Methods for predicting tumor behavior and metastatic risk in epithelial malignancies

Abstract

Provided herein are methods for predicting tumor behavior and metastatic risk in carcinomas, the method comprising an analysis of RNA expression levels of a set of epigenetically activated genes and / or a set of epigenetically silenced genes. In some instances, the method further comprises administration of adjuvant therapy to a subject identified as having a high metastatic risk for a carcinoma.

Description

Attorney Docket: 54094.4001 / WO METHODS FOR PREDICTING TUMOR BEHAVIOR AND METASTATIC RISK IN EPITHELIAL MALIGNANCIES CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 669755, filed onJuly 11, 2024, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Most patients with solid malignancies die from complications of metastasis. This is trueacross several, diverse tumor types with few exceptions (e.g., glioblastoma). Due to the major clinical burden of metastasis, there is an urgent need to develop therapies targeting the metastasis cascade. However, the ability to develop such therapies is currently hindered by an incomplete understanding of the molecular mechanisms of metastasis.

[0003] Lineage tracing studies utilizing patient samples have demonstrated that, across multipletumor types, metastases develop from distinct subclones within heterogeneous primary tumors, suggesting the existence of subclones with an intrinsic advantage in progressing through the metastasis cascade. The exact nature of this advantage is unclear, although it is likely to involve molecular pathways that confer metastatic subclones with a superior ability to navigate the metastasis cascade. In patient samples, metastatic and non-metastatic subclones are genetically similar, suggesting that metastatic advantage is maintained through epigenetic mechanisms (generally, all mechanisms that propagate information through the cell cycle). The epigenetic landscape of a cell serves to constrain its molecular pathway activities, collectively known as cell states. Therefore, epigenetic analysis should be an effective way of identifying key molecular pathways regulating metastatic potential.

[0004] However, identifying the molecular pathways that enable metastasis by analyzing theeventual lesion has been challenging. Metastatic cells can change their cell state within the spectrum of what is allowable given their epigenetic landscape. Indeed, cancer cells are exposed to varying environmental contexts inherent to progression through the metastasis cascade. Therefore, the observed cell states and active molecular pathways in a metastatic tumor may not be the same as those that enabled the founder of a metastatic tumor to successfully progress 1 4125-3810-3132.7Attorney Docket: 54094.4001 / WO through the cascade. As such, there remains an unmet need for identifying cell state fate of tumor samples with respect to their metastatic potential in patients. BRIEF SUMMARY OF THE INVENTION

[0005] In one aspect, the present disclosure provides a method for identifying metastatic risk of acarcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises: (i) RNA expression levels of a plurality of genes listed in Table 1, and (ii) RNA expression levels of a plurality of genes listed in Table 2; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, (ii) gene identity and ranking of the plurality of genes listed in Table 1, and (iii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

[0006] In a further embodiment and in accordance with the above, the method further comprisesadministering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk. In yet a further embodiment, the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof.

[0007] In a further embodiment and in accordance with any of the above, the carcinomacomprises: (i) a pancreatic carcinoma, (ii) a colon carcinoma, (iii) a breast carcinoma, or (iv) a prostate carcinoma. In yet a further embodiment, the carcinoma comprises a pancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic 2 4125-3810-3132.7Attorney Docket: 54094.4001 / WO cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In yet other further embodiments, the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In yet other further embodiments, the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In yet other further embodiments, the carcinoma comprises a prostate carcinoma, wherein the prostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

[0008] In a further embodiment and in accordance with any of the above, the method furthercomprises: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In yet a further embodiment, the first threshold is a fragments per kilobase million (FPKM) value of 1.00.

[0009] In a further embodiment and in accordance with any of the above, the method furthercomprises extracting the plurality of nucleic acid molecules from the tumor sample from the human subject. In yet a further embodiment, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules. In still a further embodiment, the assay of step (a) is performed on at least a portion of a plurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of the plurality of RNA molecules. In an even further embodiment, the method further comprises, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules. 3 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0010] In a further embodiment and in accordance with any of the above, the tumor samplecomprises: (i) a malignant sample, (ii) a benign sample, or (iii) a mixed sample, whereas in other embodiments, the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more healthy cells.

[0011] In a further embodiment and in accordance with any of the above, the method furthercomprises obtaining the tumor sample from the human subject.

[0012] In a further embodiment and in accordance with any of the above, the assay comprises:(i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. In yet a further embodiment, the assay comprises a sequencing assay, and the sequencing assay comprises a high-throughput sequencing assay. In still a further embodiment, the high-throughput sequencing assay comprises: (i) RNA-sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq). In an even further embodiment, the sequencing assay produces a plurality of nucleic acid sequencing reads, and the method further comprises aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In still yet a further embodiment, the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

[0013] In a further embodiment and in accordance with any of the above, the assay comprises:(i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. In yet a further embodiment, the assay comprises a spatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

[0014] In a further embodiment and in accordance with any of the above, the first single samplescoring algorithm comprises singscore.

[0015] In a further embodiment and in accordance with any of the above, the method furthercomprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk.

[0016] In a further embodiment and in accordance with any of the above, RNA expression levelsare determined for at least 300 genes selected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2. In other embodiments, RNA expression 4 4125-3810-3132.7Attorney Docket: 54094.4001 / WO levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1, and RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. In some further embodiments, RNA expression levels are determined for each of the genes listed in Table 1, and RNA expression levels are determined for each of the genes listed in Table 2.

[0017] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels.

[0018] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (a), aliquoting at least a portion of the plurality of nucleic acid molecules.

[0019] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (a) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C.

[0020] In a further embodiment and in accordance with any of the above, (i) the assay of step (a)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4; (ii) the ranked list of step (b) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4; (iii) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) an enrichment of at least a subset of the plurality of genes listed in Table 2, (3) a depletion of at least a subset of the plurality of genes listed in Table 4, (4) a depletion of at least a subset of the plurality of genes listed in Table 1, or (5) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In yet a further embodiment, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk. In still a further embodiment, the 5 4125-3810-3132.7Attorney Docket: 54094.4001 / WO method further comprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0021] In a further embodiment and in accordance with any of the above, the assay of step (a)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (f) providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment, the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method. In still a further embodiment, the method further comprises, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0022] In a further embodiment and in accordance with any of the above, the tumor sample wasobtained at a first time point, and the method further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample 6 4125-3810-3132.7Attorney Docket: 54094.4001 / WO was obtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment and in accordance with any of the foregoing, the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof. In still a further embodiment, the second time point is subsequent to the first time point. In an even further embodiment, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In still yet a further embodiment, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point; whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point; whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point; whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point.

[0023] In a further embodiment and in accordance with any of the above, the first time pointoccurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions.

[0024] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises a plurality of genes listed in Table 1; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes 7 4125-3810-3132.7Attorney Docket: 54094.4001 / WO listed in Table 1, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies a depletion of at least a subset of the plurality of genes listed in Table 1; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

[0025] In a further embodiment and in accordance with the above, the method further comprisesadministering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk. In still a further embodiment, the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof.

[0026] In a further embodiment and in accordance with any of the above, the carcinomacomprises: (i) a pancreatic carcinoma, (ii) a colon carcinoma, (iii) a breast carcinoma, or (iv) a prostate carcinoma. In yet a further embodiment, the carcinoma comprises a pancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In yet other further embodiments, the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In yet other further embodiments, the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In yet other further embodiments, the carcinoma comprises a prostate carcinoma, wherein the prostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional 8 4125-3810-3132.7Attorney Docket: 54094.4001 / WO cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

[0027] In a further embodiment and in accordance with any of the above, the method furthercomprises: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In yet a further embodiment, the first threshold is a fragments per kilobase million (FPKM) value of 1.00.

[0028] In a further embodiment and in accordance with any of the above, the method furthercomprises extracting the plurality of nucleic acid molecules from the tumor sample from the human subject. In yet a further embodiment, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules. In still a further embodiment, the assay of step (a) is performed on at least a portion of a plurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of the plurality of RNA molecules. In an even further embodiment, the method further comprises, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules.

[0029] In a further embodiment and in accordance with any of the above, the tumor samplecomprises: (i) a malignant sample, (ii) a benign sample, or (iii) a mixed sample, whereas in other embodiments, the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more healthy cells.

[0030] In a further embodiment and in accordance with any of the above, the method furthercomprises obtaining the tumor sample from the human subject.

[0031] In a further embodiment and in accordance with any of the above, the assay comprises:(i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. In yet a further embodiment, the assay comprises a sequencing assay, and the sequencing assay comprises a high-throughput sequencing assay. In still a further embodiment, the high-throughput sequencing assay comprises: (i) RNA-sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq). In an even further embodiment, the sequencing assay produces a plurality of nucleic acid sequencing reads, and the method further comprises aligning 9 4125-3810-3132.7Attorney Docket: 54094.4001 / WO at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In still yet a further embodiment, the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

[0032] In a further embodiment and in accordance with any of the above, the assay comprises:(i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. In yet a further embodiment, the assay comprises a spatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

[0033] In a further embodiment and in accordance with any of the above, the first single samplescoring algorithm comprises singscore.

[0034] In a further embodiment and in accordance with any of the above, the method furthercomprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk.

[0035] In a further embodiment and in accordance with the above, RNA expression levels of theplurality of genes listed in Table 1 is determined for at least 122 of the genes listed in Table 1. In yet a further embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In still a further embodiment, RNA expression levels are determined for each of the genes listed in Table 1.

[0036] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels.

[0037] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (a), aliquoting at least a portion of the plurality of nucleic acid molecules.

[0038] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (a) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C.

[0039] In a further embodiment and in accordance with any of the above, (i) the assay of step (a)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) 10 4125-3810-3132.7Attorney Docket: 54094.4001 / WO RNA expression levels of a plurality of genes listed in Table 4; (ii) the ranked list of step (b) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4; (iii) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) a depletion of at least a subset of the plurality of genes listed in Table 4, (3) a depletion of at least a subset of the plurality of genes listed in Table 1, (4) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In yet a further embodiment, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk. In still a further embodiment, the method further comprises prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0040] In a further embodiment and in accordance with any of the above, the assay of step (a)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (f) providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment, the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method. In still a further embodiment, 11 4125-3810-3132.7Attorney Docket: 54094.4001 / WO the method further comprises, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0041] In a further embodiment and in accordance with any of the above, the tumor sample wasobtained at a first time point, and the method further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment and in accordance with any of the foregoing, the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof. In still a further embodiment, the second time point is subsequent to the first time point. In an even further embodiment, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In still yet a further embodiment, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point, whereas in other 12 4125-3810-3132.7Attorney Docket: 54094.4001 / WO embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point.

[0042] In a further embodiment and in accordance with any of the above, the first time pointoccurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions.

[0043] In a further embodiment and in accordance with any of the above, (i) the assay of step (a)further determines RNA expression levels of a plurality of genes listed in Table 2; (ii) the ranked list of step (b) further includes the plurality of genes listed in Table 2; (iii) gene identity and ranking of the plurality of genes listed in Table 2 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (1) outputting a second metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the second metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; and (2) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the second metric. In yet a further embodiment, RNA expression levels are determined for at least 300 genes selected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2. In other embodiments, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1, and RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. In still a further embodiment, RNA expression levels are determined for each of the genes listed in Table 1, and RNA expression levels are determined for each of the genes listed in Table 2.

[0044] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises a plurality of genes listed 13 4125-3810-3132.7Attorney Docket: 54094.4001 / WO in Table 2; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies an enrichment of at least a subset of the plurality of genes listed in Table 2; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

[0045] In a further embodiment and in accordance with the above, the method further comprisesadministering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk. In still a further embodiment, the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof.

[0046] In a further embodiment and in accordance with any of the above, the carcinomacomprises: (i) a pancreatic carcinoma, (ii) a colon carcinoma, (iii) a breast carcinoma, or (iv) a prostate carcinoma. In yet a further embodiment, the carcinoma comprises a pancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In yet other further embodiments, the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In yet other further embodiments, the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In yet other further embodiments, the carcinoma comprises a prostate carcinoma, wherein the prostate 14 4125-3810-3132.7Attorney Docket: 54094.4001 / WO carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

[0047] In a further embodiment and in accordance with any of the above, the method furthercomprises: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In yet a further embodiment, the first threshold is a fragments per kilobase million (FPKM) value of 1.00.

[0048] In a further embodiment and in accordance with any of the above, the method furthercomprises extracting the plurality of nucleic acid molecules from the tumor sample from the human subject. In yet a further embodiment, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules. In still a further embodiment, the assay of step (a) is performed on at least a portion of a plurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of the plurality of RNA molecules. In an even further embodiment, the method further comprises, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules.

[0049] In a further embodiment and in accordance with any of the above, the tumor samplecomprises: (i) a malignant sample, (ii) a benign sample, or (iii) a mixed sample, whereas in other embodiments, the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more healthy cells.

[0050] In a further embodiment and in accordance with any of the above, the method furthercomprises obtaining the tumor sample from the human subject.

[0051] In a further embodiment and in accordance with any of the above, the assay comprises:(i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. In yet a further embodiment, the assay comprises a sequencing assay, and the sequencing assay comprises a high-throughput sequencing assay. In still a further embodiment, the high-throughput sequencing assay comprises: (i) RNA-sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq). In an even further embodiment, the sequencing assay 15 4125-3810-3132.7Attorney Docket: 54094.4001 / WO produces a plurality of nucleic acid sequencing reads, and the method further comprises aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In still yet a further embodiment, the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

[0052] In a further embodiment and in accordance with any of the above, the assay comprises:(i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. In yet a further embodiment, the assay comprises a spatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

[0053] In a further embodiment and in accordance with any of the above, the first single samplescoring algorithm comprises singscore.

[0054] In a further embodiment and in accordance with any of the above, the method furthercomprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk.

[0055] In a further embodiment and in accordance with the above, RNA expression levels of theplurality of genes listed in Table 1 is determined for at least 141 genes listed in Table 2. In yet a further embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 182 of the genes listed in Table 2. In still a further embodiment, RNA expression levels are determined for each of the genes listed in Table 2.

[0056] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels.

[0057] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (a), aliquoting at least a portion of the plurality of nucleic acid molecules.

[0058] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (a) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. 16 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0059] In a further embodiment and in accordance with any of the above, (i) the assay of step (a)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4; (ii) the ranked list of step (b) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4; (iii) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) an enrichment of at least a subset of the plurality of genes listed in Table 2, (3) a depletion of at least a subset of the plurality of genes listed in Table 4, (4) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In yet a further embodiment, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk. In still a further embodiment, the method further comprises prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0060] In a further embodiment and in accordance with any of the above, the assay of step (a)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (f) providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk 17 4125-3810-3132.7Attorney Docket: 54094.4001 / WO and / or a classical cell state. In yet a further embodiment, the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method. In still a further embodiment, the method further comprises, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0061] In a further embodiment and in accordance with any of the above, the tumor sample wasobtained at a first time point, and the method further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment and in accordance with any of the foregoing, the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof. In still a further embodiment, the second time point is subsequent to the first time point. In an even further embodiment, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In still yet a further embodiment, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point, whereas in other 18 4125-3810-3132.7Attorney Docket: 54094.4001 / WO embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point.

[0062] In a further embodiment and in accordance with any of the above, the first time pointoccurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions.

[0063] In a further embodiment and in accordance with any of the above, (i) the assay of step (a)further determines RNA expression levels of a plurality of genes listed in Table 1; (ii) the ranked list of step (b) further includes the plurality of genes listed in Table 1; (iii) gene identity and ranking of the plurality of genes listed in Table 1 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (1) outputting a second metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the second metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; and (2) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the second metric. In yet a further embodiment, RNA expression levels are determined for at least 300 genes selected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2. In other embodiments, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1, and RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. In still a further embodiment, RNA expression levels are determined for each of the genes listed in Table 1, and RNA expression levels are determined for each of the genes listed in Table 2.

[0064] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) extracting a plurality of RNA molecules from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein: (i) the tumor sample comprises: (1) a malignant sample, (2) a benign sample, (3) a mixed sample, or 19 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (4) a mixture of cells comprising one or more cells suspected of being cancerous and one or more healthy cells, and (ii) the carcinoma comprises: (1) a pancreatic carcinoma, (2) a colon carcinoma, (3) a breast carcinoma, or (4) a prostate carcinoma; (b) synthesizing a plurality of cDNA molecules using at least a portion of the plurality of RNA molecules; (c) performing an assay on at least a portion of the plurality of cDNA molecules, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises: (i) RNA expression levels of a plurality of genes listed in Table 1, and (ii) RNA expression levels of a plurality of genes listed in Table 2, wherein: (1) the assay comprises: (A) RNA-seq, (B) a microarray, or (C) a spatial transcriptomic assay, (2) the plurality of genes listed in Table 1 comprises 157 or more of the genes listed in Table 1, and (3) the plurality of genes listed in Table 2 comprises 182 or more of the genes listed in Table 2; (d) generating a ranked list of genes using determined RNA expression levels; (e) providing (i) the ranked list of genes, (ii) gene identity and ranking of the plurality of genes listed in Table 1, and (iii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm, wherein the first single sample scoring algorithm comprises singscore; (f) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; (g) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric; and (h) administering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk for the carcinoma, wherein the adjuvant therapy comprises: (1) chemotherapy, (2) hormonal therapy, (3) targeted therapy, (4) immunotherapy, or (5) a combination thereof.

[0065] In a further embodiment and in accordance with the above, the carcinoma comprises apancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) 20 4125-3810-3132.7Attorney Docket: 54094.4001 / WO undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In some other embodiments, the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In some other embodiments, the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In some other embodiments, the carcinoma comprises a prostate carcinoma, wherein the prostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

[0066] In a further embodiment and in accordance with any of the above, the method furthercomprises: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In yet a further embodiment, the first threshold is a fragments per kilobase million (FPKM) value of 1.00.

[0067] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules.

[0068] In a further embodiment and in accordance with any of the above, the method furthercomprises obtaining the tumor sample from the human subject.

[0069] In a further embodiment and in accordance with any of the above, the assay comprises asequencing assay, and the sequencing assay comprises a high-throughput sequencing assay. In yet a further embodiment, the high-throughput sequencing assay comprises: (i) RNA-sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC- seq). In still a further embodiment, the sequencing assay produces a plurality of nucleic acid 21 4125-3810-3132.7Attorney Docket: 54094.4001 / WO sequencing reads, and the method further comprises aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In an even further embodiment, the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

[0070] In a further embodiment and in accordance with any of the above, the assay comprises aspatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

[0071] In a further embodiment and in accordance with any of the above, the method furthercomprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk. In yet a further embodiment, RNA expression levels are determined for each of the genes listed in Table 1 and Table 2.

[0072] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the generating of step (d), normalizing individual instances of determined RNA expression levels.

[0073] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c), aliquoting at least a portion of the plurality of nucleic acid molecules.

[0074] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C.

[0075] In a further embodiment and in accordance with any of the above, (1) the assay of step (c)further determines: (A) RNA expression levels of a plurality of genes listed in Table 3, and (B) RNA expression levels of a plurality of genes listed in Table 4; (2) the ranked list of step (d) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4; (3) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (i) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the 22 4125-3810-3132.7Attorney Docket: 54094.4001 / WO plurality of genes listed in Table 3, (2) a depletion of at least a subset of the plurality of genes listed in Table 4, or (3) any combination thereof; and (j) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In yet a further embodiment, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk. In still a further embodiment, the method further comprises, prior to the generating of step (d), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0076] In a further embodiment and in accordance with any of the above, the assay of step (c)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (i) providing: (1) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (2) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (j) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (2) a depletion of at least a subset of the plurality of genes listed in Table 4; and (k) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment, the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method. In still a further embodiment, the method further comprises, prior to the providing of step (i), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0077] In a further embodiment and in accordance with any of the above, the tumor sample wasobtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (b)-(e) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the 23 4125-3810-3132.7Attorney Docket: 54094.4001 / WO human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (k) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (l) repeating steps (b)-(i) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (m) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (l) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (m) repeating steps (b)-(j) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (n) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment and in accordance with any of the foregoing, the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof.

[0078] In a further embodiment and in accordance with any of the above, the second time pointis subsequent to the first time point. In yet a further embodiment, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In still a further embodiment, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point.

