Detection of high-risk primary tumor subclones to inform adjuvant therapy and monitoring tests

Abstract

Methods are provided for identifying genomic variations associated with metastatic potential in a sample. In some embodiments, lymphovascular invasion (LVI) status is determined for a formalin-fixed paraffin-embedded (FFPE) tissue sample, and genomic material from an LVI-positive sample is sequenced to identify genomic variations associated with metastatic potential. In some embodiments, a sample from surgical resection material and / or FFPE tissue is dissociated into a single-cell composition, cells expressing Ki67 are enriched, and genomic material from the enriched cells is sequenced to identify genomic variations. The identified genomic variations may be used for adjuvant therapy selection, recurrence monitoring, and / or inclusion in a molecular residual disease panel.

Description

Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) DETECTION OF HIGH-RISK PRIMARY TUMOR SUBCLONES TO INFORM ADJUVANT THERAPY AND MONITORING TESTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of the filing date of United States Provisional Patent Application No. 63 / 789,810 filed on April 16, 2025, the disclosure of which is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE DISCLOSURE

[0002] Cancer is a disease marked by the uncontrolled proliferation of abnormal cells. In normal tissue, cells divide and organize within the tissue in response to signals from surrounding cells, resulting in normal cellular behavior that is carefully orchestrated by the tissue context. Cancer cells do not respond to growth-limiting contextual cues from the surrounding tissue, and they often harbor genetic alterations that drive them to proliferate and form a tumor. As the growth of a tumor progresses, genetic and phenotypic alterations continue to accumulate, allowing populations of cancer cells to overcome additional "checkpoints," such as an anti-tumor immune response, and manifesting as a more high-risk growth phenotype of the cancer cells. If left untreated, metastasis, the spread of cancer cells to distant areas of the body by way of the lymphatic system or bloodstream, may ensue. Metastasis results in the formation of secondary tumors at multiple sites, damaging healthy tissue. Most cancer death is caused by such secondary tumors. Timely diagnosis and treatment of cancer enhances the likelihood of a successful outcome.

[0003] Across cancers that are treated by surgery with curative intent, there is a need to determine the appropriate adjuvant treatment that will prevent recurrence of the primary tumor. Primary tumor features (stage, grade, etc.) are often used for risk assessment, but other biomarker approaches can also inform treatment. In breast cancer, for example, gene expression tests will be employed to determine the risk of recurrence, and patients with a score indicating high risk of recurrence will receive adjuvant chemotherapy rather than adjuvant anti-endocrine therapy. Therapy escalation / de-escalation decisions are not trivial, especially for cancers like head and neck cancer, where the site of the primary tumor greatly impacts patient function and well-being.

[0004] Recurrences can be seeded by subclones from the primary tumor which contain additional alterations that make them resistant to treatment, or harbor other high-risk features. DueAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) to inadequate tumor sampling and tests that are not sensitive enough, it is currently impossible to consistently identify whether a patient harbors high-risk subclones in the primary tumor, and what variations they contain; these subclones only emerge and are detectable later in the patient journey in the metastatic disease, often a significant amount of time after decisions are made about escalation / de-escalation of adjuvant treatment. Increasingly, therapy decisions may also be impacted by assessment of molecular residual disease (MRD), through detection of tumor cells / DNA from longitudinal blood samples.BRIEF SUMMARY OF THE INVENTION

[0005] A first aspect of the present disclosure is a method of identifying one or more high-risk variants or variations (referred to herein as "variations") associated with cells harboring metastatic potential in one or more formalin-fixed paraffin-embedded (FFPE) tissue samples, comprising: determining a lymphovascular invasion (LVI) status in each of the one or more FFPE tissue samples; sequencing genomic material derived from the one or more FFPE tissue samples determined to be positive for LVI to provide one or more sequencing data sets; identifying the one or more high-risk genomic variations in the one or more sequencing data sets; and determining at least one clinical decision based on the identification of the one or more high-risk genomic variations.

[0006] In some embodiments, the LVI status is determined by staining the one or more FFPE tissue samples with a morphological stain. In some embodiments, the LVI status is determined by virtually staining the one or more FFPE tissue samples. In some embodiments, the LVI status is determined visually (such as after staining with a primary stain, such as hematoxylin and eosin). In some embodiments, the LVI status is determined using a trained classifier.

[0007] In some embodiments, the method further comprises adding the identified one or more high-risk genomic variations or the genomic variation associated with cells harboring metastatic potential to a molecular residual disease (MRD) panel.

[0008] In some embodiments, the determined clinical decision comprises disease prognosis, predicting recurrence of disease, inclusion of subjects of clinical trials, or therapeutic treatment strategy. In some embodiments, the determined clinical decision is to escalate therapy. In some embodiments, the determined clinical decision is to deescalate therapy.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0009] In some embodiments, the sequencing comprises exome sequencing. In some embodiments, the sequencing comprises next-generation sequencing. In some embodiments, the next-generation sequencing comprises pyrosequencing, nanopore sequencing, sequencing-by-synthesis, sequencing-by-ligation, and single molecule real-time sequencing.

[0010] In some embodiments, the method further comprises detecting, characterizing, or identifying one or more low prevalence genetic events in the generated one or more sequencing data sets. In some embodiments, the one or more low prevalence genetic events are selected from a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene.

[0011] A second aspect of the present disclosure is a method of identifying one or more high-risk genomic variations or a genomic variation associated with cells harboring metastatic potential in a single cell composition, comprising: obtaining one or more surgical resections or residual surgical material; homogenizing the one or more surgical resections or the residual surgical material to provide a homogenized sample; dissociating the homogenized sample into the single cell composition; staining cells in the single cell composition for the expression of the Ki67 biomarker; sorting the cells expressing the Ki67 biomarker into a first population of cells; sequencing genomic material derived from the cells in the first population of cells to provide one or more sequencing data sets; identifying the one or more high-risk genomic variations in the one or more sequencing data sets; and determining at least one clinical decision based on the identification of the one or more high-risk genomic variations.

[0012] In some embodiments, the method further comprises adding the identified one or more high-risk genomic variations or the genomic variation associated with cells harboring metastatic potential to a molecular residual disease (MRD) panel. In some embodiments, the determined clinical decision comprises disease prognosis, predicting recurrence of disease, inclusion of subjects of clinical trials, and / or therapeutic treatment strategy. In some embodiments, the determined clinical decision is to escalate therapy. In some embodiments, the determined clinical decision is to deescalate therapy.

[0013] In some embodiments, the sequencing comprises exome sequencing. In some embodiments, the sequencing comprises next-generation sequencing. In some embodiments, the next-generation sequencing comprises pyrosequencing, nanopore sequencing, sequencing-by-synthesis, sequencing-by-ligation, and single molecule real-time sequencing.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0014] In some embodiments, the method further comprises detecting, characterizing, or identifying one or more low prevalence genetic events in the generated one or more sequencing data sets. In some embodiments, the one or more low prevalence genetic events are selected from a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene.

[0015] A third aspect of the present disclosure is a method of identifying one or more high-risk genomic variations or a genomic variation associated with cells harboring metastatic potential in a single cell composition, comprising: obtaining one or more formalin-fixed paraffin-embedded (FFPE) tissue samples; homogenizing the one or more FFPE tissue samples to provide a homogenized sample; dissociating the homogenized sample into the single cell composition; staining cells in the single cell composition for the expression of the Ki67 biomarker; sorting the cells expressing the Ki67 biomarker into a first population of cells; sequencing genomic material derived from the cells in the first population of cells to provide one or more sequencing data sets; identifying the one or more high-risk genomic variations in the one or more sequencing data sets; and determining at least one clinical decision based on the identification of the one or more high-risk genomic variations. In some embodiments, the sorting comprises fluorescence-activated cell sorting (FACS).

[0016] In some embodiments, the method further comprises adding the identified one or more high-risk genomic variations or the genomic variation associated with cells harboring metastatic potential to a molecular residual disease (MRD) panel.

