3 (e.g., the SCLC is subtyped based on which of ASCL1, NEURODI, and POU2F3 is most highly expressed relative to one another). In some embodiments, the SCLC subtype is an inflamed SCLC subtype (SCLC-1), and is optionally characterized by low activity (e.g., low expression) of ASCL1, NEURODI, and POU2F3 (e.g., low relative to activity in a healthy subject, an average subject having SCLC, and / or one or more alternative SCLC subtypes) and / or an inflamed gene signature.

[0052] In some embodiments, the activity of the one or more transcription factors is determined by measuring transcription factor binding. In some embodiments, the activity of the one or more transcription factors is assessed using a method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and / or (iv) DNA methylation. In some embodiments, the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, and pan-acetylation. In someembodiments, the histone modification assay detects H3K4me3 modifications. In some embodiments, the histone modification assay detects H3K27ac modifications. In some embodiments, the one or more genomic loci arc selected from those provided Table 4.

[0053] In some embodiments, the present disclosure provides a method of treatment in which a subject has previously been determined to have mEGFRm LU AD prior to determining SCLC / LUAD status using a method described herein, wherein the method comprises: administering an SCLC therapy (e.g., as described herein) if the subject has been determined to have SCLC using a method described herein, and administering a LU AD therapy (e.g., as described herein), if the subject has been determined to have LU AD using a method described herein.

[0054] In another aspect, the present disclosure provides a method of treating a subject having lung cancer, the method comprising: administering a lung cancer therapy to the subject based on the SCLC / LUAD status of the lung cancer, wherein the SCLC / LUAD status of the lung cancer has been determined using any one of the aforementioned methods of determining SCLC / LUAD status. In some embodiment, the method further comprises determining the SCLC / LUAD status of the lung cancer using any one of the aforementioned methods of determining SCLC / LUAD status. In some embodiments, the lung cancer has been determined to be SCLC cancer and the cancer therapy is a SCLC cancer therapy. In some embodiments, the SCLC cancer has been subtyped, and the method comprises administering an SCLC therapy to the subject based on the SCLC subtype (e.g., an SCLC therapy is administered that has been shown to provide an improved benefit for that SCLC subtype as compared to other therapeutics commonly administered to subjects having SCLC).

[0055] In some embodiments, an SCLC therapy comprises administering (i) an agent that targets DLL3 (e.g., Tarlatlamab), and / or (ii) a PD-L1 inhibitor in combination with platinum-etoposide chemotherapy or PARP inhibitors.

[0056] In some embodiments, if a lung cancer has been determined to be EGFRm LU AD at higher risk for SCLC transformation (e.g., exhibits loss of TP53 and / or RBI), a method comprises administering platinum / etoposide chemotherapy in combination with Osimertinib.

[0057] In some embodiments, the SCLC cancer has been subtyped on the basis of increased ASCL1 activity e.g., expression), and the SCLC therapy is one that has been associated with providing an improved therapeutic benefit in subjects diagnosed with an ASCL1subtype of SCLC (e.g., improved relative to alternative therapeutics that are commonly administered to subjects having SCLC). In some embodiments, the SCLC therapy is a BCL2 apoptosis regulator, a BCL2 inhibitor, a DLL3 inhibitor (e.g., rovalpituzumab tesirine), an LSD1 inhibitor, and / or a therapeutic targeting CEACAM5 (e.g., labetuzumab govitecan).

[0058] In some embodiments, the SCLC cancer has been subtyped on the basis of increased NEURODI activity (e.g., expression), and the SCLC therapy is one that has been associated with providing an improved therapeutic benefit in subjects diagnosed with a NEURODI subtype of SCLC (e.g., improved relative to alternative therapeutics that are commonly administered to subjects having SCLC). In some embodiments, the SCLC therapy is an Aurora kinase inhibitor, a Somatostatin receptor 2 (SSTR2) inhibitor (e.g., lanreotide), a therapeutic targeting SSTR2 (e.g., PEN-221), or an immunotherapy (e.g., durvalumab) coadministered with platinum-etoposide.

[0059] In some embodiments, the SCLC cancer has been subtyped on the basis of increased POU2F3 activity (e.g., expression), and the SCLC therapy is one that has been associated with an improved therapeutic benefit in subjects diagnosed with a POU2F3 subtype of SCLC. In some embodiments, the SCLC therapy comprises an insulin like growth factor 1 receptor inhibitor (optionally without chemotherapy), cisplatin, a PARP inhibitor, an antimetabolite (e.g., an anti-folate or nucleoside analog), and / or durvalumab (optionally administered without platinum-etoposide).

[0060] In some embodiments, the SCLC cancer has been subtyped on the basis of increased YAP1 activity (e.g., expression), and the SCLC therapy is one that has been associated with an improved therapeutic benefit in subjects diagnosed with a YAP1 subtype of SCLC. In some embodiments, the SCLC therapy comprises durvalumab co-administered with platinum-etoposide.

[0061] In some embodiments, the SCLC subtype is an inflamed SCLC subtype (SCLC-I) and is optionally characterized by (i) low activity (e.g., low expression) of ASCL1, NEURODI, and POU2F3 (e.g., low relative to activity in a healthy subject, an average subject having SCLC, and / or one or more alternative SCLC subtypes), and / or (ii) is characterized by poor response to immune checkpoint blockade. In some embodiments, the SCLC therapy comprises an anti-PD-L1 therapeutic and a chemotherapeutic, immune checkpoint blockade, a Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib, an EMT-inhibitor (e.g., HDACi (e.g., mocetinostat)), a MICAinhibitor (e.g., IPH43), and / or an immunotherapy (e.g., durvalumab) co-administered with platinum-ctopo side.

[0062] In some embodiments, the lung cancer has been determined to be LU AD cancer and the cancer therapy is a LU AD cancer therapy. In some embodiments, the LU AD cancer therapy comprises administering a selective EGFR tyrosine kinase inhibitor (e.g., Osimertinib).

[0063] In another aspect, the present disclosure provides a method of monitoring the SCLC / LUAD status of a lung cancer in a subject, and optionally treating the lung cancer, the method comprising; determining the SCLC / LUAD status of the lung cancer using any one of the aforementioned methods of determining SCLC / LUAD status at first and second time points. In some embodiments, the subject is being treated with a therapeutic agent that can lead to transformation from LU AD cancer (or more generally a NSCLC cancer) to SCLC cancer, e.g., where the subject has epidermal growth factor receptor (EGFR) mutant LU AD cancer and is being treated with a tyrosine kinase inhibitor (TKI), the subject has anaplastic lymphoma kinase (ALK)-positive LUAD cancer and is being treated with an ALK inhibitor, or the subject has wild-type EGFR or ALK LUAD cancer and is being treated with immunotherapy. In some embodiments, the method further comprises administering a lung cancer therapy, optionally a SCLC cancer therapy or LUAD cancer therapy, to the subject based on the SCLC / LUAD status of the lung cancer at the second time point, optionally wherein the type, dose and / or frequency of administration of the cancer therapy is adjusted based on the SCLC / LUAD status of the lung cancer at the second time point.

[0064] In another aspect, the present disclosure provides a method of treating a subject having a lung cancer, the method comprising: (i) administering an SCLC therapeutic agent to the subject, wherein the subject has been determined to have a validated epigenetic profile indicative of an SCLC cancer based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject; or (ii) administering a LUAD therapeutic agent to the subject, wherein the subject has been determined to have a validated epigenetic profile indicative of LUAD cancer based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject, wherein the presence of the validated epigenetic profile has been determined using a validated classifier, wherein the validated classifier has been obtained by: (a) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one ormore transcription factors, and / or DNA methylation in biological samples obtained from a first cohort of subjects who have previously been determined to have an SCLC cancer (e.g., de novo SCLC or transformed SCLC); (b) determining a genomic profde of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a second cohort of healthy subjects or subjects who have previously been determined to have LUAD cancer; (c) comparing the genomic profile determined in step (a) and the genomic profile determined in step (b), to identify genomic loci that have statistically different histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“differential loci”); (d) training a classifier on histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels in the differential loci to distinguish between (i) samples from one or more biological samples obtained from the first cohort, and (ii) samples from one or more biological samples obtained from the second cohort, to identify samples having a profile of histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“epigenetic profile”) that indicates that the samples are likely obtained from the first cohort; and (e) obtaining the validated classifier by validating the classifier from step (d) on a third cohort comprising an independent and group of subjects with SCLC and LUAD cancers and selecting a threshold such that the validated classifier predicts SCLC cancers, with an area under the receiver operating characteristic (AUROC) greater than 0.5 e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95), wherein subjects falling within the group of predicted SCLC cancers display the validated epigenetic profile and subjects that do not fall within the group of SCLC cancers lack the validated epigenetic profile.

[0065] In some embodiments, the differential loci in step (c) were identified by comparing the genomic profile of one or more histone modifications and / or DNA methylation in (i) one or more biological samples from the first cohort and (ii) one or more biological samples from the second cohort.

[0066] In some embodiments, the classifier in step (d) was trained on histone modification and / or DNA methylation levels in (i) one or more biological samples from the first cohort and (ii) one or more biological samples from the second cohort.

[0067] In some embodiments, the validated classifier in step (e) was validated using liquid biopsy samples from the third cohort.

[0068] In some embodiments, the classifier in step (d) was trained on two or more histone modification levels in the differential loci. In some embodiments, the two or more histone modification levels comprise H3K4me3 and H3K27ac modification levels.

[0069] In some embodiments, the classifier in step (d) was trained on one or more histone modification levels and DNA methylation in the differential loci. In some embodiments, the one or more histone modification levels comprise H3K4me3 and / or H3K27ac modification levels. In some embodiments, the classifier in step (d) was trained using ridge regression, elastic-net regression, or lasso regression. In some embodiments, the one or more histone modification levels comprise H3K4me3 and / or H3K27ac modification levels. In some embodiments, the one or more histone modification levels comprise H3K4me3 and H3K27ac modification levels. In some embodiments, the biological sample is a liquid biopsy sample, e.g., a plasma sample, serum sample, or urine sample.

[0070] In another aspect, the present disclosure provides a kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-3. In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 1, 2, 3, or 4 genomic loci in Table 4. In some embodiments, the kit comprises reagents for quantifying H3K4me3 or H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5. In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5.

[0071] In some embodiments, the kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones. In some embodiments, the kit comprises one or more methyl-binding domains for use in MBD-seq.

[0072] In some embodiments, the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the kit comprises reagents for library preparation for sequencing. In some embodiments, the kit comprises reagents for sequencing. In some embodiments, the kit comprises instructions for determining if a subject has SCLC cancer or LU AD cancer, optionally instructions for determining if a subject has a subtype of SCLC cancer characterized by increased activity (e.g., expression) of ASCL1, NEURODI, YAP1, and / or POU2F3.

[0073] In another aspect, the present disclosure provides a non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method of any one of the aforementioned methods of determining SCLC / LUAD status.

[0074] In another aspect, the present disclosure provides a computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of the aforementioned methods of determining SCLC / LUAD status.

[0075] In another aspect, the present disclosure provides a system for determining the SCLC / LUAD status of a lung cancer in a subject, the system comprising a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium and / or a computer system of the present disclosure. In some embodiments, the sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample. In some embodiments, the system further comprises a sample preparation device. In some embodiments, the sample preparation device is configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample. In some embodiments, the sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample. In some embodiments, the one or more genomic loci are selected from Tables 1-3. In some embodiments, the device comprises reagents for quantifying H3K4me3, e.g., for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the device comprises reagents for quantifying H3K27ac, e.g., for at least 5, 10, 20,30, 40, or 50 genomic loci in Table 2. In some embodiments, the device comprises reagents for quantifying DNA methylation, e.g., for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the reagents comprise one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones. In some embodiments, the reagents comprise one or more methyl-binding domains for use in MBD-seq. In some embodiments, the device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample. In some embodiments, the device comprises reagents for library preparation for sequencing. In some embodiments, the sequencer comprises reagents for sequencing.

[0076] In some embodiments, a method is for determining SCLC and / or LU AD status of a cancer in a subject (e.g., patient). The method may include receiving (e.g., by a processor of a computing device) one or more genomic profiles of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation for the subject. The method may further include determining whether the subject has an epigenetic profile indicative of an SCLC or LU AD by classifying (e.g., by the processor) the genomic profile using classifier.

[0077] In some embodiments, a classifier has been trained using one or more genomic profiles of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation for one or more biological samples obtained from cohorts of subjects who have previously have been determined to have an SCLC (e.g., de novo SCLC or transformed SCLC) or LUAD. In some embodiments, genomic profiles are for differential loci corresponding to statistically different histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels between one or more biological samples obtained from cohorts of subjects who have previously have been determined to have an SCLC (e.g., de novo SCLC or transformed SCLC) or LUAD.

[0078] In some embodiments, a classifier has been trained based on two or more histone modification levels in the differential loci. In some embodiments, a genomic profile comprises two or more histone modification levels. In some embodiments, such two or more histone modification levels comprise H3K4me3 and H3K27ac modification levels.

[0079] In some embodiments, a genomic profile comprises one or more histone modification levels and DNA methylation. In some embodiments, a classifier has been trainedbased on one or more histone modification levels and DNA methylation in the differential loci. In some embodiments, such one or more histone modification levels comprise H3K4mc3 and / or H3K27ac modification levels. In some embodiments, such one or more histone modification levels comprise H3K4me3 and H3K27ac modification levels.

[0080] In some embodiments, a classifier has been trained with data derived from plasma. In some embodiments, such a classifier has been trained with data derived from liquid biopsy samples.

[0081] In some embodiments, a classifier is a validated classifier. In some embodiments, a classifier has been validated by selecting a threshold such that the validated classifier predicts SCLC cancers with an area under the receiver operating characteristic (AUROC) greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95). In some embodiments, a classifier has been validated on a cohort of an independent group of subjects with an SCLC (e.g., de novo SCLC or transformed SCLC) and LU AD, wherein subjects falling within a group of predicted SCLC (e.g., de novo SCLC or transformed SCLC) cancers display a validated epigenetic profile and subjects that do not fall within the group of predicted SCLC cancers lack the validated epigenetic profile. In some embodiments, a classifier has been validated using liquid biopsy sample data.

[0082] A non-transitory computer readable storage medium may be encoded with a computer program, where the program may comprise instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method for determining SCLC and / or LU AD of a cancer in a subject (e.g., patient). A computer system may include a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform a method for determining SCLC and / or LU AD of a cancer in a subject (e.g., patient).

[0083] In some embodiments, a method of treating a subject having a cancer includes administering an SCLC therapeutic agent to the subject, wherein the subject has been determined to have a validated epigenetic profile indicative of an SCLC based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject. In some embodiments, presence of the validated epigenetic profile has beendetermined using a classifier (e.g., a validated classifier) according to a method for determining SCLC and / or LU AD of a cancer in a subject (e.g., patient).

[0084] In some embodiments, method of treating a subject having a cancer includes administering a LU AD therapeutic agent to the subject, wherein the subject has been determined to have a validated epigenetic profile indicative of LU AD based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject. In some embodiments, presence of the validated epigenetic profile has been determined using a classifier (e.g., a validated classifier) according to a method for determining SCLC and / or LU AD of a cancer in a subject (e.g., patient).BRIEF DESCRIPTION OF THE DRAWING

[0085] Fig. 1 shows representative ROC curves for exemplary SCLC / LUAD status classifiers that were generated in accordance with Example 2. As shown, different classifiers were generated using genomic loci from Tables 1-3 for different modifications, namely (i) H3K4me3 modifications, (ii) H3K27ac modifications, (iii) DNA methylation (MBD) or (iv) all of the above (combined). AUC for individual modifications was calculated based on SCLC / LUAD ratio for each modification individually. AUC for the combined modifications was calculated on the fitted values from a logistic regression. Each individual modification and the combination of all three were able to correctly classify SCLC from LUAD. AUC values were 0.85 for H3K4me3 modifications, 0.83 for H3K27ac modifications, 0.9 for DNA methylation (MBD) and 0.92 for the combination of all three.

[0086] Fig. 2 shows representative, non-limiting graphs that demonstrate the accuracy of SCLC / LUAD status (based on AUCROC) determination using the classifiers that were generated in accordance with Example 2. As described in Example 3, average AUC and 95% confidence intervals are shown from 500 repeated samplings of regions used to calculate SCLC / LUAD ratios. As shown, the different sampling used different subsets of the genomic loci in Table 1 (H3K4me3), Table 2 (H3K27ac), Table 3 (MBD) and Tables 1-3 (combined).

[0087] Fig. 3 shows representative, non-limiting graphs that demonstrate the accuracy of SCLC / LUAD status (based on AUCROC) determination using the classifiers that were generated in accordance with Example 2. As described in Example 4, the widths of genomic loci in Tables 1-3 were increased / decreased and AUCs calculated on re-computed SCLC / LUAD ratios. Asshown, increasing or decreasing the classifier input genomic region width by up to 50% had little effect on predictive performance.

[0088] Fig. 4 shows the results of experiments performed in accordance with Example 5 where different machine learning (ML) approaches were used to generate different SCLC / LUAD status classifiers using the genomic loci in Tables 1-3. The results show the average (95% CI) AUC from 50 repeats of 5-fold cross validation. The background adjusted counts normalized to library size in each individual genomic locus were provided to three different ML algorithms instead of being used to calculate SCLC / LUAD ratio scores. As shown, the different ML approaches (glmnet, Random forest and SVM) yielded similar predictive performance.

[0089] Fig. 5 shows normalized H3K4me3 cfChlP-seq signal at the DLL3 promoter stratified by cancer type. As shown, H3K4me3 cfChlP-seq signal was highest for Merkel cell carcinoma, neuroendocrine prostate cancer (NEPC), melanoma and small cell lung cancer (SCLC). Lower and upper hinges indicate 25thand 75thpercentiles; whiskers extend to 1.5 x the inter-quartile ranges (IQR).

[0090] Fig. 6 shows heatmaps of quantified and scaled enhancer and promoter activity at established SCLC subtype driver genes (columns) in SCLC patient plasma samples(rows). Unsupervised clustering of enhancer activity showed distinct groups of SCLC samples, suggesting the ability to infer SCLC subtypes using the epigenomic -based liquid biopsy platform. The observed distribution of SCLC subtypes is within the expected distribution of 70% SCLC-A, 11% SCLC-N, 17% SCLC-P and 2% SCLC-Y3(p=0.2).

[0091] Fig. 7 shows patient- specific promoter signals for genes that are associated with differential transcriptional biology across SCLC and LUAD. As shown in the figure, clear differences were seen between SCLC and LUAD. Samples were grouped by histology and sorted by ichorCNA ctDNA fraction estimate within each group.

[0092] Fig. 8. shows the ability of a classifier that uses enhancer, promoter and methylation signal quantified at loci determined to be differential between SCLC and NSCLC in cell lines (e.g., loci described herein) to distinguish between subjects having SCLC and LUAD.5-fold cross validation was performed using regularized logistic regression to estimate the predictive performance of classifying SCLC and LUAD. Selected features comprise 65% promoters, 23% enhancers and 13% methylation. As shown in Fig. 8, predictive performanceremained high even for samples with ctDNA levels below the ichorCNA limit of detection, indicating that the assay is sensitive below 3% ctDNA.

[0093] Fig. 9 shows results from an in silico experiment to determine the limit of detection of assays described herein. In silico plasma samples were created to simulate lower ctDNA levels by diluting the high ctDNA profiles of 17 LU AD and 12 SCLC plasma samples with each of 24 healthy plasma samples. Regularized logistic regression was used to classify in silico mixtures using a leave-one-out scheme in which all mixtures generated from a given pair of cancer and healthy samples were held out and then predicted based on a model fit to the remaining mixture samples. As shown Fig. 9, AUC estimates from these simulations remain above 0.90 even at 0.5% ctDNA and above 0.80 at 0.4% ctDNA.

[0094] Fig. 10 is a block diagram of an example network environment for use in the methods and systems described herein, according to illustrative embodiments of the present disclosure.

[0095] Fig. 11 is a block diagram of an example computing device and an example mobile computing device, for use in illustrative embodiments of the present disclosure.

[0096] Figure 12. Overview of an experimental approach to perform comprehensive epigenomic profiling of lung cancer patient-derived xenografts (PDXs) and multi-analyte epigenomic profiling of cfDNA from 1 mL of patient plasma and non-invasively detect SCLC transformation in patients with EGFRm LUAD. A person of skill in the art will understand that one or more of (e.g., all of) the analytes indicated in Figure 1 can be used in a method described in the present disclosure. A person of skill in the art will also understand that analytes can be assessed in any order, to the extent permitted by experimental protocols.

[0097] Figure 13. Comprehensive epigenomic profiling of LU AD, tSCLC, and de novo SCLC reveals widespread epigenomic reprogramming in small transformation. (A) Principal component analysis (PCA) plots of the ATAC, H3K27ac ChIP, H3K4me3 ChIP, H3K27me3 ChIP, MeDIP, and RNA sequencing data reveal clustering of tSCLC tumors with de novo SCLC tumors and their distinction from LUAD tumors. (B) Representative epigenomic data from an EGFRm tSCLC, EGFRm LUAD, and de novo SCLC PDX, showing gain of signal in markers of active gene transcription (ATAC-seq, H3K27ac ChlP-seq, H3K4me3 ChIP-seq, gene body DNA methylation) and loss of signal with repressive marks (H3K27me3 ChlP-seq) at neural lineage-defining genes in tSCLC. Each track depicts signal intensity for the indicated epigenetic mark in the indicated sample.