[0079] In a further embodiment and in accordance with any of the above, the first time pointoccurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions. 24 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0080] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) extracting a plurality of RNA molecules from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein: (i) the tumor sample comprises: (1) a malignant sample, (2) a benign sample, (3) a mixed sample, or (4) a mixture of cells comprising one or more cells suspected of being cancerous and one or more healthy cells, and (ii) the carcinoma comprises: (1) a pancreatic carcinoma, (2) a colon carcinoma, (3) a breast carcinoma, or (4) a prostate carcinoma; (b) synthesizing a plurality of cDNA molecules using at least a portion of the plurality of RNA molecules; (c) performing an assay on at least a portion of the plurality of cDNA molecules, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises RNA expression levels of a plurality of genes listed in Table 1, wherein: (1) the assay comprises: (A) RNA-seq, (B) a microarray, or (C) a spatial transcriptomic assay, and (2) the plurality of genes listed in Table 1 comprises 157 or more of the genes listed in Table 1; (d) generating a ranked list of genes using determined RNA expression levels; (e) providing (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes listed in Table 1, as input to a first single sample scoring algorithm, wherein the first single sample scoring algorithm comprises singscore; (f) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies a depletion of at least a subset of the plurality of genes listed in Table 1; (g) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric; and (h) administering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk for the carcinoma, wherein the adjuvant therapy comprises: (1) chemotherapy, (2) hormonal therapy, (3) targeted therapy, (4) immunotherapy, or (5) a combination thereof.

[0081] In a further embodiment and in accordance with the above, the carcinoma comprises apancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, 25 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In some other embodiments, the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In some other embodiments, the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In some other embodiments, the carcinoma comprises a prostate carcinoma, wherein the prostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

[0082] In a further embodiment and in accordance with any of the above, the method furthercomprises: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In yet a further embodiment, the first threshold is a fragments per kilobase million (FPKM) value of 1.00.

[0083] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules.

[0084] In a further embodiment and in accordance with any of the above, the method furthercomprises obtaining the tumor sample from the human subject.

[0085] In a further embodiment and in accordance with any of the above, the assay comprises asequencing assay, and the sequencing assay comprises a high-throughput sequencing assay. In yet a further embodiment, the high-throughput sequencing assay comprises: (i) RNA-sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC- 26 4125-3810-3132.7Attorney Docket: 54094.4001 / WO seq). In still a further embodiment, the sequencing assay produces a plurality of nucleic acid sequencing reads, and the method further comprises aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In an even further embodiment, the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

[0086] In a further embodiment and in accordance with any of the above, the assay comprises aspatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

[0087] In a further embodiment and in accordance with any of the above, the method furthercomprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk. In yet a further embodiment, RNA expression levels are determined for each of the genes listed in Table 1 and Table 2.

[0088] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the generating of step (d), normalizing individual instances of determined RNA expression levels.

[0089] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c), aliquoting at least a portion of the plurality of nucleic acid molecules.

[0090] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C.

[0091] In a further embodiment and in accordance with any of the above, (1) the assay of step (c)further determines: (A) RNA expression levels of a plurality of genes listed in Table 3, and (B) RNA expression levels of a plurality of genes listed in Table 4; (2) the ranked list of step (d) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4; (3) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (i) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample 27 4125-3810-3132.7Attorney Docket: 54094.4001 / WO scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) a depletion of at least a subset of the plurality of genes listed in Table 4, or (3) any combination thereof; and (j) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In yet a further embodiment, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk. In still a further embodiment, the method further comprises, prior to the generating of step (d), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0092] In a further embodiment and in accordance with any of the above, the assay of step (c)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (i) providing: (1) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (2) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (j) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (2) a depletion of at least a subset of the plurality of genes listed in Table 4; and (k) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment, the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method. In still a further embodiment, the method further comprises, prior to the providing of step (i), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0093] In a further embodiment and in accordance with any of the above, the tumor sample wasobtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (b)-(e) on at least a portion of the plurality of nucleic acid molecules 28 4125-3810-3132.7Attorney Docket: 54094.4001 / WO from the one or more biological samples; and (k) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (k) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (l) repeating steps (b)-(i) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (m) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (l) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (m) repeating steps (b)-(j) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (n) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment and in accordance with any of the foregoing, the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof.

[0094] In a further embodiment and in accordance with any of the above, the second time pointis subsequent to the first time point. In yet a further embodiment, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In still a further embodiment, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point. 29 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0095] In a further embodiment and in accordance with any of the above, the first time pointoccurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions.

[0096] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) extracting a plurality of RNA molecules from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein: (i) the tumor sample comprises: (1) a malignant sample, (2) a benign sample, (3) a mixed sample, or (4) a mixture of cells comprising one or more cells suspected of being cancerous and one or more healthy cells, and (ii) the carcinoma comprises: (1) a pancreatic carcinoma, (2) a colon carcinoma, (3) a breast carcinoma, or (4) a prostate carcinoma; (b) synthesizing a plurality of cDNA molecules using at least a portion of the plurality of RNA molecules; (c) performing an assay on at least a portion of the plurality of cDNA molecules, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises RNA expression levels of a plurality of genes listed in Table 2, wherein: (1) the assay comprises: (A) RNA-seq, (B) a microarray, or (C) a spatial transcriptomic assay, and (2) the plurality of genes listed in Table 2 comprises 182 or more of the genes listed in Table 2; (d) generating a ranked list of genes using determined RNA expression levels; (e) providing (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm, wherein the first single sample scoring algorithm comprises singscore; (f) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies an enrichment of at least a subset of the plurality of genes listed in Table 2; (g) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric; and (h) administering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk for the carcinoma, wherein the adjuvant therapy comprises: (1) chemotherapy, (2) hormonal therapy, (3) targeted therapy, (4) immunotherapy, or (5) a combination thereof.

[0097] In a further embodiment and in accordance with the above, the carcinoma comprises apancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal 30 4125-3810-3132.7Attorney Docket: 54094.4001 / WO adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In some other embodiments, the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In some other embodiments, the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In some other embodiments, the carcinoma comprises a prostate carcinoma, wherein the prostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

[0098] In a further embodiment and in accordance with any of the above, the method furthercomprises: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In yet a further embodiment, the first threshold is a fragments per kilobase million (FPKM) value of 1.00.

[0099] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules.

[0100] In a further embodiment and in accordance with any of the above, the method furthercomprises obtaining the tumor sample from the human subject. 31 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0101] In a further embodiment and in accordance with any of the above, the assay comprises asequencing assay, and the sequencing assay comprises a high-throughput sequencing assay. In yet a further embodiment, the high-throughput sequencing assay comprises: (i) RNA-sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC- seq). In still a further embodiment, the sequencing assay produces a plurality of nucleic acid sequencing reads, and the method further comprises aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In an even further embodiment, the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

[0102] In a further embodiment and in accordance with any of the above, the assay comprises aspatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

[0103] In a further embodiment and in accordance with any of the above, the method furthercomprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk. In yet a further embodiment, RNA expression levels are determined for each of the genes listed in Table 1 and Table 2.

[0104] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the generating of step (d), normalizing individual instances of determined RNA expression levels.

[0105] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c), aliquoting at least a portion of the plurality of nucleic acid molecules.

[0106] In a further embodiment and in accordance with any of the above, the method furthercomprises, prior to the assay of step (c) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C.

[0107] In a further embodiment and in accordance with any of the above, (1) the assay of step (c)further determines: (A) RNA expression levels of a plurality of genes listed in Table 3, and (B) RNA expression levels of a plurality of genes listed in Table 4; (2) the ranked list of step (d) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 32 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 4; (3) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (i) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) a depletion of at least a subset of the plurality of genes listed in Table 4, or (3) any combination thereof; and (j) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In yet a further embodiment, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk. In still a further embodiment, the method further comprises, prior to the generating of step (d), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0108] In a further embodiment and in accordance with any of the above, the assay of step (c)further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (i) providing: (1) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (2) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (j) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (2) a depletion of at least a subset of the plurality of genes listed in Table 4; and (k) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment, the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method. In still a further embodiment, the method further comprises, prior to the providing of step (i), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4. 33 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0109] In a further embodiment and in accordance with any of the above, the tumor sample wasobtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (b)-(e) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (k) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (l) repeating steps (b)-(i) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (m) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. In some other further embodiments, the tumor sample was obtained at a first time point, and the method further comprises: (l) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (m) repeating steps (b)-(j) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (n) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In yet a further embodiment and in accordance with any of the foregoing, the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof.

[0110] In a further embodiment and in accordance with any of the above, the second time pointis subsequent to the first time point. In yet a further embodiment, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In still a further embodiment, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point, whereas in other embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point, whereas in other 34 4125-3810-3132.7Attorney Docket: 54094.4001 / WO embodiments the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point.

[0111] In a further embodiment and in accordance with any of the above, the first time pointoccurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] The novel features of the invention are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Fig.”, “FIG.”, “Figure”, “Figures”, “Figs.”, and “FIGs.” herein) of which:

[0113] Fig. 1 (Figs. 1A-1E) shows an overview of an exemplary methodology for the isolation ofprimary PDAC subclones with high and low metastatic potential. Fig.1A shows a schematic depicting an exemplary process for producing monoclonal KPC lines labeled with a plurality of barcodes and experimental design for in vivo metastasis competition assays. Fig.1B shows representative photographs and light micrographs of hematoxylin and eosin (H&E) stained formalin-fixed, paraffin-embedded (FFPE) sections of liver and peritoneal metastases generated by transplantation of KPC cells into syngeneic immunocompetent C57BL / 6J mice. Scale bar indicates 500 μm on low magnification images, and 50 μm on high magnification inset images. Fig.1C shows fractions of metastases in which at least one assigned barcode was observed for each monoclonal cell line tested for each mouse, as well as mean across mice (bars with error bars representing standard error of the mean (S.E.M.). Fig.1D shows histograms depicting distributions of tumor clonalities. Fig.1E shows fractions of metastases in which each subclone was observed.

[0114] Fig. 2 (Figs. 2A-2F) shows differentially accessible peaks specific to metastasis-highPDAC subclones and their link to genes involved in inflammation, survival, differentiation, and motility pathways. Fig.2A shows principal component analysis plots based on normalized accessibility of a group-specific shared peak set. KPC-1-derived lines are shown on the left panel, and KPC-2-derived lines are shown on the right panel. Fig.2B shows a pie chart illustrating the fraction of total peaks in a consensus peak set found to have significantly 35 4125-3810-3132.7Attorney Docket: 54094.4001 / WO different accessibility between metastasis-high and metastasis-low cell lines when controlling for parental group status using the generalized linear model feature of DESeq2. A false-discovery rate (FDR) cutoff of 0.05 was used. Fig.2C shows averaged peak ATAC-seq signals for peaks with decreased accessibility (left panel) and increased accessibility (right panel) in metastasis- high cell lines. Fig.2D shows a heatmap depicting the normalized accessibility for each differential peak (row) for each sample (column). Samples were clustered based on Pearson correlation. Fig.2E and Fig.2F show bar plots demonstrating most significant gene ontology (GO) pathways when applying Genomic Regions Enrichment of Annotation Tool (GREAT) to significant peaks with increased (Fig.2E) or decreased (Fig.2F) accessibility in metastasis-high cell lines. The default settings for GREAT were used.

[0115] Fig. 3 (Figs. 3A-3E) shows an identification of epigenetically activated genes andepigenetically silenced genes specific to metastasis-high PDAC subclones. Fig.3A shows a heatmap depicting normalized expression for each differentially expressed gene for each KPC monoclonal cell line. A generalized linear model was used to calculate the differential expression of each gene between metastasis-high and metastasis-low KPC lines while controlling for parental group status. An FDR cutoff of 0.05 was used. Fig.3B shows a scatterplot depicting, for each significantly differentially accessible peak (see, e.g., Fig.2), that peak’s differential accessibility between metastasis-high and metastasis-low cell lines on the X-axis, and the nearest gene’s differential expression between metastasis-high and metastasis-low cell lines on the Y- axis. The color of the peak indicates whether the nearest gene is significantly differentially expressed. Fig.3C shows a diamond plot depicting the top 50 most downregulated genes and the top 50 most upregulated genes in metastasis-high cell lines relative to metastasis-low cell lines. The position of the gene label on the Y-axis indicates that gene’s differential expression. The genes are arranged by rank from most downregulated on the left to most upregulated on the right. Above each gene label are arranged diamonds representing significantly differentially accessible peaks for which the noted gene is the closest gene. The color of the diamond indicates that peak’s differential accessibility. Fig.3D and Fig.3E show signal tracks depicting chromatin accessibility and gene expression of genomic regions containing Il18r1 (Fig.3D) or Mnx1 (Fig. 3E), representative epigenetically activated and silenced genes, respectively, broken down by metastatic potential. To perform this analysis, normalized signal tracks were first generated for each monoclonal KPC cell line. Then, signal tracks for metastasis-high and metastasis-low lines 36 4125-3810-3132.7Attorney Docket: 54094.4001 / WO were separately averaged to produce consensus signal tracks, as shown in Fig.3D and Fig.3E. Asterisks indicate peaks identified to be differentially accessible between metastasis-high and metastasis-low cell lines.

[0116] Fig. 4 (Figs. 4A-4C) shows differential transcription factor activity in metastasis-high andmetastasis-low PDAC subclones. Fig.4A shows differential footprint scores between metastasis- high and metastasis-low monoclonal KPC cell lines for all significant JASPAR202 non- redundant vertebrate transcription factors (TFs; top panel; Bonferroni-adjusted p-value < 0.05) and the top 20 TFs in either direction (i.e., activated or silenced; bottom panel). Fig.4B shows the top 10 most enriched TF motifs in metastasis-low (top panel) and metastasis-high (bottom panel) associated peaks. Fig.4C shows differential expression values for all TFs significantly differentially expressed between metastasis-high and metastasis-low cell lines as defined by FDR < 0.05.

[0117] Fig. 5 shows principal component analysis (PCA) plots based on normalized accessibilityof a consensus peak set shared across Alonso-Curbelo et al. samples (left panel), and metastasis- high and metastasis-low KPC monoclonal cell lines (right panel). The left panel was generated first using data from the Alonso-Curbelo et al. samples only. Then, these same PCA loadings were used to cluster the metastasis-high and metastasis-low KPC cell lines.

[0118] Fig. 6 (Figs. 6A and 6B) shows differences between the epigenetically activated andsilenced genes specific to metastasis-high PDAC subclones and basal- and classical-subtype defining genes. Fig.6A shows an “UpSet” plot depicting unique overlaps between genes epigenetically activated in metastasis-high PDAC subclones in mice (“Metastasis-High”) and basal-subtype defining genes as identified in Collisson et al. (“Collisson-QM”), Moffitt et al. (“Moffitt-Basal”), and Bailey et al. (“Bailey-Squamous”). Fig.6B shows an UpSet plot depicting unique overlaps between genes epigenetically silenced in metastasis-high PDAC subclones in mice (“Metastasis-Low”) and classical-subtype defining genes as identified in Collisson et al. (“Collisson-Classical”), Moffitt et al. (“Moffitt-Classical”), and Bailey et al. (“Bailey-Pancreatic Progenitor”).

[0119] Fig. 7 (Figs. 7A and 7B) shows survival probability for patients stratified according toMetScore (“High” vs. “Low”). Fig.7A shows box plots depicting distributions of MetScores for primary and metastatic samples in the indicated human patient datasets. P-values were calculated using Wilcoxon rank sum tests. * < 0.05; ** < 0.001; *** < 0.0001. Fig.7B shows Kaplan-Meier 37 4125-3810-3132.7Attorney Docket: 54094.4001 / WO curves depicting overall survival for PDAC patients in the noted datasets stratified by MetScore. Patients in each dataset were ranked by MetScore, and the top half were defined as “High”, and the bottom half was considered as “Low”. P-values were calculated using log-rank tests.

[0120] Fig. 8 (Figs. 8A-8D) shows the isolation of primary PDAC subclones with high and lowliver colonization potential. Fig.8A shows a schematic overview of barcoding and expansion of monoclonal KPC cell lines, followed by in vivo metastasis competition assays. Fig.8B shows representative gross pathology (bottom right and bottom left inset images) and H&E-stained FFPE sections of metastatic tumor at low magnification and high magnification (top right and top left inset images). Scale bars: 500^μm (low magnification) and 50^μm (high magnification insets). Fig.8C shows histograms showing the distribution of tumor clonalities (defined as the number of unique subclones per tumor) across four experiments. Fig.8D shows the detected frequency of each subclone across metastases in the four experiments. Points represent individual mice; bars indicate group means ± S.E.M. Sample sizes for Fig.8C and Fig.8D: KPC-1 liver, 27 metastases from 3 mice; KPC-1 peritoneum, 24 metastases from 5 mice; KPC-2 liver, 46 metastases from 5 mice; KPC-2 peritoneum, 60 metastases from 5 mice.

[0121] Fig. 9 (Figs. 9A-9H) shows the identification of gene programs defining metastasis-highand metastasis-low states. Fig.9A shows averaged ATAC-seq signal profiles for peaks with significantly greater accessibility in met-low (left panel) or met-high (right panel) subclones (FDR < 0.05). Each lines represents a subclone, colored by metastatic potential (red = high, blue = low). Figs.9B and 9C show heatmaps showing scaled, log-transformed normalized accessibility (Fig.9B) or expression (Fig.9C) for differentially accessible peaks (Fig.9B) or differentially expressed genes (Fig.9C) (FDR < 0.05). Subclones (columns) are clustered by Pearson correlation. Fig.9D shows a scatterplot showing the relationship between differential chromatin accessibility (x-axis) and differential gene expression (y-axis) for each significantly differential peak. Points are shown in grayscale shading based on whether the nearest gene is differentially expressed. Fig.9E shows a dot plot (top panel) of enriched KEGG pathways among met-high genes (FDR < 0.05), and a pie chart (bottom panel) showing the proportion of these pathways manually classified as inflammation-related. Fig.9F shows a dot plot (top panel) of enriched Gene Ontology (GO) terms among met-low genes (FDR < 0.05), and a pie chart (bottom panel) showing the proportion of pathways related to development, motility, or Wnt signaling. Fig.9G shows a heatmap of scaled, log-transformed normalized expression for 38 4125-3810-3132.7Attorney Docket: 54094.4001 / WO module genes across met-high and met-low subclones. Fig.9H shows a rank-ordered plot of differential binding scores (DBS) for significant transcription factor motifs (n = 499; two-sided T test Bonferroni-adjusted P < 0.05). Motifs were selected from the JASPAR CORE vertebrates nonredundant set and filtered for expression in KPC subclones (n = 411 total). Positive DBS values indicate increased binding in met-high subclones; negative values indicate increased binding in met-low subclones. Motifs associated with inflammation or development are highlighted.

[0122] Fig. 10 (Figs. 10A-10E) shows that the metastatic potential axis is independent ofnormal-to-PDAC and classical-basal axes. Fig.10A shows a principal component analysis of normalized ATAC-seq signal across a consensus peak set. Fig.10B shows a scatterplot showing each gene in the met-high and met-low gene sets. X-axis: log2fold change between primary PDAC and normal pancreas. Y-axis: log2 fold change between met-high and met-low subclones. Grayscale shading indicates gene module membership as defined in Fig.9G. Fig.10C shows predicted basal-like subtype probabilities for met-high and met-low subclones based on the PurIST classifier. Values < 0.5 indicate likely classical subtype. Fig.10D shows bar plots showing single-sample gene set enrichment analysis (ssGSEA) scores (left panel) and average TPM (right panel) for classical and basal marker genes across met-high and met-low subclones. Bars represent means; whiskers, ±S.E.M. Marker genes were selected from the top 25 exemplar classical and basal genes in Moffitt et al., 2015, with known mouse orthologs. P-values from two-sided Wilcoxon rank-sum tests; NS, not significant (P > 0.05). Fig.10E shows a heatmap of scaled, log-transformed expression for the marker genes used in Fig.10D across met-high and met-low subclones.