[0017] In some embodiments, the determined clinical decision comprises disease prognosis, predicting recurrence of disease, inclusion of subjects of clinical trials, and / or therapeutic treatment strategy. In some embodiments, the determined clinical decision is to escalate therapy. In some embodiments, the determined clinical decision is to deescalate therapy.

[0018] In some embodiments, the sequencing comprises exome sequencing. In some embodiments, the sequencing comprises next-generation sequencing. In some embodiments, the next-generation sequencing comprises pyrosequencing, nanopore sequencing, sequencing-by-synthesis, sequencing-by-ligation, and single molecule real-time sequencing.

[0019] In some embodiments, the method further comprises detecting, characterizing, or identifying one or more low prevalence genetic events in the generated one or more sequencing data sets. In some embodiments, the one or more low prevalence genetic events are selected fromAttorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene.

[0020] In some embodiments, the dissociation comprises a non-enzymatic dissociation process. In some embodiments, the one or more FFPE samples are tissue sections / tissue curls. In some embodiments, the one or more FFPE samples are tumor blocks, tissue cores, or tissue biopsies.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 provides a flowchart illustrating a method of identifying whether one or more samples harbor one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential and / or making a clinical decision based on the identification of the one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential.

[0022] FIG. 2 provides a flowchart illustrating a method of identifying one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential in a homogenized sample, where the one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential are identified in a population of cells expressing the Ki67 biomarker. In the embodiment of FIG. 2, the homogenized sample is derived from one or more surgical resection samples or residual surgical material.

[0023] FIG. 3 provides a flowchart illustrating a method of identifying one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential in a homogenized sample, where the one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential are identified in a population of cells expressing the Ki67. In the embodiment of FIG. 3, the homogenized sample is derived from one or more formalin-fixed paraffin-embedded samples.

[0024] FIG. 4 provides a flowchart illustrating a method of identifying one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential in one or more samples, where the samples used for identifying the one or more high-risk genomic variations and / or one or more genomic variations associated with cells harboring metastatic potential are determined to be positive for lymphovascular invasion.Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application)DETAILED DESCRIPTION

[0025] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0026] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0027] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0028] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0029] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one elementAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0030] As used herein, the term "biological sample," "tissue sample," "specimen" or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0031] As used herein, the term "biomarker" refers to any molecule or group of molecules found in a biological sample that can be used to characterize the biological sample or a subjectAttorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) from which the biological sample is obtained. For example, a biomarker may be a molecule or group of molecules whose presence, absence, or relative abundance is characteristic of a particular cell or tissue type or state; or characteristic of a particular pathological condition or state; or indicative of the severity of a pathological condition, the likelihood of progression or regression of the pathological condition, and / or the likelihood that the pathological condition will respond to a particular treatment. As another example, the biomarker may be a cell type or a microorganism (such as a bacterium, mycobacterium, fungus, virus, and the like), or a substituent molecule or group of molecules thereof.

[0032] As used herein, "clinical decision" may refer to gathering information and integrating this information to draw diagnostic conclusions and / or determine one or more treatments to administer to a subject, such as a mammalian subject, such as a human patient. Such diagnostic conclusions may include the disease from which a subject suffers and what testing should be performed on the subject. In some embodiments, a clinical decision may also include determining disease prognosis, predicting recurrence of disease, predicting targets of therapy of disease, inclusion of subjects in clinical trials, or a determination of a therapeutic treatment strategy for at least one subject. In some embodiments, the "clinical decision" comprises determining whether a subject should receive escalated adjuvant therapy, de-escalated adjuvant therapy, targeted adjuvant therapy, longitudinal molecular residual disease (MRD) monitoring, intensified surveillance, and / or subject-specific recurrence monitoring, based at least in part on whether an identified genomic variation is clonal, subclonal, present in a metastasis-seeding subclone, present in a proliferating subclone, enriched in an LVI-positive sample, and / or enriched in a Ki67-positive population.

[0033] As used herein, the term "next generation sequencing" refers to sequencing technologies having high-throughput sequencing as compared to traditional Sanger- and capillary electrophoresis-based approaches, wherein the sequencing process is performed in parallel, for example producing thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. These technologies produce shorter reads (anywhere from about 25 - about 500 bp) but many hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, CA) include, but are not limited to iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0034] It is believed that the Illumina next-generation sequencing technology uses clonal amplification and sequencing by synthesis (SBS) chemistry to enable rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into a nucleic acid chain. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence. A non-limiting example of a sequencing device available from ThermoFisher Scientific (Waltham, MA) includes the Ion Personal Genome Machine™ (PGM™) System.

[0035] It is believed that Ion Torrent sequencing measures the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. A non-limiting example of a sequencing device available from Pacific Biosciences (Menlo Park, CA) includes the PacBio Sequel Systems. A non-limiting example of a sequencing device available from Roche (Pleasanton, CA) is the Roche 454. Next-generation sequencing methods may also include nanopore sequencing methods. In general, three nanopore sequencing approaches have been pursued: strand sequencing in which the bases of DNA are identified as they pass sequentially through a nanopore, exonuclease-based nanopore sequencing in which nucleotides are enzymatically cleaved one-by-one from a DNA molecule and monitored as they are captured by and pass through the nanopore, and a nanopore sequencing by synthesis (SBS) approach in which identifiable polymer tags are attached to nucleotides and registered in nanopores during enzyme-catalyzed DNA synthesis. Common to all these methods is the need for precise control of the reaction rates so that each base is determined in order.

[0036] Strand sequencing requires a method for slowing down the passage of the DNA through the nanopore and decoding a plurality of bases within the channel; ratcheting approaches, taking advantage of molecular motors, have been developed for this purpose. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to guarantee its capture and its transit through the pore at a rate slow enough to obtain a valid ionic current signal. In addition, both methods rely on distinctions among the four natural bases, two relatively similar purines and two similar pyrimidines.

[0037] The nanopore SBS approach utilizes synthetic polymer tags attached to the nucleotides that are designed specifically to produce unique and readily distinguishable ionic current blockade signatures for sequence determination. In some embodiments, sequencing of nucleic acid molecules via nanopore sequencing includes preparing nanopore sequencingAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) complexes and determining polynucleotide sequences. Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052, and PCT Publication Nos. WO2014 / 074727, W02006 / 028508, WO2012 / 083249, and WO / 2014 / 074727, the disclosures of which are hereby incorporated by reference herein in their entireties. In some embodiments, tagged nucleotides may be used in the determination of the polynucleotide sequences (see, e.g., PCT Publication No. WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402, the disclosures of which are hereby incorporated by reference herein in their entireties).

[0038] Analysis of the data generated by sequencing is performed using software and / or statistical algorithms that perform various data conversions, e.g., conversion of signal emissions into base calls, conversion of base calls into consensus sequences for a nucleic acid template, etc. Such software, statistical algorithms, and the use of such are described in detail, in U.S. Patent Application Publication Nos. 2009 / 0024331 2017 / 0044606 and in PCT Publication No. WO / 2018 / 034745, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0039] As used herein, the term "sequencing" refers to the determination of the order and position of bases in a nucleic acid molecule. More particularly, the term "sequencing" refers to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNA sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modern automated DNA sequencing instruments.

[0040] As used herein, the term "subclonal mutation" refers to a mutation present in less than 100% of cancer cells, typically less than 50%. A subclonal mutation can be present in the majority of a tumor (i.e., less than 100%, but greater than 50%) or in the minority of the tumor (i.e., less than 50%). In some embodiments, different tumor cell populations that arise as a result of tumor heterogeneity are called "subclones," the progeny of a mutant cell arising in a clone. The prevalence of subclones within a tumor may vary. Certain subclones comprise the majority of the tumor but decrease over time and / or following certain treatments. Other subclones are initiallyAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) undetectable but later become abundant. Multiple subclones can exist simultaneously and vary in their prevalence over time it takes for the tumor to grow large enough to be detectable. The term "low prevalence events" or "low prevalence genetic events" within a tumor refers to rare events or rare genetic events (such as mutations) that occur at a rate of about 10 to about 1%, about 1 to about0.1%, aboutO.l to about 0.01%, aboutO.Ol to about 0.001%, aboutO.001 to about 0.0001%, about 0.0001 to about 0.00001%, about 0.00001 to about 0.000001%, or below about 0.000001%.