[0098] Figure 14. Comparative analysis identifies a robust set of highly differential epigenomic features between LU AD and SCLC. (A) Heatmap of normalized H3K27ac tag densities at differential H3K27ac sites between LU AD and SCLC tumors (FDR- adjusted P < 0.001 and log2 fold-change > 2) located ±2 kb from peak center. (B) Volcano plot showing overlap of the log2 fold-change differentially expressed genes between LU AD and SCLC PDXs with respective differential H3K27ac peaks enriched in LUAD (blue) and SCLC (red). Two-sided p-values were corrected for multiple hypothesis testing (FDR-adjusted P < 0.05).Abbreviations: LUAD, lung adenocarcinoma; SCLC, small cell lung cancer; tSCLC, transformed SCLC; EGFRm, EGFR mutant.

[0099] Figure 15. Non-invasive detection of tSCLC via tissue-informed epigenomic cfDNA analysis. Box plots show cfDNA SCLC Risk Scores for plasma samples from patients with EGFRm LUAD and EGFRm tSCLC based on H3K27ac cfChlP-seq analysis (A), H3K4me3 cfChlP-seq analysis (B), cfDNA methylation analysis (C), or cfDNA chromatin accessibility analysis (D). Box plots show the interquartile range and median value for each dataset with whiskers representing the larger value of 1.5 times the interquartile range or the largest value in the dataset. P-values were calculated using Mann- Whitney test. Corresponding ROC curves with the AUROC are included. Abbreviations: cfDNA, cell-free DNA; LUAD, lung adenocarcinoma; SCLC, small cell lung cancer; tSCLC, transformed SCLC; EGFRm, EGFR mutant; AUROC, area under the receiver operating characteristic curve.

[0100] Figure 16. Integrating multiple epigenomic cfDNA analytes improves non-invasive detection of tSCLC. (A) Venn diagram showing the overlap of differential H3K27ac, DNA methylation, and open chromatin sites between SCLC and LUAD PDXs. (B) Box plot shows cfDNA SCLC Risk Scores for plasma samples from patients with EGFRm LUAD and EGFRm tSCLC based on an integrated epigenomic classifier incorporating H3K27ac, DNA methylation, and chromatin accessibility analysis. Box plot shows the interquartile range and median value for each dataset with whiskers representing the larger value of 1.5 times the interquartile range or the largest value in the dataset. P-value was calculated using Mann-Whitney test. ROC curve with the AUROC is included. Optimal cut-off was calculated using Youden index. (C) Correlation of SCLC Risk Score with estimated cfDNA tumor fraction inpatients with EGFRm tSCLC and EGFRm LUAD. Abbreviations: cfDNA, cell-free DNA;LU AD, lung adenocarcinoma; SCLC, small cell lung cancer; tSCLC, transformed SCLC;EGFRm, EGFR mutant; ROC, receiver operative characteristic; AUROC, area under the receiver operating characteristic curve.

[0101] Figure 17. Patient vignettes highlight the ability to non-invasively detect small cell transformation in patients with EGFRm LUAD through cfDNA epigenomic profiling.Longitudinal assessment of the integrated epigenomic cfDNA SCLC Risk Score in two patients who EGFRm LUAD who experienced biopsy-proven SCLC. Abbreviations: EGFRm, EGFR mutant; cfDNA, cell-free DNA; LUAD, lung adenocarcinoma; SCLC, small cell lung cancer.

[0102] Figure 18. Gene expression for ASCL1, NEURODI, POU2F3, and YAP1 in SCLC PDXs. Abbreviations: SCLC, small cell lung cancer; PDX, patient-derived xenograft; FPKM, fragments per kilobase million.

[0103] Figure 19. Epigenomic datasets generated from lung cancer PDXs.Abbreviations: PDX, patient-derived xenograft; LUAD, lung adenocarcinoma; SCLC, small cell lung cancer; tSCLC, transformed SCLC.

[0104] Figure 20. Unsupervised hierarchical clustering of lung adenocarcinoma (LUAD), transformed small cell lung cancer (SCLC), and de novo SCLC patient-derived xenografts (PDXs) for ATAC-seq (A), H3K27ac ChlP-seq (B), H3K4me3 ChlP-seq (C), H3K27me3 ChlP-seq (D), and MeDIP-seq (E) data.

[0105] Figure 21. Venn diagram showing overlap of lung adenocarcinoma (LUAD), transformed small cell lung cancer (SCLC), and de novo SCLC patient-derived xenografts (PDX) H3K27ac ChlP-seq (A), H3K4me3 ChlP-seq (B), ATAC-seq (C), and MeDIP-seq (D) peaks.

[0106] Figure 22. Number of differential (A) H3K27ac, (B) H3K4me3, (C) DNA methylation, and (D) open chromatin sites enriched in lung adenocarcinoma (LUAD) or small cell lung cancer (SCLC) patient-derived xenografts before and after removal of peaks also present in white blood cells (WBCs).

[0107] Figure 23. Volcano plot showing overlap of the log2 fold-change differentially expressed genes between LUAD and SCLC PDXs with respective differential (A) H3K4me3 ChlP-seq and (B) ATAC-seq peaks enriched in lung adenocarcinoma (LUAD; blue) and smallcell lung cancer (SCLC; red) patient-derived xenografts. Two-sided p-values were corrected for multiple hypothesis testing (FDR-adjusted P < 0.05).

[0108] Figure 24. Epigenomic datasets generated from cfDNA plasma samples from patients with metastatic lung cancer. Abbreviations: cfDNA, cell-free DNA; LUAD, lung adenocarcinoma; SCLC, small cell lung cancer; tSCLC, transformed SCLC; EGFRm, EGFR mutant.

[0109] Figure 25. Representative epigenomic data from an EGFRm tSCLC and EGFRm LUAD showing the distribution of H3K27ac and H3K4me3 signal intensity near INSMI, a neural lineage-defining gene. Grey bars indicate the nearest enhancers characterized as Elite GeneHancer and Elite GeneHancer-gene association for INSMI.

[0110] Figure 26. H3K27ac signal at select genes in representative cfDNA samples from a healthy cancer-free control, a patient with metastatic EGFRm lung adenocarcinoma (LUAD), and a patient with metastatic EGFRm transformed small cell lung cancer (tSCLC). Samples from cancer patients were selected based on high estimated cfDNA tumor content from low-pass whole genome sequencing data: 55% for the EGFRm LUAD patient and 46% for the EGFRm tSCLC patient. Each track depicts signal intensity for the indicated epigenetic mark in the indicated sample. Each sample is scaled to the peak signal intensity in GAPDH for that sample.

[0111] Figure 27. Classification of LUAD and SCLC cell lines based on H3K27ac ChlP-seq data. (A) Dot plot shows SCLC Risk Scores generated using the PDX-derived classifier for LUAD (n=20) and SCLC (n=13) cell lines stratified by molecular subtype. The table to the right of the graph shows the Mann- Whitney Test P values for notable comparisons. Receiver operating characteristic (ROC) curves are shown with the area under the ROC curve (AUROC) for (B) LUAD versus all SCLC cell lines and (C) LUAD versus the SCLC-P, SCLC-A, and SCLC-N cell lines (excluding SCLC-Y cell lines). Abbreviations: LUAD, lung adenocarcinoma; SCLC, small cell lung cancer; Y, YAP1, P, POU2F3, A, ASCLF, N, NEURODI.

[0112] Figure 28. Number of base pairs covered by differential H3K27ac, DNA methylation, and open chromatin sites enriched in lung adenocarcinoma (LUAD) or small cell lung cancer (SCLC) patient-derived xenografts (PDXs) after removal of peaks also present in white blood cells.DETAILED DESCRIPTION

[0113] The present disclosure is based, at least in part, on the demonstration that the SCLC / LUAD status of a lung cancer in a subject can be determined by detecting and quantifying the presence of histone modifications and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from the subject. The present disclosure also encompasses methods where chromatin accessibility and / or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and / or DNA methylation. The present disclosure is also based, at least in part, on the demonstration that genomic loci that are differentially modified based on different types of histone modifications (e.g., histone methylation marks such as H3K4me3 and histone acetylation marks such as H3K27ac) and / or DNA methylation can be combined into multimodal classifiers to determine SCLC / LUAD status. These new monomodal and multimodal classifiers provide minimally invasive ways of determining SCLC / LUAD status that are more accurate, objective, and comprehensive than the current tissue-based approaches. No liquid biopsy platform to date has been able to provide actionable resolution on a transcriptionally regulated phenotype relevant for therapy such as SCLC / LUAD status.SCLC / LUAD status and lung cancer

[0114] About 80% to 85% of lung cancers are non-small cell lung cancer (NSCLC). The main subtypes of NSCLC are adenocarcinoma (LU AD), squamous cell carcinoma (SCC), and large cell carcinoma (LCC). These subtypes, which start from different types of lung cells, are grouped together as NSCLC because their treatment and prognoses are often similar. Some types of lung cancer can be further subtyped on the basis of oncogenes that drive the cancer. For example, one type of LU AD is mEGFR (mutated Epidermal growth factor receptor) LUAD, in which a mutation in the EGFR receptor results in the receptor being constitutively rather than being activated only in the presence an endogenous cognate ligand. Exemplary mutations include E19del and L858R.

[0115] About 10% to 15% of all lung cancers arc small cell lung cancer (SCLC), a type of neuroendocrine tumor with high malignancy and poor prognosis. Besides de novo SCLC, there is transformed SCLC, which has similar characteristics of pathological morphology,molecular characteristics, clinical manifestations and drug sensitivity. However, de novo SCLC and transformed SCLC have different pathogenesis and tumor microenvironment. SCLC transformation is one of the mechanisms of resistance to chemotherapy, immunotherapy, and targeted therapy in NSCLC. It usually occurs in epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (LU AD) after treatment with tyrosine kinase inhibitors (TKIs) (Sequist et al., Sei Transl Med (2011) 3:75ra26). SCLC transformation can also occur in anaplastic lymphoma kinase (ALK)-positive lung cancer after treatment with ALK inhibitors and in wild-type EGFR or ALK NSCLC treated with immunotherapy (Ferrer et al., J Thorac Oncol (2019) 14:130-134). Chemotherapy was previously used to treat transformed SCLC, yet it is associated with an unsatisfactory prognosis.

[0116] SCLC cancers can be treated with any SCLC cancer therapies, e.g., those disclosed herein. LU AD cancers can be treated with any LU AD cancer therapies, e.g., those disclosed herein.SCLC cancer therapies

[0117] Currently approved SCLC cancer therapies include chemotherapy and immunotherapy.Chemotherapy

[0118] Chemotherapy is typically part of the treatment for small cell lung cancer (SCLC). This is because SCLC has usually already spread by the time it is found, so other treatments such as surgery or radiation therapy would not reach all areas of cancer.

[0119] For subjects with limited stage SCLC, chemotherapy is often given with radiation therapy. This is known as chemoradiation. For subjects with extensive stage SCLC, chemotherapy with or without immunotherapy is usually the main treatment. Sometimes radiation therapy is given as well. Generally, SCLC is treated with combinations of chemotherapeutic agents. The combinations used most often are cisplatin and etoposide, carboplatin and etoposide, cisplatin and irinotecan, or carboplatin and irinotecan.Topotecan and lurbinectedin are chemotherapeutic agents that might be used by themselves in subjects with SCLC that has spread, especially if they have already tried cisplatin or carboplatin.

[0120] Chemotherapeutic agents for lung cancer are typically administered intravenously, either as an injection over a few minutes or as an infusion over a longer period of time.

[0121] Medical practitioners administer chemotherapy in cycles, with each period of treatment followed by a rest period to give the subject time to recover from the effects of the chemotherapeutic agents. Cycles are most often 3 or 4 weeks long, and initial treatment is typically 4 to 6 cycles. The schedule varies depending on the chemotherapeutic agents used. For example, some chemotherapeutic agents are given only on the first day of the chemotherapy cycle. Others are given for a few days in a row, or once a week. Then, at the end of the cycle, the chemotherapy schedule repeats to start the next cycle.

[0122] For advanced cancers, the initial chemotherapy combination is often given for 4 to 6 cycles, sometimes in combination with immunotherapy. Beyond this, medical practitioners might also recommend extending treatment with a single immunotherapy, for subjects who have had a good response to their initial chemotherapy or have had no worsening of their cancer.

[0123] If the cancer progresses (gets worse) during treatment or returns after treatment is finished, other chemotherapeutic agents may be tried. The choice of chemotherapeutic agents depends to some extent on how soon the cancer begins to grow again. The longer it takes for the cancer to return, the more likely it is to respond to further treatment. If cancer returns more than 6 months after treatment, it might respond again to the same chemotherapeutic agents that were given the first time. If the cancer comes back sooner, or if it keeps growing during treatment, further treatment with the same chemotherapeutic agents is unlikely to be helpful. If further chemotherapy is given, most medical practitioners prefer treatment with a single, different chemotherapeutic agent to help limit side effects. Topotecan and lurbinectedin are most often used, although other chemotherapeutic agents might also be tried.Immunotherapy

[0124] An important part of the immune system is its ability to keep itself from attacking normal cells in the body. To do this, it uses “checkpoints” or proteins on immune cells that need to be turned on (or off) to start an immune response. Cancer cells sometimes use checkpoints to avoid being attacked by the immune system. Therapeutic agents that target these checkpoints can be used to treat some subjects with small cell lung cancer (SCLC).

[0125] Atezolizumab and durvalumab are exemplary checkpoint inhibitors that target PD-L1, a protein related to PD-1 that is found on some tumor cells and immune cells.Camrelizumab is an exemplary checkpoint inhibitor that targets PD- 1. Blocking of these proteins can help boost the immune response against cancer cells. These therapeutic agents can be used as part of the first-line treatment for advanced SCLC, along with etoposide and a platinum chemotherapy (e.g., carboplatin or cisplatin). Either therapeutic agent can then be continued alone as maintenance therapy. This combination of PD-L1 immunotherapy with chemotherapy also seems to help some subjects with SCLC live longer. These therapeutic agents are administered as an intravenous (IV) infusion, typically every 2, 3 or 4 weeks.Other SCLC cancer therapies

[0126] While the sections above focus on FDA approved SCLC cancer therapies, many other SCLC cancer therapies are being developed and / or assessed in clinical trials (e.g., see Giunta et al., Front Med (Lausanne) (2022) 9:924853, the entire contents of which are incorporated herein by reference).

[0127] For example, Poly ADP-ribose polymerase (PARP) inhibitors (PARPi) such as olaparib, fluzoparib and talazoparib have been approved in ovarian cancer, prostate cancer and / or breast cancer and are currently under investigation in SCLC, given their potential of enhancing cytotoxic response to chemotherapy, radiotherapy, and immunotherapy (Barayan et al., J Thorac Dis (2020) 12:6240-6252). Exemplary combination therapies under clinical assessment for first-line therapy in combination with platinum chemotherapy (e.g., carboplatin or cisplatin) including durvalumab and Olaparib. The combinations of camrelizumab and fluzoparib, and atezolizumab and talazoparib are being assessed for maintenance therapy of SCLC. The combination of durvalumab and ceralasertib, an ATR inhibitor (ATRi), and the combination of durvalumab and AZD2811, Aurora Kinase B inhibitors (AurKBi), are also being investigated for maintenance therapy of SCLC.

[0128] Other new immunomodulatory agents under investigation could are also being assessed for their ability to potentiate the effect of immune checkpoint inhibitors like atezolizumab and durvalumab that target PD-L1 though their effect on specific immune targets like: LAG3, expressed on activated T and NK cells (Goldberg and Drake, Curr Top Microbiol Immunol (2011) 344:269-278); TIGIT, upregulated by activated T cells and regulatory cells(Chauvin and Zarour, J Immunother Cancer (2020) 8:57); ILT4, expressed in myeloid cells (Gao et al., Biochim Biophys Acta Rev Cancer (2018) 1869:278-285); and CD27, involved in T cell proliferation and differentiation to memory and effector cells (Starzer and Berghoff, ESMO Open (2020) 4(S3):e000629).

[0129] Delta-like ligand 3 (DLL3) has emerged as an attractive tumor- specific target uniquely overexpressed on the cell surface of SCLC and other high-grade neuroendocrine cancers (NECs) (Saunders et al., Sci Transl Med (2015) 7:302ral36 and Rudin et al., J Hematol Oncol (2023) 16(1 ):66). Rovalpituzumab tesirine is an antibody-drug conjugate (ADC) comprising a DLL3-specific humanized monoclonal antibody (SC 16) conjugated to a membrane-permeable pyrrolobenzodiazepine (PBD) dimer toxin via a lysosomal, protease-sensitive dipeptide linker (Saunders et al., Sci Transl Med (2015) 7:302ral36). Binding of rovalpituzumab tesirine to cell surface DLL3 causes internalization of the ADC-target complex by endocytosis. Rovalpituzumab tesirine’s valine-alanine linker is subsequently cleaved by lysosome-associated cathepsin B, releasing PBD into the cytoplasm. PBD then enters the nucleus, cross-links DNA, and induces tumor cell death by apoptosis. In some embodiments, a DLL3-targeted therapeutic, e.g., an ADC such as rovalpituzumab tesirine can be used to treat a subject that has been determined to have a cancer in accordance with a method of the present disclosure, e.g., a subject determined to have SCLC. In some embodiments, such methods may involve a further step of detecting or quantifying the presence of an H3K4me3 modification at the DLL3 promoter in accordance with methods of the present disclosure, e.g., in a plasma sample, e.g., within genomic locus chrl9:39, 988, 452-39, 990, 287 (hgl9) or one or more subregions thereof, e.g., via cfChlP-seq. In some embodiments such methods include a step of administering a DLL3-targeted therapeutic, e.g., an ADC such as rovalpituzumab tesirine to the subject. In some embodiments, the present disclosure encompasses methods that involve a step of detecting or quantifying the presence of an H3K4me3 modification at the DLL3 promoter, e.g., in a plasma sample, e.g., within genomic locus chrl9:39, 988, 452-39, 990, 287 (hg 19) or one or more subregions thereof, e.g., via cfChlP-seq where the subject is not or has not previously been determined to have SCLC in accordance with a method of the present disclosure, e.g., where the subject is independently diagnosed as having SCLC or where the subject has a non-SCLC cancer, e.g., Merkel cell carcinoma, neuroendocrine prostate cancer (NEPC) or melanoma. In some embodiments, detection or quantification of an H3K4me3 modification at the DLL3 promoter,e.g., in a plasma sample, e.g., within genomic locus chr 19:39,988,452-39,990,287 (hg 19) or one or more subregions thereof, e.g., via cfChIP-seq may be used for the selection of a DLL3-targeted therapeutic, e.g., an ADC such as rovalpituzumab tesirine to treat such a subject. In some embodiments such methods include a step of administering a DLL3-targeted therapeutic, e.g., an ADC such as rovalpituzumab tesirine to the subject.

[0130] It is to be understood that these other SCLC cancer therapies can also be used in treatment methods of the present disclosure.LUAD cancer therapies

[0131] Currently approved LUAD cancer therapies include chemotherapy, immunotherapy and targeted therapies.Chemotherapy

[0132] Not all subjects with non-small cell lung cancer (NSCLC), e.g., LUAD will need chemotherapy, but depending on the cancer's stage and other factors, chemotherapy may be recommended in the following situations. Before surgery (neoadjuvant) chemotherapy may be used (sometimes with radiation therapy) to try to shrink a tumor to remove it with less extensive surgery. After surgery (adjuvant) chemotherapy may be used (sometimes with radiation therapy) to try to kill any cancer cells that might have been left behind or have spread but can't be seen even on imaging tests. For locally advanced NSCLC (e.g., LUAD), chemotherapy along with radiation therapy is sometimes given as the main treatment for more advanced cancers that have grown into nearby structures so that surgery is not an option or for subjects who aren’t healthy enough for surgery. For metastatic (stage IV) NSCLC (e.g., LUAD) chemotherapy may be given for lung cancer that has spread to areas outside the lung such as the bones, liver, or adrenal gland.

[0133] The chemotherapeutic agents most often used for NSCLC (e.g., LUAD) include cisplatin, carboplatin, paclitaxel, albumin-bound paclitaxel (nab-paclitaxel), docetaxel, gemcitabine, vinorelbine, etoposide and pemetrexed. Combinations of two chemotherapeutic agents are often used to treat early-stage lung cancer. If a combination is used, it often includes cisplatin or carboplatin plus one other chemotherapeutic agent. Sometimes other combinations that do not include these chemotherapeutic agents, such as gemcitabine with vinorelbine or paclitaxel, may be used.

[0134] Chemotherapeutic agents for lung cancer are typically administered intravenously, either as an injection over a few minutes or as an infusion over a longer period of time.

[0135] Medical practitioners administer chemotherapy in cycles, with each period of treatment followed by a rest period to give the subject time to recover from the effects of the chemotherapeutic agents. Cycles are most often 3 or 4 weeks long, and initial treatment is typically 4 to 6 cycles. The schedule varies depending on the chemotherapeutic agents used. For example, some chemotherapeutic agents are given only on the first day of the chemotherapy cycle. Others are given for a few days in a row, or once a week. Then, at the end of the cycle, the chemotherapy schedule repeats to start the next cycle.

[0136] Adjuvant and neoadjuvant chemotherapy is often administered for 3 to 4 months, depending on the chemotherapeutic agents used. The length of treatment for advanced lung cancer is based on how well it is working for the subject in question.

[0137] For advanced cancers, the initial chemotherapy combination is often given for 4 to 6 cycles. Some medical practitioners now recommend giving treatment beyond this with a single chemotherapy or targeted therapy, in subjects who have had a good response to their initial chemotherapy or have had no worsening of their cancer. Continuing this treatment, known as maintenance therapy, seems to help keep the cancer in check and help some subjects live longer.