[0123] Fig. 11 (Figs. 11A-11E) shows that the metastatic potential axis is conserved betweenmouse and human PDAC. Fig.11A shows box plots showing MetScore distribution in primary and metastatic tumors across human PDAC cohorts (left panel: PACA-US; right panel: PACA- CA). Points represent individual tumors. P-values were calculated using two-sided Wilcoxon rank-sum tests. *P < 0.05, ***P < 0.001. Fig.11B shows a dot plot of MetScores for matched primary and metastatic tumors from rapid autopsy patients in the PACA-US cohort. Lines connect samples from the same patient. P-values derived from a linear mixed-effects model with anatomic site as a fixed effect and patient as a random effect. ***P < 0.001. Fig.11C shows a violin plot showing MetScore distributions across single tumor-enriched epithelial cells derived 39 4125-3810-3132.7Attorney Docket: 54094.4001 / WO from primary tumors and metastases. P-value calculated using a generalized linear model with donor type as a fixed effect and donor as a random effect. ***P < 0.001. Fig.11D shows UMAP plots of tumor-enriched epithelial cells colored by Louvain cluster (first panel), MetScore (second panel), and donor type (third panel). For MetScore, values above the 90thpercentile and below the 10th percentile were capped. Fig.11D further shows violin plots showing MetScore distributions across Louvain clusters (top right panel), and a stacked bar plot showing the proportion of primary and metastasis-derived cells within each cluster (bottom right panel). Fig. 11E shows Kaplan-Meier curves for overall survival stratified by MetScore (High = top 50%; Low = bottom 50%) in each dataset (first panel: PACA-US; second panel: PACA-CA; third panel: TCGA-PAAD). P-values from log-rank tests. Fig.11E further shows a Forest plot (right panel) showing estimated hazard ratios and 95% confidence intervals derived from observed and expected event counts in each group using log-rank statistics. A pooled hazard ratio (diamond symbol) was calculated via fixed-effects meta-analysis using inverse-variance weighting of log hazard ratios.

[0124] Fig. 12 (Figs. 12A-12G) shows that MetScore complements classical-basal subtyping instratifying human PDAC. Fig.12A shows box plots showing MetScore distributions in primary and metastatic tumors from the PACA-US cohort, stratified by molecular subtype as annotated by Moffitt et al., 2015. Points represent individual tumors. P-values were calculated using two- sided Wilcoxon rank-sum tests. *P < 0.05, ***P < 0.001. Fig.12B shows a scatterplot showing the relationship between MetScore (y-axis) and scB / scC (x-axis) across single tumor-enriched epithelial cells. Grey line shows linear regression fit. Pearson’s correlation coefficient (r) is indicated. Fig.12C shows UMAPs of tumor-enriched epithelial cells colored by Louvain cluster (first panel), MetScore (second panel), and scB / scC (third panel). Fig.12C further shows violin plots showing the distributions of MetScore (top right panel) and scB / scC (bottom right panel) across Louvain clusters. Fig.12D shows violin plots showing distributions of MetScore (left panel) and scB / scC (right panel) across primary tumor- and metastasis-derived epithelial cells. P- value calculated using a generalized linear model with donor type as a fixed effect and donor as a random effect. ***P < 0.001; NS, not significant. Fig.12E shows dot plots (left panel) showing median classification accuracy (dot) and interquartile range (whiskers) across 10-fold cross- validation for logistic regression models predicting donor type (primary vs. metastasis) using either MetScore or scB / scC. P-values calculated using one-sided one-sample proportion tests 40 4125-3810-3132.7Attorney Docket: 54094.4001 / WO comparing model accuracy to the no-information rate (dashed line). ***P <0.001. NS, not significant. Fig.12E further shows precision-recall curves for models trained on MetScore or scB / scC (right panel). Dashed line indicates expected performance of a random classifier. Fig. 12F shows a forest plot showing hazard ratios (HRs; squares) and 95% confidence intervals (whiskers) for high MetScore (top 50%) and basal subtype (PurIST predicted probability > 0.5) with respect to overall survival in Cox proportional hazards models adjusted for both variables. Analyses were conducted separately in the PACA-US, PACA-CA, and TCGA-PAAD cohorts. Pooled HRs were calculated using a fixed-effects meta-analysis model based on inverse-variance weighting of log-transformed hazard ratios. *P < 0.05, **P < 0.01, ***P < 0.001. Fig.12G shows a Kaplan-Meier curve showing overall survival of patients stratified by MetScore (High > 50thpercentile; Low ≤ 50thpercentile) and classical-basal subtype (PurIST). Cohorts were pooled and survival differences compared using a log-rank test.

[0125] Fig. 13 (Figs. 13A-13E) shows a shared cell state axis underlies metastatic potentialacross human carcinomas. Fig.13A shows box plots showing MetScore distributions in primary and metastatic tumors across the indicated carcinoma cohorts (first panel: Colon MCC; second panel: Colon Consort.; third panel: Breast AURORA; fourth panel: Breast MBC; fifth panel: Prostate Michigan). Fig.13B shows a box plot showing MetScore distributions in primary and metastatic tumors across the TCGA-SKCM cohort. Points represent individual tumors. P-values were calculated using two-sided Wilcoxon rank-sum tests. *P < 0.05, ***P < 0.001. Fig.13C shows a Kaplan-Meier curve showing overall survival stratified by MetScore (High = top 50%; Low = bottom 50%) in the pMMR COAD TCGA dataset. Fig.13D shows a Kaplan-Meier curve showing overall survival stratified by MetScore (High = top 50%; Low = bottom 50%) in the HR+ Breast IDC METABRIC dataset. P-values calculated using log-rank tests. Fig.13E shows Kaplan-Meier curves for overall survival in stage III mismatch repair-proficient (pMMR) colon adenocarcinoma (COAD) patients in the TCGA stratified by receipt of post-operative therapy (yes vs. no) within low MetScore (bottom 50%; left panel) and high MetScore (top 50%; right panel) groups. P-values from log-rank tests.

[0126] Fig. 14 (Figs. 14A-14E) shows that c-Fos is a positive functional mediator of PDAC livercolonization. Fig.14A shows a schematic illustrating the design of a targeted in vivo shRNA screen in KPC-1_Hi2 cells to identify functional mediators of metastatic colonization. Fig.14B shows a volcano-style plot showing each gene included in the screen. X-axis: mean log₂ fold 41 4125-3810-3132.7Attorney Docket: 54094.4001 / WO change in shRNA abundance between primary tumor and liver metastasis conditions (averaged across all shRNAs per gene). Y-axis: −log₁₀ weighted combined p-value from a linear model. Each gene was targeted by three independent shRNAs; control non-targeting shRNAs were included. Primary tumor samples (n = 6 tumors, 6 mice); liver metastases (n = 36 tumors, 4 mice). Fig.14C shows a bar plot showing qRT-PCR-based quantification of Fos mRNA in KPC- 2_HiB cells expressing either shRNAs targeting Fos or a non-targeting control. Rps29 was used as a housekeeping gene. Bars represent mean ± S.E.M. from technical replicates (n = 3). P-value from one-sided Wilcoxon rank-sum test. *P < 0.05. Fig.14D shows histograms (left panel: shFos #1 (n = 32 tumors, 3 mice); right shFos #2 (n = 56 tumors, 5 mice)) showing thedistribution of shFos representation [shFos / (shFos + shControl)] in liver metastases following intrasplenic injection of mixed populations. Dashed lines indicate pre-injection (blue) and post- injection (red) medians. Samples with low sequencing coverage were excluded. P-values from one-sided one-sample Wilcoxon signed-rank tests. Fig.14E shows line plots showing in vitro proliferation of KPC-2_HiB cells expressing Fos-targeting (left panel: shFos #1; right panel: shFos #2) or control shRNAs over time, measured using a CyQUANT assay. Cell counts normalized to 24-hour values. Points represent mean ± S.E.M. from technical replicates (n = 4). P-value calculated using a linear mixed-effects model testing the interaction between time and shRNA. NS, not significant.

[0127] Fig. 15 (Figs. 15A-15C) shows data related to the isolation of primary PDAC subcloneswith high and low liver colonization potential. Fig.15A shows in vitro growth curves for KPC-1- (left panel) and KPC-2-derived (right panel) barcoded monoclonal lines. Points represent the mean average of four technical replicates with error bars representing the S.E.M. Fig.15B shows the fraction of three 10-cm dishes in which each subclone was observed after 28 days of passaging (left panel: KPC-1-derived; right panel: KPC-2-derived). Fig.15C shows agarose gel electrophoresis of PCR products from genotyping assays for the presence of the Pdx1-Cre transgene (top panel) and for recombination of the Lsl-KrasG12D(middle panel) and Lsl-TrpR172Halleles (bottom panel).

[0128] Fig. 16 pie charts showing the fraction of peaks significantly differentially accessiblebetween met-high and met-low subclones that overlap candidate cis-regulatory elements (cCREs) identified by the ENCODE project (left panel) and the breakdown of overlaps by cCRE type (right panel). Some peaks overlap multiple cCREs. 42 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0129] Fig. 17 shows principal component analysis (PCA) based on scaled, normalizedaccessibility of a consensus ATAC-seq peak set. The left panel shows PCA considering the WT, Kras, WT inflamed, Kras inflamed, and PDAC samples only. The right panel compares the metastasis-low and metastasis-high subclones on the same PCA axes as the left panel.

[0130] Fig. 18 (Figs. 18A-18D) shows that the metastasis-high state is largely maintained duringPDAC liver colonization. Fig.18A shows a schematic illustrating the experimental design for assessing epigenomic changes pre- and post-liver colonization. Fig.18B shows bar plots showing the proportion of reads aligning to mutant and wild-type alleles at the Kras (left panel) and Trp53 (right panel) loci in pre-colonization samples (n = 4 biological replicates; aliquots of cultured cells) and post-colonization liver metastases (n = 3 biological replicates; individual mice). Fig.18C shows an alluvial plot depicting the directionality of chromatin accessibility changes for met-high and met-low peaks between pre- and post-colonization samples (FDR < 0.05). Fig.18D shows a bar plot displaying the top enriched GO Biological Process pathways identified by GREAT analysis of met-high peaks that remained unchanged or gained accessibility following liver colonization.

[0131] Fig. 19 (Figs. 19A-19D) shows that the metastatic potential axis is conserved betweenmouse and human PDAC. Fig.19A shows pie charts depicting fractions of genes with significantly increased or decreased expression (FDR < 0.05) in metastases versus primary tumors in the specified cohorts that are met-high genes, met-low genes, or neither (first panel: PACA-US increased; second panel: PACA-US decreased; third panel: PACA-CA increased; fourth panel: PACA-CA decreased). Fig.19B shows box plots showing MetScoreRNAonlydistributions in primary and metastatic tumors across human PDAC cohorts (left panel: PACA- US; right panel: PACA-CA). Points represent individual tumors. P-values were calculated using two-sided Wilcoxon rank-sum tests. *P < 0.05. NS, not significant. Fig.19C shows swam plots depicting MetScores for all metastatic samples in the indicated cohort stratified by anatomic site left panel: PACA-US; right panel: PACA-CA). Each point represents a tumor. Fig.19D shows Kaplan-Meier curves for overall survival stratified by MetScoreRNAonly(High = top 50%; Low = bottom 50%) in each dataset (left panel: PACA-US; middle panel: PACA-CA; right panel: TCGA-PAAD). P-values from log-rank tests.

[0132] Fig. 20 shows a dot plot (left panel) showing median classification accuracy (dot) andinterquartile range (whiskers) across 10-fold cross-validation for logistic regression models 43 4125-3810-3132.7Attorney Docket: 54094.4001 / WO predicting donor type (primary vs. metastasis) using either MetScore, scB / scC, or both MetScore and scB / scC. P-value was calculated using a two-sided paired t-test on fold wise performance differences and adjusted for multiple hypothesis testing using the Bonferroni method. ***P < 0.001. Fig.20 further shows precision -recall curves (right panel) for models trained on MetScore, scB / scC, or both MetScore and scB / scC. Dashed line indicates expected performance of a random classifier. DETAILED DESCRIPTION OF THE INVENTION

[0133] The description is presented to enable one of ordinary skill in the art to make and use theinvention and is provided in the context of a patent application and its requirements. The section headings used herein are for organization purposes only and are not to be construed as limiting the subject matter described. While various embodiments of the invention(s) of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention(s). It should be understood that various alternatives to the embodiments of the invention(s) described herein may be employed in practicing any one of the inventions(s) set forth herein.

[0134] All patents, published patent applications, other publications, and sequences fromGenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology. I. Definitions

[0135] Unless defined otherwise, technical, and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For purposes of the present disclosure, the following terms are defined below. The definitions provided are intended to apply to a given term, as well as other derivative linguistic re-phrasings and grammatical equivalents of the term.

[0136] As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeablyto refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The terms include ribonucleic acid (RNA) molecules, deoxyribonucleic acid (DNA) molecules, complementary deoxyribonucleic acid (cDNA) molecules derived from RNA, 44 4125-3810-3132.7Attorney Docket: 54094.4001 / WO and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form or analog of either type of nucleotide, or combinations thereof.

[0137] As used herein, the term “cell-free nucleic acids” refers to nucleic acid molecules foundcirculating in blood plasma or in another biological fluid from a subject (such as, for example, urine, cerebrospinal fluid (CSF), stool, ascites, and the like). Cell-free nucleic acids may be cell- free ribonucleic acid molecules (cfRNA), cell-free deoxyribonucleic acid molecules (cfDNA), or combinations thereof.

[0138] As used herein, the term “reference genome” can refer to any particular known,sequenced, or characterized genome, whether partial or complete, of any organism or virus that may be used to reference identified sequences from a subject. Unless the context clearly dictates otherwise, “a reference genome” is a human reference genome. Exemplary reference genomes used for human subject include, but are not limited to: (i) NCBI build 34 (University of California, Santa Cruz (UCSC) equivalent: hg16), (ii) NCBI build 35 (UCSC equivalent: hg17), (iii) NCBI build 36.1 (UCSC equivalent: hg18), (iv) GRCh37 (UCSC equivalent: hg19), (v) GRCh38 (UCSC equivalent: hg38), (vi) T2T-CHM13, and others known in the relevant art.

[0139] As used herein, the term “transcriptome” can refer to a collection of all messenger RNAmolecules expressed from genes of an organism or to the collection of genes transcribed into RNA molecules in an organism (or, in one or more cells, tissues, or organs of an organism). Generally, references to “transcriptome” can include protein-coding genes (alone), or to all genes (i.e., protein-encoding and noncoding genes). For example, the term “human transcriptome” can refer to a collection of all messenger RNA molecules expressed from genes of Homo sapiens, including protein-coding and / or noncoding genes, or to the collection of genes transcribed into RNA molecules in Homo sapiens (or, in one or more cells, tissue, or organs of a human). For human reference genome GRCh38, it is known that there are approximately 58,000 genes (encompassing approximately 198,000 transcripts), of which there are approximately 19,950 protein-coding genes (encompassing approximately 80,000 transcripts). Unless the context clearly dictates otherwise, references to “a human transcriptome” is generally meant to refer to the collection of (approximately) 19,950 protein-encoding genes within the human genome. Due to various factors, such as, for example, epigenetic modifications, chromatin states, and the like, a first cell / tissue / organ / tumor sample / biological sample obtained from a human subject can 45 4125-3810-3132.7Attorney Docket: 54094.4001 / WO exhibit differential patterns of gene transcription compared to a second cell / tissue / organ / tumor sample / biological sample obtained from the same human subject. As a first nonlimiting example, in a tumor sample determined to be of a high metastatic risk, such a tumor sample may exhibit an enrichment of epigenetically activated genes (such as, for example, a plurality of the genes listed in Table 2 and / or Table 3), and / or a depletion of epigenetically silenced genes (such as, for example, a plurality of the genes listed in Table 1 and / or Table 4). As a separate nonlimiting example, in a tumor sample determined to be of a low metastatic risk, such a tumor sample may exhibit an enrichment of a plurality of genes listed in Table 1 and / or Table 4, and / or a depletion of a plurality of genes listed in Table 2 and / or Table 3.

[0140] As used herein, the terms “complementary” or “complementarity” refers to specific basepairing between nucleotides or nucleic acids. Base pairing may be “perfectly complementary” (i.e., 100% complementary) or “partially complementary” (i.e., < 100% complementary).

[0141] The terms “patient,” “subject,” “individual,” and the like may be used interchangeablyherein. As used herein, these terms can generally refer to a member of any invertebrate or vertebrate species. Accordingly, the term “subject” is intended to encompass any member of the Kingdom Animalia, including, but not limited to the phylum Chordata (e.g., members of Classes Osteichthyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals)), and all Orders and Families encompassed therein. In some embodiments, the presently disclosed subject matter relates to human subjects.

[0142] Similarly, all “genes,” gene names, and gene products disclosed herein are intended tocorrespond to “orthologs” from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, the genes and / or gene products disclosed herein are also intended to encompass “homologous genes” (or “homologs”) and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.

[0143] As used herein, the term “gene” can refer to the segment of DNA involved in producingor encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding 46 4125-3810-3132.7Attorney Docket: 54094.4001 / WO segments (exons). Genes are defined by symbol and nomenclature for the human gene as assigned by the HUGO Gene Nomenclature Committee. As used herein, the term “gene identity” refers to a name or label assigned as an identifier for a gene (e.g., the gene encoding the breast cancer type 1 susceptibility protein in humans has gene identity “BRCA1,” whereas the gene encoding the homologous protein in mice has gene identity “Brca1”). Table 1: Epigenetically Silenced Genes (174; Homo sapiens) (i) FSTL1 (lix) HMGCS2 (cxvii) ST3GAL3 (ENSG00000163430) (ENSG00000134240) (ENSG00000126091)47 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (xxi) GULP1 (lxxix) SERINC5 (cxxxvii) TLN2 (ENSG00000144366) (ENSG00000164300) (ENSG00000171914) (xxii) PPARA (lxxx) TTC27 (cxxxviii) PATZ14125-3810-3132.7Attorney Docket: 54094.4001 / WO (xlviii) ITGB8 (cvi) SFRP4 (clxiv) SEMA3A (ENSG00000105855) (ENSG00000106483) (ENSG00000075213) (xlix) CCDC3 (cvii) TEAD1 (clxv) ARHGAP44Table 2: Epigenetically Activated Genes (202; Homo sapiens) (i) VMA21 (lii) IRAK1 (ciii) MAGED1 (clv) SERPINB5 (ENSG00000160131) (ENSG00000184216) (ENSG00000179222) (ENSG00000206075)49 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (xiv) PHLDB2 (lxv) RHOF (cxvi) SHROOM2 (clxvii) CREB3L3 (ENSG00000144824) (ENSG00000139725) (ENSG00000146950) (ENSG00000060566) (xv) SLC6A8 (lxvi) FAM50A (cxvii) ACKR3 (clxviii) DDIT44125-3810-3132.7Attorney Docket: 54094.4001 / WO (xli) TRPM1 (xcii) NGF (cxliv) FAM199X (cxciv) ARHGAP40 (ENSG00000134160) (ENSG00000134259) (ENSG00000123575) (ENSG00000124143) (xlii) CFAP69 (xciii) PIR (cxlv) ARAF (cxcv) SLC4A11Table 3: Basal-Subtype Defining Genes (14; Homo sapiens) (i) CD109 (ENSG00000156535) (viii) CHST6 (ENSG00000183196)Table 4: Classical-Subtype Defining Genes (14; Homo sapiens) (viii) PLA2G10 (i) GPR160 (ENSG00000173890)51 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (iv) TMEM45B (xi) FAM3D (ENSG00000198643) (ENSG00000151715 )[01, uence or nucleic acidsequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence. Homologs may arise through speciation events (giving rise to “orthologs”), through gene duplication events (giving rise to “paralogs”), or through horizontal gene transfer events. Homologs may be identified by phylogenetic methods, through identification of common functional domains in aligned nucleic acid or protein sequences, or through sequence comparisons. Most often, homologs will have functional, structural, and / or genomic similarities.

[0145] As used herein, the term “ortholog” refers to a gene (or a protein encoded by a gene) thatis part of a group of genes (or part of a group of proteins) that are predicted to have evolved from a common ancestral gene by speciation. Typically, orthologs retain the same or similar function despite differences in their primary structure.