[0041] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In some embodiments, the tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, the tumor is a solid tumor or a non-solid or soft tissue tumor. In some embodiments, a solid tumor includes any cancer of body tissues other than blood, bone marrow, or the lymphatic system. In some embodiments, solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Non-limiting examples of solid tumors of epithelial origin include tumors of the gastrointestinal tract, colon, colorectal breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, nasopharynx, skin, uterus, male genital organ, urinary organs (e.g., urothelium carcinoma, dysplastic urothelium carcinoma, transitional cell carcinoma), bladder, and skin. In some embodiment, solid tumors of non-epithelial origin include sarcomas, pituitary tumors, and bone tumors.

[0042] Non-limiting examples of hematological malignancies include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease).

[0043] As used herein, the terms "tumor sample" or "tumor tissue" encompass samples prepared from a tumor or from a sample potentially including or suspected of comprising cancer cells, or to be tested for the potential presence of cancer cells, such as a lymph node. As used herein, the term "tumor" refers to a mass or a neoplasm, which itself is defined as an abnormal new growth of cells that may grow more rapidly than normal cells and may continue to grow if not treated. Tumor sizes can vary widely. A tumor may be solid, or fluid filled. A tumor canAttorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) refer to benign, or malignant (capable of metastasis) growths. Some tumors can contain neoplastic cells that are benign) and, simultaneously, contain malignant cancer cells (such as adenocarcinoma). For purposes of the disclosure, tumors include primary tumors and metastatic tumors.

[0044] OVERVIEW

[0045] The present disclosure is directed to methods of identifying cells with metastatic potential in a primary tumor. The present disclosure is also directed to methods of identifying one or more high-risk genomic variations within one or more samples or a homogenized sample derived from one or more samples, such as one or more samples derived from a patient diagnosed with cancer, or such as a patient receiving or having already received neoadjuvant therapy. The present disclosure is also directed to methods of determining whether one or more samples harbor one or more high-risk genomic variations. In some embodiments, the confirmed presence / absence of one or more high-risk genomic variations (e.g., high-risk subclones) may alone be a predictive / prognostic biomarker that informs downstream clinical decisions, e.g., adjuvant therapy escalation / de-escalation decisions. In some embodiments, targeted adjuvant therapy approaches may also be informed by the targets of therapy identified by the one or more high-risk genomic variations (e.g., high-risk subclones). In some embodiments, the one or more high-risk genomic variations, or any genomic variation associated with the primary tumor cells harboring metastatic potential, may be added to tumor-informed MRD panels for monitoring. In addition, integrating information from one or more identified high-risk genomic variations (e.g., high-risk subclones) and MRD may be more informative than either feature alone, or they can both be assessed by a sequencing test for a potential bundled product.

[0046] With reference to FIG. 1, in some embodiments one or more samples derived from a patient (step 110) are analyzed to determine whether they harbor lymphovascular invasion (step 115), i.e., are positive for lymphovascular invasion. In these embodiments, if the one or more samples are determined to harbor lymphovascular invasion, the genetic material from the cells in the one or more samples are sequenced (step 116), such as by next generation sequencing. Following sequencing, high-risk genomic variations, or any genomic variation associated with the cells harboring metastatic potential, may be identified (step 117) and used to inform one or more clinical decisions (step 119), e.g., whether to continue or cease adjuvant therapy, etc. In someAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) embodiments, the one or more high-risk genomic variations or genomic variation associated with clones with metastatic potential may be added to a monitoring panel (step 118).

[0047] Again, with reference to FIG. 1, in other embodiments one or more aliquots of a homogenized sample (see step 111) (such as a homogenized sample derived from one or more samples (step 110), such as derived from a patient receiving or having already received neoadjuvant therapy), are stained for the presence of the Ki67 biomarker (step 112). In these embodiments, those cells which stain positive for the Ki67 biomarker are isolated / sorted (step 113); and then genetic material from those cells is sequenced, such as by next generation sequencing (step 114). Following sequencing, high-risk genomic variations, or any genomic variation associated with the cells harboring metastatic potential, may be identified (step 117) and used to inform one or more clinical decisions (step 119), e.g., whether to continue or cease adjuvant therapy, etc. In some embodiments, the one or more high-risk genomic variations, or any genomic variation associated with the cells harboring metastatic potential, may be added to a monitoring panel (step 118).

[0048] In some embodiments, the methods disclosed herein are configured to identify actionable subclonal drivers, metastasis-seeding subclones, proliferating subclones, treatmentresistant subclones, and / or combinations thereof from representative samples of fixed tumor tissue. In some embodiments, the identified genomic variations are further characterized according to clonality, cancer cell fraction, and / or whether the variations are associated with a metastasisseeding lineage.

[0049] Yet other embodiments of the present disclosure are depicted in FIGS. 2, 3, and 4. For instance, and with reference to FIG. 2, in some embodiments the sample is a surgical resection or residual surgical material, such as a surgical resection or residual surgical material derived from a primary tumor, such as from a patient who has undergone or is currently undergoing neoadjuvant therapy (step 211). In other embodiments, and with reference to FIG. 3, the sample includes one or more FFPE samples, such as one or more FFPE samples from a patient undergoing or who has undergone neoadjuvant therapy (step 311 or step 411). In some embodiments, FFPE samples are utilized when there is no residual surgical material available. In some embodiments, multiple FFPE tissue samples, each derived from a different tumor section of a tumor of the same subject, are combined and collectively homogenized and / or dissociated according to the methods described herein. For example, in some embodiments, and to address spatial heterogeneity across multipleAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) blocks taken from the same patient, one or more FFPE tissue samples are combined from multiple FFPE blocks containing tissue removed from the subject.

[0050] In some embodiments, the surgical resection, the residual surgical material, or the one or more FFPE samples are homogenized to provide a homogenized sample (step 212 or step 312). Methods of homogenization are described herein. Subsequently, the homogenized sample is dissociated, such as into a composition comprising single cells (step 213 or step 313). Methods of dissociating cells from homogenized samples are described herein. Next, the dissociated cells are stained for the expression of the Ki67 biomarker (step 214 or step 314). Cells which stain positive for the expression of the Ki67 biomarker are isolated or sorted (such as with FACS) into a population of cells expressing the Ki67 biomarker (step 215 or step 315). The population of cells expressing the Ki67 biomarker is then sequenced to provide one or more sets of sequencing data (step 216 or step 316). The one or more sequencing data sets can then be analyzed to determine whether the population of cells expressing the Ki67 biomarker includes one or more high-risk genomic variations and / or one or more biomarkers of high-risk disease (step 217 or step 317). Finally, a clinical decision may be made based on the presence of the one or more high-risk genomic variations, or any genomic variation associated with the cells harboring metastatic potential, in the homogenized sample. For instance, a decision may be made as to whether to escalate or deescalate therapy (step 219 or step 319). Alternatively, or in addition, a decision may be made regarding treatment with one or more targeted therapies. Additionally, one or more high-risk genomic variations, or any genomic variation associated with the cells harboring metastatic potential, may be added to a MRD panel for monitoring (step 218 or step 318).

[0051] In some embodiments, a first enriched population comprising Ki67-positive cells is analyzed in parallel with a second enriched population, e.g., a population comprising cytokeratin-positive cells derived from the same homogenized sample. In some embodiments, comparison of the genomic variations present in the first enriched population and the second enriched population identifies one or more proliferating high-risk subclones, actionable subclonal drivers, and / or metastasis-seeding subclones that are enriched in the Ki67-positive population relative to the second enriched population, e.g., a cytokeratin-positive population.