[0138] If the initial chemotherapy treatment for advanced lung cancer is no longer working, the medical practitioner may recommend second-line treatment with a single chemotherapeutic agent such as docetaxel or pemetrexed, or with immunotherapy or a targeted therapy.Immunotherapy

[0139] An important part of the immune system is its ability to keep itself from attacking normal cells in the body. To do this, it uses “checkpoints” or proteins on immune cells that need to be turned on (or off) to start an immune response. Cancer cells sometimes use checkpoints to avoid being attacked by the immune system. Therapeutic agents that target these checkpoints can be used to treat some subjects with non-small cell lung cancer (NSCLC, e.g., LU AD).PD-1 and PD-L1 inhibitors

[0140] Nivolumab, pembrolizumab, and cemiplimab target PD- 1, a protein on T cells that normally helps keep these cells from attacking other cells in the body. By blocking PD-1, these therapeutic agents boost the immune response against cancer cells. This can shrink some tumors or slow their growth.

[0141] Atezolizumab and durvalumab target PD-L1, a protein related to PD-1 that is found on some tumor cells and immune cells. Blocking this protein can help boost the immune response against cancer cells. This can shrink some tumors or slow their growth.

[0142] These immunotherapies can be used in different situations to treat NSCLC, e.g., LUAD. In some cases, before one of these therapeutic agents can be used, lab tests might need to be done on the cancer cells to show they have at least a certain amount of the PD-L1 protein.

[0143] Nivolumab can be used along with chemotherapy as a first treatment before surgery (known as neoadjuvant treatment) in some subjects with early-stage NSCLC, e.g., LUAD.

[0144] Pembrolizumab, atezolizumab, or cemiplimab can be used (sometimes with chemotherapy) as part of the first treatment in some subjects with metastatic NSCLC, e.g., LUAD.

[0145] Nivolumab can be an option as part of the first treatment in some subjects with metastatic NSCLC, e.g., LU AD, along with CTLA-4 inhibitor ipilimumab, which is described below. Similarly, durvalumab can be given along with the CTLA-4 inhibitor tremelimumab. Chemotherapy is often given along with these treatments as well.

[0146] Nivolumab, pembrolizumab, and atezolizumab can also be used in subjects with certain types of advanced NSCLC, e.g., LU AD whose cancer starts growing again after chemotherapy or other treatments.

[0147] For subjects with Stage III NSCLC, e.g., LU AD patients who cannot have surgery or chemotherapy with radiation, pembrolizumab or cemiplimab can be given as the first treatment.

[0148] Durvalumab can be used in subjects with Stage III NSCLC, e.g., LU AD patients whose cancer cannot be removed with surgery and has not gotten worse after they have received chemotherapy with radiation (chemoradiation). The goal of treatment with this therapeutic agent (also called consolidation therapy) is to keep the cancer from getting worse for as long as possible.

[0149] Atezolizumab or pembrolizumab can be used in subjects with some earlier stages of NSCLC, e.g., LU AD patients who have already been treated with surgery followed by chemotherapy. This is known as adjuvant therapy.

[0150] All of these therapeutic agents are given as an intravenous (IV) infusion.Depending on the therapeutic agent, they might be given every 2, 3, 4, or 6 weeks.CTLA-4 inhibitors

[0151] Ipilimumab and tremelimumab are also immunotherapies that boost the immune response, but they block CTLA-4, another protein on T cells that normally helps keep them in check. These therapeutic agents are used along with a PD- 1 inhibitor (ipilimumab with nivolumab, and tremelimumab with durvalumab); they are not used alone. They might be an option as part of the first treatment for certain types of advanced NSCLC, e.g., LU AD, most often along with chemotherapy as well.

[0152] These therapeutic agents are given by intravenous (IV) infusion, usually once every 3 or 6 weeks.Targeted therapies

[0153] As researchers have learned more about the changes in non-small cell lung cancer (NSCLC), e.g., LU AD cells that help them grow, they have developed therapeutic agents to specifically target these changes. Targeted therapies work differently from standard chemotherapy. They sometimes work when chemotherapy does not, and they often have different side effects. At this time, targeted therapeutic agents are most often used for advanced lung cancers, either along with chemotherapy or by themselves.Therapeutic agents that target tumor blood vessel growth (angiogenesis)

[0154] For tumors to grow, they need to form new blood vessels to keep them nourished. This process is called angiogenesis. Some targeted therapeutic agents called angiogenesis inhibitors, block this new blood vessel growth:

[0155] Bevacizumab is used to treat advanced NSCLC, e.g., LUAD. It is an antibody that targets vascular- endothelial growth factor (VEGF), a protein that helps new blood vessels to form. This therapeutic agent is often used with chemotherapy for a time. Then if the cancerresponds, the chemotherapy may be stopped and bevacizumab given by itself until the cancer starts growing again.

[0156] Ramucirumab can also be used to treat advanced NSCLC, e.g., LUAD. This therapeutic agent is an antibody that targets a VEGF receptor. It helps stop the formation of new blood vessels. This therapeutic agent is often combined with chemotherapy, typically after another treatment stops working.

[0157] Either of these therapeutic agents might also be used along with the targeted therapeutic agent erlotinib (see below) as the first treatment in subjects whose cancer cells have certain EGFR gene mutations.Therapeutic agents that target cells with KRAS gene changes

[0158] Some NSCLCs, e.g., LUADs have changes in the KRAS gene that cause them to make an abnormal form of the KRAS protein. This abnormal protein helps the cancer cells grow and spread.

[0159] About 1 in 8 subjects (13%) with NSCLC, e.g., LUAD have a specific type of KRAS gene change called a KRAS G12C mutation. NSCLCs, e.g., LUADs with this mutation are often resistant to other targeted therapeutic agents such as EGFR inhibitors (see below).

[0160] Sotorasib and adagrasib are therapeutic agents known as KRAS inhibitors. They work by attaching to the KRAS G12C protein, which helps keep cancer cells from growing. One of these therapeutic agents may be helpful if the subject has advanced NSCLC, e.g., LUAD, and the cancer cells are found to have the KRAS G12C mutation.

[0161] These therapeutic agents are administered as pills, typically once or twice a day.Therapeutic agents that target cells with EGFR gene changes

[0162] Epidermal growth factor receptor (EGFR) is a protein on the surface of cells. It normally helps the cells grow and divide. Sometimes NSCLC, e.g., LUAD cells have high levels of overactive EGFR, which makes them grow faster. Therapeutic agents called EGFR inhibitors can block the signal from EGFR that tells the cells to grow. Some of these therapeutic agents can be used to treat NSCLC, e.g., LUAD.

[0163] EGFR inhibitors used in NSCLC, e.g., LUAD with EGFR gene mutations include erlotinib, afatinib, gefitinib, osimertinib and dacomitinib.

[0164] For advanced NSCLC, e.g., LU AD one of these therapeutic agents is often used as the first treatment for advanced NSCLCs, e.g., LUADs that have certain mutations in the EGFR gene. Most of these therapeutic agents are used alone, although erlotinib can also be used along with a targeted therapeutic agent that affects new blood vessel growth (see above).

[0165] For earlier stage NSCLC, e.g., LU AD osimertinib can also be used as an adjuvant (additional) treatment after surgery for some earlier stage lung cancers with certain EGFR gene mutations.

[0166] All of these therapeutic agents are administered as pills.

[0167] A subset of EGFR inhibitors can be used to target cells with the T790M mutation. EGFR inhibitors can often shrink tumors for several months or more. But eventually these therapeutic agents stop working for most subjects, usually because the cancer cells develop another mutation in the EGFR gene. One such mutation is known as T790M. Osimertinib is an EGFR inhibitor that often works against cells with the T790M mutation.

[0168] A subset of EGFR inhibitors can be used to target cells with an exon 20 mutation. While the EGFR inhibitors listed above can help many subjects whose cancer cells have EGFR gene mutations, they do not help everyone. For example, cancer cells with an EGFR gene change known as an exon 20 insertion mutation are much less likely to affected by these therapeutic agents. However, other therapeutic agents that target cancer cells with an exon 20 mutation are now available. Amivantamab is a bispecific antibody that targets two proteins that help cancer cells grow: EGFR and MET. This therapeutic agent is given as an intravenous infusion.Mobocertinib is a therapeutic agent that targets the EGFR protein in a slightly different way. This therapeutic agent is administered as pills, typically once a day. These therapeutic agents can be used to treat advanced NSCLC, e.g., LU AD when the cancer cells have an exon 20 mutation, typically after chemotherapy has been tried.Therapeutic agents that target cells with ALK gene changes

[0169] About 5% of NSCLCs, e.g., LUADs have a rearrangement in a gene called ALK. This change is often seen in subjects who do not smoke (or subjects who are light smokers) who are younger and who have the adenocarcinoma subtype of NSCLC, i.e., LUAD. The ALK gene rearrangement produces an abnormal ALK protein that causes the cells to grow and spread. Therapeutic agents that target the abnormal ALK protein include crizotinib, ceritinib, alectinib,brigatinib, and lorlatinib. These therapeutic agents can often shrink tumors in subjects whose advanced lung cancers have an ALK gene change. Although they can help after chemotherapy has stopped working, they are often used instead of chemotherapy in subjects whose cancers have an ALK gene rearrangement. These therapeutic agents are administered as pills.Therapeutic agents that target cells with ROS1 gene changes

[0170] About 1% to 2% of NSCLCs, e.g.. LUADs have a rearrangement in a gene called R OS1. This change is most often seen in subjects who have the adenocarcinoma subtype of NSCLC, i.e., LU AD and whose tumors are also negative for ALK, KRAS and EGFR mutations. The ROS1 gene rearrangement is similar to the ALK gene rearrangement, and some therapeutic agents can work on cells with either ALK or ROS1 gene changes. Therapeutic agents that target the abnormal ROS1 protein include crizotinib, ceritinib, lorlatinib, lorbrena, and entrectinib. These therapeutic agents can often shrink tumors in subjects whose advanced lung cancers have a ROS1 gene change. Crizotinib or ceritinib might be used as first treatment, instead of chemotherapy, and lorlatinib may be used when crizotinib or ceritinib have stopped working. Entrectinib can be used in subjects with metastatic NSCLC, e.g., LU AD that has a ROS1 gene change. These therapeutic agents are administered as pills.Therapeutic agents that target cells with BRAF gene changes

[0171] In some NSCLCs, e.g., LUADs, the cells have changes in the BRAF gene. Cells with these changes make an altered BRAF protein that helps them grow. Dabrafenib is a type of therapeutic agent known as a BRAF inhibitor, which attacks the BRAF protein directly.Trametinib is known as a MEK inhibitor because it attacks the related MEK proteins. These therapeutic agents can be used together to treat metastatic NSCLC, e.g., LU AD if it has a certain type of BRAF gene change. These therapeutic agents are administered as pills or capsules each day.Therapeutic agents that target cells with RET gene changes

[0172] In a small percentage of NSCLCs, e.g., LUADs, the cells have certain changes in the RET gene that cause them to make an abnormal form of the RET protein. This abnormal protein helps the cells grow. Selpercatinib and pralsetinib are therapeutic agents known as RETinhibitors. They work by attacking the RET protein. These therapeutic agents can be used to treat advanced NSCLC, e.g., LU AD if the cancer cells have certain types of RET gene changes. These therapeutic agents are administered by mouth as capsules, typically once or twice a day.Therapeutic agents that target cells with MET gene changes

[0173] In some NSCLCs, e.g., LUADs, the cells have changes in the MET gene that cause them to make an abnormal form of the MET protein. This abnormal protein helps the cells grow and spread.

[0174] Capmatinib and tepotinib are types of therapeutic agents known as MET inhibitors. They work by attacking the MET protein. These therapeutic agents can be used to treat metastatic NSCLC, e.g., LU AD if the cancer cells have certain types of MET gene changes. Capmatinib is administered as pills, typically twice a day. Tepotinib is also administered as pills, but usually once a day.Therapeutic agents that target cells with HER2 gene changes

[0175] In a small percentage of NSCLCs, e.g., LUADs, the cancer cells have certain changes in the HER2 gene that help them grow. Trastuzumab deruxtecan is an antibody-drug conjugate (ADC). It is composed of an antibody that targets the HER2 protein (trastuzumab), which is linked to a chemotherapeutic agent (deruxtecan). The antibody acts like a homing signal by attaching to the HER2 protein on cancer cells, bringing the chemotherapy directly to them. This therapeutic agent can be used to treat NSCLC, e.g., LU AD that cannot be removed by surgery or that has spread, if the cancer cells have certain types of HER2 gene changes, and if at least one other treatment has already been tried. This therapeutic agent is infused into a vein (IV). It is typically given once every 3 weeks.Therapeutic agents that target cells with NTRK gene changes

[0176] A very small number of NSCLCs, e.g., LUADs have changes in one of the NTRK genes. Cells with these gene changes can lead to abnormal cell growth and cancer. Larotrectinib and entrectinib target and disable the proteins made by the NTRK genes. These therapeutic agents can be used in subjects with advanced lung cancer that is still growing despiteother treatments and whose tumor has an NTRK gene change. These therapeutic agents are administered as pills, once or twice daily.Other LOAD cancer therapies

[0177] While the sections above focus on FDA approved LU AD cancer therapies, many other LU AD cancer therapies are being developed and / or assessed in clinical trials (e.g., see Guo et al., Front Oncol (2022) 12:945102, the entire contents of which are incorporated herein by reference). It is to be understood that these other LU AD cancer therapies can also be used in treatment methods of the present disclosure.Subjects and Samples

[0178] A sample analyzed using methods, kits and systems provided herein can be any biological sample including any processed sample that includes circulating tumor DNA (ctDNA) derived from a biological sample. In various embodiments, a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a mammalian subject. In various embodiments, a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a human subject.

[0179] In various instances, a human subject is a subject diagnosed or seeking diagnosis as having, diagnosed as, or seeking diagnosis as at risk of having, and / or diagnosed as or seeking diagnosis as at immediate risk of having, a lung cancer, e.g., SCLC cancer, etc. In various instances, a human subject is a subject identified as needing SCLC / LUAD status screening. In certain instances, a human subject is a subject identified as needing SCLC / LUAD status screening by a medical practitioner.

[0180] The subject may not have undergone previous treatments for cancer, such as the treatments recited in this disclosure. In other embodiments, the subject has undergone previous treatments for cancer, such as the treatments recited in this disclosure.

[0181] In various embodiments a subject has one or more biomarkers and / or risk factors for cancer, e.g., lung cancer, e.g., SCLC cancer, etc. In certain embodiments, a human subject is identified as in need of SCLC / LUAD status screening based on an initial cancer diagnosis, e.g., a lung cancer diagnosis. In various instances, a human subject is a subject not yet diagnosed ashaving, not at risk of having, not at immediate risk of having, not diagnosed as having, and / or not seeking diagnosis for a cancer, e.g., a lung cancer.

[0182] In various embodiments, a sample from a subject, e.g., a human can be obtained from a liquid biopsy. In certain embodiments, a sample and / or reference is obtained from serum, plasma, or urine. In certain embodiments, the sample is serum. In certain embodiments, a sample comprises circulating tumor DNA (ctDNA). In certain embodiments, a sample is derived from about 1 mL of blood obtained from the subject. In certain embodiments, a sample is derived from about 0.5-5 mL of blood obtained from the subject, e.g., about 0.5 to about 2 mL, about 0.5 to 1.75 mL, about 0.5 to 1.5 mL, about 0.75 to 1.25 mL, about 0.9 to 1.1 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, or about 5 mL of blood.

[0183] In various embodiments, a sample is a sample of cell-free DNA (cfDNA). cfDNA is typically found in human biofluids (e.g., plasma, serum, or urine) in short, double-stranded fragments. The concentration of cfDNA is typically low, but can significantly increase under particular conditions, including without limitation pregnancy, autoimmune disorders, myocardial infarction, and cancer. Circulating tumor DNA (ctDNA) is the component of cell-free DNA specifically derived from cancer cells. ctDNA can be present in human biofluids bound to leukocytes and erythrocytes or not bound to leukocytes and erythrocytes. Various tests for detection of tumor-derived ctDNA are based on detection of genetic or epigenetic modifications that are characteristic of cancer (e.g., of a relevant cancer). Genetic or epigenetic factors characteristic of cancer can include, without limitation, oncogenic or cancer-associated mutations in tumor-suppressor genes, activated oncogenes, chromosomal disorders, histone modifications (e.g., histone methylation and / or histone acetylation), chromatin accessibility, binding of one or more transcription factors and / or DNA methylation.

[0184] In various embodiments, ctDNA comprises less than 30%, less than 20%, or less than 10% of the cfDNA in the liquid biopsy sample obtained from the subject, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less than 1% of the cfDNA in the sample. In some embodiments, the percentage of ctDNA in the liquid biopsy sample is assessed using ichorCNA which estimates the percentage of ctDNA in a sample probabilistically (see Adalsteinsson et al., Nat Commun (2017) 8(1): 1324 the entire contents of which are incorporated herein by reference).

[0185] cfDNA and ctDNA can provide a real-time or nearly real time metric of status of a source tissue. cfDNA and ctDNA demonstrate a half-life in blood of about 2 hours, such that a sample taken at a given time provides a relatively timely reflection of the status of a source tissue.

[0186] In some embodiments, a method comprises isolating DNA (e.g., cfDNA) from a liquid biopsy sample. Various methods of isolating nucleic acids from a sample (e.g., of isolating cfDNA from blood or plasma) are known in the art. Nucleic acids can be isolated using, without limitation, standard DNA purification techniques, by direct gene capture (e.g., by clarification of a sample to remove assay-inhibiting agents and capturing a target nucleic acid, if present, from the clarified sample with a capture agent to produce a capture complex and isolating the capture complex to recover the target nucleic acid).

[0187] Reagents and protocols for obtaining and analyzing cfDNA and ctDNA, such as circulating in blood or other tissue, are commercially available as described in the Examples and well-known in the art (see, for example, Anker et al., Cancer and Metastasis Rev (1999) 18:65-73; Wua et al., Clin Chim Acta (2002) 321:77-87; Fiegl et al., Cancer Res (2005) 15:1141-1145; Pathak et al., Clin Chem (2006) 52:1833-1842; Schwarzenbach et al., Clin Cancer Res (2009) 15:1032-1038; Schwarzenbach et al., Nat Rev Cancer (2011) 11:426-437) the contents of each of which is separately incorporated herein by reference in their entirety).

[0188] In various embodiments, samples can be collected from individuals repeatedly over a period of time (e.g., once daily, weekly, monthly, annually, biannually, etc.). In various embodiments, such samples can be used to verify results from earlier detections and / or to identify an alteration in biological pattern because of, for example, disease progression, resistance to therapy, treatment, remission, and the like. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three-month intervals according to the present disclosure. In various embodiments, samples can be collected for monitoring over time beginning at or at certain clinically determined stages, such as at resistance to a therapy, before radiographic progression, after radiographic progression, and / or at tissue biopsy. In addition, the SCLC / LUAD status obtained at different points in time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.

[0189] Samples include materials prepared by processes including, without limitation, steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants, desalting, concentration and / or extraction of sample nucleic acids, and / or amplification of sample nucleic acids (e.g., by PCR or other nucleic acid amplification techniques). Samples also include materials prepared by techniques that isolate, e.g., nucleosomes or transcription factors and / or nucleic acids associated with nucleosomes or transcription factors.

[0190] Removal from a sample of proteins that are not desirable for a relevant purpose or context (e.g., high abundance, uninformative, or undetectable proteins) can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and / or electrodialysis. High affinity reagents include antibodies or other reagents e.g., aptamers) that selectively bind to high abundance proteins. Sample preparation can also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filters include membranes that separate molecules based on size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration. Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.Electrodialysis is a procedure which uses an electromembrane or semipermeable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermeable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.

[0191] Separation and purification in the present disclosure may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip). Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used forelectrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.

[0192] Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and / or base and an organic such as an alcohol or acetonitrile.

[0193] Capillary isotachophoresis (CITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free- solution CE (FSCE), is based on differences in the electrophoretic mobility of the analytes, determined by the charge on the analytes, and the frictional resistance the analytes encounter during migration, which is often directly proportional to the size of the analytes. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.

[0194] Separation and purification techniques used in the present disclosure can include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

[0195] In some embodiments, whole blood is collected from a subject, and a plasma layer is separated by centrifugation. cfDNA may be then extracted from the plasma using methods known in the art.Histone Modifications, Chromatin Accessibility and Transcription Factor Binding

[0196] Histone methylation is understood to increase or decrease expression of associated coding sequences, depending on which histone residue is methylated. Histone methylation is an essential modification that can cause monomethylation (mel), dimethylation (me2), and trimethylation (me3) of several amino acids, thus directly affecting heterochromatin formation, gene imprinting, X chromosome inactivation, and gene transcriptional regulation. Histone methyltransferases promote monomethylation, dimethylation, or trimethylation of histones while histone demethylases promote demethylation of histones. In general, lysine (Lys or K), arginine (Arg or R), and rarely histidine (His or H) are the most common histone methyl acceptors. Histone methylation only occurs at specific lysine and arginine sites of histone H3 and H4. In histone H3, lysine 4, 9, 26, 27, 36, 56, and 79 and arginine 2, 8, and 17 can be methylated. By comparison, histone H4 has fewer methylation sites, in which only lysine 5, 12, and 20 and arginine 3 can be methylated. Histone methylation is often associated with transcriptional activation or inhibition of downstream genes. The methylation of histone H3K4, R8, R17, K26, K36, K79, H4R3, and K12 can activate gene transcription. However, the methylation of histone H3K9, K27, K56, H4K5, and K20 can inhibit gene transcription. Thus, for example, H3K4 methylation generally activates gene expression, while H3K27 methylation generally represses gene expression.