[0146] As used herein, the term “paralog” refers to homologs in the same species that haveevolved by genetic duplication of a common ancestral gene. In many cases, paralogs exhibit related but not always identical functions. To the extent that a particular species has evolved multiple related genes (or proteins encoded by the genes) from an ancestral DNA sequence shared with another species, the term ortholog can encompass the term paralog.

[0147] As used herein, the terms “treatment,” “treating,” and the like, are used herein togenerally mean obtained a desired pharmacologic and / or physiologic effect in response to one or more medical interventions, such as, for example, administration of one or more therapies, one or more surgical procedures, and the like. The effect may be prophylactic, in terms of completely or partially preventing a disease, condition, or symptoms thereof, and / or may be therapeutic in terms of a partial or complete cure for a disease or condition and / or an adverse effect, such as a symptom, attributable to the disease or condition. "Treatment" as used herein covers any treatment of a disease or condition of a subject and includes: (a) preventing the disease or 52 4125-3810-3132.7Attorney Docket: 54094.4001 / WO condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).

[0148] As used herein, the term “therapy” refers to one or more medications administered as afirst line of treatment to a subject in need thereof at a first point in time to effectuate treatment of a disease, disorder, or condition (such as, for example, cancer), or to effectuate treatment of a symptom of the disease / disorder / condition, wherein the therapy is known to effectuate treatment of the disease / disorder / condition or a symptom thereof. Non-limiting examples of therapy include: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, as well as others known in the relevant (including combinations and / or sub-combinations as appropriate for a particular oncological paradigm). Further, a therapy for a disease (such as, for example, cancer) may comprise one or more therapeutic interventions. As used herein, the term “therapeutic intervention” refers to one or more of: (i) surgical debulking, debridement, and other surgical procedures for removing part of or all of a tumor / cancerous lesion, (ii) photodynamic therapy, (iii) therapeutic hyperthermia, (iv) radiation therapy, as well as combinations thereof. As will be appreciated by those skilled in the relevant art, suitability of a given therapy for a disease / disorder / condition is generally indicated by the specific clinical scenario and the therapy has been validated in one or more clinical trials.

[0149] As used herein, the term “adjuvant therapy” refers to one or more therapies or therapeuticinterventions administered to a subject in need thereof subsequent to the administration of a first therapy, wherein the adjuvant therapy is known to effectuate treatment of a disease / disorder / condition (or a symptom thereof) for which the subject received the first therapy. Non-limiting examples of adjuvant therapies include: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, as well as others known in the relevant (including combinations and / or sub-combinations as appropriate for a particular oncological paradigm). To help clarify, by way of a nonlimiting example, a human subject suffering from colon adenocarcinoma may receive a first therapy (e.g., surgical excision of the adenocarcinoma alone or in combination with a first chemotherapeutic agent). Subsequent to the first therapy, it may be determined that the first therapy is insufficient to resolve the disease / disorder / condition for which the human subject is being treated based on, for example, a determination that the 53 4125-3810-3132.7Attorney Docket: 54094.4001 / WO human subject has a high metastatic risk. As such, the human subject can then receive adjuvant therapy (such as, for example, adjuvant oxaliplatin-based chemotherapy). As will be appreciated by those skilled in the relevant art, suitability of a given adjuvant therapy for a disease / disorder / condition is generally indicated by the specific clinical scenario and the adjuvant therapy has been validated in one or more clinical trials.

[0150] As used herein, the term “cancer” refers to a broad group of various diseasescharacterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring cells or tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0151] As used herein, the term “tumor” refers to any neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Generally, a “tumor sample” may comprise “a malignant sample,” “a benign sample,” or “a mixed sample.” In some instances, a biological sample may be suspected of being a “tumor sample.” In such instances, the biological samples comprise a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more healthy (“normal”) cells. Subsequent histopathological analysis of a prospective “tumor sample” can elucidate whether the “tumor sample” is a “malignant,” “benign,” or “mixed” sample.

[0152] As used herein, the term “a malignant sample” generally refers to a biological sampleobtained from a subject, wherein cells comprising the biological sample are substantially cancerous cells. As used herein, the term “a benign sample” generally refers to a biological sample obtained from a subject, wherein cells comprising the biological sample are characterized by an unregulated or a dysregulated cellular growth, but are not cancerous. As used herein, the term “a mixed sample” generally refers to a biological sample obtained from a subject, wherein cells comprising the biological sample are a mixture of cancerous cells and normal cells (or, “healthy” cells). As used herein, the term “a normal sample” generally refers to a biological sample obtained from a subject, wherein cells comprising the biological sample are characterized by physiologically appropriate cellular growth and are not cancerous.

[0153] As used herein, the term “pre-cancerous” refers to a condition or a cellular growth thattypically precedes or develops into a cancer.

[0154] As used herein, the terms “non-metastatic” or “localized,” which can be usedinterchangeably, is meant a cancer that is benign or that remains at a primary site and has 54 4125-3810-3132.7Attorney Docket: 54094.4001 / WO typically not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Conversely, the term “metastatic” generally refers to a cancer that is not benign (i.e., malignant) and has penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. As used herein, the term “primary site” generally refers to a first portion of an anatomic region, an anatomic subregion, an organ system, an organ, or a tissue identified as containing one or more cells known to be or suspected of being cancerous. As used herein, the term “secondary site” generally refers to a second portion of an anatomic region, an anatomic subregion, an organ system, an organ, or a tissue that is distinct from the “primary site” and is identified as containing one or more cells known to be or suspected of being cancerous.

[0155] As used herein, the term “carcinoma” refers to a cancer arising from an epithelial tissueof the skin or of the lining of internal organs and passageways. Like other types of cancer, carcinomas are abnormal cells that divide in an unregulated or dysregulated manner. Nonlimiting examples of carcinomas include: (1) pancreatic carcinomas (such as, for example, pancreatic ductal adenocarcinoma), (2) colon carcinomas (such as, for example, colon adenocarcinoma), (3) breast carcinomas (such as, for example, invasive ductal carcinoma), (4) prostate carcinomas (such as, for example, prostate adenocarcinoma), and others known in the relevant art.

[0156] As used herein, the singular forms “a,” “an”, and “the” include plural referents unless thecontext clearly dictates otherwise (such as, for example, by reciting: “exactly one of [x],” “a singular instance of [x],” and the like). Thus, for example, reference to “a nucleic acid molecule” includes mixtures of nucleic acid molecules; reference to “a biological” includes two or more biological samples, and the like. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0157] As used herein, the term “a plurality of [x]” refers to two or more instances of a recitedterm unless the context clearly dictates otherwise. For example, “a plurality of genes listed in Table 1” refers to two or more genes set forth in Table 1, “a plurality of genes listed in Table 2” refers to two or more genes set forth in Table 2, etc.

[0158] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each ofthe two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A (alone)”, and “B (alone)”. 55 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0159] As used herein, the term “about” a value (or parameter) refers to ±10% of a stated value.When referring to a range of values (or parameters), the term “about” refers to +10% of the upper limit and -10% of the lower limit of a stated range of values. When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the scope of the present disclosure. Where the stated range includes upper and / or lower limits, ranges excluding either of those included limits are also included in the present disclosure.

[0160] As used herein, the term “all or substantially all of [x]” refers to the entirety (i.e., 100%)of a term or at least about 95% of a term. For example, “all or substantially all of genes comprising a human transcriptome” refers to all genes encompassed by the human transcriptome (as is defined above) or at least about 95% of all genes encompassed by the human transcriptome, “all or substantially all of the genes listed in Table 2” refers to all genes set forth in Table 2 or at least about 95% of the genes set forth in Table 2, and the like.

[0161] As used herein, the use of ordinal terms, such as, for example, “first,” “second,” “third,”“fourth”, “fifth,” etc., to modify an element of the disclosure does not by itself connote any priority, precedence, or order of one element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one element having a certain name (e.g., a metric) from another element having a same name but for the use of the ordinal term to distinguish the elements (e.g., “a first metric,” “a second metric,” “a third metric,” “a fourth metric,” etc.).

[0162] It is appreciated that certain features of the disclosure, which are, for clarity, described inthe context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. 56 4125-3810-3132.7Attorney Docket: 54094.4001 / WO II. Overview

[0163] The present invention is directed to methods for determining metastatic risk ofcarcinomas (i.e., cancers arising from an epithelial tissue of the skin or of the lining of internal organs and passageways) via an analysis of RNA expression levels in a human transcriptome. Specifically, enrichment of a plurality of genes listed in Table 2 and / or depletion of a plurality of genes listed in Table 1 is indicative of a high metastatic risk for carcinomas, whereas enrichment of a plurality of genes listed in Table 1 and / or depletion of a plurality of genes listed in Table 2 is indicative of a low metastatic risk for carcinomas. In response to a determination that a human subject has a high metastatic risk for carcinoma, adjuvant therapy can be administered at a clinically actionable point, prior to the human subject succumbing to metastatic disease and / or sequelae of their carcinoma.

[0164] Various genomic approaches have been postulated as surrogates for determining cell statefate of tumors (e.g., metastatic potential). However, such approaches, by virtue of nucleic acid molecule analyzed (i.e., DNA instead of RNA), usually can only serve as indirect measurements of cell state fate because the individual cells comprising a tumor are often heterogeneous with respect to genetic expression (i.e., a plurality of cells within a tumor may share similar genetic sequences, but they may exhibit different transcriptomes due to various factors known in the relevant art, such as, for example, epigenetic modifications), whereas the methods described herein represent direct measurements of cell state fate / metastatic risk because they analyze expressed genetic elements as opposed to potential cell states encoded by DNA. As such, the utilization of RNA in lieu of DNA to determine cell state fate / metastatic risk represents an unconventional approach in the field of cancer diagnostics.

[0165] Separately, most genomic approaches require paired analysis of a corresponding normal(“healthy”) sample to accurately diagnose cancer. Contrary to such genomic analysis, the methods described herein do not require comparative analysis of a normal sample (yet another unconventional element of the disclosure).

[0166] Current methods for determining metastatic risk of cancer utilize cancer-specific genesets (or, in some instances, a two or more gene sets to further subtype the cancer; e.g., a first panel to determine that a patient has leukemia and one or more panels to subtype the leukemia). Conversely, the methods described herein represent a set of genetic markers capable of 57 4125-3810-3132.7Attorney Docket: 54094.4001 / WO characterizing epithelial cancers. Phrased differently, the combination of genes listed in Table 1 and Table 2 have been found to be pan-epithelial cancer markers, whereas current methods are cancer-specific markers, further demonstrating the unconventionality of the approaches described herein.

[0167] Further, current methods for determining metastatic risk of cancer consist of a list of“bad” genes (i.e., genes indicative of cancer), whereas the methods described herein utilize a list of “bad” genes (i.e., genes indicative of high metastatic risk; see, e.g., Table 2 and / or Table 3) and a list of “good” genes (i.e., genes indicative of low metastatic risk; see, e.g., Table 1 and / or Table 4). By determining which genes are enriched and which are depleted from the list of “good” and “bad” genes, a more accurate metastatic risk score can be determined compared to conventional methodologies, underscoring yet another element of the disclosure that is not routine or well-understood. III. Methods of Use

[0168] Provided herein are methods for identifying metastatic risk of a carcinoma in a subject,such as, for example, a human subject, who is known to have or is suspected of having a carcinoma. Such methods are also generally suitable for: (1) prognosing metastatic risk of a carcinoma, (2) identifying a carcinoma patient (such as, for example, a human subject) as likely to experience recurrence of the carcinoma following administration of a first therapy, (3) monitoring and / or detecting of early relapse or metastasis of a carcinoma, (4) analyzing a biological sample from a subject known to have or is suspected of having a carcinoma, and / or (5) identifying cell state of a plurality of cells from a tumor sample from a subject known to have or is suspected of having a carcinoma, according to the aspects and embodiments described herein. A. Depletion of Epigenetically Silenced Genes and / or Enrichment of Epigenetically Activated Genes

[0169] In one aspect, the present disclosure provides a method for identifying metastatic risk of acarcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels of a plurality of genes comprising a human transcriptome, wherein RNA expression levels of the 58 4125-3810-3132.7Attorney Docket: 54094.4001 / WO plurality of genes of the human transcriptome comprises: (i) RNA expression levels of a plurality of genes listed in Table 1, and (ii) RNA expression levels of a plurality of genes listed in Table 2; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, (ii) gene identity and ranking of the plurality of genes listed in Table 1, and (iii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

[0170] In some embodiments, the plurality of genes comprising a human transcriptomecomprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the genes comprising the human transcriptome. In some embodiments, the plurality of genes comprising the human transcriptome comprises about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100% of the human transcriptome. In some embodiments, the plurality of genes comprising the human transcriptome comprises about all (i.e., ~100%) or about substantially all (i.e., at least about 95%) genes encoding the human transcriptome.

[0171] In some embodiments, the method further comprises administering adjuvant therapy tothe human subject if the human subject is identified as having a high metastatic risk. In some further embodiments, the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. It is appreciated that a person of ordinary skill in the art will be able to readily identify and select an appropriate adjuvant therapy for administration to the human subject in accordance with standards of care for a given carcinoma.

[0172] Examples of suitable chemotherapy include, but are not limited to: (i) FOLFIRINOX(i.e., a combination of the drugs leucovorin calcium (folinic acid), fluorouracil, irinotecan 59 4125-3810-3132.7Attorney Docket: 54094.4001 / WO hydrochloride, and oxaliplatin), (ii) FOLFOX (i.e., a combination of the drugs leucovorin calcium (folinic acid), fluorouracil, and oxaliplatin), (iii) FOLFOX-4, (iv) FOLFOX-6, (v) modified FOLFOX-6 (mFOLFOX-6), (vi) FOLFOX-7, (vii) gemcitabine and / or nab-paclitaxel (Abraxane ®), (viii) AC-T (i.e., a combination of doxorubicin hydrochloride (Adriamycin) and cyclophosphamide, followed by treatment with paclitaxel), (ix) TC (i.e., a combination of docetaxel and cyclophosphamide), (x) AC (i.e., a combination of doxorubicin and cyclophosphamide), (xi) docetaxel, (xii) carboplatin, (xiii) fludarabine, (xiv) capecitabine, (xv) irinotecan, (xvi) trifluridine and / or tipiracil, (xvii) epirubicin, (xviii) ixabepilone, (xix) eribulin, (xx) vinorelbine, (xxi) cabazitaxel, (xxii) mitoxantrone, (xxiii) estramustine, and others known in the relevant art.

[0173] Examples of suitable hormonal therapy include, but are not limited to: (i) tamoxifen, (ii)anastrozole, (iii) exemestane, (iv) letrozole, (v) goserelin, (vi) leuprorelin (leuprolide), (vii) triptorelin, (viii) bicalutamide (Casodex), (ix) nilutamide (Nilandron), (x) flutamide (Eulexin), (xi) enzalutamide, (xii) apalutamide, (xiii) darolutamide, (xiv) cyproterone acetate, (xv) ketoconazole, (xvi) aminoglutethimide, and others known in the relevant art.

[0174] Examples of suitable targeted therapy include, but are not limited to: (i) Olaparib(Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), (vi) Larotrectinib (Vitrakvi ®), (vii) Palbociclib, (viii) ribociclib, (ix) abemaciclib, (x) bevacizumab, (xi) ramucirumab, (xii) ziv-aflibercept, (xiii) fruquintinib, (xiv) rucaparib, (xv) talazoparib, (xvi) niraparib and / or abiraterone, and others known in the relevant art.

[0175] Examples of suitable immunotherapy include, but are not limited to: (i) checkpointinhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0176] Generally, the carcinoma can comprise: (i) adenocarcinoma, (ii) squamous cellcarcinoma, (iii) adenosquamous carcinoma, (iv) anaplastic carcinoma, (v) large cell carcinoma, (vi) small cell carcinoma, (vii) spindle cell carcinoma, (viii) giant cell carcinoma, (ix) sarcomatoid carcinoma, (x) pleomorphic carcinoma, or others known in the relevant art. Further, 60 4125-3810-3132.7Attorney Docket: 54094.4001 / WO a carcinoma may be defined by anatomic origin of the carcinoma (such as, for example, the pancreas, the colon (e.g., ascending, transverse, descending, sigmoid), one or both breasts, the prostate, etc.).

[0177] In some embodiments, the carcinoma comprises: (i) a pancreatic carcinoma (i.e., acarcinoma of the pancreas), (ii) a colon carcinoma (i.e., a carcinoma of the colon), (iii) a breast carcinoma (i.e., a carcinoma of breast tissue), or (iv) a prostate carcinoma (i.e., a carcinoma of the prostate).

[0178] In some further embodiments, the carcinoma is a pancreatic carcinoma, wherein thepancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In instances where the carcinoma is a pancreatic carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the pancreatic carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for pancreatic carcinomas include, but are not limited to: (i) FOLFIRINOX, (ii) gemcitabine and / or nab-paclitaxel, (iii) carboplatin, (iv) fludarabine, and others known in the relevant art. Examples of suitable hormonal therapy for pancreatic carcinomas include, but are not limited to: (i) cyproterone acetate, (ii) ketoconazole, (iii) aminoglutethimide, (iv) abarelix, (v) degarelix, (vi) leuprolide, (vii) triptorelin, (viii) buserelin, and others known in the relevant art. Examples of suitable targeted therapy for pancreatic carcinomas include, but are not limited to: (i) Olaparib (Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), (vi) Larotrectinib (Vitrakvi ®), and others known in the relevant art. Examples of suitable immunotherapy for pancreatic carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) 61 4125-3810-3132.7Attorney Docket: 54094.4001 / WO immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0179] In some other further embodiments, the carcinoma is a colon carcinoma, wherein thecolon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In instances where the carcinoma is a colon carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the colon carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for colon carcinomas include, but are not limited to: (i) FOLFOX, (ii) capecitabine, (iii) irinotecan, (iv) oxaliplatin, (v) trifluridine and / or tipiracil, and others known in the relevant art. Examples of suitable hormonal therapy for colon carcinomas include, but are not limited to: (i) estrogen, (ii) estrogen and progestin, and others known in the relevant art. Examples of suitable targeted therapy for colon carcinomas include, but are not limited to: (i) bevacizumab, (ii) ramucirumab, (iii) ziv-aflibercept, (iv) fruquintinib, and others known in the relevant art. Examples of suitable immunotherapy for colon carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0180] In some other further embodiments, the carcinoma is a breast carcinoma, wherein thebreast carcinoma comprises: (i) invasive (or, infiltrating) ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In instances where the carcinoma is a breast carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the breast carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for breast 62 4125-3810-3132.7Attorney Docket: 54094.4001 / WO carcinomas include, but are not limited to: (i) AC-T, (ii) TC, (iii) AC, (iv) doxorubicin, (v) epirubicin, (vi) paclitaxel, (vii) docetaxel, (viii) 5-fluorouracil (5-FU), (viii) capecitabine, (ix) cyclophosphamide, (x) carboplatin, (xi) ixabepilone, (xii) eribulin, (xiii) vinorelbine, and others known in the relevant art. Examples of suitable hormonal therapy for breast carcinomas include, but are not limited to: (i) tamoxifen, (ii) anastrozole, (iii) exemestane, (iv) letrozole, (v) goserelin, and others known in the relevant art. Examples of suitable targeted therapy for breast carcinomas include, but are not limited to: (i) Palbociclib, (ii) ribociclib, (iii) abemaciclib, and others known in the relevant art. Examples of suitable immunotherapy for breast carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0181] In some other further embodiments, the carcinoma is a prostate carcinoma, wherein theprostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate. In instances where the carcinoma is a prostate carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the prostate carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for prostate carcinomas include, but are not limited to: (i) docetaxel, (ii) cabazitaxel, (iii) mitoxantrone, (iv) estramustine, (v) carboplatin, and others known in the relevant art. Examples of suitable hormonal therapy for prostate carcinomas include, but are not limited to: (i) leuprorelin (leuprolide), (ii) goserelin, (iii) triptorelin, (iv) bicalutamide (Casodex), (v) nilutamide (Nilandron), (vi) flutamide (Eulexin), (vii) enzalutamide, (viii) apalutamide, (ix) darolutamide, and others known in the relevant art. Examples of suitable targeted therapy for prostate carcinomas include, but are not limited to: (i) rucaparib, (ii) Olaparib, (iii) talazoparib, (iv) niraparib and / or abiraterone, and others known in the relevant art. Examples of suitable immunotherapy for prostate carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer 63 4125-3810-3132.7Attorney Docket: 54094.4001 / WO vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0182] In some embodiments, the method further comprises: (i) comparing determined RNAexpression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In some further embodiments, the first threshold is a fragments per kilobase million (FPKM) value of 1.00. In some other further embodiments, the first threshold may be a FPKM value of about 1.00. Alternatively, in some embodiments, the first threshold is an average signal value above background for a “blank” or “control” sample.