[0052] In some embodiments, a genomic variation identified in the Ki67-positive population but absent from, or present at a lower cancer cell fraction in, a second enriched population (such as a cytokeratin-positive population) is classified as associated with aAttorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) proliferating subclone. In some embodiments, the proliferating subclone is a high-risk subclone, a treatment-resistant subclone, and / or a metastasis-seeding subclone.

[0053] With reference to FIG. 4, in some embodiments a lymphovascular invasion status of the one or more samples, such as the one or more FFPE samples, is determined (step 412). In some embodiments, a visual assessment of the one or more samples may be made (after staining the one or more samples) to ascertain whether the one or more samples are positive for LVI. In other embodiments, the assessment may be made using a digital pathology workflow (after staining of the one or more samples), such as with a machine learning algorithm or trained classification engine / trained classifier. In other embodiments, the one or more samples may be virtually stained and then visually assessed or assessed with a digital pathology workflow. Subsequently, the one or more FFPE samples identified as being positive for (or harboring) LVI are sequenced to provide one or more sets of sequencing data (step 413). In some embodiments, the one or more samples identified as being positive for LVI are pooled or homogenized prior to sequencing. The one or more sequencing data sets can then be analyzed to determine whether the one or more samples positive for LVI include one or more high-risk biomarkers (step 414). Finally, a clinical decision may be made based on the presence of the one or more high-risk genomic variations, or any genomic variation associated with the cells harboring metastatic potential, in the one or more samples. For instance, a decision may be made as to whether to escalate or deescalate therapy (step 416). Alternatively, or in addition, a decision may be made regarding treatment with one or more targeted therapies. Additionally, one or more high-risk genomic variations, or any other genomic feature present in the LVI-containing FFPE sample, may be added to a MRD panel for monitoring (step 415).

[0054] In some embodiments, the one or more FFPE samples determined to be positive for LVI are selected because LVLpositive regions are enriched for metastasis-seeding subclones relative to LVLnegative regions from the same primary tumor. In some embodiments, one or more genomic variations identified in the LVLpositive samples are incorporated into a tumor-informed MRD panel and / or used to guide escalation of adjuvant therapy. In some embodiments, one or more genomic variations identified in an LVLpositive sample and / or in a Ki67-positive population are selected for inclusion in a subject-specific tumor-informed MRD assay, optionally together with one or more genomic variations identified in a matched metastatic sample.

[0055] Samples / Tissue SamplesAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0056] The one or more samples or tissue samples may, in some embodiments, be derived from tumor tissue (cancerous or non-cancerous tumor tissue), a metastatic lesion, normal tissue, a lymph node, or any combination thereof. In some embodiments, the samples are derived from a subject previously diagnosed with cancer. In other embodiments, samples are derived from a subject at risk of developing cancer, such as the result of a genetic mutation or prior cancer. In yet other embodiments, the samples are derived from normal tissue adjacent to tumor tissue.

[0057] In some embodiments, the samples of the present disclosure are derived from one or more biopsy samples; derived from residual surgical material; and / or derived from surgical resection materials. In some embodiments, the samples are derived from a patient undergoing neoadjuvant therapy. In other embodiments, the samples are derived from a patient having previously received neoadjuvant therapy. In some embodiments, the samples are derived from a patient expected to undergo therapy, such as neoadjuvant therapy.

[0058] In some embodiments, the sample is derived from a fixed tissue specimen, e.g., a tissue specimen fixed in 10% neutral buffered formalin or another aldehyde-based fixative. As such, in some embodiments, the samples of the present disclosure are derived from one or more fixed samples or fixed biopsy samples; and / or derived from fixed residual surgical material. In some embodiments, the one or more samples may be from the same tumor specimen but derived from different spatial locations of that tumor specimen. In some embodiments, the sample is derived from a frozen tissue specimen.

[0059] In other embodiments, the sample is derived from a fixed tissue specimen, but not one that has been embedded in paraffin, e.g., the sample may be fixed but not embedded in paraffin or derived from a paraffin-embedded sample. In yet other embodiments, the sample is derived from a formalin-fixed paraffin-embedded (FFPE) tissue specimen or cytology specimen. In some embodiments, the FFPE samples are biopsy samples. In other embodiments, the FFPE samples are tissue curls. In some embodiments, the FFPE samples are tissue sections which at least 10 microns thick, such as between about 10 and about 30 microns in thickness. In other embodiments, the one or more FFPE samples are entire FFPE tissue blocks. In some embodiments, the FFPE blocks can range from needle biopsies, which are a few micrograms, to blocks that are 3 cm * 2.5 cm x 0.4 cm. In other embodiments, the one or more FFPE samples are tissue cores.

[0060] Homogenized Tissue SamplesAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0061] In some embodiments, the sample comprises a homogenized sample, e.g., a homogenized sample derived from one or more samples which have been blended, such as blended together. In some embodiments, the homogenized sample is derived from one or more fixed samples, such as surgical resection samples and / or residual surgical material. In other embodiments, the sample comprises dissociated cells derived from a fixed tissue sample or from a homogenized fixed tissue sample.

[0062] In some embodiments one or more input samples, e.g., one or more fixed or unfixed samples, one or more fixed or unfixed biopsy samples, or fixed or unfixed residual surgical material, are obtained, and then the one or more input samples are homogenized to form a homogenate. Cells within the homogenate are then dissociated to form the homogenized sample. Following sufficient dissociation of the input sample, the subpopulations of cells (including tumor cells) that were originally spatially segregated within the original sample are substantially uniformly distributed throughout the homogenized sample. That is, as a result of homogenizing the input sample, any heterogeneity of cells within the input sample is substantially uniformly distributed within the resulting homogenized sample or a portion or fraction thereof, such that the homogenized sample (or any portion or fraction thereof) substantially uniformly expresses the heterogeneity of the input sample (or one or more input samples) from which it was derived.

[0063] In some embodiments, and as a result of homogenizing the input sample, any aliquot removed from the homogenized sample comprises one or more populations of subclones at a proportion (or ratio) at which they existed within the input sample. Said another way, the disclosed homogenization methods generate a homogenized sample that contains subclones at the proportion at which they existed within the solid tumor. In some embodiments, any sample taken from the homogenate will contain each subclone in the same proportion as present in the original tumor (i.e., the original input sample).

[0064] Because the sample generated by the disclosed methods (e.g., homogenization and / or dissociation) is representative (or substantially representative) of the tumor as a whole, even low prevalence subclones in a sample can be detected, in addition to all other subclones that exist at higher prevalence rates. In some embodiments, any aliquot removed from the homogenized sample comprises one or more populations of subclones, such as one or more populations of low prevalence subclones, at the proportion at which they existed within the input sample.

[0065] Input Sample for HomogenizationAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0066] In some embodiments, the homogenized sample is derived from a tumor (cancerous or non-cancerous), a metastatic lesion, normal tissue, whole blood, a lymph node, or any combination therefore (collectively the "input sample"). In some embodiments, the homogenized sample is derived from a surgical resection or residual surgical material (such as material from which biopsies have been collected which would otherwise be destroyed). In some embodiments, the homogenized samples disclosed herein are obtained by homogenization and dissociation of one or more putative normal tissue specimens, e.g., derived from a subject at risk of developing cancer as the result of a genetic mutation or prior cancer, or adjacent normal tissue from a surgical resection for use as a control sample. Any of these samples may be fixed (such as using one or more aldehyde-based fixatives). In some embodiments, the input sample is derived from one or more FFPE samples, including those described herein.

[0067] In some embodiments, the residual surgical tumor sample is a specimen that would have otherwise been destroyed. In some embodiments, an entire obtained residual surgical tumor sample is fixed in an aldehyde-based fixative but not paraffin embedded, and wherein all the fixed but not paraffin embedded residual surgical sample is homogenized. In some embodiments, an entire obtained residual surgical tumor sample is fixed in an aldehyde-based fixative, and wherein all the fixed residual surgical sample is homogenized. In some embodiments, residual surgical tumor sample is not derived from tissue specimens used for diagnostic purposes. In some embodiments, residual surgical tumor sample is not derived from tissue specimens used in TNM staging.