[0197] Histone acetylation occurs predominantly at lysine residues and is generally understood to increase expression of associated coding sequences. Without wishing to be bound by any theory, acetylation of lysine residues is thought to neutralize lysine’s positive charge and thereby cause histones to drift away from DNA, which has a negative charge. The released structure facilitates access to transcriptional machinery such as transcription factors and RNA polymerase II. Histone acetylation and deacetylation are generally catalyzed by histone acetyltransferases (HATs) and HDACs, respectively. Acetyl-CoA can be a source and co-factor of acetylation. In regulatory regions, HATs can acetylate histones and recruit HAT-containing complexes to activate the transcriptional process. For instance, H3K9ac and H3K27ac levels can be associated with promoter and enhancer activities. Furthermore, H3K27ac enhances not only the kinetics of transcriptional activation, but also accelerates the transition of RNA polymerase II from the initiation state to the elongation state.Differential modification of a genomic locus (e.g., differential histone methylation and / or differential histone acetylation) can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0198] Chromatin accessibility can refer to the degree to which nuclear macromolecules are able to physically contact DNA and is determined in part by the occupancy and modification status of nucleosomes. Modified histones can regulate chromatin accessibility through a variety of mechanisms, such as altering transcription factor (TF) binding through steric hindrance and modulating nucleosome affinity for active chromatin remodelers. The topological organization of nucleosomes across the genome is non-uniform: while histones can be densely arranged within facultative and constitutive heterochromatin, histones can be depleted at regulatory loci, including within enhancers, insulators and transcribed gene bodies. Active regulatory elements of the genome are generally accessible.

[0199] Differential accessibility of a genomic locus can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0200] A reference can be a value or set of values that are predetermined or derived from a sample or set of samples. A reference can be a sample or set of samples. A reference value can be a predetermined threshold value, a value that varies in accordance with circumstances (e.g., according to patient subpopulation, age, weight, or other variables), or a ratio. Reference ratios can be ratios relating to the modification and / or accessibility of multiple loci within individual samples and / or references, or across or between samples and / or references. In various embodiments, a reference can have or represent a normal, non-diseased state. In some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, a reference can have or represent a diseased state, e.g., a lung cancer, stage of lung cancer, orsubtype of lung cancer, e.g., SCLC or LU AD cancer. In some embodiments, a reference can represent SCLC cancer based on IHC testing. In some embodiments, a reference can represent LU AD cancer based on IHC testing. In some embodiments, a reference can correspond to a subject having lung cancer and / or a lung cancer subtype, e.g., SCLC or LU AD cancer.

[0201] In some embodiments, a reference is a predetermined threshold. In some embodiments, the predetermined threshold has previously been shown to be capable of distinguishing LU AD and SCLC subjects (e.g., distinguish with an AUROC of greater than 0.5). In some embodiments, a reference is a measurement from a liquid biopsy sample. In some embodiments, a reference is a measurement from liquid biopsy samples obtained from a cohort of subjects. In some embodiments, a reference is a normalized sample. In some embodiments, a reference is a measurement obtained from liquid biopsy samples obtained from a cohort of subjects who have previously been determined to have lung cancer, including, e.g., LU AD and / or SCLC.

[0202] In certain instances, a reference is a non-contemporaneous sample from the same source, e.g., a prior sample from the same source, e.g., from the same subject. In certain instances, a reference for the modification status of one or more genomic loci (e.g., one or more differentially modified genomic loci) can be the modification status of the one or more genomic loci (e.g., one or more differentially modified genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., SCLC cancer or LU AD cancer). In certain instances, a reference for the accessibility status of one or more genomic loci (e.g., one or more differentially accessible genomic loci) can be the accessibility status of the one or more genomic loci (e.g., one or more differentially accessible genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., SCLC cancer or LU AD cancer).

[0203] In some illustrative but non-limiting embodiments of the present disclosure differential modification or differential accessibility can refer to a differential (e.g., between a sample and a reference) with an absolute log2(fold-change) that is greater than or equal to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or more, or any range in between, inclusive, e.g., as measured according to an assay provided herein.

[0204] Enhancers are genomic loci that can be differentially modified or differentially accessible in and / or between conditions, diseases, and other states. Enhancers are cis-actingDNA regulatory regions that are thought to bind trans-acting proteins that contribute to expression patterns of associated genes. Chromatin ImmunoPrecipitation sequencing (ChlP-scq) of histone modifications (e.g., acetylation) have identified millions of enhancers in mammalian genomes. The number of active enhancers in any given cell type is estimated to be in the tens of thousands. Certain transcription factors (TFs), sometimes referred to as “master” transcription factors, associate with active enhancers with important impacts on gene expression and cell function. Certain such transcription factors preferentially associate with enhancers that regulate genes required for establishing cell identity and function, including enhancer domains known as “super-enhancers”. Moreover, master TFs can participate in inter-connected auto-regulatory circuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function to maintain cell state and / or cell survival.Techniques for Detecting and Quantifying Histone Modifications and Transcription Factor Binding

[0205] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying histone modifications and / or transcription factor binding. In some embodiments, the methods, kits and systems of present disclosure involve the detection and quantification of histone modifications and / or transcription factor binding in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. Chromatin ImmunoPrecipitation (ChIP) is one technique of molecular biology useful in detecting and quantifying histone modifications and transcription factor binding in samples. CUT& RUN or CUT& Tag are other more recent techniques that can also be used to detect and quantify histone modifications and transcription factor binding sites.

[0206] ChIP can involve various steps including one or more of fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. ChIP has become a very widely used tissue-based technique for determining the in vivo location of binding sites of various transcription factors and histones. Because the proteins are captured at the sites of their binding with DNA, ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniques such as PCR and real-time PCR, PCR with single-stranded conformational polymorphism,Southern blot analysis, Western blot analysis, cloning, and microarray. The resulting versatility has increased the potential of this technique.

[0207] ChIP of tissue samples usually involves cross-linking of the chromatin-bound proteins by formaldehyde, followed by sonication or nuclease treatment to obtain small DNA fragments. Immunoprecipitation can be then carried out using specific antibodies to the DNA-binding protein of interest. The DNA can be then released from the proteins and analyzed using various methods. ChIP has also been used to study RNA-protein interactions. X-ChIP methods utilize fixed chromatin fragmented by sonication, while the N-ChIP methods utilize native chromatin, which can be unfixed and nuclease digested.

[0208] The first step of the technique can be the cross-linking of DNA and proteins. Formaldehyde is one of the most used cross-linking agents. One advantage of using formaldehyde can be the ease of reversibility of the cross-links and its ability to form bonds that span approximately 2 angstroms. This means that formaldehyde can bind molecules in close association with each other. Generally, formaldehyde can be added to the medium in the cell culture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues has also been done. Other cross-linking agents that have been used include chemicals such as methylene blue and acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, and ultraviolet (UV) light and lasers.

[0209] Harvested chromatin can be sonicated in one or more sonication cycles. DNA can be typically broken into to 100-500 bp fragments to pinpoint the location of the DNA sequence of interest. An alternative to sonication can be nuclease digestion of the chromatin, e.g., in N-ChlP methods. Purification of chromatin can be achieved using a cesium chloride (CsCl) gradient centrifugation.

[0210] Chromatin can be enriched for a particular histone modification using an agent that binds the histone modification (e.g., immunoprecipitating using one or more antibodies that bind a target epitope).

[0211] For example, an antibody used in ChIP can selectively bind a particular transcription factor or one or more particular histone modifications, such as one or more particular histone acetylation modifications or histone methylation modifications. In some embodiments, an antibody used to bind a target epitope can be a “pan” antibody (e.g., a panacetylation antibody, a pan-methylation antibody, an antibody that binds a group of histonemodifications associated with increased transcription activation, and / or an antibody that binds a group of histone modifications associated with increased transcription repression). The antibody against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated. Immunosorbants commonly used to separate the antigen- antibody complex from the lysate include salmon sperm DNA-protein A-Sepharose®, protein G, magnetic beads, and other engineered immunoprecipitation systems known to those of skill in the art.

[0212] Immunoprecipitated DNA can be eluted. Once the DNA of interest is isolated, many detection and quantification methods can be used to study the isolated gene fragments. Commonly utilized methods include PCR, real-time PCR, slot blot hybridization, microarray techniques, and deep or next-generation sequencing. ChlP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. ChlP-seq can be used to map DNA-binding proteins, e.g., transcription factor binding sites and histone modifications in a genome-wide manner.

[0213] Cell-free Chromatin ImmunoPrecipitation sequencing (cfChlP-seq) involves applying ChlP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples including cfDNA such as plasma samples including cfDNA e.g., see Sadeh et al., Nat Biotechnol (2021) 39: 586-598 and Jang et al., Life Sei Alliance (2023) 6(12):e202302003 the entire contents of each of which are incorporated herein by reference). In some embodiments, cfChlP-seq uses antibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and / or H3K27ac) and / or transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject. Without limitation, exemplary antibodies that bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham, MA) and C15410003 (available from Diagenode in Denville, NJ) and exemplary antibodies that bind H3K27ac include ab21623 or ab4729 (both available from Abeam in Cambridge, UK) and C15210016 (available from Diagenode in Denville, NJ).

[0214] In some embodiments, the antibodies or antibody fragments can be covalently coupled to beads, e.g., epoxy beads. In some embodiments, the antibodies or antibody fragments can be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads. After washing, a cfDNA library is then typically prepared from the captured cfDNA. Library preparation can be done on-bead or after releasingthe captured cfDNA by digestion of bound histones, e.g., using proteinase K. The cfDNA library is then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation sequencing (NGS) as is known in the art. The reads are then analyzed, e.g., aligned and / or counted using standard bioinformatic techniques as is known in the art. A cfChlP-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference. In some embodiments, histone modifications at a given genomic loci can be quantified using sequencing data. E.g., in some embodiments, histone modifications can be quantified by counting the number of sequence reads that fall within a genomic loci (e.g., have at least one nucleotide overlapping with a genomic loci). In some embodiments, non-uniquely mapped and / or redundant sequence reads are discarded prior to quantifying histone modifications. In some embodiments, when quantifying histone modifications, sequence reads that fall within high noise regions of the genome are ignored.

[0215] In some embodiments, sequence reads are adjusted on the basis of sequencing depth prior to counting. Adjusting on the basis of sequencing depth can include, e.g., quantile normalizing sequence reads to a common reference distribution. In some embodiments, sequence reads are adjusted on the basis of ChIP quality prior to counting. In some embodiments, sequence reads are normalized relative to aggregate counts across a set of regions (e.g., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 or more regions) previously determined to have DNAse hypersensitivity in most cell types. In some embodiments, an estimate of local background signal is subtracted from the count of sequence reads at each genomic loci.

[0216] CUT& Tag involves antibody-based binding of a target protein, e.g., transcription factor or histone modification of interest, where antibody incubation is directly followed by the shearing of the chromatin and library preparation (see Kaya-Okur et al., Nat Comm (2019) 10:1930). CUT& Tag assays take advantage of a Tn5 transposase that is fused with Protein A to direct the enzyme to the antibody bound to its target on chromatin. Tn5 transposase is pre-loaded with sequencing adapters (generating the assembled pA-Tn5 adapter transposome) to carry out antibody-targeted tagmentation. In a typical CUT& Tag assay samples are incubated with an antibody immobilized on Concanavalin A-coated magnetic beads to facilitate subsequent washing steps. Cells can be incubated with a primary antibody specific for the target protein of interest followed by incubation with a secondary antibody. Samples can then be incubated withassembled transposomes, which consist of Protein A fused to the Tn5 transposase enzyme that is conjugated to NGS adapters. After incubation, unbound transposomc can be washed away using stringent conditions. Tn5 is a Mg2+-dependent enzyme so Mg2+can be added to activate the reaction, which results in the chromatin being cut close to the protein binding site and simultaneous addition of the NGS adapter DNA sequences. Chromatin cleavage and library preparation can be achieved in one single step.

[0217] CUT& RUN is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing (see Skene and Henikoff, Elife (2017) 6:1-35, Skene et al., Nat Protoc (2018) 13:1006-1019). As only targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT& RUN has low background levels. In an example CUT& RUN assay, a sample is incubated with an antibody or antibody fragment that binds the target protein, e.g., transcription factor or histone modification of interest. The sample is then incubated with Protein- A-MNase after which CaCE can be added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the target protein. The protein-A-MNase reaction can be quenched by adding chelating agents (EDTA and EGTA). Cleaved DNA fragments are then liberated, extracted, and used to construct a sequencing library.

[0218] A person of skill in the art is aware of suitable DNA sequencing technologies for use in methods described herein that comprise a sequencing step. Suitable DNA sequencing technologies include, e.g., next generation sequencing (NGS) approaches. Additional steps that are required to prepare DNA for sequencing via an appropriate sequencing approach can be incorporated into methods described herein. For example, in some embodiments, a method described herein comprises attaching (e.g., ligating) DNA adapters to cfDNA. In some embodiments, DNA adapters can be attached prior to, during, or after enrichment for a histone modification. In some embodiments, a method comprises amplifying cfDNA after attaching DNA adapters.Techniques for Detecting and Quantifying Chromatin Accessibility

[0219] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying chromatin accessibility. In some embodiments, the methods, kits and systems of the present disclosure involve the detectionand quantification of chromatin accessibility in samples, e.g... in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. ATAC-seq (Assay for Transposase Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde- Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and DNase hypersensitivity assays are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples. Sono-Seq is another alternative method that could be used (see Auerbach et al., Proc Natl Acad USA (2009) 106(35):14926-14931).

[0220] DNase hypersensitivity assays can use the non-specific DNA endonuclease Deoxyribonuclease I (DNase I), which selectively digests accessible DNA regions. DNase I hypersensitivity sites (DHS) identified by DNase-seq include open chromatin regulatory regions. A typical DNase hypersensitivity assay can include a first step in which nuclei are isolated from cells using lysis buffer, and nuclei are digested using DNase I. DNA fragment sizes are measured to identify optimal digestion using gel electrophoresis. Biotinylated linkers can be ligated to the ends of digested DNA after polishing to make blunt ends, and the DNA can then be isolated. DNA with biotinylated linker can be digested by restriction endonuclease Mmel and captured by streptavidin coated Dynabeads® to generate short tags to which a second sequencing adaptor can be ligated. A second linker can be ligated and amplified to generate a library for sequencing. A DNase-seq bioinformatic pipeline can include, e.g.. alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.

[0221] MNase-seq determines chromatin accessibility with micrococcal nuclease (MNase) that preferentially digests nucleosome-free, protein-unbound DNA. A typical MNase-seq assay can include a first step in which nuclei are isolated from either native or crosslinked chromatin and digested using MNase with titration. In vivo formaldehyde crosslinking step that is designed to capture the interaction between proteins and DNA. This crosslinking allows bound proteins to shield their associated DNA from digestion by MNase. Following crosslinking, samples are digested with MNase, which can be specifically activated by addition of Ca2+ to the buffer. Digestion can be halted by chelating the reaction, at which point the samples are RNase treated, crosslinks are reversed, and proteins are digested away from the chromatin. DNA can then be isolated via a phenol-chloroform extraction. Uncut DNA is purified andmononucleosome bands are isolated and excised through gel electrophoresis. Isolated DNA can be amplified by adding adapters to generate a library, and sequenced. MNase-seq primarily sequences regions of DNA bound by histones or other proteins. Therefore, it indirectly determines which regions of DNA are accessible by directly determining which regions are bound to nucleosomes or proteins.

[0222] FAIRE-seq is a method in which nucleosome-depleted regions of DNA (NDRs) are isolated from chromatin. A typical FAIRE-seq assay can include a first step in which cells are fixed using formaldehyde so that histones are crosslinked to interacting DNA. Crosslinked chromatin can then be sheared by sonication that generates protein-free DNA and protein-crosslinked DNA fragments. Protein-free DNA can be isolated using a phenol-chloroform extraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA stays in aqueous phase. Highly crosslinked DNA remains in the organic phase and the non-crosslinked DNA is pulled to the aqueous phase. Non-crosslinked DNA from the aqueous phase can then be amplified and sequenced. Reads enriched in the sequencing pool tend to have lower nucleosome and transcription factor binding and are therefore inferred to come from accessible regions.

[0223] NOMe-seq is a method to identify nucleosome-depleted regions of DNA (NDRs) with M. CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins. Unlike CmpG, GpCmin the human genome does not occur naturally in most cell types. GpCmlevels at open chromatin regions can be compared to background signals and used to detect and quantify NDRs. A typical NOMe-seq protocol can include a step in which samples are treated with M. CviPI and S-adenosylhomocysteine (SAM) to methylate accessible GpC sites. M. CviPI treated DNA can be sheared using a sonicator, so that DNA fragments can be sequenced. DNA is treated with bisulfite, which converts unmethylated cytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected. A library is generated using adapters and sequenced. Accessible chromatin is expected to have high levels of GpCmbut low levels of CmpG. Therefore, NOMe-seq identifies NDRs using the two separate methylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.

[0224] ATAC-seq uses hyperactive Tn5 transposase that preferentially cuts accessible chromatin regions and simultaneously inserts adapters to the fragmented region (Buenrostro et al., Nat Methods (2013) 10(12): 1213- 1218 the entirety of which is incorporated herein byreference). A typical ATAC-seq assay can include a first step in which samples are incubated with Tn5 transposase. DNA can then be isolated and purified. DNA fragmented and tagged by Tn5 transposase can be purified and then amplified to generate a library and sequenced for analysis.Techniques for Detecting and Quantifying DNA Methylation

[0225] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying DNA methylation. In some embodiments, the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq) are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples. Reduced representation bisulfite sequencing (RRBS) is another alternative method that could be used (see Meissner et al., Nucleic Acids Res (2005) 33(18):5868-5877). Illumina Infinium arrays could also be used to detect and quantify DNA methylation.

[0226] DNA methylation typically refers to the methylation of the 5’ position of cytosine (mC) by DNA methyltransferases (DNMT). It is a major epigenetic modification in humans and many other species. In mammals, most DNA methylations occur within the context of CpG dinucleotides. DNA methylation is thought to be a repressive chromatin modification. Aberrant methylation can lead to many diseases including cancers (Robertson, Nat Rev Genet (2005) 6:597-610 and Bergman and Cedar, Nat Struct Mol Biol (2013) 20:274-281).

[0227] Bisulfite sequencing (BS-Seq) or Whole-Genome Bisulfite Sequencing (WGBS) is a well-established protocol to detect methylated cytosines in genomic DNA. In this method, genomic DNA is treated with sodium bisulfite and then sequenced, providing single-base resolution of methylated cytosines in the genome. Upon bisulfite treatment, unmethylated cytosines are deaminated to uracils which, upon sequencing, are converted to thymidines. Simultaneously, methylated cytosines resist deamination and are read as cytosines. The location of the methylated cytosines can then be determined by comparing treated and untreated sequences.

[0228] In some embodiments, methylated DNA can be sequenced using a method that comprises enriching for cfDNA that comprises methylated DNA. Enrichment can be accomplished e.g., using an agent that selectively binds methylated DNA (e.g., an antibody as in MeDIP-seq or a methyl-CpG-Binding Domain (MBD), as in MBD-seq). In some embodiments, an agent that binds methylated DNA is attached (e.g., via a covalent or noncovalent bond) to a physical support (e.g., a bead, a magnetic bead, an agarose bead, or a magnetic epoxy bead), wherein the attaching can be prior to, during, or after incubation with a sample.

[0229] MeDIP-seq was first reported by Weber et al., Nat Genet (2005) 37:853-862. In a typical MeDIP-seq protocol, antibody or antibody-fragment that binds 5 -methylcytidine (5mC) is used to enrich methylated DNA fragments, then these fragments are sequenced and analyzed. If using 5mC-specific antibodies or antibody fragments, methylated DNA is isolated from genomic DNA via immunoprecipitation. Anti-5mC antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing.

[0230] Methyl-CpG-Binding Domain sequencing (MBD-seq) is similar to MeDIP-seq except that it uses methyl binding domain (MBD) proteins instead of antibodies or antibody fragments to bind methylated DNA. In a typical MBD-seq protocol, genomic DNA is first sonicated and incubated with tagged MBD proteins that can bind methylated cytosines. The protein-DNA complex is then precipitated with antibody-conjugated beads that are specific to the MBD protein tag, followed by DNA purification and sequencing.

[0231] In some embodiments, DNA methylation at a given genomic loci can be quantified by sequencing methylated DNA. For example, in some embodiments, DNA methylation at a genomic loci can be quantified by counting the number of sequence reads that overlap with the genomic loci (e.g., comprise at least one nucleotide that overlaps with the genomic loci).

[0232] A person of skill in the art is aware of suitable DNA sequencing technologies for use in methods described herein that comprise a sequencing step. Suitable DNA sequencing technologies include, e.g., next generation sequencing (NGS) approaches. Additional steps that are required to prepare DNA for sequencing via an appropriate sequencing approach can be incorporated into methods described herein. For example, in some embodiments, a method described herein comprises attaching (e.g., ligating) DNA adapters to cfDNA. In someembodiments, DNA adapters can be attached prior to, during, or after enrichment for a histone modification.Classifiers

[0233] In some embodiments, the present disclosure provides methods for obtaining a classifier, e.g., a validated classifier that can be used to determine SCLC / LUAD status. In some embodiments, a subject is determined to have a validated epigenetic profile indicative of an SCLC or LU AD cancer based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject, wherein the presence of the validated epigenetic profile has been determined using a validated classifier.