[0183] In some embodiments, the method further comprises extracting the plurality of nucleicacid molecules from the tumor sample from the human subject. It is expressly contemplated that “extracting” nucleic acid molecules can involve subsequent isolation and / or purification step(s). Nucleic acid molecules may be extracted, isolated, and / or purified according to any method known to those skilled in the relevant art, such as, for example, organic extraction and precipitation methods (e.g., using TRIzol reagent or phenol / chloroform), silica-based nucleic acid isolation (e.g., using PureLink or GeneJET columns), paramagnetic bead-based nucleic acid purification, and the like. In some further embodiments, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules, whereas in some other further embodiments, the plurality of nucleic acid molecules comprises a plurality of deoxyribonucleic acid (DNA) molecules. Nonlimiting examples of RNA molecules include: (i) total cellular RNA, (ii) ribosomal RNA (rRNA), (iii) cell-free RNA (cfRNA), (iv) circulating tumor RNA (ctRNA), (v) messenger RNA (mRNA; or fragments thereof), (vi) poly(A)-tailed mRNA (or fragments thereof), (vii) transfer RNA (tRNA; or fragments thereof), (viii) mitochondrial RNA, and the like. Nonlimiting examples of DNA molecules include: (i) genomic DNA (or fragments thereof), (ii) cell-free DNA (cfDNA), (iii) circulating tumor DNA (ctDNA), (iv) mitochondrial DNA (or fragments thereof), (v) cDNA synthesized from RNA, and the like. 64 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0184] In some embodiments, the assay of step (a) is performed on at least a portion of aplurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of a plurality of RNA molecules. In some embodiments, the method further comprises, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules. The plurality of cDNA molecules may be synthesized according to any method known to those skilled in the relevant art.

[0185] In some embodiments, the tumor sample comprises: (i) a malignant sample, (ii) a benignsample, or (iii) a mixed sample. In some other embodiments, the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more normal (“healthy”) cells. In some embodiments, the method further comprises obtaining the tumor sample from the human subject. The tumor sample can be obtained according to any method known in the relevant art, including, but not limited to: (i) biopsy, (ii) fine needle aspirate, (iii) tissue brushing, (iv) tissue washing, (v) excision of a tissue (e.g., a “punch” biopsy), and the like, as well as combinations thereof. Further, as will be appreciated by those skilled in the relevant art, the tumor sample can be obtained from any anatomic region, anatomic subregion, organ system, organ, tissue, etc. suitable for a given carcinoma (e.g., obtaining a biopsy from a portion of the colon in the instance of a colon carcinoma).

[0186] In some embodiments, the assay comprises: (i) a sequencing assay, (ii) a microarray, or(iii) a spatial transcriptomic assay. In some further embodiments, the assay comprises a sequencing assay and the sequencing assay comprises a high-throughput sequencing assay. In still some further embodiments, the high-throughput sequencing assay comprises: (i) ribonucleic acid-sequencing (RNA-seq), (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq), or another high-throughput sequencing method known in the relevant art. In some other further embodiments, the assay comprises a spatial transcriptomic assay. A number of platforms have been developed for performing spatial transcriptomics. Examples for situ hybridization transcriptomics include, but are not limited to: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, and (vi) hybridization-based in situ sequencing (HybISS) (see, e.g., J. Liu, et al., Life Sci Alliance.2022 Dec 16;6(1):e202201701; S. He, et al., Nat Biotechnol.2022 Dec; 40(12): 1794-1806; S. M. Salas, et al., bioRxiv 2023.02.13.528102; for more on HybISS, see D. Gyllborg, et al., Nucleic 65 4125-3810-3132.7Attorney Docket: 54094.4001 / WO Acids Res.2020 Nov 4;48(19):e112; each of which are incorporated by reference in their entirety, with particularity for relevant disclosure pertaining to spatial transcriptomic assays).

[0187] A summary of the differentially accessible open chromatin regions between met-high andmet-low subclones, including: gene name, gene symbol, Ensembl ID, chromosome number, start and end points, fold change, P-value, FDR, etc., which provides data supporting an embodiment of the use of inventions described herein wherein the assay comprises ATAC-seq, can be found in Supp. Table 4 of Handler et al. (2024) ‘Identifying a gene signature of metastatic potential by linking pre-metastatic state to ultimate metastatic fate’, bioRxiv (Cold Spring Harbor Laboratory) [Preprint]. Available at: https: / / doi.org / 10.1101 / 2024.08.14.607813.

[0188] In instances where the assay comprises a sequencing assay, the sequencing assayproduces a plurality of nucleic acid sequencing reads, and the method can further comprise aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In some further embodiments, the reference genome comprises: (i) NCBI build 34 (University of California, Santa Cruz (UCSC) equivalent: hg16), (ii) NCBI build 35 (UCSC equivalent: hg17), (iii) NCBI build 36.1 (UCSC equivalent: hg18), (iv) GRCh37 (UCSC equivalent: hg19), (v) GRCh38 (UCSC equivalent: hg38), (vi) T2T-CHM13, or others known in the relevant art.

[0189] As will be appreciated by those skilled in the relevant, the first single sample scoringalgorithm may comprise any single sample, scoring algorithm known in the relevant art, such as, for example, singscore (see, e.g., Foroutan, M., Bhuva, D.D., Lyu, R. et al. Single sample scoring of molecular phenotypes. BMC Bioinformatics 19, 404 (2018). https: / / doi.org / 10.1186 / s12859- 018-2435-4, or https: / / www.bioconductor.org / packages / release / bioc / html / singscore.html, both of which are incorporated by reference in their entirety), a k Top Scoring Pairs (kTSP)-based method, and others known in the relevant art. In some preferred embodiments, the first single sample scoring algorithm comprises singscore. In some further embodiments, the method further comprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk. In still some further embodiments, the probabilistic determination identifies the subject as having a “high” metastatic risk, whereas in some other further embodiments, the probabilistic determination identifies the subject as having a “low” metastatic risk.

[0190] In some embodiments, RNA expression levels of the plurality of genes listed in Table 1 isdetermined for at least about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, 66 4125-3810-3132.7Attorney Docket: 54094.4001 / WO about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the genes listed in Table 1 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least about 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 1 is determined for 100% of the genes listed in Table 1.

[0191] In some embodiments, RNA expression levels of the plurality of genes listed in Table 1 isdetermined for at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the genes listed in Table 1 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 1 is determined for 100% of the genes listed in Table 1.

[0192] In some embodiments, RNA expression levels of the plurality of genes listed in Table 1 isdetermined for at least about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, 67 4125-3810-3132.7Attorney Docket: 54094.4001 / WO about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173 or more of the genes listed in Table 1. In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 1 is determined for each of the genes listed in Table 1 (i.e., for all 174 genes listed in Table 1).

[0193] In some embodiments, RNA expression levels of the plurality of genes listed in Table 1 isdetermined for at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 or more of the genes listed in Table 1. In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 1 is determined for each of the genes listed in Table 1 (i.e., for all 174 genes listed in Table 1).

[0194] In some embodiments, RNA expression levels of the plurality of genes listed in Table 2 isdetermined for at least about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more of the genes listed in Table 2 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 2 is determined for 68 4125-3810-3132.7Attorney Docket: 54094.4001 / WO at least about 90% or more of the genes listed in Table 2 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 2 is determined for 100% of the genes listed in Table 2.

[0195] In some embodiments, RNA expression levels of the plurality of genes listed in Table 2 isdetermined for at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the genes listed in Table 2 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 2 is determined for 100% of the genes listed in Table 2.

[0196] In some embodiments, RNA expression levels of the plurality of genes listed in Table 2 isdetermined for at least about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, about 200, about 201 or more of the genes listed in Table 2. In a preferred embodiment, RNA expression of the plurality of genes listed in Table 2 is determined for at least about 182 of the genes listed in Table 2. In a more preferred embodiment, RNA expression levels of the plurality 69 4125-3810-3132.7Attorney Docket: 54094.4001 / WO of the genes listed in Table 2 is determined for each of the genes listed in Table 2 (i.e., for all 202 genes listed in Table 2).

[0197] In some embodiments, RNA expression levels of the plurality of genes listed in Table 2 isdetermined for at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 or more of the genes listed in Table 2. In a preferred embodiment, RNA expression of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 2 is determined for each of the genes listed in Table 2 (i.e., for all 202 genes listed in Table 2).

[0198] In some embodiments, RNA expression levels are determined for at least 300 genesselected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2. In some further embodiments: (1) about 30% of the at least 300 genes are genes listed in Table 1 and about 70% of the at least 300 genes are genes listed in Table 2, (2) about 40% of the at least 300 genes are genes listed in Table 1 and about 60% of the at least 300 genes are genes listed in Table 2, (3) about 50% of the at least 300 genes are genes listed in Table 1 and about 50% of the at least 300 genes are genes listed in Table 2, (4) about 55% of the at least 300 genes are genes listed in Table 1 and about 45% of the at least 300 genes are genes listed in Table 2, or (5) about 58% of the at least 300 genes are genes listed in Table 1 and about 42% of the at least 300 genes are genes listed in Table 2.

[0199] In some embodiments, the method further comprises, prior to the generating of step (b),normalizing individual instances of determined RNA expression levels. In some further embodiments, normalizing individual instances of determined RNA expression levels comprises (i) normalization of raw sequencing counts by gene length, total library size, and / or sequencing depth, (ii) quantile normalization, (iii) trimmed mean of M-value (TMM) normalization, (iv) batch correction methods (such as, for example, Limma, ComBat, etc.), or other normalization methods known in the relevant art (see, e.g., Liu, X. et al. (2019) ‘Normalization methods for the 70 4125-3810-3132.7Attorney Docket: 54094.4001 / WO analysis of unbalanced transcriptome data: A Review’, Frontiers in Bioengineering and Biotechnology, 7. doi:10.3389 / fbioe.2019.00358, the contents of which are incorporated by reference in their entirety with particularity for discussion pertaining to normalization methods.

[0200] In some embodiments, the method further comprises, prior to the assay of step (a),aliquoting at least a portion of the plurality of nucleic acid molecules.

[0201] In some embodiments, the method further comprises, prior to the assay of step (a),storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. In some preferred embodiments, the plurality of nucleic acid molecules is stored at a temperature that is less than or equal to -80°C. Alternatively, or in addition, the method may further comprise, subsequent to the assay of step (a), storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. In some preferred embodiments, the plurality of nucleic acid molecules is stored at a temperature that is less than or equal to -80°C.

[0202] In some embodiments, the tumor sample was obtained at a first time point, and themethod further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0203] In some embodiments, the assay of step (a) further determines: (1) RNA expressionlevels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4. In some further embodiments, the ranked list of step (b) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4. In still some further embodiments, (1) gene identity and ranking of the plurality of genes listed in Table 3, and (2) gene identity and ranking of the plurality of genes listed in Table 4, are also provided as input 71 4125-3810-3132.7Attorney Docket: 54094.4001 / WO to the first single sample scoring algorithm. In yet some further embodiments, the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) an enrichment of at least a subset of the plurality of genes listed in Table 2, (3) a depletion of at least a subset of the plurality of genes listed in Table 4, (4) a depletion of at least a subset of the plurality of genes listed in Table 1, or (5) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In some further embodiments, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk (e.g., high metastatic risk, low metastatic risk). In still some further embodiments, the method further comprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0204] In some embodiments, the tumor sample was obtained at a first time point, and themethod further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0205] In some embodiments, the assay of step (a) further determines: (1) RNA expressionlevels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4. In some further embodiments, the method further comprises: (f) 72 4125-3810-3132.7Attorney Docket: 54094.4001 / WO providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In some further embodiments, the first single sample classifier algorithm comprises: (i) a k Top Scoring Pairs (kTSP)-based method (see, e.g., Moffitt, R.A. et al. (2015) ‘Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma’, Nature Genetics, 47(10), pp. 1168–1178. Doi:10.1038 / ng.3398, the contents of which are incorporated by reference in their entirety), (ii) singscore, or other single sample classifier algorithms known in the relevant art. In still some further embodiments, the method further comprises, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0206] In some further embodiments, the tumor sample was obtained at a first time point, andthe method described above further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples. 73 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0207] In some embodiments, the second time point is subsequent to the first time point, whereasin other embodiments, the first time point may be subsequent to the second time point. In some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) after the earlier time point.

[0208] In some embodiments, the second time point is subsequent to the first time point. In someembodiments, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the administration of the first therapy.

[0209] In some embodiments, the second time point is subsequent to the first time point. In someembodiments, the first time point occurs prior to one or more therapeutic interventions, and the second time point occurs subsequent to the one or more therapeutic interventions. In some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 74 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 16, 17, 18, 19, 20 or more week(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the one or more therapeutic interventions. B. Depletion of Epigenetically Silenced Genes

[0210] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels of a plurality of genes comprising a human transcriptome, wherein RNA expression levels of the plurality of genes of the human transcriptome comprises a plurality of genes listed in Table 1; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes listed in Table 1, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies a depletion of at least a subset of the plurality of genes listed in Table 1; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

[0211] In some embodiments, the plurality of genes comprising a human transcriptomecomprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the genes comprising the human transcriptome. In some embodiments, the plurality of genes comprising the human transcriptome comprises about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100% of the human transcriptome. In some embodiments, the plurality of genes comprising the human transcriptome comprises about 75 4125-3810-3132.7Attorney Docket: 54094.4001 / WO all (i.e., ~100%) or about substantially all (i.e., at least about 95%) genes encoding the human transcriptome.

[0212] In some embodiments, the method further comprises administering adjuvant therapy tothe human subject if the human subject is identified as having a high metastatic risk. In some further embodiments, the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. It is appreciated that a person of ordinary skill in the art will be able to readily identify and select an appropriate adjuvant therapy for administration to the human subject in accordance with standards of care for a given carcinoma.

[0213] Examples of suitable chemotherapy include, but are not limited to: (i) FOLFIRINOX(i.e., a combination of the drugs leucovorin calcium (folinic acid), fluorouracil, irinotecan hydrochloride, and oxaliplatin), (ii) FOLFOX (i.e., a combination of the drugs leucovorin calcium (folinic acid), fluorouracil, and oxaliplatin), (iii) FOLFOX-4, (iv) FOLFOX-6, (v) modified FOLFOX-6 (mFOLFOX-6), (vi) FOLFOX-7, (vii) gemcitabine and / or nab-paclitaxel (Abraxane ®), (viii) AC-T (i.e., a combination of doxorubicin hydrochloride (Adriamycin) and cyclophosphamide, followed by treatment with paclitaxel), (ix) TC (i.e., a combination of docetaxel and cyclophosphamide), (x) AC (i.e., a combination of doxorubicin and cyclophosphamide), (xi) docetaxel, (xii) carboplatin, (xiii) fludarabine, (xiv) capecitabine, (xv) irinotecan, (xvi) trifluridine and / or tipiracil, (xvii) epirubicin, (xviii) ixabepilone, (xix) eribulin, (xx) vinorelbine, (xxi) cabazitaxel, (xxii) mitoxantrone, (xxiii) estramustine, and others known in the relevant art.

[0214] Examples of suitable hormonal therapy include, but are not limited to: (i) tamoxifen, (ii)anastrozole, (iii) exemestane, (iv) letrozole, (v) goserelin, (vi) leuprorelin (leuprolide), (vii) triptorelin, (viii) bicalutamide (Casodex), (ix) nilutamide (Nilandron), (x) flutamide (Eulexin), (xi) enzalutamide, (xii) apalutamide, (xiii) darolutamide, (xiv) cyproterone acetate, (xv) ketoconazole, (xvi) aminoglutethimide, and others known in the relevant art.

[0215] Examples of suitable targeted therapy include, but are not limited to: (i) Olaparib(Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), (vi) Larotrectinib (Vitrakvi ®), (vii) Palbociclib, (viii) ribociclib, (ix) abemaciclib, 76 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (x) bevacizumab, (xi) ramucirumab, (xii) ziv-aflibercept, (xiii) fruquintinib, (xiv) rucaparib, (xv) talazoparib, (xvi) niraparib and / or abiraterone, and others known in the relevant art.

[0216] Examples of suitable immunotherapy include, but are not limited to: (i) checkpointinhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0217] Generally, the carcinoma can comprise: (i) adenocarcinoma, (ii) squamous cellcarcinoma, (iii) adenosquamous carcinoma, (iv) anaplastic carcinoma, (v) large cell carcinoma, (vi) small cell carcinoma, (vii) spindle cell carcinoma, (viii) giant cell carcinoma, (ix) sarcomatoid carcinoma, (x) pleomorphic carcinoma, or others known in the relevant art. Further, a carcinoma may be defined by anatomic origin of the carcinoma (such as, for example, the pancreas, the colon (e.g., ascending, transverse, descending, sigmoid), one or both breasts, the prostate, etc.).

[0218] In some embodiments, the carcinoma comprises: (i) a pancreatic carcinoma (i.e., acarcinoma of the pancreas), (ii) a colon carcinoma (i.e., a carcinoma of the colon), (iii) a breast carcinoma (i.e., a carcinoma of breast tissue), or (iv) a prostate carcinoma (i.e., a carcinoma of the prostate).

[0219] In some further embodiments, the carcinoma is a pancreatic carcinoma, wherein thepancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In instances where the carcinoma is a pancreatic carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the pancreatic carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for pancreatic carcinomas include, but are not limited to: (i) FOLFIRINOX, (ii) gemcitabine and / or nab-paclitaxel, (iii) 77 4125-3810-3132.7Attorney Docket: 54094.4001 / WO carboplatin, (iv) fludarabine, and others known in the relevant art. Examples of suitable hormonal therapy for pancreatic carcinomas include, but are not limited to: (i) cyproterone acetate, (ii) ketoconazole, (iii) aminoglutethimide, (iv) abarelix, (v) degarelix, (vi) leuprolide, (vii) triptorelin, (viii) buserelin, and others known in the relevant art. Examples of suitable targeted therapy for pancreatic carcinomas include, but are not limited to: (i) Olaparib (Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), (vi) Larotrectinib (Vitrakvi ®), and others known in the relevant art. Examples of suitable immunotherapy for pancreatic carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0220] In some other further embodiments, the carcinoma is a colon carcinoma, wherein thecolon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In instances where the carcinoma is a colon carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the colon carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for colon carcinomas include, but are not limited to: (i) FOLFOX, (ii) capecitabine, (iii) irinotecan, (iv) oxaliplatin, (v) trifluridine and / or tipiracil, and others known in the relevant art. Examples of suitable hormonal therapy for colon carcinomas include, but are not limited to: (i) estrogen, (ii) estrogen and progestin, and others known in the relevant art. Examples of suitable targeted therapy for colon carcinomas include, but are not limited to: (i) bevacizumab, (ii) ramucirumab, (iii) ziv-aflibercept, (iv) fruquintinib, and others known in the relevant art. Examples of suitable immunotherapy for colon carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or 78 4125-3810-3132.7Attorney Docket: 54094.4001 / WO prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0221] In some other further embodiments, the carcinoma is a breast carcinoma, wherein thebreast carcinoma comprises: (i) invasive (or, infiltrating) ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In instances where the carcinoma is a breast carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the breast carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for breast carcinomas include, but are not limited to: (i) AC-T, (ii) TC, (iii) AC, (iv) doxorubicin, (v) epirubicin, (vi) paclitaxel, (vii) docetaxel, (viii) 5-fluorouracil (5-FU), (viii) capecitabine, (ix) cyclophosphamide, (x) carboplatin, (xi) ixabepilone, (xii) eribulin, (xiii) vinorelbine, and others known in the relevant art. Examples of suitable hormonal therapy for breast carcinomas include, but are not limited to: (i) tamoxifen, (ii) anastrozole, (iii) exemestane, (iv) letrozole, (v) goserelin, and others known in the relevant art. Examples of suitable targeted therapy for breast carcinomas include, but are not limited to: (i) Palbociclib, (ii) ribociclib, (iii) abemaciclib, and others known in the relevant art. Examples of suitable immunotherapy for breast carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0222] In some other further embodiments, the carcinoma is a prostate carcinoma, wherein theprostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate. In instances where the carcinoma is a prostate carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the prostate 79 4125-3810-3132.7Attorney Docket: 54094.4001 / WO carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for prostate carcinomas include, but are not limited to: (i) docetaxel, (ii) cabazitaxel, (iii) mitoxantrone, (iv) estramustine, (v) carboplatin, and others known in the relevant art. Examples of suitable hormonal therapy for prostate carcinomas include, but are not limited to: (i) leuprorelin (leuprolide), (ii) goserelin, (iii) triptorelin, (iv) bicalutamide (Casodex), (v) nilutamide (Nilandron), (vi) flutamide (Eulexin), (vii) enzalutamide, (viii) apalutamide, (ix) darolutamide, and others known in the relevant art. Examples of suitable targeted therapy for prostate carcinomas include, but are not limited to: (i) rucaparib, (ii) Olaparib, (iii) talazoparib, (iv) niraparib and / or abiraterone, and others known in the relevant art. Examples of suitable immunotherapy for prostate carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0223] In some embodiments, the method further comprises: (i) comparing determined RNAexpression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In some further embodiments, the first threshold is a fragments per kilobase million (FPKM) value of 1.00. In some other further embodiments, the first threshold may be a FPKM value of about 1.00. Alternatively, in some embodiments, the first threshold is an average signal value above background for a “blank” or “control” sample.