[0068] In some embodiments, the input sample (e.g., the tumor or other tissue sample utilized) has a volume of at least about 1 cm3, at least about 2 cm3, at least about 3 cm3, at least about 4 cm3, at least about 5 cm3, at least about 6 cm3, at least about 7 cm3, at least about 8 cm3, at least about 9 cm3, at least about 10 cm3, at least about 15 cm3, at least about 20 cm3, at least about 25 cm3, at least about 50 cm3, at least about 100 cm3, at least about 250 cm3, at least about 500 cm3, at least about 1,000 cm3, at least about 2,500 cm3, at least about 5,000 cm3, at least about 7,500 cm3, at least about 10,000 cm3 or larger. In some embodiments, the input sample (e.g., the tumor or other tissue sample utilized) may have a width at the widest point of at least about 0.5 cm, at least about 1 cm, at least about 1.5 cm, at least about 2 cm, at least about 2.5 cm, at least about 3 cm, at least about 3.5 cm, at least about 4 cm, at least about 4.5 cm, at least aboutAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) 5 cm, at least about 6 cm, at least about 7 cm, at least about 10 cm, at least about 25 cm, at least about 50 cm or larger.

[0069] In some embodiments, the sample comprises at least about 100 to about 200; 200 to about 1,000; about 1,000 to about 5,000; about 10,000 to about 100,000; about 100,000 to about 1,000,000; about 1,000,000 to about 5,000,000; about 5,000,000 to about 1,000,000,000; about 1,000,000,000 to about 5,000,000,0000, or more cells, optionally from spatially distinct regions of the tumor. Generally, there are between about 500,000,000 cells to about 1 billion cells in a tumor portion having a diameter of about 1 cm. For example, an excisional sample such as a biopsy having about a 2 cm diameter may comprise between about 3 to about 5 billion cells.

[0070] Homogenization

[0071] Methods of preparing a homogenate from one or more tissue samples are described in United States Patent Publication No. 2020 / 0049599, the disclosure of which is hereby incorporated by reference herein in its entirety. For instance, and in some embodiments, a tumor sample (e.g., a surgical resection or residual surgical material), lymph node sample, blood sample, and / or other tissue sample is homogenized through a mechanical dissociation method, such as by placing the input sample into a mechanical shearing apparatus, e.g., a blender or an ultra sonicator. Alternatively, the input sample may be chemically dissociated, such as through enzymatic dissociation. In yet other embodiments, the input sample may be homogenized through any combination of mechanical dissociation and chemical dissociation. For instance, in some embodiments, the formation of a homogenized sample comprises mechanically dissociating (or shearing) an obtained input sample to provide a homogenized sample; and then dissociating cells or nuclei from the homogenized sample to provide the homogenized sample.

[0072] In some embodiments, homogenization may be affected by a method which preserves the integrity of the cells within the sample, i.e., the bulk of the cells within the homogenized sample or samples are not lysed and whereby the resultant homogenate and portions thereof are "representative" of the sample or samples. Therefore, the cells within the sample or a portion thereof reflect the percentages of the different cell types within the entirety of the tissue sample or samples, e.g., a solid tumor or a lymph node. This may be accomplished, for example, by mechanical dissociation of the tumor sample or a portion thereof (such as mechanical dissociation performed with or without the addition of liquid to the tumor sample or a portion thereof) and / or chemical or enzymatic dissociation of the tumor sample or a portion thereof (suchAttorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) as treatment with an enzyme that selectively or preferentially or primarily acts upon extracellular matrix proteins as compared to membrane-associated proteins). Alternatively, homogenization of the input sample may result in the dissociation of the cells from the input sample while still generating a sample that is representative of the starting tissue, such as a whole tumor. In some embodiments, the homogenized input sample may optionally be further dissociated and / or treated to remove or isolate specific types of molecules such as specific cell types, proteins, nucleic acids, or lipids, and the like thereby generating other homogenized samples which may be used in diagnostic and therapeutic methods.

[0073] In some embodiments, mechanical dissociation comprises cutting, scraping, or scratching the tissue into small pieces. In some embodiments, the minced tissue is washed in medium to separate the cells from the tissue. Optionally, the minced tissue may be agitated and / or sonicated to loosen the cells. In some embodiments, the homogenization processes comprise the use of a mechanical process, non-limiting examples of such include mortar and pestle, a dounce homogenizer or tissue grinder, a hand held electronic rotary blade tissue homogenizer (such as Omni-TH available from Thomas Scientific), a bead beating homogenizer (such as a bullet blender or a Burton Precellys 24 Tissue Homogenizer or a Bead Ruptor available from OMNI), optionally at a speed of between about 100 and about 75,000 RPM for rotational homogenizers or a speed of about 0.5 m / s to about 2.5 m / s for bead beaters, and for a length of about 30 second to about 5 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 30 minutes, or about 30 minutes to about 60 minutes. In some embodiments, the homogenate is created by manual dicing using a scalpel or knife. In some embodiments, the homogenization further comprises cell conditioning, where the cell conditioning comprises adjusting pH and / or heat and subsequently treating the sample with a cell conditioning buffer.

[0074] Enzymatic dissociation is the process of using enzymes to digest tissue pieces thereby releasing cells from tissue. Many different types of enzymes may be used in this process and, as the skilled artisan will appreciate, certain enzymes are more effective with certain tissue types. The skilled artisan will also appreciate any enzymatic dissociation process may use one or more enzymes in combination with each other, or one or more enzymes in combination with other chemical and / or mechanical dissociation methods. Non-limiting examples of suitable enzymes include, but are not limited to, collagenase, trypsin, elastase, hyaluronidase, papain, DNase 1, neutral protease, and trypsin inhibitor. In some embodiments, the enzyme is selected from a groupAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) consisting of interstitial collagenase, Gelatinase- A, Stromelysin 1, Matrilysin, Neutrophil collagenase, Gelatinase-B, Stromelysin 2, Stromelysin 3, Macrophage metalloelastase, Collagenase 3, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, Collagenase 4, Enamelysin, X-MMP, CA-MMP, MT5-MMP, MT6-MMP, Matrilysin-2, MMP-22, endoproteinase, trypsin, chymotrypsin, endoproteinase Asp-N, endoproteinase Arg-C, endoproteinase Glu-C (V8 protease), endoproteinase Lys-C, pepsin, thermolysin, elastase, papain, proteinase K, subtilisin, clostripain, exopeptidase, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, carboxypeptidase Y, cathepsin C, acylamino-acid-releasing enzyme, and pyroglutamate aminopeptidase.

[0075] Collagenase is a proteolytic enzyme used to digest proteins found in the extracellular matrix. Unique to enzymatic proteases, collagenase can attack and degrade the triplehelical native collagen fibrils that are commonly found in connective tissue. There exist four basic collagenase types, namely: Type 1, which is suitable for use in epithelial, liver, lung, fat and adrenal tissue cell specimens; Type 2, which is suitable for use in heart, bone, muscle, thyroid and cartilage tumor originating tissues given its high proteolytic activity; Type 3, which is suitable for use in mammary cells given its low proteolytic activity; and Type 4: which is suitable for islets and other research protocols where receptor integrity is important, given its tryptic activity.

[0076] Trypsin is described as a pancreatic serine (an amino acid) protease that has specificity for peptide bonds that involve the carboxyl group of arginine and lysine amino acids. It is considered one of the most highly specific proteases. Trypsin alone is not usually effective for tissue dissociation because it shows minimal selectivity to extracellular proteins. It is usually combined with other enzymes such as collagenase or elastase.

[0077] Elastase is another pancreatic serine protease, which has specificity for peptide bonds that are next to neutral amino acids. It is unique among proteases in its ability to hydrolyze native elastin. Elastase can also be found in blood components and bacteria. In some embodiments, it is suitable for isolation of Type II cells from lung tissue.