[0234] For illustration purposes and without limitation, in an exemplary embodiment of the present disclosure, the validated classifier may be obtained by:

[0235] (a) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a first cohort of subjects who have previously been determined to have an SCLC cancer (e.g., de novo SCLC or transformed SCLC);

[0236] (b) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a second cohort of healthy subjects or subjects who have previously been determined to have LU AD cancer;

[0237] (c) comparing the genomic profile determined in step (a) and the genomic profile determined in step (b), to identify genomic loci that have statistically different histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“differential loci”);

[0238] (d) training a classifier on histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels in the differential loci to distinguish between (i) samples from one or more biological samples obtained from the first cohort, and (ii) samples from one or more biological samples obtained from the second cohort, to identify samples having a profile of histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“epigenetic profile”) that indicates that the samples are likely obtained from the first cohort; and

[0239] (e) obtaining the validated classifier by validating the classifier from step (d) on a third cohort comprising an independent and blinded group of subjects with SCLC and LU AD cancers and selecting a threshold such that the validated classifier predicts SCLC cancers, with an area under the receiver operating characteristic (AUROC) greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95), wherein subjects falling within the group of predicted SCLC cancers display the validated epigenetic profile and subjects that do not fall within the group of SCLC cancers lack the validated epigenetic profile.

[0240] A person of ordinary skill will appreciate that other methods can be used to obtain a classifier, e.g., a validated classifier that can be used to determine SCLC / LUAD status and that the present disclosure is not limited to classifiers obtained in accordance with this method.Exemplary Genomic Loci

[0241] The present disclosure includes the identification of exemplary genomic loci that are differentially modified and / or differentially accessible in SCLC vs. LU AD cancer. See Tables 1-3 which show the chromosomal coordinates of each genomic locus and whether they are correlated with SCLC or LUAD cancer (genomic loci in columns with “Genomic locus (SCLC)” in the header correlate with SCLC while genomic loci with “Genomic locus (LUAD)” in the header correlate with LUAD cancer). The genomic loci are sorted based on their chromosomal coordinates which are based on human genome build hgl9.

[0242] The present disclosure is not limited to methods that use the exact same chromosomal coordinates that are recited in Tables 1-13. The present disclosure encompasses methods that use any of the genomic loci in Table 1-13 and also subregions thereof, i.e., references herein to methods that involve detecting and / or quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci of Table 1-13 encompasses methods that detect these marks anywhere within these genomic loci including within any subregions. For example, where Table 2 references chrl:110800810-110801156 as a genomic locus for detecting and / or quantifying H3K27ac modification, this encompasses methods that detect and / or quantify H3K27ac modification at any position or sub-region of chrl: 110800810- 110801156, e.g., methods that detect and / or quantify H3K27ac modification within chrl:110800910-110801056,etc. In some embodiments, a subregion may span at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-13. In some embodiments, a subregion may span less than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-13. In some embodiments, a subregion may have the same central coordinate as a genomic locus recited in Tables 1-13. In some embodiments, a subregion may have a different central coordinate as a genomic locus recited in Tables 1-13. It is also to be understood that the lower / upper coordinates of the genomic loci in Tables 1-13 are approximate and that the present disclosure encompasses methods where any one or more of the genomic loci are expanded by increasing the size of the genomic locus by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or up to 50% in one or both directions.

[0243] In some embodiments a classifier is generated using a set of differentially modified and / or differentially accessible genomic loci that are correlated with SCLC and a set of differentially modified and / or differentially accessible loci that are correlated with LU AD cancer. Sequence reads that fall into each selected genomic locus are analyzed and counted, e.g., as described herein including the Examples. In some embodiments, counts from genomic loci that are correlated with SCLC are aggregated and counts from genomic loci that are correlated with LU AD cancer are aggregated. In some embodiments, a ratio of the aggregated SCLC and LU AD cancer counts is used to determine SCLC / LUAD status. Other ways of using the genomic loci and related sequencing data to generate and apply a classifier to determine SCLC / LUAD status are described herein and known in the art, e.g., without limitation, methods that use a learning statistical classifier system or a combination of learning statistical classifier systems.

[0244] In some embodiments, exemplary genomic loci from one or more of Tables 1-13 are used in a monomodal classifier, e.g., a classifier that uses a single histone modification (e.g., H3K4me3 or H3K27ac) or DNA methylation at one or more genomic loci for purposes of determining SCLC / LUAD status. In some embodiments, exemplary genomic loci from any one of Table 1-13, or any combination thereof, are used in combination in a multimodal classifier, e.g., a classifier that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or one or more histone modifications (e.g., H3K4me3 and / or H3K27ac) and DNA methylation at one or more genomic loci for purposes of determining SCLC / LUAD status.

[0245] In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor at one or more loci provided in one or more of Tables 1-13. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 800, 1,000, 1,500, 2,000, 3,000, 4,000, or more loci listed in one or more of Tables 1-13 (e.g., 1-200, 5-200, 10-200, 15-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 1-150, 5-150, 10-150, 15-150, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 1-100, 5-100, 10-100, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70- 100, 80-100, 90-100). In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor at each of the loci provided in one or more of Tables 1-13. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor for at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in one or more of Tables 1-13. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor for at least a percent of loci identified in Table 13 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%.Differential H3K4me3 modification

[0246] Genomic loci demonstrating differential H3K4 methylation (in particular H3K4 trimethylation, H3K4me3) in SCLC vs. LU AD cancer are provided in Table 1 which shows the chromosomal coordinates of each genomic locus and whether they are correlated with SCLC or LU AD cancer (genomic loci in columns with “Genomic locus (SCLC)” in the header correlate with SCLC while genomic loci with “Genomic locus (LU AD)” in the header correlate with LU AD cancer). The genomic loci are sorted based on their chromosomal coordinates which are based on human genome build hgl9.

[0247] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Table 1 be assessed for H3K4me3 modification.Instead, a subset of loci may be assessed for H3K4me3 modification. Subsets of the genomic loci of Table 1 can be selected (e.g., for use in determining SCLC / LUAD status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Table 1, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining SCLC / LUAD status. See also the Examples of the present disclosure for experiments showing that informative classifiers can be generated using many different combinations of the loci. The present disclosure particularly includes, among other things, subsets of the genomic loci of Table 1, which have an absolute log2(fold-change) of 6.0 or higher, 5.5 or higher, 5.0 or higher, 4.5 or higher, 4.0 or higher, 3.5 or higher, 3.0 or higher, 2.5 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher. The present disclosure also includes subsets of the genomic loci of Table 1, which have an absolute log2(fold-change) of 6.0 or higher, 5.5 to less than 6.0, 5.0 to less than 5.5, 4.5 to less than 5.0, 4.0 to less than 4.5, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, or 0.6 to less than 0.8.

[0248] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have aparticular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 1 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 1 (e.g., about 1 to about 1,000, about 5 to about 3,000, about 10 to about 1000, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 1 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 1 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0249] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthysubject or a subject with LUAD cancer) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Table 1). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 10 loci identified in Table 1 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 25 loci identified in Table 1 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 50 loci identified in Table 1 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 10 loci identified in Table 1 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 25 loci identified in Table 1 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 50 loci identified in Table 1 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer).

[0250] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Table 1 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer).In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 25 loci identified in Table 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Table 1 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 25 loci identified in Table 1 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 50 loci identified in Table 1 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 1 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0251] In various embodiments, differentially H3K4me3 modified refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / ormode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16-fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. In various embodiments, an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold. 1.2-fold to 4.0-fold. 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6.

[0252] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 loci identified in Table 5 (or any subset thereof), e.g., of those loci listed as H3K4me3 loci, are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 5 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 15 and an upper bound selected from 10, 15, and 20 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained orderived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 15 or 20 loci identified in Table 5 (e.g., about 1 to about 20, about 2 to about 20, about 5 to about 20, about 5, about 10, about 15, about 20 loci) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 5 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 5 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0253] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 loci identified in Table 12 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 12 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, or 135 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 12 (e.g., about 1 to about 135, about 5 to about 135, about 10 to about 135, about 1 to about 135, about 1 to about 135, about 1 to about 135, about 1 to about 135, about 1 to about 135, about 1 to about 100,about 2 to about 135, about 5 to about 135, about 10 to about 135, about 20 to about 135, about 25 to about 135, about 50 to about 135, about 20 to about 135, about 50 to about 135, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, or about 135) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular’ SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 12 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 12 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0254] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 loci identified in Table 13 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 loci identified in Table 13 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 13 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). Incertain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least a percent of loci identified in Table 13 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC).Differential H3K27ac modification

[0255] Genomic loci demonstrating differential H3K27ac modification in SCLC vs. LU AD cancer are provided in Table 2 which shows the chromosomal coordinates of each genomic locus and whether they are correlated with SCLC or LU AD cancer (genomic loci in columns with “Genomic locus (SCLC)” in the header correlate with SCLC while genomic loci with “Genomic locus (LUAD)” in the header correlate with LU AD cancer). The genomic loci are sorted based on their chromosomal coordinates which are based on human genome build hgl9.

[0256] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Table 2 be assessed for H3K27ac modification.Instead, a subset of loci may be assessed for H3K27ac modification. Subsets of the genomic loci of Table 2 can be selected (e.g., for use in determining SCLC / LUAD status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Table 2, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining SCLC / LUAD status. See also the Examples of the present disclosure for experiments showing that informative classifiers can be generated using many different combinations of the loci. The present disclosure particularly includes, among other things, subsets of the genomic loci of Table 2, which have an absolute log2(fold-change) of 6.0 or higher, 5.5 or higher, 5.0 or higher, 4.5 or higher, 4.0 or higher, 3.5 or higher, 3.0 or higher, 2.5 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 orhigher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher. The present disclosure also includes subsets of the genomic loci of Tabic 2, which have an absolute log2(fold-change) of 6.0 or higher, 5.5 to less than 6.0, 5.0 to less than 5.5, 4.5 to less than 5.0, 4.0 to less than 4.5, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, or 0.6 to less than 0.8.

[0257] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 (or any subset thereof) are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 2 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 is found to be H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 2 (e.g., about 1 to about 1,000, about 5 to about 3,000, about 10 to about 1000, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are H3K27ac modified ascompared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 2 are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 2 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0258] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Table 2). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 10 loci identified in Table 2 is H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 25 loci identified in Table 2 is H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 50 loci identified in Table 2 is H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have aparticular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 10 loci identified in Table 2 arc H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 25 loci identified in Table 2 are H3K27ac modified as compared to a reference (e.g.. a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 50 loci identified in Table 2 are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer).

[0259] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Table 2 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 (or any subset thereof) in total are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 25 loci identified in Table 2 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 (or any subset thereof) in total are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Table 2 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 (or any subset thereof) in total are H3K27ac modified as compared to a reference (e.g., asample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 25 loci identified in Table 2 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 (or any subset thereof) in total are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status e.g., SCLC status) if at least five of the top 50 loci identified in Table 2 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 2 (or any subset thereof) in total are H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer).

[0260] In various embodiments, differentially H3K27ac modified refers to an acetylation status characterized by an increase or decrease in a value measuring acetylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16-fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. In various embodiments, an increase or decrease in a value measuring acetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1,0-fold to 4.0-fold. 1,2-fold to 4.0-fold. 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6.

[0261] Genomic loci demonstrating differential H3K27ac modification in different subtypes of SCLC are provided in Table 4, which shows chromosomal coordinates of each genomic locus and which subtype they are associated with. In some embodiments, a method described herein comprises assessing H3K27ac modifications at 1, 2, 3, or 4 of the genomic loci listed in Table 4.

[0262] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, or 7 loci identified in Table 5 (or any subset thereof), e.g., of those loci listed as H3K27ac loci, are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if 1, 2, 3, 4, 5, 6, or 7 loci of Table 5 arc differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0263] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or 5500 loci identified in Table 6 (or any subset thereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 6 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or5500 is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 6 (e.g., about 1 to about 5500, about 5 to about 5500, about 10 to about 5500, about 1 to about 1000, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 6 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0264] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 loci identified in Table 7 (or any subset thereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject withSCLC). Tn certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least a number of loci identified in a Table 7 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 7 (e.g., about 1 to about 5000, about 5 to about 5000, about 10 to about 5000, about 1 to about 1000, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 7 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least a percent of loci identified in Table 7 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC).Differential DNA methylation

[0265] Genomic loci demonstrating differential DNA methylation in SCLC vs. LU AD cancer are provided in Table 3 which shows the chromosomal coordinates of each genomic locus and whether they are correlated with SCLC or LU AD cancer (genomic loci in columns with “Genomic locus (SCLC)” in the header correlate with SCLC while genomic loci with “Genomic locus (LU AD)” in the header correlate with LUAD cancer). The genomic loci are sorted based on their chromosomal coordinates which are based on human genome build hgl9.

[0266] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Table 3 be assessed for DNA methylation. Instead, a subset of loci may be assessed for DNA methylation. Subsets of the genomic loci of Table 3 can be selected (e.g., for use in determining SCLC / LUAD status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Table 3, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining SCLC / LUAD status. See also the Examples of the present disclosure for experiments showing that informative classifiers can be generated using many different combinations of the loci. The present disclosure particularly includes, among other things, subsets of the genomic loci of Table 3, which have an absolute log2(fold-change) of 6.0 or higher, 5.5 or higher, 5.0 or higher, 4.5 or higher, 4.0 or higher, 3.5 or higher, 3.0 or higher, 2.5 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher. The present disclosure also includes subsets of the genomic loci of Table 3, which have an absolute log2(fold-change) of 6.0 or higher, 5.5 to less than 6.0, 5.0 to less than 5.5, 4.5 to less than 5.0, 4.0 to less than 4.5, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to lessthan 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, or 0.6 to less than 0.8.

[0267] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5,000 loci identified in Table 3 (or any subset thereof) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 3 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 3 (e.g., about 1 to about 1,000, about 5 to about 3,000, about 10 to about 1000, about 25 to about 200, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of lociidentified in Table 3 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 3 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0268] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 3 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Table 3). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 10 loci identified in Table 3 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 25 loci identified in Table 3 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 50 loci identified in Table 3 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 10 loci identified in Table 3 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUADstatus (e.g., SCLC status) if at least five of the top 25 loci identified in Table 3 are differentially DNA methylated as compared to a reference e.g., a sample from a healthy subject or a subject with LU AD cancer). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 50 loci identified in Table 3 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer).

[0269] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Table 3 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 3 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 25 loci identified in Table 3 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 3 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Table 3 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 3 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 25 loci identified in Table 3 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 3 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least five of the top 50 loci identified in Table 3 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, or 3000 loci identified in Table 3 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0270] In various embodiments, differentially DNA methylated refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16-fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16-fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6. In various embodiments, an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold. 1.2-fold to 4.0-fold. 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, le-2, 5e-3, le-3, 5e-4, le-4, 5e-5, le-5, 5e-6, or le-6.

[0271] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, or 4 loci identified in Table 5 (or any subset thereof), e.g., of those loci listed as MBD genomic loci, are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status e.g., SCLC status) if 1, 2, 3, or 4 loci of Table 5 are differentially methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0272] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 650 loci identified in Table 8 (or any subset thereof) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 8 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 650 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD cancer). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 8 (e.g., about 1 to about 650, about 5 to about 650, about 10 to about 650, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130,about 135, about 140, about 145, or about 150 loci) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 8 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD cancer). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 8 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference e.g., a sample from a healthy subject or a subject with LU AD cancer).

[0273] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, or 600 loci identified in Table 9 (or any subset thereof) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least a number of loci identified in a Table 9 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 9 (e.g., about 1 to about 600, about 5 to about 600, about 10 to about 600, about 1 to about 550, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 9 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if at least a percent of loci identified in Table 9 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC).Differential chromatin accessibility or transcription factor binding

[0274] Genomic loci provided in Tables 1-13 can also demonstrate differential chromatin accessibility or transcription factor binding in SCLC cancer vs. LUAD cancer.

[0275] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0276] In some embodiments, without wishing to be limited to any particular- scientific theory, chromatin accessibility corresponds and / or is correlated with H3K4me3 modifications. As a result, in some embodiments, SCLC / LUAD status may be determined by detecting andquantifying chromatin accessibility at one or more genomic loci in Table 1 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.

[0277] In some embodiments, without wishing to be limited to any particular' scientific theory, chromatin accessibility corresponds and / or is correlated with H3K27ac modifications. As a result, in some embodiments, SCLC / LUAD status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Table 2 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications.

[0278] In some embodiments, without wishing to be limited to any particular' scientific theory, chromatin accessibility corresponds and / or is correlated with DNA methylation. As a result, in some embodiments, SCLC / LUAD status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Table 3 in accordance with the section above discussing exemplary genomic loci with differential DNA methylation.

[0279] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0280] In some embodiments, without wishing to be limited to any particular' scientific theory, binding of RNA pol II corresponds and / or is correlated with H3K4me3 modifications. As a result, in some embodiments, SCLC / LUAD status may be determined by detecting and quantifying binding of RNA pol II at one or more genomic loci in Table 1 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.

[0281] In some embodiments, without wishing to be limited to any particular scientific theory, binding of p300, mediator complex, cohesin complex or RNA pol II corresponds and / or is correlated with H3K27ac modifications. As a result, in some embodiments, SCLC / LUAD status may be determined by detecting and quantifying binding of p300, mediator complex, cohesin complex or RNA pol II at one or more genomic loci in Table 2 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications.

[0282] In some embodiments, without wishing to be limited to any particular' scientific theory, binding of NKX2-1, ASCL1, POU2F3, NEURODI, YAP1, MYC, SOX2, or HNF4a,corresponds and / or is correlated with histone methylation (e.g., H3K4me3), histone acetylation (e.g., H3K27ac) or DNA methylation. As a result, in some embodiments, SCLC / LUAD status may be determined by detecting and quantifying binding of NKX2-1, ASCL1, POU2F3, NEURODI, YAP1, MYC, SOX2, or HNF4a at one or more genomic loci in Tables 1-3 in accordance with the sections above discussing exemplary genomic loci with differential histone methylation (e.g., H3K4me3), histone acetylation (e.g., H3K27ac) or DNA methylation.

[0283] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 loci identified in Table 10 (or any subset thereof) have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a number of loci identified in a Table 10 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 is found to have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 10 (e.g., about 1 to about 5000, about 5 to about 5000, about 10 to about 5000, about 1 to about 1000, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) have differentialchromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with LU AD). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 10 have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., SCLC status) if at least a percent of loci identified in Table 10 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with LUAD).

[0284] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 loci identified in Table 11 (or any subset thereof) have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LUAD status) if at least a number of loci identified in a Table 11 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500, 3000, 3500, 4000, or 4500 is found to have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular’ SCLC / LUAD status (e.g., LUAD status) if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Table 11 (e.g., about 1 to about 4500, about 5 to about 4500, about 10 to about 4500, about 1 to about 1000, about 1 to about 900, about 1 to about 800, about 1 to about 700, about 1 to about 600, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200,, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) have differential chromatin accessibility as compared to a reference (e.g.. a sample from a healthy subject or a subject with SCLC). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Table 11 have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular SCLC / LUAD status (e.g., LU AD status) if at least a percent of loci identified in Table 11 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to have differential chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject or a subject with SCLC).Applications

[0285] Methods, kits and systems of the present disclosure include analysis of differentially modified and / or differentially accessible genomic loci to determine the SCLC / LUAD status of a lung cancer. Methods, kits and systems of the present disclosure can be used in any of a variety of applications. For example, methods, kits and systems of the present disclosure can be used in detecting and / or treating cancers based on SCLC / LUAD status.Methods, kits and systems of the present disclosure can also be used to detect or determine resistance of a lung cancer, e.g., LU AD cancer to a therapy or transformation of a lung cancer, e.g., from LU AD to SCLC.

[0286] In various embodiments, methods, kits and systems of the present disclosure can be applied to an asymptomatic human subject. As used herein, a subject can be referred to as “asymptomatic” if the subject does not report, and / or demonstrate by non-invasively observableindicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or cancer screening), sufficient characteristics of cancer to support a medically reasonable suspicion that the subject is likely suffering from cancer, e.g., lung cancer. Detection of early-stage cancer can be achieved using methods, kits and systems of the present disclosure, with attendant medical benefits including potential for early treatment and attendant improvement in therapeutic outcomes.

[0287] In various embodiments, methods, kits, and systems of the present disclosure can be applied to a human subject that has increased susceptibility for lung cancer (including SCLC and / or LU AD). Exemplary factors that increase susceptibility for lung cancer include a history of smoking, exposure to secondhand smoke, exposure to certain toxins, and family history.

[0288] In various embodiments, methods, kits and systems of the present disclosure can be applied to a symptomatic human subject. As used herein, a subject can be referred to as “symptomatic” if the subject report, and / or demonstrates by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or cancer screening), sufficient characteristics of cancer to support a medically reasonable suspicion that the subject is likely suffering from lung cancer. For example, in various embodiments a sample from a subject, optionally where the subject has a lung cancer that is of unknown SCLC / LUAD status, can be assayed according to one or more embodiments of the present disclosure to determine if the lung cancer is SCLC cancer or LU AD cancer. In various embodiments a sample from a subject, where the subject has a lung cancer that is known or suspected of having SCLC cancer (or LU AD cancer), can be assayed according to one or more embodiments of the present disclosure to determine if the lung cancer is in fact SCLC cancer (or LU AD cancer).