[0224] In some embodiments, the method further comprises extracting the plurality of nucleicacid molecules from the tumor sample from the human subject. It is expressly contemplated that “extracting” nucleic acid molecules can involve subsequent isolation and / or purification step(s). Nucleic acid molecules may be extracted, isolated, and / or purified according to any method known to those skilled in the relevant art, such as, for example, organic extraction and 80 4125-3810-3132.7Attorney Docket: 54094.4001 / WO precipitation methods (e.g., using TRIzol reagent or phenol / chloroform, silica-based nucleic acid isolation (e.g., using PureLink or GeneJET columns), paramagnetic bead-based nucleic acid purification, and the like. In some further embodiments, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules, whereas in some other further embodiments, the plurality of nucleic acid molecules comprises a plurality of deoxyribonucleic acid (DNA) molecules. Nonlimiting examples of RNA molecules include: (i) total cellular RNA, (ii) ribosomal RNA (rRNA), (iii) cell-free RNA (cfRNA), (iv) circulating tumor RNA (ctRNA), (v) messenger RNA (mRNA; or fragments thereof), (vi) poly(A)-tailed mRNA (or fragments thereof), (vii) transfer RNA (tRNA; or fragments thereof), (viii) mitochondrial RNA, and the like. Nonlimiting examples of DNA molecules include: (i) genomic DNA (or fragments thereof), (ii) cell-free DNA (cfDNA), (iii) circulating tumor DNA (ctDNA), (iv) mitochondrial DNA (or fragments thereof), (v) cDNA synthesized from RNA, and the like.

[0225] In some embodiments, the assay of step (a) is performed on at least a portion of aplurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of a plurality of RNA molecules. In some embodiments, the method further comprises, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules. The plurality of cDNA molecules may be synthesized according to any method known to those skilled in the relevant art.

[0226] In some embodiments, the tumor sample comprises: (i) a malignant sample, (ii) a benignsample, or (iii) a mixed sample. In some other embodiments, the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more normal (“healthy”) cells. In some embodiments, the method further comprises obtaining the tumor sample from the human subject. The tumor sample can be obtained according to any method known in the relevant art, including, but not limited to: (i) biopsy, (ii) fine needle aspirate, (iii) tissue brushing, (iv) tissue washing, (v) excision of a tissue (e.g., a “punch” biopsy), and the like, as well as combinations thereof. Further, as will be appreciated by those skilled in the relevant art, the tumor sample can be obtained from any anatomic region, anatomic subregion, organ system, organ, tissue, etc. suitable for a given carcinoma (e.g., obtaining a biopsy from a portion of the colon in the instance of a colon carcinoma).

[0227] In some embodiments, the assay comprises: (i) a sequencing assay, (ii) a microarray, or(iii) a spatial transcriptomic assay. In some further embodiments, the assay comprises a 81 4125-3810-3132.7Attorney Docket: 54094.4001 / WO sequencing assay and the sequencing assay comprises a high-throughput sequencing assay. In still some further embodiments, the high-throughput sequencing assay comprises: (i) ribonucleic acid-sequencing (RNA-seq), (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq), or another high-throughput sequencing method known in the relevant art. In some other further embodiments, the assay comprises a spatial transcriptomic assay. A number of platforms have been developed for performing spatial transcriptomics. Examples for situ hybridization transcriptomics include, but are not limited to: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, and (vi) hybridization-based in situ sequencing (HybISS) (see, e.g., J. Liu, et al., Life Sci Alliance.2022 Dec 16;6(1):e202201701; S. He, et al., Nat Biotechnol.2022 Dec; 40(12): 1794-1806; S. M. Salas, et al., bioRxiv 2023.02.13.528102; for more on HybISS, see D. Gyllborg, et al., Nucleic Acids Res.2020 Nov 4;48(19):e112; each of which are incorporated by reference in their entirety, with particularity for relevant disclosure pertaining to spatial transcriptomic assays).

[0228] A summary of the differentially accessible open chromatin regions between met-high andmet-low subclones, including: gene name, gene symbol, Ensembl ID, chromosome number, start and end points, fold change, P-value, FDR, etc., which provides data supporting an embodiment of the use of inventions described herein wherein the assay comprises ATAC-seq, can be found in Supp. Table 4 of Handler et al. (2024) ‘Identifying a gene signature of metastatic potential by linking pre-metastatic state to ultimate metastatic fate’, bioRxiv (Cold Spring Harbor Laboratory) [Preprint]. Available at: https: / / doi.org / 10.1101 / 2024.08.14.607813.

[0229] In instances where the assay comprises a sequencing assay, the sequencing assayproduces a plurality of nucleic acid sequencing reads, and the method can further comprise aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In some further embodiments, the reference genome comprises: (i) NCBI build 34 (University of California, Santa Cruz (UCSC) equivalent: hg16), (ii) NCBI build 35 (UCSC equivalent: hg17), (iii) NCBI build 36.1 (UCSC equivalent: hg18), (iv) GRCh37 (UCSC equivalent: hg19), (v) GRCh38 (UCSC equivalent: hg38), (vi) T2T-CHM13, and others known in the relevant art.

[0230] As will be appreciated by those skilled in the relevant, the first single sample scoringalgorithm may comprise any single sample, scoring algorithm known in the relevant art, such as, for example, singscore (see, e.g., Foroutan, M., Bhuva, D.D., Lyu, R. et al. Single sample scoring of molecular phenotypes. BMC Bioinformatics 19, 404 (2018). https: / / doi.org / 10.1186 / s12859- 82 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 018-2435-4, or https: / / www.bioconductor.org / packages / release / bioc / html / singscore.html, both of which are incorporated by reference in their entirety), a k Top Scoring Pairs (kTSP)-based method, and others known in the relevant art. In some preferred embodiments, the first single sample scoring algorithm comprises singscore. In some further embodiments, the method further comprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk. In still some further embodiments, the probabilistic determination identifies the subject as having a “high” metastatic risk, whereas in some other further embodiments, the probabilistic determination identifies the subject as having a “low” metastatic risk.

[0231] In some embodiments, RNA expression levels of the plurality of genes listed in Table 1 isdetermined for at least about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the genes listed in Table 1 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least about 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 1 is determined for 100% of the genes listed in Table 1.

[0232] In some other embodiments, RNA expression levels of the plurality of genes listed inTable 1 is determined for at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the genes listed in Table 1 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 90% or more of the genes listed in Table 1 (again, rounded to the nearest 83 4125-3810-3132.7Attorney Docket: 54094.4001 / WO corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 1 is determined for 100% of the genes listed in Table 1.

[0233] In some other embodiments, RNA expression levels of the plurality of genes listed inTable 1 is determined for at least about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173 or more of the genes listed in Table 1. In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 1 is determined for each of the genes listed in Table 1 (i.e., for all 174 genes listed in Table 1).

[0234] In some other embodiments, RNA expression levels of the plurality of genes listed inTable 1 is determined for at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 or more of the genes listed in Table 1. In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 1 is determined for each of the genes listed in Table 1 (i.e., for all 174 genes listed in Table 1). 84 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0235] In some embodiments, the method further comprises, prior to the generating of step (b),normalizing individual instances of determined RNA expression levels. In some further embodiments, normalizing individual instances of determined RNA expression levels comprises (i) normalization of raw sequencing counts by gene length, total library size, and / or sequencing depth, (ii) quantile normalization, (iii) trimmed mean of M-value (TMM) normalization, (iv) batch correction methods (such as, for example, Limma, ComBat, etc.), or other normalization methods known in the relevant art (see, e.g., Liu, X. et al. (2019) ‘Normalization methods for the analysis of unbalanced transcriptome data: A Review’, Frontiers in Bioengineering and Biotechnology, 7. doi:10.3389 / fbioe.2019.00358, the contents of which are incorporated by reference in their entirety with particularity for discussion pertaining to normalization methods.

[0236] In some embodiments, the method further comprises, prior to the assay of step (a),aliquoting at least a portion of the plurality of nucleic acid molecules.

[0237] In some embodiments, the method further comprises, prior to the assay of step (a),storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. In some preferred embodiments, the plurality of nucleic acid molecules is stored at a temperature that is less than or equal to -80°C. Alternatively, or in addition, the method may further comprise, subsequent to the assay of step (a), storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. In some preferred embodiments, the plurality of nucleic acid molecules is stored at a temperature that is less than or equal to -80°C.

[0238] In some embodiments, the tumor sample was obtained at a first time point, and themethod further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known 85 4125-3810-3132.7Attorney Docket: 54094.4001 / WO in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0239] In some embodiments, the assay of step (a) further determines: (1) RNA expressionlevels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4. In some further embodiments, the ranked list of step (b) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4. In still some further embodiments, (1) gene identity and ranking of the plurality of genes listed in Table 3, and (2) gene identity and ranking of the plurality of genes listed in Table 4, are also provided as input to the first single sample scoring algorithm. In yet some further embodiments, the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) a depletion of at least a subset of the plurality of genes listed in Table 4, (3) a depletion of at least a subset of the plurality of genes listed in Table 1, or (4) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In some further embodiments, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk (e.g., high metastatic risk, low metastatic risk). In still some further embodiments, the method further comprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0240] In some embodiments, the tumor sample was obtained at a first time point, and themethod further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a 86 4125-3810-3132.7Attorney Docket: 54094.4001 / WO tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0241] In some embodiments, the assay of step (a) further determines: (1) RNA expressionlevels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4. In some further embodiments, the method further comprises: (f) providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In some further embodiments, the first single sample classifier algorithm comprises: (i) a k Top Scoring Pairs (kTSP)-based method (see, e.g., Moffitt, R.A. et al. (2015) ‘Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma’, Nature Genetics, 47(10), pp. 1168–1178. Doi:10.1038 / ng.3398, the contents of which are incorporated by reference in their entirety), (ii) singscore, or other single sample classifier algorithms known in the relevant art. In still some further embodiments, the method further comprises, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0242] In some embodiments, the tumor sample was obtained at a first time point, and themethod described above further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a 87 4125-3810-3132.7Attorney Docket: 54094.4001 / WO classical cell state. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0243] In some embodiments, the second time point is subsequent to the first time point, whereasin other embodiments, the first time point may be subsequent to the second time point. In some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) after the earlier time point.

[0244] In some embodiments, the second time point is subsequent to the first time point. In someembodiments, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the administration of the first therapy. 88 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0245] In some embodiments, the second time point is subsequent to the first time point. In someembodiments, the first time point occurs prior to one or more therapeutic interventions, and the second time point occurs subsequent to the one or more therapeutic interventions. In some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the one or more therapeutic interventions.

[0246] In some embodiments, the assay of step (a) further determines RNA expression levels ofa plurality of genes listed in Table 2. In some further embodiments, the ranked list of step (b) further includes the plurality of genes listed in Table 2. In still some further embodiments, gene identity and ranking of the plurality of genes listed in Table 2 are also provided as input to the first single sample scoring algorithm. In yet some further embodiments, the method further comprises: (1) outputting a second metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the second metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; and (2) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the second metric.

[0247] In some further embodiments, RNA expression levels of the plurality of genes listed inTable 2 is determined for at least about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, 89 4125-3810-3132.7Attorney Docket: 54094.4001 / WO about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more of the genes listed in Table 2 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 2 is determined for at least about 90% or more of the genes listed in Table 2 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 2 is determined for 100% of the genes listed in Table 2.

[0248] In some other further embodiments, RNA expression levels of the plurality of geneslisted in Table 2 is determined for at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the genes listed in Table 2 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 2 is determined for 100% of the genes listed in Table 2.

[0249] In some other further embodiments, RNA expression levels of the plurality of geneslisted in Table 2 is determined for at least about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 90 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, about 200, about 201 or more of the genes listed in Table 2. In a preferred embodiment, RNA expression of the plurality of genes listed in Table 2 is determined for at least about 182 of the genes listed in Table 2. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 2 is determined for each of the genes listed in Table 2 (i.e., for all 202 genes listed in Table 2).

[0250] In some other further embodiments, RNA expression levels of the plurality of geneslisted in Table 2 is determined for at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 or more of the genes listed in Table 2. In a preferred embodiment, RNA expression of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 2 is determined for each of the genes listed in Table 2 (i.e., for all 202 genes listed in Table 2).

[0251] In some further embodiments, RNA expression levels are determined for at least 300genes selected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2. In some further embodiments: (1) about 30% of the at least 300 genes are genes listed in Table 1 and about 70% of the at least 300 genes are genes listed in Table 2, (2) about 40% of the at least 300 genes are genes listed in Table 1 and about 60% of the at least 300 genes are genes listed in Table 2, (3) about 50% of the at least 300 genes are genes listed in Table 1 and about 50% of the at least 300 genes are genes listed in Table 2, (4) about 55% of the at least 300 genes are genes listed in Table 1 and about 45% of the at least 300 genes are genes listed in Table 2, or (5) about 58% of the at least 300 genes are genes listed in Table 1 and about 42% of the at least 300 genes are genes listed in Table 2. 91 4125-3810-3132.7Attorney Docket: 54094.4001 / WO C. Enrichment of Epigenetically Activated Genes

[0252] In another aspect, the present disclosure provides a method for identifying metastatic riskof a carcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels a plurality of genes comprising a human transcriptome, wherein RNA expression levels of the plurality of genes of the human transcriptome comprises a plurality of genes listed in Table 2; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies an enrichment of at least a subset of the plurality of genes listed in Table 2; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

[0253] In some embodiments, the plurality of genes comprising a human transcriptomecomprises at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the genes comprising the human transcriptome. In some embodiments, the plurality of genes comprising the human transcriptome comprises about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100% of the human transcriptome. In some embodiments, the plurality of genes comprising the human transcriptome comprises about all (i.e., ~100%) or about substantially all (i.e., at least about 95%) genes encoding the human transcriptome.

[0254] In some embodiments, the method further comprises administering adjuvant therapy tothe human subject if the human subject is identified as having a high metastatic risk. In some further embodiments, the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. It is appreciated that a person of ordinary skill in the art will be able to readily identify and select an appropriate 92 4125-3810-3132.7Attorney Docket: 54094.4001 / WO adjuvant therapy for administration to the human subject in accordance with standards of care for a given carcinoma.

[0255] Examples of suitable chemotherapy include, but are not limited to: (i) FOLFIRINOX(i.e., a combination of the drugs leucovorin calcium (folinic acid), fluorouracil, irinotecan hydrochloride, and oxaliplatin), (ii) FOLFOX (i.e., a combination of the drugs leucovorin calcium (folinic acid), fluorouracil, and oxaliplatin), (iii) FOLFOX-4, (iv) FOLFOX-6, (v) modified FOLFOX-6 (mFOLFOX-6), (vi) FOLFOX-7, (vii) gemcitabine and / or nab-paclitaxel (Abraxane ®), (viii) AC-T (i.e., a combination of doxorubicin hydrochloride (Adriamycin) and cyclophosphamide, followed by treatment with paclitaxel), (ix) TC (i.e., a combination of docetaxel and cyclophosphamide), (x) AC (i.e., a combination of doxorubicin and cyclophosphamide), (xi) docetaxel, (xii) carboplatin, (xiii) fludarabine, (xiv) capecitabine, (xv) irinotecan, (xvi) trifluridine and / or tipiracil, (xvii) epirubicin, (xviii) ixabepilone, (xix) eribulin, (xx) vinorelbine, (xxi) cabazitaxel, (xxii) mitoxantrone, (xxiii) estramustine, and others known in the relevant art.

[0256] Examples of suitable hormonal therapy include, but are not limited to: (i) tamoxifen, (ii)anastrozole, (iii) exemestane, (iv) letrozole, (v) goserelin, (vi) leuprorelin (leuprolide), (vii) triptorelin, (viii) bicalutamide (Casodex), (ix) nilutamide (Nilandron), (x) flutamide (Eulexin), (xi) enzalutamide, (xii) apalutamide, (xiii) darolutamide, (xiv) cyproterone acetate, (xv) ketoconazole, (xvi) aminoglutethimide, and others known in the relevant art.

[0257] Examples of suitable targeted therapy include, but are not limited to: (i) Olaparib(Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), (vi) Larotrectinib (Vitrakvi ®), (vii) Palbociclib, (viii) ribociclib, (ix) abemaciclib, (x) bevacizumab, (xi) ramucirumab, (xii) ziv-aflibercept, (xiii) fruquintinib, (xiv) rucaparib, (xv) talazoparib, (xvi) niraparib and / or abiraterone, and others known in the relevant art.

[0258] Examples of suitable immunotherapy include, but are not limited to: (i) checkpointinhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art. 93 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0259] Generally, the carcinoma can comprise: (i) adenocarcinoma, (ii) squamous cellcarcinoma, (iii) adenosquamous carcinoma, (iv) anaplastic carcinoma, (v) large cell carcinoma, (vi) small cell carcinoma, (vii) spindle cell carcinoma, (viii) giant cell carcinoma, (ix) sarcomatoid carcinoma, (x) pleomorphic carcinoma, or others known in the relevant art. Further, a carcinoma may be defined by anatomic origin of the carcinoma (such as, for example, the pancreas, the colon (e.g., ascending, transverse, descending, sigmoid), one or both breasts, the prostate, etc.).

[0260] In some embodiments, the carcinoma comprises: (i) a pancreatic carcinoma (i.e., acarcinoma of the pancreas), (ii) a colon carcinoma (i.e., a carcinoma of the colon), (iii) a breast carcinoma (i.e., a carcinoma of breast tissue), or (iv) a prostate carcinoma (i.e., a carcinoma of the prostate).