[0078] Hyaluronidase is a polysaccharidase, this enzyme is often used for dissociation of tissues, typically when combined with a cruder protease such as collagenase. It has affinity for bonds found in about all connective tissues.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0079] Papain is a sulfhydryl protease, it has wide specificity and so can degrade most protein substrates more thoroughly than pancreatic proteases, i.e., trypsin or elastase. Papain is frequently used to isolate neuronal materials from tissues.

[0080] Deoxyribonuclease I (DNase I) is frequently included in enzymatic cell isolation procedures to digest nucleic acids that leak into the dissociation medium and can increased viscosity and recovery problems. Without wishing to be bound by any theory, it is believed that DNasel will not damage intact cells.

[0081] Neutral protease, such Dispase® (available from Worthington Biochemical), is a bacterial enzyme with mild proteolytic activity, Dispase® is useful for isolating primary and secondary cell cultures because of its ability to maintain cell membrane integrity. It has been found to more efficiently dissociate fibroblast-like cells as compared to epithelial-like cells. It is inhibited by EDTA.

[0082] A trypsin inhibitor is derived from the soybean. It inactivates trypsin and so is sometimes used for specific cell isolation protocols.

[0083] Dissociation

[0084] In some embodiments and depending on the mechanical and / or biochemical dissociation process applied to the sample to generate the homogenized sample, the cell clusters may comprise more than one cell to thousands of cells. In some embodiments, the clusters can be dissociated (decreased in size and / or number of cells contained therein) by the application of further processing methods, e.g., by further mechanical and / or biochemical dissociation and / or by size exclusion, depending on the subsequent assay to be performed using the homogenized sample (for example, IHC requiring cell clusters containing tens to thousands of cells, or FACS or flow cytometry requiring single cells or fragments of cells).

[0085] For instance, in some embodiments, the samples may be further homogenized; and dissociation may be performed using one or more enzymes, such as any of the enzymes noted herein for chemical dissociation. In other embodiments, single cells are non-enzymatically dissociated from an obtained FFPE tissue sample to provide a single cell composition or a composition substantially including intact single cells. In some embodiments, single cells are non-enzymatically dissociated from an FFPE sample using one or more mechanical dissociation processes.Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application)

[0086] As used herein, "dissociation" is a process whereby an overall size, weight, and / or complexity of an obtained tissue sample is reduced. "Mechanical dissociation" refers to a dissociation of an obtained tissue sample using one or more mechanical sources and / or with one or more sources that each apply physical forces to the obtained tissue sample. In some embodiments, the one or more mechanical dissociation techniques include applying physical forces onto the obtained tissue sample such as shearing, slicing, cutting, vibration, pressure, crushing to pull apart or tear or otherwise separate the tissue sample into smaller pieces. In other embodiments, the one or more mechanical dissociation techniques include homogenizing, shearing, cutting, mincing, scraping, or scratching the obtained tissue sample. In some embodiments, at least one mechanical dissociation process comprises the use of a mortar and pestle, a dounce homogenizer, a tissue grinder, a Waring blender, a mortar and pestle, trituration with a glass Pasteur pipette, vortexing, a hand-held electronic rotary blade tissue homogenizer, or a bead beating homogenizer.

[0087] In some embodiments, mechanical dissociation may take place in several steps, where during each step the size, weight, and / or complexity of the mechanically dissociated sample is further reduced. For instance, in some embodiments, and depending on the type of mechanical dissociation technique applied, an initial mechanical dissociation process may result in a mechanically dissociated sample which includes a mixture of cell clusters, clumps of cells, and / or individual cells. In these embodiments, the mechanically dissociated sample comprising the clusters, clumps, and / or individual cells may then be further mechanically dissociated using one or more mechanical dissociation processes to provide a further dissociated solution, i.e., one where the size, weight, and / or complexity of any clusters and / or clumps of cells is reduced. In some embodiments, mechanical dissociation of an obtained FFPE tissue sample is performed in the presence of one or more fluids, such as one or more buffers and / or one or more non-enzymatic cell conditioning agents. Suitable buffers include borate buffers, phosphate buffers, citrate buffers, tris buffers, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and combinations thereof. In some embodiments, the mechanical dissociation of one or more obtained FFPE tissue samples is performed in the presence of distilled water. In some embodiments, any resulting single cell composition includes the cells and a fluid, such as water, a buffer, and / or a cell conditioning agent. In some embodiments, any resulting single cell composition includes the cells and a fluid, such as water, a buffer, and / or a cell conditioning agent; but is free from any exogeneous enzymes.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0088] In some embodiments, a mechanically dissociated sample may be further processed. In some embodiments, the further processing may include performing one or more filtration processes, agitation processes, sonication processes, separation processes, centrifugation processes, and / or lateral flow processes. In some embodiments, the one or more dissociation processes include at least one mechanical dissociation process, at least one filtration process, and at least one agitation or sonication process. Such further processing steps may be conducted without introducing any exogeneous enzymes to the mechanically dissociated sample.

[0089] In some embodiments, the preparation of the homogenized sample further comprises filtering or sizing the homogenate or a portion or fraction thereof, which may result in obtaining single cells or small cell clusters, such as doublets or triplets.

[0090] In some embodiments, the cellular componentry of the homogenized sample may optionally be separated by one or multiple filtration steps. For example, following homogenization and optional disassociation of the homogenate through physical and / or biochemical means, the disassociated sample may be optionally filtered through a 1 -micron filter to remove all intact cellular material. To the extent that single cells derived from the homogenized sample are obtained following filtration, such cells may be analyzed using fluorescent activated cell sorting (FACS) and flow cytometry analysis (such as described herein).

[0091] Samples Positive for Lymphovascular Invasion

[0092] In some embodiments, one or more samples are independently analyzed for lymphovascular invasion. For instance, if four samples have been derived from a tumor specimen, each of the four samples may be independently analyzed to determine if they harbor lymphovascular invasion. Lymphovascular invasion (LVI) is defined as the invasion of vessel walls (blood vessels and / or lymphatic vessels) by tumor cells and / or the presence of tumor emboli within an endothelial-lined space and this may be considered as the initial image (e.g., a whole slide image, a field-of-view, etc.) finding for lymph node metastasis and other types of organ metastases.

[0093] Methods of assessing lymphovascular invasions are known in the art (see, e.g., Peters, E.E.M., Nucci, M.R., Gilks, C.B., McCluggage, W.G. and Bosse, T. (2025), Practical guidance for assessing and reporting lymphovascular space invasion (LVSI) in endometrial carcinoma. Histopathology, 86: 173-182. https: / / doi.org / 10.llll / his.15272). For instance, in some embodiments, the one or more tumor cells or endothelial cells are stained with aAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) morphological stain to evaluate for LVI. In some embodiments, samples are assessed for LVI by morphologically staining the tumor sample with hematoxylin and eosin. In other embodiments, samples are assessed for LVI by staining with one or more of hematoxylin and eosin (H&E) staining, elastic staining, Factor VIII staining, Ulex europaeus I agglutinin staining, CD31, CD34, D2-40, ERG, and D2-40 (podoplanin) immunohistochemical (IHC) stains, among others. (See, e.g., Gonzalez J, Bahmad HF, Ocejo S, Abreu A, Popp M, Gogola S, Fernandez V, Recine M, Poppiti R. The Usefulness of Elastin Staining to Detect Vascular Invasion in Cancer. Int J Mol Sci. 2023 Oct 17;24(20): 15264. doi: 10.3390 / ijms242015264. PMID: 37894944; PMCID: PMC 10607141).

[0094] In some embodiments, the one or more tissue samples are morphologically stained (e.g., with H&E) and evaluated by a pathologist. In other embodiments, the one or more tissue samples are morphologically stained (e.g., with H&E) and evaluated using a digital pathology workflow or algorithm, such as one based on machine learning or using a trained image classifier. In yet other embodiments, the one or more tissue samples are virtually stained (morphologically and / or for one or more biomarkers), and the virtually stained images are utilized for histological examination. For instance, in some embodiments, the samples are virtually morphologically stained (i.e., where a virtually stained image manifests the appearance of an unstained biological specimen as if it were stained with a morphological stain). In some embodiments, the samples are virtually stained according to the methods described in U.S. Patent No. 11,482,320 and in U.S. Publication No. 2024 / 0046473, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0095] Subsequently, genetic material derived from cells within the one or more samples determined to be positive for LVI are then sequenced, such as described herein. In some embodiments, the sequencing comprises next-generation sequencing. In some embodiments, the sequencing comprises exome sequencing.