[0289] In some embodiments, methods, kits and systems of the present disclosure can be used to determine that a subject has SCLC cancer that correlates with a prior determination based on IHC testing. In some embodiments, methods, kits and systems of the present disclosure can be used to determine that a subject has LU AD cancer that correlates with a prior determination based on IHC testing.

[0290] In some embodiments, methods, kits and systems of the present disclosure can be used to validate or confirm a prior determination that a subject has SCLC cancer, optionally SCLC cancer that correlates with IHC testing. In some embodiments, methods, kits and systemsof the present disclosure can be used to validate or confirm a prior determination that a subject has LU AD cancer, optionally LU AD cancer that correlates with IHC testing.

[0291] In some embodiments, methods, kits and systems of the present disclosure are used to identify and detect new SCLC or LU AD related categories that are independent of IHC testing. For example, instead of training the classifier on samples from cohorts that were defined based on IHC testing, classifiers are trained on samples from cohorts that are defined based on whether they respond or do not respond to a particular therapeutic agent. The resulting classifiers are then used to identify subjects that are more likely to respond to the therapeutic agent independent of any IHC testing. It is therefore to be understood that the term “SCLC / LUAD status” as used herein is not limited to SCLC and LU AD status based on IHC or other histological testing but can encompass any SCLC or LU AD related categories including whether a subject will or will not respond to a particular therapeutic agent.

[0292] Those of skill in the art will appreciate that regular, preventive, and / or prophylactic screening to determine SCLC / LUAD status improves diagnosis of cancer, including and / or particularly early-stage cancer. Thus, the present disclosure provides, among other things, methods, kits and systems particularly useful for the diagnosis and treatment of early-stage cancer. Generally, and particularly in embodiments in which SCLC cancer detection in accordance with the present disclosure is carried out annually, and / or in which a subject is asymptomatic at time of detecting, methods, kits and systems of the present disclosure are especially likely to detect early-stage SCLC cancer including transformed SCLC. In various embodiments, detecting in accordance with methods, kits and systems of the present disclosure reduces cancer mortality, e.g., by early cancer diagnosis. In various embodiments, detecting in accordance with methods, kits and systems of the present disclosure is performed when a subject with LU AD cancer (or more generally a NSCLC cancer) is being treated with a therapeutic agent that can lead to transformation from LU AD cancer (or more generally a NSCLC cancer) to SCLC cancer, e.g., in epidermal growth factor receptor (EGFR) mutant LU AD cancer after treatment with tyrosine kinase inhibitors (TKIs), in anaplastic lymphoma kinase (ALK)-positive LU AD cancer after treatment with ALK inhibitors, in wild-type EGFR or ALK LU AD cancer treated with immunotherapy.

[0293] In various embodiments SCLC / LUAD status determination in accordance with the present disclosure is performed once for a given subject or multiple times for a given subject.In various embodiments, SCLC / LUAD status determination in accordance with the present disclosure is performed on a regular basis, e.g., every six months, annually, every two years, every three years, every four years, every five years, or every ten years.

[0294] In various embodiments, methods, kits and systems disclosed herein provide a determination of SCLC / LUAD status. In other instances, methods, kits and systems disclosed herein will be indicative of SCLC / LUAD status but not definitive for SCLC / LUAD status. In various instances in which methods, kits and systems of the present disclosure are used to determine SCLC / LUAD status, the same can be followed by a further confirmatory assay, which further assay can confirm, support, undermine, or reject a determination resulting from a prior determination, e.g., a determination in accordance with the present disclosure. As used herein, a confirmatory assay can be a test that is currently recognized by medical practitioners, e.g., based on IHC or other histological testing.

[0295] In various embodiments, SCLC / LUAD status determination according to one or more methods, kits and / or systems disclosed herein is followed by treatment of cancer. In various embodiments, treatment of cancer includes administration of a therapeutic regimen including one or more cancer therapies provided herein, including without limitation one or more of an SCLC or LU AD therapy, surgery, radiation, endocrine therapy, chemotherapy, and / or immunotherapy. In various embodiments, treatment of cancer includes administration of a therapeutic regimen including one or more treatments provided herein as available, appropriate, and / or preferred for a particular SCLC / LUAD status.

[0296] In various embodiments, methods, kits and systems can be used to determine whether a particular subject and / or cancer is likely to be and / or is characterized as responsive to a SCLC or LUAD therapeutic agent. In some such embodiments, methods, kits and systems can be followed by treatment of the subject with a SCLC or LUAD therapeutic agent.

[0297] In various embodiments, methods, kits and systems can be used to determine whether a particular subject and / or cancer is likely to be and / or is characterized as resistant to, non-responsive to, or not recommended treatment with a SCLC or LUAD therapeutic agent. In some such embodiments, methods, kits and systems can be followed by treatment with one or more of surgery and / or radiation, HER2-targeted therapy (if HER2-positive), endocrine therapy (if positive for a hormone receptor such as estrogen receptor), chemotherapy and immunotherapy instead of a SCLC or LUAD therapeutic agent.

[0298] Responsiveness can refer to the ability or likelihood of a therapy to cause a reduction in tumor size or inhibit tumor growth or metastasis. Responsiveness can refer to improvement in prognosis (e.g., increased time to cancer recurrence or increased life expectancy, e.g., overall survival, recurrence-free survival, metastasis-free survival, or disease-free survival). Responsiveness can refer to achievement of a treatment benefit, including e.g., improvement in one or more symptoms of cancer, e.g., lung cancer. Responsiveness can be measured quantitatively (e.g., as in the case of tumor size; as in the case of measurement of histone modification, chromatin accessibility, transcription factor binding, or DNA methylation at one or more genomic loci; or as in the calculation of clinical benefit (CBR)), or qualitatively (e.g., by measures such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria). Resistance can refer to the inability or unlikelihood of a therapy to achieve a desired therapeutic effect (e.g., a reduction in tumor size, improvement in prognosis, or other treatment benefit such as, e.g., improvement in one or more symptoms of cancer) in a subject and / or cancer. Resistance includes both acquired and natural resistance. In certain embodiments, resistance includes the extent to which one or more desired therapeutic benefits results from administration of a therapy to a subject and / or cancer is less than that expected and / or achieved in a reference (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of benefit achieved in a reference).

[0299] In various embodiments, methods, kits and systems can be used to detect the clinical efficacy of a course of therapy for cancer, e.g., lung cancer. For example, methods and / or compositions of the present disclosure could be used to determine the presence, absence, or SCLC / LUAD status of a lung cancer in a subject over the course of treatment. Methods and / or compositions of the present disclosure could be used in conjunction with, or confirmed by, other means of determining the presence, absence, or SCLC / LUAD status of a lung cancer including, for example measurements of tumor size or character by techniques such as CT, PET, mammogram, ultrasound, palpation, histology, caliper measurement after biopsy or surgical resection, or by various qualitative, quantitative, or semi quantitative scoring systems including without limitation based on IHC testing, residual cancer burden (Symmans et al., J Clin Oncol (2007) 25:4414-4422, incorporated by reference herein in its entirety) or Miller-Payne score (Ogston et al., Breast (2003) 12:320-327, incorporated by reference herein in its entirety) in aqualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), or “clinical progressive disease” (cPD).

[0300] In some embodiments, methods, kits, and systems described herein can be used to monitor progression of disease in a subject. In some embodiments, monitoring progression entails obtaining and characterizing samples from a subject at at least a first and a second time point. In some embodiments, at the first time point, a subject has already been diagnosed with lung cancer (e.g., SCLC or LU AD). In some embodiments, at a first time point, a subject has been determined to have lung cancer and therapy is administered before or close to (e.g., the same day as) the first time point or between the first time point and the second time point; in such embodiments, determination of SCLC / LUAD status at at least the first and the second time points can be used to monitor treatment efficacy and / or determine when a change in therapy should be made. For example, in some embodiments, a subject has previously been diagnosed with LU AD at the first time point, a LU AD therapy is being or will be administered to the subject, and disease status can be monitored for transformation into SCLC, which can be useful, e.g., for determining whether a change to an SCLC therapy should be made. In some embodiments, treatment efficacy can be monitored, e.g., by using a method described herein to determine a decrease or increase in disease state signal, which can be useful, e.g., for determining whether an administered therapy is effective and / or whether a change in therapy should be made. In some embodiments, at the first time point, a lung cancer has gone into remission for a subject (e.g., the subject has minimal residual disease). In embodiments where a lung cancer has gone into remission, methods, kits, and systems described herein can be useful, e.g., for detecting reoccurrence of cancer, and can be faster, less expensive, and / or less invasive than, e.g., approaches that rely on tissue biopsies and / or imaging techniques.

[0301] In some embodiments, methods, kits and systems for SCLC / LUAD status determination provided herein can inform treatment and / or payment e.g., reimbursement for or reduction of cost of medical care, such as detecting or treatment) decisions and / or actions, e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.

[0302] In some embodiments, methods, kits and systems for SCLC / LUAD status determination provided herein can inform decision making relating to whether health insuranceproviders reimburse a healthcare cost payer or recipient (or not), e.g., for (1) SCLC / LUAD status determination itself (e.g., reimbursement for detecting otherwise unavailable, available only for periodic / regular detecting, or available only for temporally- and / or incidentally- motivated detecting); and / or for (2) treatment, including initiating, maintaining, and / or altering therapy, e.g., based on the determined SCLC / LUAD status. For example, in some embodiments, methods, kits and systems for SCLC / LUAD status determination provided herein are used as the basis for, to contribute to, or support a determination as to whether a reimbursement or cost reduction will be provided to a healthcare cost payer or recipient. In some instances, a party seeking reimbursement or cost reduction can provide results of SCLC / LUAD status determination conducted in accordance with the present disclosure together with a request for such reimbursement or reduction of a healthcare cost. In some instances, a party making a determination as to whether or not to provide a reimbursement or reduction of a healthcare cost will reach a determination based in whole or in part upon receipt and / or review of results of SCLC / LUAD status determination conducted in accordance with the present disclosure.

[0303] In various embodiments, SCLC / LUAD status determination using methods, kits and systems disclosed herein can be used in classifying subjects, samples, and / or tumors (e.g., lung cancer subjects, samples, and / or tumors). In various embodiments, methods, kits and systems disclosed herein can be used to generate a set of subjects, samples, and / or tumors identified according to the present methods, kits and systems each classified as corresponding to a particular SCLC / LUAD status, and optionally using two or more of such classified subjects, samples, and / or tumors to identify biomarkers that distinguish the classes (i.e., distinguish the subjects, samples, and / or tumors according to their class, e.g., according to their SCLC / LUAD status).

[0304] For illustration purposes and without limitation, in an exemplary assay of the present disclosure, one or more samples obtained from a subject (e.g., a liquid biopsy sample including cfDNA, e.g., a plasma sample including cfDNA) are analyzed by a method comprising enriching for cfDNA comprising a particular histone modification, wherein enriching is performed by a method that comprises incubating the sample with a reagent that specifically binds the histone modification being enriched for, and sequencing the enriched cfDNA. One example of such an assay is ChlP-seq for a histone modification (e.g., H3K4me3 and / or H3K27ac). Sequence reads (e.g., ChlP-seq sequence reads) can be aligned to human genomebuild hg19, e.g., using the Burrows-Wheeler Aligner (BWA). Non-uniquely mapping and redundant reads are optionally discarded.

[0305] To provide one example of peak calling, MACS v2.1.1.20140616 can be used for sequence (e.g., ChlP-seq) peak calling with a q-value (FDR) threshold of 0.01. Sequence (e.g., ChlP-seq) data quality can optionally be evaluated by any of one or more of a variety of measures, including total peak number, FRiP (fraction of reads in peak) score, number of high-confidence peaks (e.g., enriched > ten-fold over background), and percent of peak overlap with “blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019) 9( 1 ):9354). If the sequence (e.g., ChlP-seq) data quality is below a particular threshold, the data may be discarded and the assay repeated. Sequence (e.g., ChlP-seq) peaks that overlap with selected genomic loci that are differentially modified as provided herein for the relevant histone modification (Tables 1-2) can then be used to determine SCLC / LUAD status. The number of reads overlapping the selected genomic loci for the relevant histone modification can be summed, e.g., in some embodiments all the genomic loci that are differentially modified with an absolute log2(fold-change) > 4.0 are selected. In some embodiments, the average number of reads in the local background of each ChlP-seq peak is subtracted to improve signal to noise. In some embodiments, a sequence read density for one or more histone modifications can be calculated by a method that comprises (1) summing background adjusted sequence counts at one or more genomic loci and dividing the resulting sum by the total number of kilobases of the one or more genomic loci, or (2) for each genomic loci, determining the ratio of background adjusted fragment counts to the number of kilobases of the genomic loci, and then summing the ratios for each loci. In some embodiments, a method comprises determining an SCLC / LUAD ratio score, e.g., by a method that comprises (a) calculating an SCLC sequence read density, calculating a LU AD sequence read density, and dividing the SCLC sequence read density by the LU AD sequence read density. In some embodiments, an SCLC sequence read density can be determined by a method that comprises calculating sequence read density using one or more genomic loci with increased epigenetic modifications in sample(s) obtained from one or more subjects with SCLC as compared to one or more sample(s) obtained from subjects with LUAD. In some embodiments, a LU AD sequence read density can be determined by a method that comprises calculating sequence read density using one or more genomic loci with increased epigenetic modifications in sample(s) obtained from one or more subjects with LUAD as compared to oneor more sample(s) obtained from subjects with SCLC. An SCLC / LUAD ratio score for determined for one or more histone modifications. In some embodiments an SCLC / LUAD ratio score is determined for H3K4me3 modifications. In some embodiments, an SCLC / LUAD ratio score is determined for H3K27ac modifications. In some embodiments, an SCLC / LUAD ratio score is determined for methylated DNA. In some embodiments, an SCLC / LUAD ratio score is determined for H3K4me3 modifications and H3K27ac modifications, H3K4me3 and methylated DNA, or H3K27ac and methylated DNA. In some embodiments, an SCLC / LUAD ratio score is determined for each of H3K4me3 modifications, H3K27ac modifications, and methylated DNA. In some embodiments, two or more SCLC / LUAD ratio scores for different epigenetic modifications can be combined. In some embodiments, each ratio score can be combined using fitted values that have been determined using a logistic regression.

[0306] The data can then be log2-transformed and quantile normalized to match the distribution of the data used to train a classifier. Normalized data can be used as input into a classifier that was trained using the same histone modification(s) and selected genomic loci. The classifier can then use inputted data to determine SCLC / LUAD status of a subject’s cancer. It will be appreciated that this or similar approaches can be applied to assays of the present disclosure that quantify chromatin accessibility, transcription factor binding and / or DNA methylation.

[0307] In some embodiments, multiple epigenetic markers (e.g., one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation) can be quantified for a single sample. In such embodiments, two or more assays for assessing the epigenetic markers can be performed in sequence (meaning a single sample can be probed for each modification in sequence) or in parallel (meaning that a single sample can be divided into multiple fractions, and then each fraction analyzed to quantifying an epigenetic modification). In some embodiments, H3K4me3 and H3K27ac histone modifications; H3K4me3 modifications and DNA methylation; H3K27ac modifications and DNA methylation; or H3K4me3 modifications, H3K27ac histone modifications, and DNA methylation are quantified in a single sample.

[0308] For the avoidance of any doubt, those of skill in the art will appreciate from the present disclosure that methods, kits and systems for SCLC / LUAD status determination of thepresent disclosure are at least for in vitro use. Accordingly, all aspects and embodiments of the present disclosure can be performed and / or used at least in vitro.

[0309] Those of skill in the ail will also appreciate that, in certain embodiments, methods of the present disclosure can be implemented on and / or in conjunction with a computer program and computer system. In some embodiments, methods of the present disclosure can be implemented on and / or in conjunction with a non-transitory computer readable storage medium encoded with the computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method. A computer system can also store and manipulate data generated by methods of the present disclosure that comprise a plurality of genomic locus modification status and / or accessibility status changes / profiles, which data can be used by a computer system in implementing methods disclosed herein. In certain embodiments, a computer system (i) receives modification status and / or accessibility status data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate references), e.g., to determine SCLC / LUAD status. In certain embodiments, a computer system (i) compares the genomic locus modification and / or accessibility status to a reference; and (ii) outputs an indication of whether the modification status and / or accessibility status of the genomic locus is significantly different from the reference and / or provides a determination regarding SCLC / LUAD status.

[0310] Numerous types of computer systems can be used to implement methods of the present disclosure according to knowledge possessed by a skilled artisan in the bioinformatics and / or computer arts. Several software components can be loaded into memory during operation of such a computer system. The software components can comprise both software components that are standard in the art and components that are special to the present disclosure (e.g., dCHIP software described in Lin et al., Bioinformatics (2004) 20:1233-1240, incorporated herein by reference in its entirety; radial basis machine learning algorithms (RBM) known in the art). Methods of the present disclosure can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms. Such packages include, e.g., Matlab from Mathworks (Natick, MA), Mathematica from Wolfram Research (Champaign, IL), S-Plus fromMathSoft (Seattle, WA), R from R Foundation for Statistical Computing (Vienna, Austria), Python from Python Software Foundation (Wilmington, DE), or Perl from Perl Foundation (Holland, MI). In certain embodiments, a computer system comprises a database for storage of genomic locus modification status and / or accessibility status data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time. In addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan.

[0311] As demonstrated in the Examples, various algorithms can be applied to the comparison, between samples and references, of the modification status and / or accessibility status of genomic loci that are differentially modified in SCLC or LU AD cancers. In various embodiments, an algorithm can be a single learning statistical classifier system. Other suitable statistical algorithms are well known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., a panel of genomic loci of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those described in the Examples and also those using inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C& RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certainembodiments, methods of the present disclosure can include sending classification results to a medical practitioner, e.g., an oncologist.

[0312] In various embodiments, the area under the receiver operating characteristic (AUROC) for determining if a subject has a particular status (e.g., SCLC cancer vs. LUAD cancer) is greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95).Formulation and Administration of Therapeutic Agents

[0313] The present disclosure includes methods where a therapeutic agent or regimen is administered to a subject based on the SCLC / LUAD status of a lung cancer. In general, the therapeutic agent or regimen provided herein will be available, appropriate, and / or preferred for the determined SCLC / LUAD status. Those of skill in the art will be aware of recommended and / or governmentally approved formulations and / or dosages for various therapeutic agents provided herein.

[0314] The present disclosure includes pharmaceutical compositions for delivery of one or more therapeutic agents to a subject. As disclosed herein, a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art. A suitable means of administration can be selected based on the age and condition of a subject.

[0315] Pharmaceutical composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms. Pharmaceutical composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application.Accordingly, the compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or a non-parenteral mode. As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection or infusion.

[0316] In some embodiments, the compositions provided herein are present in unit dosage form, which unit dosage form can be suitable for self-administration. Such a unit dosageform may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.

[0317] A pharmaceutical composition of the present disclosure can be in an injectable or infusible form. For example, the present disclosure includes sterile formulations for injection or infusion, which can be formulated in accordance with conventional pharmaceutical practices. Sterile solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Solutions can be formulated, e.g., using distilled water, physiological saline, or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, or sodium chloride as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, an alcohol such as ethanol and / or a polyalcohol such as propylene glycol or polyethylene glycol, and / or a nonionic surfactant such as polysorbate 80™ or HCO-50, and the like. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin. In particular instances, a pharmaceutical composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage, e.g., at 2-8°C (e.g., 4°C).

[0318] In various embodiments, a pharmaceutical composition of the present disclosure can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0319] In various instances, a pharmaceutical composition can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, without limitation, any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0320] In certain embodiments, compositions can be formulated with a carrier that will protect the therapeutic agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, poly glycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art. See, e.g., J. R. Robinson (1978) “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0321] Route of administration can be parenteral, for example, administration by injection. Administration by injection can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. Administration can be systemic or local. In certain embodiments, a composition described herein can be therapeutically delivered to a subject by way of local administration. As used herein, “local administration” or “local delivery,” can refer to delivery that does not rely upon transport of the composition or therapeutic agent to its intended target tissue or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or therapeutic agent or by injection or implantation of a device containing the composition or therapeutic agent. In certain embodiments, following local administration in the vicinity of a target tissue or site, the composition or therapeutic agent, or one or more components thereof, may diffuse to an intended target tissue or site that is not the site of administration.

[0322] A pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. Examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procainehydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.

[0323] In various embodiments, subcutaneous administration can be accomplished by means of a device, such as a syringe, a prefilled syringe, an auto-injector e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with subcutaneous infusion sets, or other device for combining with a therapeutic agent for subcutaneous injection.

[0324] An injection system of the present disclosure may employ a delivery pen as described in U. S. Pat. No. 5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the art. Such devices can include at least one injection needle, are typically pre-filled with one or more therapeutic unit doses of a solution that includes the therapeutic agent and are useful for rapidly delivering solution to a subject with as little pain as possible. One medication delivery pen includes a vial holder into which a vial of a therapeutic or other medication may be received. The pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and / or indicate the dosage of medication that is injected into the user. See, e.g., U. S. Pat. No. 6,192,891. In some embodiments, the needle of the pen device is disposable and the kits include one or more disposable replacement needles. Pen devices suitable for delivery of any one of the presently featured compositions are also described in, e.g., U. S. Pat. Nos. 6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described in, e.g., U. S. Pat. No. 7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLYIM, manufactured by Scandinavian Health Ltd.