[0261] In some further embodiments, the carcinoma is a pancreatic carcinoma, wherein thepancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells. In instances where the carcinoma is a pancreatic carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the pancreatic carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for pancreatic carcinomas include, but are not limited to: (i) FOLFIRINOX, (ii) gemcitabine and / or nab-paclitaxel, (iii) carboplatin, (iv) fludarabine, and others known in the relevant art. Examples of suitable hormonal therapy for pancreatic carcinomas include, but are not limited to: (i) cyproterone acetate, (ii) ketoconazole, (iii) aminoglutethimide, (iv) abarelix, (v) degarelix, (vi) leuprolide, (vii) triptorelin, (viii) buserelin, and others known in the relevant art. Examples of suitable targeted therapy for pancreatic carcinomas include, but are not limited to: (i) Olaparib (Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), (vi) Larotrectinib (Vitrakvi ®), and others known in the relevant art. Examples of suitable 94 4125-3810-3132.7Attorney Docket: 54094.4001 / WO immunotherapy for pancreatic carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0262] In some other further embodiments, the carcinoma is a colon carcinoma, wherein thecolon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma. In instances where the carcinoma is a colon carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the colon carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for colon carcinomas include, but are not limited to: (i) FOLFOX, (ii) capecitabine, (iii) irinotecan, (iv) oxaliplatin, (v) trifluridine and / or tipiracil, and others known in the relevant art. Examples of suitable hormonal therapy for colon carcinomas include, but are not limited to: (i) estrogen, (ii) estrogen and progestin, and others known in the relevant art. Examples of suitable targeted therapy for colon carcinomas include, but are not limited to: (i) bevacizumab, (ii) ramucirumab, (iii) ziv-aflibercept, (iv) fruquintinib, and others known in the relevant art. Examples of suitable immunotherapy for colon carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0263] In some other further embodiments, the carcinoma is a breast carcinoma, wherein thebreast carcinoma comprises: (i) invasive (or, infiltrating) ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ. In instances where the carcinoma is a breast 95 4125-3810-3132.7Attorney Docket: 54094.4001 / WO carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the breast carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for breast carcinomas include, but are not limited to: (i) AC-T, (ii) TC, (iii) AC, (iv) doxorubicin, (v) epirubicin, (vi) paclitaxel, (vii) docetaxel, (viii) 5-fluorouracil (5-FU), (viii) capecitabine, (ix) cyclophosphamide, (x) carboplatin, (xi) ixabepilone, (xii) eribulin, (xiii) vinorelbine, and others known in the relevant art. Examples of suitable hormonal therapy for breast carcinomas include, but are not limited to: (i) tamoxifen, (ii) anastrozole, (iii) exemestane, (iv) letrozole, (v) goserelin, and others known in the relevant art. Examples of suitable targeted therapy for breast carcinomas include, but are not limited to: (i) Palbociclib, (ii) ribociclib, (iii) abemaciclib, and others known in the relevant art. Examples of suitable immunotherapy for breast carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0264] In some other further embodiments, the carcinoma is a prostate carcinoma, wherein theprostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate. In instances where the carcinoma is a prostate carcinoma and the method further comprises administration of an adjuvant therapy to a human subject identified as having a high metastatic risk for the prostate carcinoma, the adjuvant therapy may comprise: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof. Examples of suitable chemotherapy for prostate carcinomas include, but are not limited to: (i) docetaxel, (ii) cabazitaxel, (iii) mitoxantrone, (iv) estramustine, (v) carboplatin, and others known in the relevant art. Examples of suitable hormonal therapy for prostate carcinomas include, but are not limited to: (i) leuprorelin (leuprolide), (ii) goserelin, (iii) triptorelin, (iv) bicalutamide (Casodex), (v) nilutamide (Nilandron), (vi) flutamide (Eulexin), (vii) enzalutamide, (viii) apalutamide, (ix) darolutamide, and others known in the relevant art. Examples of suitable targeted therapy for 96 4125-3810-3132.7Attorney Docket: 54094.4001 / WO prostate carcinomas include, but are not limited to: (i) rucaparib, (ii) Olaparib, (iii) talazoparib, (iv) niraparib and / or abiraterone, and others known in the relevant art. Examples of suitable immunotherapy for prostate carcinomas include, but are not limited to: (i) checkpoint inhibitor(s), (ii) adoptive cell therapy (such as, for example, T-cell transfer therapy), (iii) cancer vaccines (such as, for example, therapeutic and / or prophylactic cancer vaccines), (iv) monoclonal antibody (-ies; such as, for example, monospecific or bispecific antibodies), (v) immunomodulator(s), (vi) oncolytic virus(es), (vii) cytokine(s), and others known in the relevant art.

[0265] In some embodiments, the method further comprises: (i) comparing determined RNAexpression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes. In some further embodiments, the first threshold is a fragments per kilobase million (FPKM) value of 1.00. In some other further embodiments, the first threshold may be a FPKM value of about 1.00. Alternatively, in some embodiments, the first threshold is an average signal value above background for a “blank” or “control” sample.

[0266] In some embodiments, the method further comprises extracting the plurality of nucleicacid molecules from the tumor sample from the human subject. It is expressly contemplated that “extracting” nucleic acid molecules can involve subsequent isolation and / or purification step(s). Nucleic acid molecules may be extracted, isolated, and / or purified according to any method known to those skilled in the relevant art, such as, for example, organic extraction and precipitation methods (e.g., using TRIzol reagent or phenol / chloroform, silica-based nucleic acid isolation (e.g., using PureLink or GeneJET columns), paramagnetic bead-based nucleic acid purification, and the like. In some further embodiments, the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules, whereas in some other further embodiments, the plurality of nucleic acid molecules comprises a plurality of deoxyribonucleic acid (DNA) molecules. Nonlimiting examples of RNA molecules include: (i) total cellular RNA, (ii) ribosomal RNA (rRNA), (iii) cell-free RNA (cfRNA), (iv) circulating tumor RNA (ctRNA), (v) messenger RNA (mRNA; or fragments thereof), (vi) poly(A)-tailed mRNA (or fragments 97 4125-3810-3132.7Attorney Docket: 54094.4001 / WO thereof), (vii) transfer RNA (tRNA; or fragments thereof), (viii) mitochondrial RNA, and the like. Nonlimiting examples of DNA molecules include: (i) genomic DNA (or fragments thereof), (ii) cell-free DNA (cfDNA), (iii) circulating tumor DNA (ctDNA), (iv) mitochondrial DNA (or fragments thereof), (v) cDNA synthesized from RNA, and the like.

[0267] In some embodiments, the assay of step (a) is performed on at least a portion of aplurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of a plurality of RNA molecules. In some embodiments, the method further comprises, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules. The plurality of cDNA molecules may be synthesized according to any method known to those skilled in the relevant art.

[0268] In some embodiments, the tumor sample comprises: (i) a malignant sample, (ii) a benignsample, or (iii) a mixed sample. In some other embodiments, the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more normal (“healthy”) cells. In some embodiments, the method further comprises obtaining the tumor sample from the human subject. The tumor sample can be obtained according to any method known in the relevant art, including, but not limited to: (i) biopsy, (ii) fine needle aspirate, (iii) tissue brushing, (iv) tissue washing, (v) excision of a tissue (e.g., a “punch” biopsy), and the like, as well as combinations thereof. Further, as will be appreciated by those skilled in the relevant art, the tumor sample can be obtained from any anatomic region, anatomic subregion, organ system, organ, tissue, etc. suitable for a given carcinoma (e.g., obtaining a biopsy from a portion of the colon in the instance of a colon carcinoma).

[0269] In some embodiments, the assay comprises: (i) a sequencing assay, (ii) a microarray, or(iii) a spatial transcriptomic assay. In some further embodiments, the assay comprises a sequencing assay and the sequencing assay comprises a high-throughput sequencing assay. In still some further embodiments, the high-throughput sequencing assay comprises: (i) ribonucleic acid-sequencing (RNA-seq), (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq), or another high-throughput sequencing method known in the relevant art. In some other further embodiments, the assay comprises a spatial transcriptomic assay. A number of platforms have been developed for performing spatial transcriptomics. Examples for situ hybridization transcriptomics include, but are not limited to: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, and (vi) 98 4125-3810-3132.7Attorney Docket: 54094.4001 / WO hybridization-based in situ sequencing (HybISS) (see, e.g., J. Liu, et al., Life Sci Alliance.2022 Dec 16;6(1):e202201701; S. He, et al., Nat Biotechnol.2022 Dec; 40(12): 1794-1806; S. M. Salas, et al., bioRxiv 2023.02.13.528102; for more on HybISS, see D. Gyllborg, et al., Nucleic Acids Res.2020 Nov 4;48(19):e112; each of which are incorporated by reference in their entirety, with particularity for relevant disclosure pertaining to spatial transcriptomic assays).

[0270] A summary of the differentially accessible open chromatin regions between met-high andmet-low subclones, including: gene name, gene symbol, Ensembl ID, chromosome number, start and end points, fold change, P-value, FDR, etc., which provides data supporting an embodiment of the use of inventions described herein wherein the assay comprises ATAC-seq, can be found in Supp. Table 4 of Handler et al. (2024) ‘Identifying a gene signature of metastatic potential by linking pre-metastatic state to ultimate metastatic fate’, bioRxiv (Cold Spring Harbor Laboratory) [Preprint]. Available at: https: / / doi.org / 10.1101 / 2024.08.14.607813.

[0271] In instances where the assay comprises a sequencing assay, the sequencing assayproduces a plurality of nucleic acid sequencing reads, and the method can further comprise aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome. In some further embodiments, the reference genome comprises: (i) NCBI build 34 (University of California, Santa Cruz (UCSC) equivalent: hg16), (ii) NCBI build 35 (UCSC equivalent: hg17), (iii) NCBI build 36.1 (UCSC equivalent: hg18), (iv) GRCh37 (UCSC equivalent: hg19), (v) GRCh38 (UCSC equivalent: hg38), (vi) T2T-CHM13, and others known in the relevant art.

[0272] As will be appreciated by those skilled in the relevant, the first single sample scoringalgorithm may comprise any single sample, scoring algorithm known in the relevant art, such as, for example, singscore (see, e.g., Foroutan, M., Bhuva, D.D., Lyu, R. et al. Single sample scoring of molecular phenotypes. BMC Bioinformatics 19, 404 (2018). https: / / doi.org / 10.1186 / s12859- 018-2435-4, or https: / / www.bioconductor.org / packages / release / bioc / html / singscore.html, both of which are incorporated by reference in their entirety), a k Top Scoring Pairs (kTSP)-based method, and others known in the relevant art. In some preferred embodiments, the first single sample scoring algorithm comprises singscore. In some further embodiments, the method further comprises outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk. In still some further embodiments, the probabilistic determination identifies 99 4125-3810-3132.7Attorney Docket: 54094.4001 / WO the subject as having a “high” metastatic risk, whereas in some other further embodiments, the probabilistic determination identifies the subject as having a “low” metastatic risk.

[0273] In some embodiments, RNA expression levels of the plurality of genes listed in Table 2 isdetermined for at least about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more of the genes listed in Table 2 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 2 is determined for at least about 90% or more of the genes listed in Table 2 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 2 is determined for 100% of the genes listed in Table 2.

[0274] In some other embodiments, RNA expression levels of the plurality of genes listed inTable 2 is determined for at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the genes listed in Table 2 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 2 is determined for 100% of the genes listed in Table 2.

[0275] In some other embodiments, RNA expression levels of the plurality of genes listed inTable 2 is determined for at least about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, 100 4125-3810-3132.7Attorney Docket: 54094.4001 / WO about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, about 200, about 201 or more of the genes listed in Table 2. In a preferred embodiment, RNA expression of the plurality of genes listed in Table 2 is determined for at least about 182 of the genes listed in Table 2. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 2 is determined for each of the genes listed in Table 2 (i.e., for all 202 genes listed in Table 2).

[0276] In some other embodiments, RNA expression levels of the plurality of genes listed inTable 2 is determined for at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 or more of the genes listed in Table 2. In a preferred embodiment, RNA expression of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 2 is determined for each of the genes listed in Table 2 (i.e., for all 202 genes listed in Table 2).

[0277] In some embodiments, the method further comprises, prior to the generating of step (b),normalizing individual instances of determined RNA expression levels. In some further embodiments, normalizing individual instances of determined RNA expression levels comprises (i) normalization of raw sequencing counts by gene length, total library size, and / or sequencing 101 4125-3810-3132.7Attorney Docket: 54094.4001 / WO depth, (ii) quantile normalization, (iii) trimmed mean of M-value (TMM) normalization, (iv) batch correction methods (such as, for example, Limma, ComBat, etc.), or other normalization methods known in the relevant art (see, e.g., Liu, X. et al. (2019) ‘Normalization methods for the analysis of unbalanced transcriptome data: A Review’, Frontiers in Bioengineering and Biotechnology, 7. doi:10.3389 / fbioe.2019.00358, the contents of which are incorporated by reference in their entirety with particularity for discussion pertaining to normalization methods.

[0278] In some embodiments, the method further comprises, prior to the assay of step (a),aliquoting at least a portion of the plurality of nucleic acid molecules.

[0279] In some embodiments, the method further comprises, prior to the assay of step (a),storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. In some preferred embodiments, the plurality of nucleic acid molecules is stored at a temperature that is less than or equal to -80°C. Alternatively, or in addition, the method may further comprise, subsequent to the assay of step (a), storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C. In some preferred embodiments, the plurality of nucleic acid molecules is stored at a temperature that is less than or equal to -80°C.

[0280] In some embodiments, the tumor sample was obtained at a first time point, and themethod further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0281] In some embodiments, the assay of step (a) further determines: (1) RNA expressionlevels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4. In some further embodiments, the ranked list of step (b) further includes 102 4125-3810-3132.7Attorney Docket: 54094.4001 / WO the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4. In still some further embodiments, (1) gene identity and ranking of the plurality of genes listed in Table 3, and (2) gene identity and ranking of the plurality of genes listed in Table 4, are also provided as input to the first single sample scoring algorithm. In yet some further embodiments, the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, (2) an enrichment of at least a subset of the plurality of genes listed in Table 2, (3) a depletion of at least a subset of the plurality of genes listed in Table 4, or (4) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric. In some further embodiments, the method further comprises outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk (e.g., high metastatic risk, low metastatic risk). In still some further embodiments, the method further comprises, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0282] In some embodiments, the tumor sample was obtained at a first time point, and themethod further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples. 103 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0283] In some embodiments, the assay of step (a) further determines: (1) RNA expressionlevels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4. In some further embodiments, the method further comprises: (f) providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. In some further embodiments, the first single sample classifier algorithm comprises: (i) a k Top Scoring Pairs (kTSP)-based method (see, e.g., Moffitt, R.A. et al. (2015) ‘Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma’, Nature Genetics, 47(10), pp. 1168–1178. Doi:10.1038 / ng.3398, the contents of which are incorporated by reference in their entirety), (ii) singscore, or other single sample classifier algorithms known in the relevant art. In still some further embodiments, the method further comprises, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

[0284] In some embodiments, the tumor sample was obtained at a first time point, and themethod described above further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state. The one or more biological samples may be from a body fluid, cell, skin, tissue, organ, or combination thereof. Nonlimiting examples of biological samples include: blood, plasma, saliva, sputum, urine, cerebrospinal fluid (CSF), stool, a cell or a tissue biopsy, and the like. The sample may be from the skin, colon, rectum, prostate, breast, or other suitable 104 4125-3810-3132.7Attorney Docket: 54094.4001 / WO sites contingent upon the nature of the carcinoma, as are known in the relevant art. In some embodiments, the one or more biological samples comprises a plurality of biological samples.

[0285] In some embodiments, the second time point is subsequent to the first time point, whereasin other embodiments, the first time point may be subsequent to the second time point. In some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) after the earlier time point. Alternatively, or in addition to, in some further embodiments, the subsequent time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) after the earlier time point.

[0286] In some embodiments, the second time point is subsequent to the first time point. In someembodiments, the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy. In some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the administration of the first therapy. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the administration of the first therapy.

[0287] In some embodiments, the second time point is subsequent to the first time point. In someembodiments, the first time point occurs prior to one or more therapeutic interventions, and the second time point occurs subsequent to the one or more therapeutic interventions. In some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to 105 4125-3810-3132.7Attorney Docket: 54094.4001 / WO the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the one or more therapeutic interventions. Alternatively, or in addition to, in some further embodiments, the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the one or more therapeutic interventions.

[0288] In some embodiments, the assay of step (a) further determines RNA expression levels ofa plurality of genes listed in Table 1. In some further embodiments, the ranked list of step (b) further includes the plurality of genes listed in Table 1. In still some further embodiments, gene identity and ranking of the plurality of genes listed in Table 1 are also provided as input to the first single sample scoring algorithm. In yet some further embodiments, the method further comprises: (1) outputting a second metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the second metric identifies: (i) a depletion of at least a subset of the plurality of genes listed in Table 1, and / or (ii) an enrichment of at least a subset of the plurality of genes listed in Table 2; and (2) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the second metric.

[0289] In some further embodiments, RNA expression levels of the plurality of genes listed inTable 1 is determined for at least about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the genes listed in Table 1 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least about 90% or more of the genes listed in Table 1 (again, rounded to the nearest 106 4125-3810-3132.7Attorney Docket: 54094.4001 / WO corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 1 is determined for 100% of the genes listed in Table 1.

[0290] In some other further embodiments, RNA expression levels of the plurality of geneslisted in Table 1 is determined for at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the genes listed in Table 1 (rounded to the nearest corresponding integer value). In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 90% or more of the genes listed in Table 1 (again, rounded to the nearest corresponding integer value). In a more preferred embodiments, RNA expression levels of the plurality of the genes listed in Table 1 is determined for 100% of the genes listed in Table 1.

[0291] In some other further embodiments, RNA expression levels of the plurality of geneslisted in Table 1 is determined for at least about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173 or more of the genes listed in Table 1. In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 1 is determined for each of the genes listed in Table 1 (i.e., for all 174 genes listed in Table 1). 107 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0292] In some other further embodiments, RNA expression levels of the plurality of geneslisted in Table 1 is determined for at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 or more of the genes listed in Table 1. In a preferred embodiment, RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1. In a more preferred embodiment, RNA expression levels of the plurality of the genes listed in Table 1 is determined for each of the genes listed in Table 1 (i.e., for all 174 genes listed in Table 1).

[0293] In some further embodiments, RNA expression levels are determined for at least 300genes selected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2. In some further embodiments: (1) about 30% of the at least 300 genes are genes listed in Table 1 and about 70% of the at least 300 genes are genes listed in Table 2, (2) about 40% of the at least 300 genes are genes listed in Table 1 and about 60% of the at least 300 genes are genes listed in Table 2, (3) about 50% of the at least 300 genes are genes listed in Table 1 and about 50% of the at least 300 genes are genes listed in Table 2, (4) about 55% of the at least 300 genes are genes listed in Table 1 and about 45% of the at least 300 genes are genes listed in Table 2, or (5) about 58% of the at least 300 genes are genes listed in Table 1 and about 42% of the at least 300 genes are genes listed in Table 2. D. Particular Embodiments

[0294] Embodiment (A): In an exemplary embodiment, a method for identifying metastatic riskof a carcinoma comprises: (a) extracting a plurality of RNA molecules from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein: (i) the tumor sample comprises: (1) a malignant sample, (2) a benign sample, (3) a mixed sample, or (4) a mixture of cells comprising one or more cells suspected of being cancerous and one or more healthy cells, and (ii) the carcinoma comprises: (1) a pancreatic carcinoma, (2) a colon carcinoma, (3) a breast carcinoma, or (4) a prostate carcinoma; (b) synthesizing a plurality of cDNA molecules using at least a portion of the plurality of RNA molecules; (c) performing an 108 4125-3810-3132.7Attorney Docket: 54094.4001 / WO assay on at least a portion of the plurality of cDNA molecules, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises: (i) RNA expression levels of a plurality of genes listed in Table 1, and (ii) RNA expression levels of a plurality of genes listed in Table 2, wherein: (1) the assay comprises: (A) RNA-seq, (B) a microarray, or (C) a spatial transcriptomic assay, (2) the plurality of genes listed in Table 1 comprises 157 or more of the genes listed in Table 1, and (3) the plurality of genes listed in Table 2 comprises 182 or more of the genes listed in Table 2; (d) generating a ranked list of genes using determined RNA expression levels; (e) providing (i) the ranked list of genes, (ii) gene identity and ranking of the plurality of genes listed in Table 1, and (iii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm, wherein the first single sample scoring algorithm comprises singscore; (f) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; (g) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric; and (h) administering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk for the carcinoma, wherein the adjuvant therapy comprises: (1) chemotherapy, (2) hormonal therapy, (3) targeted therapy, (4) immunotherapy, or (5) a combination thereof.

[0295] Embodiment (A)(1): In some further embodiments, the carcinoma comprises apancreatic carcinoma, wherein the pancreatic carcinoma comprises pancreatic ductal adenocarcinoma.

[0296] Embodiment (A)(1)(i): In still some further embodiments, the adjuvant therapycomprises a chemotherapy, wherein the chemotherapy comprises: (i) FOLFIRINOX, (ii) gemcitabine and / or nab-paclitaxel, (iii) carboplatin, or (iv) fludarabine.