[0096] Cells Staining Positive for Ki67 Expression

[0097] In some embodiments, cells derived from a homogenized sample staining positive for Ki67 expression are sequenced.

[0098] In some embodiments, cells in the homogenized sample or a further dissociated variant, therefore, are first stained for the presence of Ki67. Non-limiting examples of suitable antibodies (clones) for detecting Ki67 are identified in the table below:Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) Specific proteindetected Name of clone VendorKi67 30-9 RocheKi67 Ki67 BioLegend Ki67 D3B5 Cell Signaling TechnologyKi67 20Raj 1 eBioscience

[0099] Following staining, the cells are sorted into at least two populations, such as a first population including those cells staining positively for the expression of Ki67; and a second population including those cells which are negative for Ki67 staining.

[0100] In some embodiments, the cells are sorted using "fluorescence-activated cell sorting" (FACS). FACS is a flow cytometry technique that allows simultaneous collection of data and sorting of cells based on one or more parameters. Methods of sorting cells using FACS are described in United States Patent Publication Nos. 2020 / 0049599, 2020 / 0011775, and 20040265835, the disclosures of which are hereby incorporated by reference herein in their entireties. In other embodiments, the single cells are sorted using magnetic-activated cell sorting. In yet other embodiments, the dissociated cells are sorted or processed using a set of buoyant particles, such as described in United States Patent No. 11,583,893, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0101] Following sorting, the population of cells staining positive for the expression of the Ki67 biomarker are sequenced. In some embodiments, the sequencing comprises next-generation sequencing. In some embodiments, the sequencing comprises exome sequencing.

[0102] In some embodiments, cells dissociated from formalin-fixed tissue are stained with an anti-Ki67 antibody and a second anti-biomarker antibody, such as an anti-cytokeratin antibody, prior to cell sorting. In some embodiments, tumor-cell gating is further refined using side scatter, DNA content, DAPI staining, exclusion of non-tumor cell populations, and / or combinations thereof.

[0103] Sequencing

[0104] The genetic material derived from (i) the one or more samples positive for lymphovascular invasion; or (ii) the population of cells derived from a homogenized sample staining positive for the expression of Ki67 may be sequenced according to any method known inAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) the art, such as with any suitable instrument including SMRT (single-molecule real-time sequencing), ion semiconductor, pyrosequencing, sequencing by synthesis, combinatorial probe anchor synthesis, and SOLiD sequencing (sequencing by ligation). Non-limiting sequencing platforms include those provided by Illumina® (e.g., the MiniSeq™, MiSeq™, NextSeq™, and / or NovaSeq™ sequencing systems); Ion Torrent™ (e.g., the Ion PGM™, Ion S5™, and / or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., the PACBIO RS II and / or Sequel II System sequencing system); ThermoFisher (e.g., a SOLID® sequencing system); or BGI Genomics (e.g., DNBSeq™ sequencing systems). See, for example U.S. Pat. Nos. 7,211,390; 7,244,559; 7,264,929; 6,255,475; 6,013,445; 8,882,980; 6,664,079; and 9,416,409; the disclosures of which are hereby incorporated by reference herein in their entireties.

[0105] In other embodiments, the obtained genetic material may be sequenced by sequencing-by-synthesis (SBS), pyrosequencing, sequencing by ligation (SBL), or sequencing by hybridization (SBH). Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568; and 6,274,320, each of which is incorporated herein by reference in its entirety). In pyrosequencing, released PPi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated can be detected via light produced by luciferase. In this manner, the sequencing reaction can be monitored via a luminescence detection system. In both SBL and SBH methods, target nucleic acids, and amplicons thereof, that are present at features of an array are subjected to repeated cycles of oligonucleotide delivery and detection. SBL methods, include those described in Shendure et al. Science 309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated herein by reference in its entirety; and the SBH methodologies are as described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor etal., Science 251(4995), 767-773 (1995); and WO 1989 / 10977, each of which is incorporated herein by reference in its entirety.

[0106] In SBS, extension of a nucleic acid primer along a nucleic acid template is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be catalyzed by a polymerase, wherein fluorescently labeled nucleotides are added to a primer (thereby extending the primer) in a template dependent fashion such that detection of theAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) order and type of nucleotides added to the primer can be used to determine the sequence of the template.

[0107] Nanopore sequencing refers to the approach in which tags that are attached to nucleotides can be distinguished by their effect on ionic currents passing through nanopores as these tagged nucleotide analogs are added to a growing (nascent) DNA strand. Measurements can be made while tagged nucleotides are still part of the ternary complex, or after their tags are released by the polymerase reaction.

[0108] Nanopore sequencing of a nucleic acid molecule may be achieved by strand sequencing and / or exosequencing of the polynucleotide sequence. In some embodiments, nanopores may be used to sequence nucleic acid molecules where a polymerized nucleic acid molecule does not pass through the nanopore during sequencing. In these embodiments, the nucleic acid molecule may be at least partially located in the vestibule of the nanopore, but not in the pore (i.e., narrowest portion) of the nanopore. The nucleic acid molecule may pass within any suitable distance from and / or proximity to the nanopore, and optionally within a distance such that byproducts released from nucleotide incorporation events, e.g., tags cleaved from tagged nucleotide analogs, are detected in the nanopore.

[0109] Nanopore sequencing utilizes different tagged nucleotide analogs each having a covalently attached tag moiety that provides an identifiable, and distinguishable signature when detected within or near a nanopore. In some embodiments, nanopore sequencing requires a set of at least the four-standard deoxy-nucleotides dA, dC, dG, and dT, wherein each nucleotide has an attached tag capable of being detected by a nanopore upon the nucleotide being incorporated by a strand extending enzyme. Examples of tagged nucleotide analogs are described in United States Patent No. 10,975,426, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0110] In some embodiments, a strand extending enzyme (e g., a DNA polymerase), such as one located in proximity to a nanopore, specifically binds a tagged nucleotide analog that is complimentary to a nucleotide of a nucleic acid molecule which is hybridized to a growing (nascent) nucleic acid strand at its active site. The strand extending enzyme (e.g., a DNA polymerase) then catalytically incorporates the complementary nucleotide moiety of the tagged nucleotide analog ("nucleotide incorporation event") to the end of the nascent nucleic acid strand. Nucleotide incorporation events are catalyzed by the enzyme, such as DNA polymerase or anyAttorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) mutant or variant thereof and use base pair interactions with a template molecule to choose amongst the available nucleotides for incorporation at each location. Completion of the catalytic incorporation event results in the release of the tag moiety and the oligophosphate moiety (minus the one phosphate incorporated into the growing strand) which then passes through the adjacent nanopore.

[0111] "Nucleotide incorporation events," as that term is used herein, means the incorporation of a tagged nucleotide analog into a growing polynucleotide chain. In some embodiments, byproducts of nucleotide incorporation events may be detected by the nanopore. In some embodiments, a byproduct may be correlated with the incorporation of a given type of modified or unmodified nucleotide. In some embodiments, the byproduct passes through the nanopore and / or generates a signal detectable in the nanopore. Released tag molecules are examples of byproducts of nucleotide incorporation events. Additional details pertaining to such nanopore-based sequencing systems and methods are described in United States Patent Nos.9,605,309 and 9,557,294, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0112] It is believed that sequencing using the SMRT platform allows the observation of single DNA polymerases reading individual molecules of DNA in real time. It is also believed that the kinetic characteristics of DNA polymerization are observable on a single-molecule basis.