[0325] In certain embodiments, administration of a therapeutic agent as described herein is achieved by administering to a subject a nucleic acid encoding a therapeutic agent described herein. Nucleic acids encoding a therapeutic agent described herein can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce therapeutic agent within cells. Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 (HSV-1), or recombinant bacterial or eukaryotic plasmids. Viral vectors can transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized, polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPCV precipitation. Examples of suitable retroviruses include adenovirus-derived vectors, adeno-associated virus (AAV), pLJ, pZIP, pWE, and pEM which are known to those skilled in the art.

[0326] In some embodiments, a composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[0327] A pharmaceutical composition can include a therapeutically effective amount of a therapeutic agent described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against a therapeutic agent. Those of skill in the art will appreciate that data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In various embodiments, the amount of active ingredient included in a pharmaceutical composition is such that a suitable dose within the designated range can be administered to subjects. The dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.

[0328] Pharmaceutical compositions including certain therapeutic agents, e.g., therapeutic antibodies, can be administered as a fixed dose, or in a milligram per kilogram (mg / kg) dose. While in no way intended to be limiting, an exemplary single dose of certain pharmaceutical compositions described herein can include certain therapeutic agents as described herein in an amount equal to, e.g., 0.001 to 1000 mg / kg, 1-1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg body weight. Exemplarydosages of a composition described herein include, without limitation, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg. The present disclosure is not limited to such ranges or dosages.

[0329] The present disclosure further includes methods of preparing pharmaceutical compositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.

[0330] In various embodiments, therapeutic agents of the present disclosure can be administered to a subject in a course of treatment that further includes administration of one or more additional therapeutic agents or therapies that are not therapeutic agents (e.g., surgery or radiation). Combination therapies of the present disclosure can include simultaneous exposure of a subject to therapeutic agents of two or more therapeutic regimens.

[0331] In certain embodiments, a therapeutic agent as described herein can be administered together with (e.g., at the same time and / or in the same composition as) an additional agent or therapy. In certain embodiments, a therapeutic agent of the present disclosure can be administered separately from an additional therapeutic agent or therapy e.g., at a different time and / or in a different composition than the additional therapeutic agent or therapy). Dosing regimens of a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination can be coordinated or independently determined. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered at the same time as therapeutic agent, on the same day as therapeutic agent, or in the same week as therapeutic agent. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered such that administration of the therapeutic agent and the additional therapeutic agent or therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of the therapeutic agent. In various embodiments, the administration frequency and / or dosage of one or more additional therapeutic agents can be the same as, similar to, or different from the administration frequency of a therapeutic agent. In some embodiments, the two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all “doses” of a firstregimen are administered prior to administration of any doses of a second regimen); in some embodiments, such therapeutic agents arc administered in overlapping dosing regimens.

[0332] In certain embodiments, administration of a therapeutic agent can be to a subject having previously received, scheduled to receive, or in the course of a treatment regimen including an additional cancer therapy. Administration of a therapeutic agent can, in some instances, improve delivery or efficacy of another therapeutic agent or therapy with which it is administered in combination.

[0333] It is contemplated that therapeutic agent combination therapies can demonstrate synergy and / or greater-than-additive effects between a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination. A therapeutic agent can be administered in any effective amount as determined independently or as determined by the joint action of therapeutic agent and any of one or more additional therapeutic agents or therapies administered. Administration of the therapeutic agent may, in some embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional therapeutic agent or therapy relative to a reference regimen for administration of additional therapeutic agent or therapy or therapy absent the therapeutic agent. In certain embodiment, a composition described herein can replace or augment other previously or currently administered therapy. For example, upon treating with therapeutic agent, administration of one or more additional therapeutic agents or therapies can cease or diminish, e.g., be administered at lower levels.Kits

[0334] The present disclosure includes kits for detecting modification and / or accessibility of one or more genomic loci. In some embodiments, the present disclosure provides kits for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci. Kits of the present disclosure can include, e.g., reagents such as buffers and / or antibodies useful in the detection and quantification of histone modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, or H3K4me3, or pan acetylation. In certain embodiments, a kit of the present disclosure can include at least oneantibody that selective binds H3K4me3 modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A kit of the present disclosure can include instructional materials disclosing or describing the use of the kit in a method of determining SCLC / LUAD status and / or treatment disclosed herein. In various embodiments, a kit of the present disclosure can include one or more therapeutic agents useful in the treatment of cancer, e.g., as disclosed herein, optionally in combination with instruction materials for treatment of lung cancer based on SCLC / LUAD status.

[0335] In some embodiments, a kit of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-13, e.g., Table 1-3.

[0336] In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 1, 2, 3, or 4 genomic loci in Table 4. In some embodiments, the kit comprises reagents for quantifying H3K4me3 or H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 6. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 7. In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 12. In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, or 18 genomic loci in Table 13. In some embodiments, the kit comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.

[0337] In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5. In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 8. In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50genomic loci in Table 9. In some embodiments, the kit comprises one or more methyl-binding domains (e.g., for use in MBD-seq). In some embodiments, the kit comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP).

[0338] In some embodiments, the kit comprises reagents for measuring chromatin accessibility for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 10. In some embodiments, the kit comprises reagents for measuring chromatin accessibility for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 11. In some embodiments, the kit comprises reagents for measuring chromatin accessibility via an ATAC-seq assay.

[0339] In some embodiments, the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the kit comprises reagents for library preparation for sequencing. In some embodiments, the kit comprises reagents for sequencing. In some embodiments, the kit comprises instructions for determining if a subject has SCLC or LU AD cancer.Systems

[0340] The present disclosure includes systems for detecting modification and / or accessibility of one or more genomic loci. In some embodiments, the present disclosure provides systems for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci. Systems of the present disclosure can include a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium and / or a computer system.

[0341] In some embodiments, the non-transitory computer readable storage medium is encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method of the present disclosure.

[0342] In some embodiments, the computer system comprises a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform a method of the present disclosure.

[0343] In some embodiments, the sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample. In some embodiments, the system also includes asample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample. The sample preparation device may include reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

[0344] Systems of the present disclosure can include, e.g., reagents such as buffers and / or antibodies useful in the detection and quantification of histone modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, or H3K4me3, or pan acetylation. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A system of the present disclosure can include instructional materials disclosing or describing the use of the system in a method of determining SCLC / LUAD status and / or treatment disclosed herein.

[0345] In some embodiments, a system of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-13, e.g., Tables 1-3.

[0346] In some embodiments, the system comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 1, 2, 3, or 4 genomic loci in Table 4. In some embodiments, the system comprises reagents for quantifying H3K4me3 or H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 6. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 7. In some embodiments, the system comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 12. In some embodiments, the system comprises reagents for quantifying H3K4me3 for at least 5, 10, or 18 genomic loci in Table 13. In someembodiments, the system comprises one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.

[0347] In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 15, 20, 25 or 30 genomic loci in Table 5. In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 8. In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 9. In some embodiments, the system comprises one or more methyl-binding domains (e.g., for use in MBD-seq). In some embodiments, the system comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP).

[0348] In some embodiments, the system comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the sequencer comprises reagents for library preparation for sequencing. In some embodiments, the sequencer comprises reagents for sequencing. In some embodiments, the system comprises instructions for determining if a subject has SCLC or LU AD cancer.

[0349] In some embodiments, the system comprises reagents for measuring chromatin accessibility for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 10. In some embodiments, the system comprises reagents for measuring chromatin accessibility for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 11. In some embodiments, the system comprises reagents for measuring chromatin accessibility via an ATAC-seq assay.

[0350] Illustrative embodiments of systems and methods disclosed herein were described above with reference to computations performed locally by a computing device. However, computations performed over a network are also contemplated. Fig. 10 shows an illustrative network environment 1000 for use in the methods and systems described herein. In brief overview, referring now to Fig. 10, a block diagram of an illustrative cloud computing environment 1000 is shown and described. The cloud computing environment 1000 may include one or more resource providers 1002a, 1002b, 1002c (collectively, 1002). Each resource provider 1002 may include computing resources. In some implementations, computing resources may include any hardware and / or software used to process data. For example, computing resources may include hardware and / or software capable of executing algorithms, computer programs,and / or computer applications. In some implementations, illustrative computing resources may include application servers and / or databases with storage and retrieval capabilities. Each resource provider 1002 may be connected to any other resource provider 1002 in the cloud computing environment 1000. In some implementations, the resource providers 1002 may be connected over a computer network 1008. Each resource provider 1002 may be connected to one or more computing device 1004a, 1004b, 1004c (collectively, 1004), over the computer network 1008.

[0351] The cloud computing environment 1000 may include a resource manager 1006. The resource manager 1006 may be connected to the resource providers 1002 and the computing devices 1004 over the computer network 1008. In some implementations, the resource manager 1006 may facilitate the provision of computing resources by one or more resource providers 1002 to one or more computing devices 1004. The resource manager 1006 may receive a request for a computing resource from a particular computing device 1004. The resource manager 1006 may identify one or more resource providers 1002 capable of providing the computing resource requested by the computing device 1004. The resource manager 1006 may select a resource provider 1002 to provide the computing resource. The resource manager 1006 may facilitate a connection between the resource provider 1002 and a particular computing device 1004. In some implementations, the resource manager 1006 may establish a connection between a particular resource provider 1002 and a particular computing device 1004. In some implementations, the resource manager 1006 may redirect a particular' computing device 1004 to a particular resource provider 1002 with the requested computing resource.

[0352] Fig. 11 shows an example of a computing device 1100 and a mobile computing device 1150 that can be used in the methods and systems described in this disclosure. The computing device 1100 is intended to represent various forms of digital computers, such as laptops, desktops, workstations, personal digital assistants, servers, blade servers, mainframes, and other appropriate computers. The mobile computing device 1150 is intended to represent various forms of mobile devices, such as personal digital assistants, cellular telephones, smartphones, and other similar' computing devices. The components shown here, their connections and relationships, and their functions, are meant to be examples only, and are not meant to be limiting.

[0353] The computing device 1100 includes a processor 1102, a memory 1104, a storage device 1106, a high-speed interface 1108 connecting to the memory 1104 and multiple high-speed expansion ports 1110, and a low-speed interface 1112 connecting to a low-speed expansion port 1114 and the storage device 1106. Each of the processor 1102, the memory 1104, the storage device 1106, the high-speed interface 1108, the high-speed expansion ports 1110, and the low-speed interface 1112, are interconnected using various busses, and may be mounted on a common motherboard or in other manners as appropriate. The processor 1102 can process instructions for execution within the computing device 1100, including instructions stored in the memory 1104 or on the storage device 1106 to display graphical information for a GUI on an external input / output device, such as a display 1116 coupled to the high-speed interface 1108. In other implementations, multiple processors and / or multiple buses may be used, as appropriate, along with multiple memories and types of memory. Also, multiple computing devices may be connected, with each device providing portions of the necessary operations (e.g., as a server bank, a group of blade servers, or a multi-processor system). Also, multiple computing devices may be connected, with each device providing portions of the necessary operations (e.g., as a server bank, a group of blade servers, or a multi-processor system). Thus, as the term is used herein, where a plurality of functions are described as being performed by “a processor”, this encompasses embodiments wherein the plurality of functions are performed by any number of processors (e.g., one or more processors) of any number of computing devices (e.g., one or more computing devices). Furthermore, where a function is described as being performed by “a processor”, this encompasses embodiments wherein the function is performed by any number of processors (e.g., one or more processors) of any number of computing devices (e.g., one or more computing devices) (e.g., in a distributed computing system).

[0354] The memory 1104 stores information within the computing device 1100. In some implementations, the memory 1104 is a volatile memory unit or units. In some implementations, the memory 1104 is a non-volatile memory unit or units. The memory 1104 may also be another form of computer-readable medium, such as a magnetic or optical disk.

[0355] The storage device 1106 is capable of providing mass storage for the computing device 1100. In some implementations, the storage device 1106 may be or contain a computer-readable medium, such as a hard disk device, an optical disk device, a flash memory or other similar solid state memory device, or an array of devices, including devices in a storage area network or other configurations. Instructions can be stored in an information carrier. The instructions, when executed by one or more processing devices (for example, processor 1102),perform one or more methods, such as those described above. The instructions can also be stored by one or more storage devices such as computer- or machine-readable mediums (for example, the memory 1104, the storage device 1106, or memory on the processor 1102).

[0356] The high-speed interface 1108 manages bandwidth-intensive operations for the computing device 1100, while the low-speed interface 1112 manages lower bandwidth-intensive operations. Such allocation of functions is an example only. In some implementations, the highspeed interface 1108 is coupled to the memory 1104, the display 1116 (e.g., through a graphics processor or accelerator), and to the high-speed expansion ports 1110, which may accept various expansion cards (not shown). In the implementation, the low-speed interface 1112 is coupled to the storage device 1106 and the low-speed expansion port 1114. The low-speed expansion port 1114, which may include various communication ports (e.g., USB, Bluetooth®, Ethernet, wireless Ethernet) may be coupled to one or more input / output devices, such as a keyboard, a pointing device, a scanner, or a networking device such as a switch or router, e.g., through a network adapter.

[0357] The computing device 1100 may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a standard server 1120, or multiple times in a group of such servers. In addition, it may be implemented in a personal computer such as a laptop computer 1122. It may also be implemented as part of a rack server system 1124. Alternatively, components from the computing device 1100 may be combined with other components in a mobile device (not shown), such as a mobile computing device 1150. Each of such devices may contain one or more of the computing device 1100 and the mobile computing device 1150, and an entire system may be made up of multiple computing devices communicating with each other.

[0358] The mobile computing device 1150 includes a processor 1152, a memory 1164, an input / output device such as a display 1154, a communication interface 1166, and a transceiver 1168, among other components. The mobile computing device 1150 may also be provided with a storage device, such as a micro-drive or other device, to provide additional storage. Each of the processor 1152, the memory 1164, the display 1154, the communication interface 1166, and the transceiver 1168, are interconnected using various buses, and several of the components may be mounted on a common motherboard or in other manners as appropriate.

[0359] The processor 1152 can execute instructions within the mobile computing device 1150, including instructions stored in the memory 1164. The processor 1152 may be implemented as a chipset of chips that include separate and multiple analog and digital processors. The processor 1152 may provide, for example, for coordination of the other components of the mobile computing device 1150, such as control of user interfaces, applications run by the mobile computing device 1150, and wireless communication by the mobile computing device 1150.

[0360] The processor 1152 may communicate with a user through a control interface 1158 and a display interface 1156 coupled to the display 1154. The display 1154 may be, for example, a TFT (Thin-Film-Transistor Liquid Crystal Display) display or an OLED (Organic Light Emitting Diode) display, or other appropriate display technology. The display interface 1156 may comprise appropriate circuitry for driving the display 1154 to present graphical and other information to a user. The control interface 1158 may receive commands from a user and convert them for submission to the processor 1152. In addition, an external interface 1162 may provide communication with the processor 1152, so as to enable near area communication of the mobile computing device 1150 with other devices. The external interface 1162 may provide, for example, for wired communication in some implementations, or for wireless communication in other implementations, and multiple interfaces may also be used.

[0361] The memory 1164 stores information within the mobile computing device 1150. The memory 1164 can be implemented as one or more of a computer-readable medium or media, a volatile memory unit or units, or a non-volatile memory unit or units. An expansion memory 1174 may also be provided and connected to the mobile computing device 1150 through an expansion interface 1172, which may include, for example, a SIMM (Single In Line Memory Module) card interface. The expansion memory 1174 may provide extra storage space for the mobile computing device 1150, or may also store applications or other information for the mobile computing device 1150. Specifically,...

Claims

CLAIMSWhat is claimed is:

1. A method of determining the SCLC / LUAD status of a lung cancer in a subject, the method comprising:quantifying, at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject, one or more epigenetic biomarkers, wherein the one or more epigenetic biomarkers comprise:(a) one or more histone modifications,(b) chromatin accessibility,(c) binding of one or more transcription factors, and(d) DNA methylation; anddetermining the SCLC / LUAD status of the lung cancer in the subject by comparing the level of the one or more epigenetic biomarkers at the one or more genomic loci to a reference;wherein the one or more genomic loci comprise (i) one or more genomic loci with an increased level of the one or more epigenetic biomarkers in subjects with SCLC as compared to subjects with LU AD, and / or (ii) one or more genomic loci with an increased level of the one or more epigenetic biomarkers in subjects with LUAD as compared to subjects with SCLC.

2. The method of claim 1, wherein:(a) the liquid biopsy sample is a plasma sample, serum sample, or urine sample; (b) the method comprises isolating cfDNA from about 1 mL of the liquid biopsy sample (e.g., plasma sample), and / or(c) the sample comprises a detectable amount of ctDNA (e.g., wherein estimated tumor fraction is >3% for the cfDNA, e.g., as determined by iChorCNA).

3. The method of claim 1 or 2, wherein the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4mel, H3K4me2, H3K4me3, and pan-acetylation (e.g., H3K4me3 and H3K27ac).

4. The method of anyone of claims 1 -3, wherein:(a) the one or more histone modifications arc quantified using an assay selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT& RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT& Tag (Cleavage Under Targets and Tagmentation) sequencing;(b) the chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay;(c) the binding of one or more transcription factors is quantified using a transcription factor binding assay that detects binding of one or more of p300, mediator complex, cohesin complex, RNA pol II, F0XA1, ESRI, PR, MYC, EN1, F0XM1, KLF4, AP-2, RARa, or RUNX1, optionally wherein the transcription factor binding assay is selected from ChlP-seq (Chromatin ImmunoPrecipitation sequencing), CUT& RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT& Tag (Cleavage Under Targets and Tagmentation) sequencing; and / or(d) DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).

5. The method of any one of claims 1-4, wherein the method comprises:(a) quantifying H3K4me3 modifications at one or more genomic loci using an assay that comprises enriching for cfDNA comprising one or more H3K4me3 modifications (e.g., using a method that comprises incubating with an agent that binds H3K4me3 modifications) and sequencing the cfDNA enriched for H3K4me3 modifications to determine a count of sequences with one or more H3K4me3 modifications;(b) quantifying H3K27ac modifications at one or more genomic loci using an assay that comprises enriching for cfDNA comprising one or more H3K27ac modifications (e.g., using a method that comprises incubating with an agent that that binds H3K27ac modifications) andsequencing the cfDNA enriched for H3K27ac modifications to determine a count of sequences with one or more H3K27ac modifications; and / or(c) quantifying methylated DNA at one or more genomic loci using an assay that comprises enriching for methylated cfDNA (e.g., using a method that comprises incubating with an agent that binds methylated DNA) and sequencing the enriched cfDNA to determine a count of sequences with one or more methylated nucleotides; andoptionally:(d) if the method comprises use of an agent that binds H3K4me3 modifications, an agent that binds H3K27ac modifications, and / or an agent that binds methylated DNA, the agent(s) are attached (e.g., via a covalent or noncovalent bond) to a physical support (e.g., a bead, a magnetic bead, an agarose bead, or a magnetic epoxy bead) prior to incubating with the sample; and / or(e) if the method comprises incubating with two or more of an agent that binds H3K4 modifications, an agent that binds H3K27ac modifications, and an agent that binds methylated DNA, the sample is incubated with the two or more agents (i) in sequence or (ii) in parallel (e.g., wherein the sample is divided into fractions and each fraction is incubated with a different agent).

6. The method of any one of claims 1-5, comprising mapping sequence reads to a reference genome, optionally wherein non-uniquely mapped and redundant sequence reads are discarded and / or peaks in high noise regions are removed.

7. The method of claim 6, wherein the one or more genomic loci correspond to sequence read peaks, wherein a sequence read peak corresponds to a region of the genome that has a higher number of sequence reads that the local background.

8. The method of any one of claims 5-7, wherein quantifying H3K4me3 modifications, H3K27ac modifications, and / or DNA methylation comprises summing the number of sequence reads having at least one nucleotide overlap with the one or more genomic loci,optionally wherein:sequence reads are adjusting on the basis of sequencing depth (e.g., quantile normalizing sequence reads to a common reference distribution) and / or ChIP quality prior to summing;sequence counts are normalized to aggregate counts in a given sample across a set of regions (e.g., 10,000 regions) previously determined to have DNAse hypersensitivity in most cell types; and / oran estimate of local background signal is subtracted from the sequence reads at each genomic loci prior to summing.

9. The method of any one of claims 1-8, wherein the reference is a predetermined threshold, a measurement from a liquid biopsy sample, a measurement from liquid biopsy samples obtained from a cohort of subjects, and / or a normalized value, wherein:the predetermined threshold and the normalized value were previously shown to distinguish LU AD and SCLC subjects (e.g., distinguish with an AUROC of greater than 0.5); the reference is a measurement from a liquid biopsy sample obtained from a cohort of subjects who have previously been determined to have LUAD or SCLC; orthe cohort of subjects had previously been determined to have lung cancer (e.g., LUAD or SCLC).

10. The method of any one of claims 5-9, comprising calculating sequence read density at the one or more genomic loci, optionally wherein sequence read density is calculated by:(a) summing background adjusted sequence counts at each of the one or more genomic loci and dividing by the sum of the kilobases of the one or more genomic loci; or (b) for each genomic loci, dividing background adjusted fragment count by the number of kilobases of the genomic loci, and then summing for each loci.

11. The method of claim 10, comprising calculating an SCLC / LUAD ratio score by a method comprising:(a) calculating an SCLC sequence read density by a method comprising summing background adjusted sequence counts at each of the one or more genomic loci with an increasedlevel of the one or more epigenetic biomarkers in sample(s) obtained from subjects with SCLC as compared to samples obtained from subjects with LU AD;(b) calculating a LU AD sequence read density by a method comprising summing background adjusted sequence counts at each of the one or more genomic loci with an increased level of the one or more epigenetic biomarkers in sample(s) obtained from subjects with LU AD as compared to samples obtained from subjects with SCLC; and(c) dividing the SCLC sequence read density by the LU AD sequence read density.