[0297] Embodiment (A)(1)(ii): In still some further embodiments, the adjuvant therapycomprises a hormonal therapy, wherein the hormonal therapy comprises: (i) cyproterone acetate, (ii) ketoconazole, (iii) aminoglutethimide, (iv) abarelix, (v) degarelix, (vi) leuprolide, (vii) triptorelin, or (viii) buserelin. 109 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0298] Embodiment (A)(1)(iii): In still some further embodiments, the adjuvant therapycomprises a targeted therapy, wherein the targeted therapy comprises: (i) Olaparib (Lynparza ®), (ii) fam-trastuzumab deruxtexcan-nxki (Enhertu ®), (iii) entrectinib (Rozlytrek ®), (iv) dabrafenib and / or trametininb (Tafinlar ® and Mekinist ®, respectively), (v) erlotinib (Tarceva ®), or (vi) Larotrectinib (Vitrakvi ®).

[0299] Embodiment (A)(1)(iv): In still some further embodiments, the adjuvant therapycomprises an immunotherapy.

[0300] Embodiment (A)(1)(v): In still some further embodiments, the adjuvant therapycomprises a combination of one or more of the adjuvant therapies of Embodiments (A)(1)(i)-(iv).

[0301] Embodiment (A)(2): In some further embodiments, the carcinoma comprises a coloncarcinoma, wherein the colon carcinoma comprises colon adenocarcinoma.

[0302] Embodiment (A)(2)(i): In still some further embodiments, the adjuvant therapycomprises a chemotherapy, wherein the chemotherapy comprises: (i) FOLFOX, (ii) capecitabine, (iii) irinotecan, (iv) oxaliplatin, or (v) trifluridine and / or tipiracil.

[0303] Embodiment (A)(2)(ii): In still some further embodiments, the adjuvant therapycomprises a hormonal therapy, wherein the hormonal therapy comprises: (i) estrogen, or (ii) estrogen and progestin.

[0304] Embodiment (A)(2)(iii): In still some further embodiments, the adjuvant therapycomprises a targeted therapy, wherein the targeted therapy comprises: (i) bevacizumab, (ii) ramucirumab, (iii) ziv-aflibercept, or (iv) fruquintinib.

[0305] Embodiment (A)(2)(iv): In still some further embodiments, the adjuvant therapycomprises an immunotherapy.

[0306] Embodiment (A)(2)(v): In still some further embodiments, the adjuvant therapycomprises a combination of one or more of the adjuvant therapies of Embodiments (A)(2)(i)-(iv).

[0307] Embodiment (A)(3): In some further embodiments, the carcinoma comprises a breastcarcinoma, wherein the breast carcinoma comprises invasive ductal carcinoma.

[0308] Embodiment (A)(3)(i): In still some further embodiments, the adjuvant therapycomprises a chemotherapy, wherein the chemotherapy comprises: (i) AC-T, (ii) TC, (iii) AC, (iv) doxorubicin, (v) epirubicin, (vi) paclitaxel, (vii) docetaxel, (viii) 5-fluorouracil (5-FU), (viii) capecitabine, (ix) cyclophosphamide, (x) carboplatin, (xi) ixabepilone, (xii) eribulin, or (xiii) vinorelbine. 110 4125-3810-3132.7Attorney Docket: 54094.4001 / WO

[0309] Embodiment (A)(3)(ii): In still some further embodiments, the adjuvant therapycomprises a hormonal therapy, wherein the hormonal therapy comprises: (i) tamoxifen, (ii) anastrozole, (iii) exemestane, (iv) letrozole, or (v) goserelin.

[0310] Embodiment (A)(3)(iii): In still some further embodiments, the adjuvant therapycomprises a targeted therapy, wherein the targeted therapy comprises: (i) Palbociclib, (ii) ribociclib, or (iii) abemaciclib.

[0311] Embodiment (A)(3)(iv): In still some further embodiments, the adjuvant therapycomprises an immunotherapy.

[0312] Embodiment (A)(3)(v): In still some further embodiments, the adjuvant therapycomprises a combination of one or more of the adjuvant therapies of Embodiments (A)(3)(i)-(iv).

[0313] Embodiment (A)(4): In some further embodiments, the carcinoma comprises a prostatecarcinoma, wherein the prostate carcinoma comprises prostate adenocarcinoma.

[0314] Embodiment (A)(4)(i): In still some further embodiments, the adjuvant therapycomprises a chemotherapy, wherein the chemotherapy comprises: (i) docetaxel, (ii) cabazitaxel, (iii) mitoxantrone, (iv) estramustine, or (v) carboplatin.

[0315] Embodiment (A)(4)(ii): In still some further embodiments, the adjuvant therapycomprises a hormonal therapy, wherein the hormonal therapy comprises: (i) leuprorelin (leuprolide), (ii) goserelin, (iii) triptorelin, (iv) bicalutamide (Casodex), (v) nilutamide (Nilandron), (vi) flutamide (Eulexin), (vii) enzalutamide, (viii) apalutamide, or (ix) darolutamide.

[0316] Embodiment (A)(4)(iii): In still some further embodiments, the adjuvant therapycomprises a targeted therapy, wherein the targeted therapy comprises: (i) rucaparib, (ii) Olaparib, (iii) talazoparib, or (iv) niraparib and / or abiraterone.

[0317] Embodiment (A)(4)(iv): In still some further embodiments, the adjuvant therapycomprises an immunotherapy.

[0318] Embodiment (A)(4)(v): In still some further embodiments, the adjuvant therapycomprises a combination of one or more of the adjuvant therapies of Embodiments (A)(4)(i)-(iv).

[0319] Embodiment (B): In an exemplary embodiment, a method for identifying metastatic riskof a carcinoma comprises: (a) extracting a plurality of RNA molecules from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein: (i) the tumor sample comprises: (1) a malignant sample, (2) a benign sample, (3) a mixed sample, or 111 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (4) a mixture of cells comprising one or more cells suspected of being cancerous and one or more healthy cells, and (ii) the carcinoma comprises: (1) a pancreatic carcinoma, (2) a colon carcinoma, (3) a breast carcinoma, or (4) a prostate carcinoma; (b) synthesizing a plurality of cDNA molecules using at least a portion of the plurality of RNA molecules; (c) performing an assay on at least a portion of the plurality of cDNA molecules, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises RNA expression levels of a plurality of genes listed in Table 1, wherein: (1) the assay comprises: (A) RNA-seq, (B) a microarray, or (C) a spatial transcriptomic assay, and (2) the plurality of genes listed in Table 1 comprises 157 or more of the genes listed in Table 1; (d) generating a ranked list of genes using determined RNA expression levels; (e) providing (i) the ranked list of genes, and (ii) gene identity and ranking of the plurality of genes listed in...

Claims

Attorney Docket: 54094.4001 / WO CLAIMS WHAT IS CLAIMED IS:

1. A method for identifying metastatic risk of a carcinoma, comprising: (a) performing an assay on at least a portion of a plurality of nucleic acid molecules extracted from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises: (i) RNA expression levels of a plurality of genes listed in Table 1, and (ii) RNA expression levels of a plurality of genes listed in Table 2; (b) generating a ranked list of genes using determined RNA expression levels; (c) providing: (i) the ranked list of genes, (ii) gene identity and ranking of the plurality of genes listed in Table 1, and (iii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm; (d) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; and (e) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric.

2. The method of claim 1, further comprising administering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk. 212 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 3. The method of claim 2, wherein the adjuvant therapy comprises: (i) chemotherapy, (ii) hormonal therapy, (iii) targeted therapy, (iv) immunotherapy, or (v) a combination thereof.

4. The method of any one of claims 1-3, wherein the carcinoma comprises: (i) a pancreatic carcinoma, (ii) a colon carcinoma, (iii) a breast carcinoma, or (iv) a prostate carcinoma.

5. The method of claim 4, wherein the carcinoma comprises a pancreatic carcinoma, wherein the pancreatic carcinoma comprises: (i) pancreatic ductal adenocarcinoma (PDAC), (ii) pancreatic squamous cell carcinoma, (iii) pancreatic adenosquamous carcinoma, (iv) pancreatic colloid carcinoma, (v) acinar cell carcinoma of the pancreas, (vi) pancreatic cystadenocarcinoma, (vii) signet ring cell carcinoma of the pancreas, (viii) pancreatic hepatoid carcinoma, (ix) undifferentiated carcinoma of the pancreas, or (x) undifferentiated carcinoma of the pancreas with osteoclast-like giant cells.

6. The method of claim 4, wherein the carcinoma comprises a colon carcinoma, wherein the colon carcinoma comprises: (i) colon adenocarcinoma, (ii) colorectal adenocarcinoma, (iii) colon mucinous adenocarcinoma, (iv) colon signet ring cell adenocarcinoma, (v) colon adenosquamous carcinoma, (vi) colon squamous cell carcinoma, or (vii) colorectal squamous cell carcinoma.

7. The method of claim 4, wherein the carcinoma comprises a breast carcinoma, wherein the breast carcinoma comprises: (i) invasive ductal carcinoma (IDC), (ii) ductal carcinoma in situ (DCIS), (iii) lobular carcinoma, (iv) mixed ductal / lobular carcinoma, (v) mucinous carcinoma, (vi) tubular carcinoma, (vii) medullary carcinoma, (viii) papillary carcinoma, or (ix) lobular carcinoma in situ.

8. The method of claim 4, wherein the carcinoma comprises a prostate carcinoma, wherein the prostate carcinoma comprises: (i) prostate adenocarcinoma, (ii) squamous cell carcinoma, (iii) transitional cell carcinoma, (iv) acinar adenocarcinoma of the prostate, (v) ductal adenocarcinoma of the prostate, or (vi) small cell carcinoma of the prostate.

9. The method of any one of claims 1-8, further comprising: (i) comparing determined RNA expression levels to a first threshold to identify a subset of genes with RNA expression levels below the first threshold; 213 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (ii) generating a modified ranked list of genes using determined RNA expression levels, wherein the modified ranked list excludes the subset of genes with RNA expression levels below the first threshold; and (iii) providing the modified ranked list of genes as input to the first single sample scoring algorithm in place of the ranked list of genes.

10. The method of claim 9, wherein the first threshold is a fragments per kilobase million (FPKM) value of 1.

00.

11. The method of any one of claims 1-10, further comprising, prior to step (a), extracting the plurality of nucleic acid molecules from the tumor sample from the human subject.

12. The method of claim 11, wherein the plurality of nucleic acid molecules comprises a plurality of ribonucleic acid (RNA) molecules.

13. The method of claim 12, wherein the assay of step (a) is performed on at least a portion of a plurality of complementary deoxyribonucleic acid (cDNA) molecules synthesized from at least a portion of the plurality of RNA molecules.

14. The method of claim 13, further comprising, prior to the assay of step (a), synthesizing the plurality of cDNA molecules from at least a portion of the plurality of RNA molecules.

15. The method of any one of claims 1-14, wherein the tumor sample comprises: (i) a malignant sample, (ii) a benign sample, or (iii) a mixed sample.

16. The method of any one of claims 1-14, wherein the tumor sample comprises a mixture of cells comprising: (i) one or more cells suspected of being cancerous, and (ii) one or more healthy cells.

17. The method of any one of claims 1-16, further comprising obtaining the tumor sample from the human subject.

18. The method of any one of claims 1-17, wherein the assay comprises: (i) a sequencing assay, (ii) a microarray, or (iii) a spatial transcriptomic assay. 214 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 19. The method of claim 18, wherein the assay comprises a sequencing assay, and the sequencing assay comprises a high-throughput sequencing assay.

20. The method of claim 19, wherein the high-throughput sequencing assay comprises: (i) RNA- sequencing (RNA-seq), or (ii) Assay for Transposable-Accessible Chromatin using Sequencing (ATAC-seq).

21. The method of claim 19 or 20, wherein the sequencing assay produces a plurality of nucleic acid sequencing reads, and the method further comprises aligning at least a portion of the plurality of nucleic acid sequencing reads to a reference genome.

22. The method of claim 21, wherein the reference genome comprises: (i) NCBI build 34, (ii) NCBI build 35, (iii) NCBI build 36.1, (iv) GRCh37, (v) GRCh38, or (vi) T2T-CHM13.

23. The method of claim 18, wherein the assay comprises a spatial transcriptomic assay, and the spatial transcriptomic assay comprises: (i) Visium HD, (ii) NanoString GeoMx, (iii) Vizgen MERSCOPE, (iv) NanoString CosMX, (v) 10xGenomics Xenium, or (vi) hybridization-based in situ sequencing (HybISS).

24. The method of any one of claims 1-23, wherein the first single sample scoring algorithm comprises singscore.

25. The method of any one of claims 1-24, further comprising outputting a second metric based on the input provided to the first single sample scoring algorithm, wherein the second metric comprises a probabilistic determination indicative of metastatic risk.

26. The method of any one of claims 1-25, wherein RNA expression levels are determined for at least 300 genes selected from a combination of the plurality of genes listed in Table 1 and the plurality of genes listed in Table 2.

27. The method of any one of claims 1-25, wherein RNA expression levels of the plurality of genes listed in Table 1 is determined for at least 157 of the genes listed in Table 1, and RNA expression levels of the plurality of genes listed in Table 2 is determined for at least 182 of the genes listed in Table 2. 215 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 28. The method of claim 27, wherein RNA expression levels are determined for each of the genes listed in Table 1, and RNA expression levels are determined for each of the genes listed in Table 2.

29. The method of any one of claims 1-28, further comprising, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels.

30. The method of any one of claims 1-29, further comprising, prior to the assay of step (a), aliquoting at least a portion of the plurality of nucleic acid molecules.

31. The method of any one of claims 1-30, further comprising, prior to the assay of step (a) storing at least a portion of the plurality of nucleic acid molecules at a temperature that is less than or equal to -20°C.

32. The method of any one of claims 1-31, wherein: (i) the assay of step (a) further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4; (ii) the ranked list of step (b) further includes the plurality of genes listed in Table 3 and the plurality of genes listed in Table 4; (iii) gene identity and ranking of the plurality of genes listed in Table 3 and gene identity and ranking of the plurality of genes listed in Table 4 are also provided as input to the first single sample scoring algorithm, and the method further comprises: (f) outputting a third metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the third metric identifies: (1) an enrichment of at least a subset of the plurality of genes listed in Table 3, 216 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (2) an enrichment of at least a subset of the plurality of genes listed in Table 2, (3) a depletion of at least a subset of the plurality of genes listed in Table 4, (4) a depletion of at least a subset of the plurality of genes listed in Table 1, or (5) any combination thereof; and (g) producing a second report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the third metric.

33. The method of claim 32, further comprising outputting a fourth metric based on the input provided to the first single sample scoring algorithm, wherein the fourth metric comprises a probabilistic determination indicative of metastatic risk.

34. The method of claim 32 or 33, further comprising, prior to the generating of step (b), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

35. The method of any one of claims 1-31, wherein the assay of step (a) further determines: (1) RNA expression levels of a plurality of genes listed in Table 3, and (2) RNA expression levels of a plurality of genes listed in Table 4, and the method further comprises: (f) providing: (i) gene identity and RNA expression levels of the plurality of genes listed in Table 3, and (ii) gene identity and RNA expression levels of the plurality of genes listed in Table 4, as input to a first single sample classifier algorithm; (g) outputting a third metric from the first single sample classifier algorithm based on the input provided to the second single sample scoring algorithm, wherein the third metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 3, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 4; and 217 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (h) producing a second report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state.

36. The method of claim 35, wherein the first single sample classifier algorithm comprises a k Top Scoring Pairs (kTSP)-based method.

37. The method of claim 35 or 36, further comprising, prior to the providing of step (f), normalizing individual instances of determined RNA expression levels of the plurality of genes listed in Table 3 and individual instances of determined RNA expression levels of the plurality of genes listed in Table 4.

38. The method of any one of claims 1-31, wherein the tumor sample was obtained at a first time point, and the method further comprises: (f) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (g) repeating steps (a)-(d) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (h) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma.

39. The method of any one of claims 32-34, wherein the tumor sample was obtained at a first time point, and the method further comprises: (h) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (i) repeating steps (a)-(f) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (j) producing an additional report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma. 218 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 40. The method of any one of claims 35-37, wherein the tumor sample was obtained at a first time point, and the method further comprises: (i) extracting a plurality of nucleic acid molecules from one or more biological samples that were obtained at a second time point; (j) repeating steps (a)-(g) on at least a portion of the plurality of nucleic acid molecules from the one or more biological samples; and (k) producing an additional report identifying the human subject as having (1) a high metastatic risk and / or a basal cell state, or (2) a low metastatic risk and / or a classical cell state.

41. The method of any one of claims 38-40, wherein the one or more biological samples comprises: (i) blood, (ii) plasma, (iii) saliva, (iv) sputum, (v) urine, (vi) cerebrospinal fluid (CSF), (vii) stool, (viii) a cell or a tissue biopsy, or (ix) a combination thereof.

42. The method of any one of claims 38-41, wherein the second time point is subsequent to the first time point.

43. The method of claim 42, wherein the first time point occurs prior to an administration of a first therapy, and the second time point occurs subsequent to the administration of the first therapy.

44. The method of claim 42 or 43, wherein the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more day(s) subsequent to the first time point.

45. The method of claim 44, wherein the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more week(s) subsequent to the first time point.

46. The method of claim 45, wherein the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more month(s) subsequent to the first time point. 219 4125-3810-3132.7Attorney Docket: 54094.4001 / WO 47. The method of claim 46, wherein the second time point is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more year(s) subsequent to the first time point.

48. The method of any one of claims 38-47, wherein the first time point occurs prior to one or more therapeutic interventions and the second time point occurs subsequent to the one or more therapeutic interventions.

49. A method for identifying metastatic risk of a carcinoma, comprising: (a) extracting a plurality of RNA molecules from a tumor sample from a human subject who is known to have or is suspected of having a carcinoma, wherein: (i) the tumor sample comprises: (1) a malignant sample, (2) a benign sample, (3) a mixed sample, or (4) a mixture of cells comprising one or more cells suspected of being cancerous and one or more healthy cells, and (ii) the carcinoma comprises: (1) a pancreatic carcinoma, (2) a colon carcinoma, (3) a breast carcinoma, or (4) a prostate carcinoma; (b) synthesizing a plurality of cDNA molecules using at least a portion of the plurality of RNA molecules; (c) performing an assay on at least a portion of the plurality of cDNA molecules, wherein the assay determines RNA expression levels of all or substantially all genes comprising a human transcriptome, wherein RNA expression levels of all or substantially all genes encoding the human transcriptome comprises: (i) RNA expression levels of a plurality of genes listed in Table 1, and (ii) RNA expression levels of a plurality of genes listed in Table 2, wherein: (1) the assay comprises: (A) RNA-seq, (B) a microarray, or (C) a spatial transcriptomic assay, (2) the plurality of genes listed in Table 1 comprises 157 or more of the genes listed in Table 1, and 4125-3810-3132.7Attorney Docket: 54094.4001 / WO (3) the plurality of genes listed in Table 2 comprises 182 or more of the genes listed in Table 2; (d) generating a ranked list of genes using determined RNA expression levels; (e) providing: (i) the ranked list of genes, (ii) gene identity and ranking of the plurality of genes listed in Table 1, and (iii) gene identity and ranking of the plurality of genes listed in Table 2, as input to a first single sample scoring algorithm, wherein the first single sample scoring algorithm comprises singscore; (f) outputting a first metric from the first single sample scoring algorithm based on the input provided to the first single sample scoring algorithm, wherein the first metric identifies: (i) an enrichment of at least a subset of the plurality of genes listed in Table 2, and / or (ii) a depletion of at least a subset of the plurality of genes listed in Table 1; (g) producing a first report identifying the human subject as having a high metastatic risk or a low metastatic risk for the carcinoma based on the first metric; and (h) administering adjuvant therapy to the human subject if the human subject is identified as having a high metastatic risk for the carcinoma, wherein the adjuvant therapy comprises: (1) chemotherapy, (2) hormonal therapy, (3) targeted therapy, (4) immunotherapy, or (5) a combination thereof. 221 4125-3810-3132.7

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