12. The method of claim 11, comprising:(a) determining an SCLC / LUAD ratio score for H3K4me3 modifications;(b) determining an SCLC / LUAD ratio score for H3K27ac modifications; and / or (c) determining an SCLC / LUAD ratio score for methylated DNA; andif each of (a)-(c) is performed, optionally combining each of the ratio scores (e.g., using fitted values determined using a logistic regression).

13. The method of any one of claims 1-12, wherein the one or more epigenetic biomarkers are quantified at one or more genomic loci listed in Tables 1-13,optionally wherein the method comprises quantifying:(a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, or 200 genomic loci listed in Table 1;(b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, or 2000 genomic loci listed in Table 2;(c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci listed in Table 3;(d) H3K4me3, H3K27ac, and / or DNA methylation for at least 5, 10, 20, or 30 genomic loci listed in Table 5(e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, 5000, or 5500 genomic loci listed in Table 6;(f) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci listed in Table 7;(g) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 600 genomic loci listed in Table 8;(h) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 600 genomic loci listed in Table 9.(i) chromatin accessibility (e.g., using ATAC-seq) for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci listed in Table 10;(j) chromatin accessibility (e.g., using ATAC-seq) for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, or 4000 genomic loci listed in Table 11;(k) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, or 100 genomic loci listed in Table 12;(l) H3K4me3 modifications for at least 5, 10, or 15 genomic loci listed in Table 13, or(m) or any combination of (a)-(l).

14. The method of any one of claims 1-13, wherein the method provides an area under the receiver operating characteristic (AUROC) for determining if a subject has SCLC vs. LU AD of greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95).

15. The method of any one of claims 1-14, wherein the subject has previously been determined to have lung cancer (e.g. EGFRm (EGFR mutant) LU AD), the subject has an increased susceptibility to lung cancer (e.g., SCLC, including de novo SCLC and transformed SCLC), and / or wherein the method further comprises determining whether the subject has lung cancer.

16. The method of any one of claims 1-15, wherein the SCLC is transformed SCLC and the LU AD is EGFRm LUAD.

17. The method of any one of claims 1-16, wherein, if the subject is determined to have SCLC, the method further comprises subtyping the SCLC by detecting increased or decreased activity (e.g., expression level) of one or more transcription factors;optionally wherein the increase or decrease is relative to average expression in a subject having SCLC and / or one or more subjects characterized by an alternative SCLC subtype.

18. The method of claim 17, wherein:(a) the SCLC subtype is characterized by increased activity of YAP1 (e.g., increased expression relative to ASCL1, NEURODI, and POU2F3 as compared to a general population of subjects with SCLC).(b) the SCLC subtype is characterized by increased activity of ASCL1, NEURODI, or POU2F3 (e.g., the SCLC is subtyped based on which of ASCL1, NEURODI, and POU2F3 is most highly expressed relative to one another).(c) the SCLC subtype is an inflamed SCLC subtype (SCLC-1), and is optionally characterized by low activity (e.g., low expression) of ASCL1, NEURODI, and POU2F3 (e.g., low relative to activity in a healthy subject, an average subject having SCLC, and / or one or more alternative SCLC subtypes) and / or an inflamed gene signature.

19. The method of claim 17 or 18, wherein activity of the one or more transcription factors is assessed by a method comprising quantifying, at one or more genomic loci in the cell-free DNA (cfDNA) from a liquid biopsy sample:(i) one or more histone modifications,(ii) chromatin accessibility,(iii) binding of one or more transcription factors (e.g., measuring binding of ASCL1, NER0D1, YAP1, and / or POU2F3 at one or more genomic loci), and / or(iv) DNA methylation,wherein the one or more genomic loci include one or more genomic loci with increased histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in sample(s) obtained from a subject with a first SCLC subtype as compared to one or more samples obtained from one or more subjects having a different SCLC subtype;optionally, wherein the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac,H3K27ac, H3K4mel, H3K4me2, H3K4me3, and pan-acetylation (e.g., H3K4me3 modifications and / or H3K27ac modifications).

20. The method of any one of claims 17-19, wherein the method comprises:(a) quantifying H3K4me3 modifications at one or more genomic loci using an assay that comprises enriching for cfDNA comprising one or more H3K4me3 modifications (e.g., an assay that comprises incubating the liquid biopsy sample with an agent that binds an H3K4me3 modification) and sequencing the cfDNA enriched for H3K4me3 modifications to determine a count of sequences comprising one or more H3K4me3 modifications;(b) quantifying H3K27ac modifications at one or more genomic loci using an assay that comprises enriching for cfDNA comprising one or more H3K27ac modifications (e.g., an assay that comprises incubating the liquid biopsy sample with an agent that binds an H3K27ac modification) and sequencing the cfDNA enriched for H3K27ac modifications to determine a count of sequences comprising one or more H3K27ac modifications; and / or;(c) quantifying methylated DNA using an assay that comprises enriching for methylated cfDNA (e.g., an assay that comprises incubating the liquid biopsy sample with an agent (e.g., an antibody or a methyl binding domain) that binds methylated DNA) and sequencing the enriched cfDNA to determine a count of sequences with one or more methylated nucleotides,optionally wherein:the agent that binds H3K4me3 modifications, the agent that binds H3K27ac modifications, and / or the agent that binds methylated DNA are attached (e.g., via a covalent or noncovalent bond) to a physical support (e.g., a bead, a magnetic bead, an agarose bead, or a magnetic epoxy bead) prior to incubating with the sample; and / or if the method comprises incubating with two or more of the agent that binds H3K4 modifications, the agent that binds H3K27ac modifications, and the agent that binds methylated DNA, the sample is (a) incubated with the two or more agents in sequence, or (b) in parallel (e.g., wherein the sample is divided into fractions and each fraction is incubated with a different agent).

21. The method of any one of claims 17-20, comprising mapping sequence reads to a reference genome, optionally wherein non-uniquely mapped and redundant sequence reads are discarded and / or high noise regions are removed.

22. The method of claim 21, wherein the one or more genomic loci correspond to sequence read peaks, wherein a sequence read peak corresponds to a region of the genome that has a higher number of sequence reads that the local background.

23. The method of any one of claims 18-22, wherein quantifying H3K4me3 modifications, H3K27ac modifications, and / or DNA methylation comprises summing the number of sequence reads having at least one nucleotide overlap the one or more genomic loci;optionally wherein:sequence reads are adjusting on the basis of sequencing depth (e.g., quantile normalizing sequence reads to a common reference distribution) and / or ChIP quality prior to summing;sequence counts are normalized to aggregate counts in a given sample across a set of regions (e.g., 10,000 regions) previously determined to have DNAse hypersensitivity in most cell types; and / oran estimate of local background signal is subtracted from the sequence reads at each genomic loci prior to summing.

24. The method of any one of claims 18-23, comprising calculating sequence read density at the one or more genomic loci, optionally wherein sequence read density is calculated by:(a) summing background adjusted sequence counts at each of the one or more genomic loci and dividing by the sum of the kilobases of the one or more genomic loci; or (b) for each genomic loci, dividing background adjusted fragment count by the number of kilobases of the genomic loci, and then summing for each loci.

25. The method of any one of claims 19-24, wherein the one or more genomic loci are selected from those provided Table 4.

26. The method of any one of claims 1 -25, wherein:(a) a biopsy of the lung cancer is not possible and / or feasible.(b) the lung cancer is metastatic lung cancer;(c) the lung cancer exhibits loss of TP53 and / or RB 1 (e.g., comprises one or more loss of function mutations); and / or(d) wherein the subject exhibits TKI resistance.

27. A method of treating a subject having lung cancer, the method comprising:administering a lung cancer therapy to the subject based on the SCLC / LUAD status of the lung cancer, wherein the SCLC / LUAD status of the lung cancer has been determined using the method of any one of claims 1-26, wherein:(a) if the lung cancer has been determined to be SCLC, the cancer therapy comprises administering an SCLC therapy; and(b) if the lung cancer has been determined to be LU AD, the cancer therapy comprises administering a LU AD therapy.

28. The method of claim 27, wherein the subject has previously been determined to have EGFRm LU AD prior to determining SCLC / LUAD status using the method of any one of claims 1-27.

29. The method of claim 28, wherein the cancer has been subtyped using a method of any one of claims 17-17, and wherein the method comprises administering an SCLC therapy to the subject based on the SCLC subtype (e.g., an SCLC therapy is administered that has been shown to provide an improved benefit for the determined SCLC subtype as compared to other therapeutics commonly administered to subjects having SCLC).

30. The method of claim 29, wherein:(a) the cancer has been subtyped on the basis of increased ASCL1 activity (e.g., expression), and the SCLC therapy is one that has been associated with providing an improved therapeutic benefit in subjects diagnosed with an ASCL1 subtype of SCLC (e.g., improved relative to alternative therapeutics that are commonly administered to subjects having SCLC),optionally wherein the SCLC therapy is a BCL2 apoptosis regulator, a BCL2 inhibitor, a DLL3 inhibitor (e.g., rovalpituzumab tcsirinc), an LSD1 inhibitor, and / or a therapeutic targeting CEACAM5 (e.g., labetuzumab govitecan);(b) the cancer has been subtyped on the basis of increased NEURODI activity (e.g., expression), and the SCLC therapy is one that has been associated with providing an improved therapeutic benefit in subjects diagnosed with a NEURODI subtype of SCLC (e.g., improved relative to alternative therapeutics that are commonly administered to subjects having SCLC), optionally wherein the SCLC therapy is an Aurora kinase inhibitor, a Somatostatin receptor 2 (SSTR2) inhibitor (e.g., lanreotide), a therapeutic targeting SSTR2 (e.g., PEN-221), or an immunotherapy (e.g., durvalumab) co-administered with platinum-etoposide;(c) the cancer has been subtyped on the basis of increased POU2F3 activity (e.g., expression), and the SCLC therapy is one that has been associated with an improved therapeutic benefit in subjects diagnosed with a POU2F3 subtype of SCLC, optionally wherein the SCLC therapy comprises an insulin like growth factor 1 receptor inhibitor (optionally without chemotherapy), cisplatin, a PARP inhibitor, an anti-metabolite (e.g., an anti-folate or nucleoside analog), and / or durvalumab (optionally administered without platinum-etoposide);(d) the subject has been subtyped on the basis of increased YAP1 activity (e.g., expression), and the SCLC therapy is one that has been associated with an improved therapeutic benefit in subjects diagnosed with a YAP1 subtype of SCLC, optionally wherein the SCLC therapy comprises durvalumab co-administered with platinum-etoposide;(e) the SCLC subtype is an inflamed SCLC subtype (SCLC-I) and is optionally characterized by (i) low activity (e.g., low expression) of ASCL1, NEURODI, and POU2F3 (e.g., low relative to activity in a healthy subject, an average subject having SCLC, and / or one or more alternative SCLC subtypes), and / or (ii) is characterized by poor response to immune checkpoint blockade; optionally wherein the SCLC therapy comprises an anti-PD-Ll therapeutic and a chemotherapeutic, immune checkpoint blockade, a Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib, an EMT-inhibitor (e.g., HDACi (e.g., mocetinostat)), a MICA inhibitor (e.g., IPH43), and / or an immunotherapy (e.g., durvalumab) co-administered with platinum-etoposide.

31. The method of claim 27, wherein:the LUAD therapy comprises administering a selective EGFR tyrosine kinase inhibitor (c.g., Osimcrtinib)); andthe SCLC therapy comprises administering (i) an agent that targets DLL3 (e.g., Tarlatlamab), and / or (ii) a PD-L1 inhibitor in combination with platinum-etoposide chemotherapy or PARP inhibitors;optionally wherein, if the lung cancer has been determined to be EGFRm LUAD at higher risk for SCLC transformation (e.g., exhibits loss of TP53 and / or RBI), the method comprises administering platinum / etoposide chemotherapy in combination with Osimertinib.

32. A method of monitoring the SCLC / LUAD status of a lung cancer in a subject, and optionally treating the lung cancer, the method comprising:determining the SCLC / LUAD status of the lung cancer using the method of any one of claims 1-26 at first and second time points.

33. The method of claim 32, wherein, prior to the first time point or between the first time point and the second time point, the subject has been administered a therapeutic agent that can lead to transformation from LUAD (or more generally a NSCLC) to SCLC, e.g., where the subject has epidermal growth factor receptor (EGFR) mutant LUAD and is being treated with a tyrosine kinase inhibitor (TKI), the subject has anaplastic lymphoma kinase (ALK)-positive LUAD and is being treated with an ALK inhibitor, or the subject has wild-type EGFR or ALK LUAD and is being treated with immunotherapy.

34. The method of claim 32 or 33, further comprising administering a lung cancer therapy, optionally a SCLC therapy or LUAD therapy, to the subject based on the SCLC / LUAD status of the lung cancer at the second time point, optionally wherein the type, dose and / or frequency of administration of the cancer therapy is adjusted based on the SCLC / LUAD status of the lung cancer at the second time point.

35. A method of treating a subject having a lung cancer, the method comprising:(i) administering an SCLC therapeutic agent to the subject if the subject has been determined to have a validated epigenetic profile indicative of an SCLC based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample (e.g., a plasma sample, serum sample, or urine sample) obtained or derived from the subject; or (ii) administering a LU AD therapeutic agent to the subject if the subject has been determined to have a validated epigenetic profile indicative of LU AD based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject,wherein the presence of the validated epigenetic profile has been determined using a validated classifier,wherein the validated classifier has been obtained by:(a) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a first cohort of subjects who have previously been determined to have an SCLC (e.g., de novo SCLC or transformed SCLC);(b) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a second cohort of healthy subjects or subjects who have previously been determined to have LU AD;(c) comparing the genomic profile determined in step (a) and the genomic profile determined in step (b), to identify genomic loci that have statistically different histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“differential loci”);(d) training a classifier on histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels in the differential loci to distinguish between (i) samples from one or more biological samples obtained from the first cohort, and (ii) samples from one or more biological samples obtained from the second cohort, to identify samples having a profile of histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“epigenetic profile”) that indicates that the samples are likely obtained from the first cohort; and(e) obtaining the validated classifier by validating the classifier from step (d) on a third cohort comprising an independent and group of subjects with SCLC and LU AD cancers and selecting a threshold such that the validated classifier predicts SCLC cancers, with an area under the receiver operating characteristic (AUROC) greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95), wherein subjects falling within the group of predicted SCLC cancers display the validated epigenetic profile and subjects that do not fall within the group of SCLC cancers lack the validated epigenetic profile.

36. The method of claim 35, wherein:(a) the differential loci in step (c) were identified by comparing the genomic profile of one or more histone modifications and / or DNA methylation in (i) one or more biological samples from the first cohort and (ii) one or more biological samples from the second cohort;(b) the classifier in step (d) was trained on histone modification and / or DNA methylation levels in (i) one or more biological samples from the first cohort and (ii) one or more biological samples from the second cohort;(c) the validated classifier in step (e) was validated using liquid biopsy samples from the third cohort; and / or(d) the classifier in step (d) was trained on one or more (e.g., two or more) histone modification levels in the differential loci and / or DNA methylation, optionally wherein the one or more histone modification levels comprise H3K4me3 and H3K27ac modification levels; and / or(e) the classifier in step (d) was trained using ridge regression, elastic net regression, or lasso regression.

37. A kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-13, e.g., Tables 1-3;optionally wherein the kit comprises reagents for quantifying:(a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, or 200 genomic loci in Table 1;(b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, or 2000 genomic loci in Table 2;(c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci in Table 3;(d) H3K4me3, H3K27ac, and / or DNA methylation for at least 5, 10, 20, or 30 genomic loci in Table 5;(e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, 5000, or 5500 genomic loci in Table 6;(f) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci in Table 7;(g) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 600 genomic loci in Table 8;(h) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 600 genomic loci in Table 9;(i) chromatin accessibility (e.g., using ATAC-seq) for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci in Table 10;(j) chromatin accessibility (e.g., using ATAC-seq) for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, or 4000 genomic loci in Table 11;(k) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, or 100 genomic loci in Table 12;(l) H3K4me3 modifications for at least 5, 10, or 15 genomic loci in Table 13, or (m) or any combination of (a)-(l).

38. The kit of claim 37, wherein the kit comprises:(a) one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones;(b) one or more methyl-binding domains for use in MBD-seq or wherein the kit comprises one or more antibodies that bind methylated DNA for use in MeDIP;(c) reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample;(d) reagents for library preparation for sequencing;(c) reagents for sequencing;(f) instructions for determining if a subject has SCLC or LU AD, optionally instructions for determining if a subject has a subtype of SCLC characterized by increased activity (e.g., expression) of ASCL1, NEUROD1, YAP1, and / or POU2F3; and / or(g) any combination of (a)-(f).

39. A non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method of any one of claims 1-26.

40. A computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of claims 1-26.

41. A system for determining the SCLC / LUAD status of a lung cancer in a subject, the system comprising a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium of claim 39 and / or a computer system of claim 40,optionally wherein the sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample.

42. The system of claim 41, further comprising a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample, wherein the sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample.

43. The system of claim 42, wherein the one or more genomic loci are selected from Tables 1-13, e.g., Tables 1-3optionally wherein the device comprises reagents for quantifying:(a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, or 200 genomic loci in Table 1;(b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, or 2000 genomic loci in Table 2.(c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci in Table 3;(d) H3K4me3, H3K27ac, and / or DNA methylation for at least 5, 10, 20, or 30 genomic loci in Table 5(e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, 5000, or 5500 genomic loci in Table 6;(f) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci in Table 7;(g) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 600 genomic loci in Table 8;(h) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, or 600 genomic loci in Table 9.(i) chromatin accessibility (e.g., using ATAC-seq) for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, 4000, or 5000 genomic loci in Table 10;(j) chromatin accessibility (e.g., using ATAC-seq) for at least 5, 10, 20, 30, 40, 50, 100, 500, 1000, 1500, 2000, 3000, or 4000 genomic loci in Table 11;(k) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, or 100 genomic loci in Table 12;(l) H3K4me3 modifications for at least 5, 10, or 15 genomic loci in Table 13, (m) or any combination of (a)-(l).

44. The system of claim 42 or 43, wherein:(a) the reagents comprise one or more antibodies for use in ChlP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones;(b) the reagents comprise one or more methyl-binding domains for use in MBD-seq; (c) the device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample;(d) the device comprises reagents for library preparation for sequencing; and / or (e) the sequencer comprises reagents for sequencing.

45. A method of determining SCLC and / or LU AD status of a cancer in a subject (e.g., patient), the method comprising;receiving (e.g., by a processor of a computing device) one or more genomic profiles of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation for a subject; anddetermining whether the subject has an epigenetic profile indicative of an SCLC or LU AD by classifying (e.g., by the processor) the genomic profile using an SCLC / LUAD classifier.

46. The method of claim 45, wherein the SCLC / LUAD classifier has been trained using one or more genomic profiles of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation for one or more biological samples obtained from one or more cohorts of subjects who have previously have been determined to have SCLC (e.g., de novo SCLC or transformed SCLC) or LU AD,optionally wherein the one or more genomic profiles used to train the SCLC / LUAD classifier are for differential loci having statistically significant differences in the levels of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation levels between one or more biological samples obtained from cohorts of subjects who have previously have been determined to have SCLC (e.g., de novo SCLC or transformed SCLC) or LUAD.

47. The method of claim 46, wherein the SCLC / LUAD classifier has been trained using:(a) genomic profiles of two or more histone modification levels in the differential loci; or(b) genomic profiles of one or more histone modifications levels and DNA methylation levels in the differential loci.

48. The method of any one of claims 45-47, comprising receiving:(a) one or more genomic profiles of two or more histone modifications, optionally wherein the two or more histone modifications comprise H3K4me3 and H3K27ac modifications;(b) one or more genomic profiles of one or more histone modifications and DNA methylation; optionally wherein the one or more histone modifications comprise H3K4me3 and / or H3K27ac modifications.

49. The method of any one of claims 45-48, wherein the SCLC / LUAD classifier has been validated by selecting a threshold such that the validated classifier predicts SCLC cancers with an area under the receiver operating characteristic (AUROC) greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95), andoptionally wherein:(a) the SCLC / LAUD classifier has been validated on a group of subjects with SCLC (e.g., de novo SCLC or transformed SCLC) or LU AD, wherein subjects falling within a group of predicted SCLC (e.g., de novo SCLC or transformed SCLC) cancers display a validated epigenetic profile and subjects that do not fall within the group of predicted SCLC cancers lack the validated epigenetic profile; and / or(b) the SCLC / LUAD classifier has been validated using liquid biopsy sample data.

50. A non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method of any one of claims 45-49.

51. A computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of claims 45-50.

52. A method of treating a subject having a cancer, the method comprising: administering an SCLC therapeutic agent to the subject, wherein the subject has been determined to have a validated epigenetic profile indicative of an SCLC based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject,wherein the presence of the validated epigenetic profile has been determined using a classifier (e.g., a validated classifier) according to a method of any one of claims 45-50.

53. A method of treating a subject having a cancer, the method comprising:administering a LUAD therapeutic agent to the subject, wherein the subject has been determined to have a validated epigenetic profile indicative of LUAD based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject,wherein the presence of the validated epigenetic profile has been determined using a classifier (e.g., a validated classifier) according to a method of any one of claims 45-50.

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