[0113] In some embodiments the incorporation of differently labeled nucleotides is observed in real time as template dependent synthesis is carried out. In particular, an individual immobilized primer / template / polymerase complex is observed as fluorescently labeled nucleotides are incorporated, permitting real time identification of each added base as it is added. In this process, label groups are attached to a portion of the nucleotide that is cleaved during incorporation. For example, by attaching the label group to a portion of the phosphate chain removed during incorporation, i.e., a P, y, or other terminal phosphate group on a nucleoside polyphosphate, the label is not incorporated into the nascent strand, and instead, natural DNA is produced. Observation of individual molecules typically involves the optical confinement of the complex within a small illumination volume. By optically confining the complex, a monitored region is created in which randomly diffusing nucleotides are present for a short period of time, while incorporated nucleotides are retained within the observation volume for longer as they are being incorporated. This results in a characteristic signal associated with the incorporation event,Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) which is also characterized by a signal profile that is characteristic of the base being added. In some embodiments, interacting label components, such as fluorescent resonant energy transfer (FRET) dye pairs, are provided upon the polymerase or other portion of the complex and the incorporating nucleotide, such that the incorporation event puts the labeling components in interactive proximity, and a characteristic signal results, that is again, also characteristic of the base being incorporated (see, e g., U.S. Pat. Nos. 6,056,661, 6,917,726, 7,033,764, 7,052,847, 7,056,676, 7,170,050, 7,361,466, 7,416,844 and Published U.S. Patent Application No. 2007-0134128, the disclosures of which are each hereby incorporated by reference herein in their entireties).

[0114] The SMRT platform uses a polymerase enzyme, a template sequence, and a primer sequence complementary to a portion of the template sequence. These components are immobilized within a confined illumination volume. The reaction mixture surrounding the complex has the four different nucleotides (A, G, T and C) each labeled with a spectrally distinguishable fluorescent label attached through its terminal phosphate group. Because the illumination volume is small, nucleotides and their associated fluorescent labels diffuse in and out of the illumination volume quickly and thus provide only short fluorescent signals. When a particular nucleotide is incorporated by the polymerase in a primer extension reaction, the fluorescent label associated with the nucleotide is retained within the illumination volume for a longer time. Once incorporated, the fluorescent label is cleaved from the base through the action of the polymerase, and the label diffuses away.

[0115] A non-limiting example of a sequencing device includes the AXELIOS platform (Roche), which is based on Roche's sequencing by expansion (SBX) technology. In SBX, sequence information from a nucleic acid template is converted into an expanded surrogate polymer molecule, sometimes referred to as an "Xpandomer," using expandable nucleotide triphosphates, after which the expanded molecule is measured as it translocates through a nanopore in a CMOS-based array. More specifically, this chemistry translates the sequence of DNA into a simple to measure surrogate molecule called an Xpandomer. Much like with polymerase chain reaction, Xpandomer synthesis is based on the natural function of DNA replication where expandable nucleoside triphosphates (X-NTPs) act as substrates for template-dependent, polymerase-based replication. Four easily differentiated X-NTPs (also called High Signal-to-Noise Reporters) are used during Xpandomer synthesis, one for each DNA base, and engineeredAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) polymerases incorporate the X-NTPs into an Xpandomer, which serves as a surrogate for the complement of the nucleic acid template. As the Xpandomer molecule transits through a nanopore, the distinct electrical signal of each base reporter is easily identifiable to enable highly accurate and high throughput nanopore-based nucleic acid sequencing. SBX is described in U.S. Patent No. 7,939,259, 9,771,614, 10,774,105, and 11,530,392, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0116] In some embodiments, genomic material is extracted from cells, such as single cells from the one or more populations of single cells prepared after cell sorting or from the tissue material. In some embodiments, a target enrichment process is performed after extraction of the genetic material. In some embodiments, genomic material is amplified within isolated individual cells. In some embodiments, the extracted genomic material is amplified, barcoded, and a sequencing library is prepared including the genomic material from an isolated single cell. In some embodiments, the sequencing library is then sequenced, such as with aNGS sequencing apparatus. In some embodiments, the sequencing provides one or more sequencing data sets. In some embodiments, the generated one or more sequence data sets are utilized to identify one or more therapeutic targets. In some embodiments, the generated one or more sequence data sets are utilized to identify one or more combination therapy targets. In some embodiments, the generated one or more sequence data sets are utilized to identify one or more immunotherapy therapy targets. In some embodiments, the generated one or more sequence data sets are utilized to determine at least one clinical decision, wherein the at least one clinical decision comprises determining disease prognosis, predicting recurrence of disease, inclusion of subjects in clinical trials, or therapeutic treatment strategy for at least one subject.

[0117] In some embodiments, sequence data generated from formalin-fixed material are filtered prior to identification of one or more genomic variations of interest. Such filtering may comprise removal of formalin-associated mutational artefacts, microhomology-induced chimeric reads (MICRs), panel-of-normal variants, orientation bias artefacts, low-confidence low-allele-frequency events, and / or combinations thereof.

[0118] In some embodiments, one or more genetic variations are identified within the one or more sequencing data sets. In some embodiments, the methods of the present disclosure further include determining whether the identified one or more genetic variations are clonal or subclonal. In some embodiments, a MRD panel may be developed based on the identified genetic variations.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) In some embodiments, the MRD panel may be used to monitor for genetic variations that may result in distant metastasis. In yet other embodiments, a clonal structure may be computed based on the identified genetic variations. In some embodiments, the computed clonal structure may be used to assess the separation between truncal variations and sub-clonal variations.

[0119] In some embodiments, the methods further comprise classifying identified genomic variations as clonal, subclonal, or indeterminate based on cancer cell fraction (CCF). In some embodiments, subclonal actionable variants, subclonal biomarkers, proliferating subclonal drivers, and / or metastasis-seeding subclones are prioritized for therapeutic decision-making, recurrence monitoring, and / or design of a subject-specific tumor-informed MRD assay.

[0120] In some embodiments, tumor mutational burden is further classified as clonal tumor mutational burden and subclonal tumor mutational burden. In some embodiments, high clonal tumor mutational burden is weighted more heavily than high subclonal tumor mutational burden in determining immunotherapy-related treatment strategy, disease prognosis, and / or recurrence monitoring.

[0121] In some embodiments, the one or more sequencing data sets are compared with sequence data from a matched metastatic sample to identify one or more genomic variations associated with a metastasis-seeding subclone. In some embodiments, one or more such genomic variations are selected for inclusion in a subject-specific tumor-informed MRD assay.

[0122] In some embodiments, the method further includes detecting, characterizing, or identifying one or more low prevalence genetic events based on the data within the one or more sequencing data sets. In some embodiments, the one or more low prevalence genetic events are selected from a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene. In some embodiments, the method further includes identifying one or more subclones. In some embodiments, the identified one or more subclones are one or more low prevalence subclones. In some embodiments, the one or more low prevalence subclones have a prevalence of less than 0.0001% within the sequenced genomic material. In some embodiments, the one or more low prevalence subclones have a prevalence of less than 0.00001% within the sequenced genomic material.Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application)

[0123] EXAMPLES

[0124] The following examples are provided to illustrate particular embodiments of the disclosure and are not intended to limit the scope of the claims.

[0125] Example 1: Identification of Actionable Subclonal Drivers via Ki67+ Enrichment in Formalin-Fixed Tissue

[0126] This example demonstrates a method for identifying rare, high-risk, and actionable subclonal variations that are typically missed by standard genomic profiling. The objective was to show that enriching for the Ki67 proliferation biomarker in single cells dissociated from homogenized residual tumor tissue enables a much higher resolution of detection for these dangerous subclones.

[0127] The methodology utilized leftover surgical tissue from breast cancer patients that was surplus to standard diagnostic requirements and had been fixed in formalin. To prepare the sample, the tissue was mechanically homogenized and dissociated into a single-cell suspension without the use of exogenous enzymes. Once suspended, the single cells were stained with antibodies against the proliferation biomarker Ki67. Fluorescence-activated cell sorting (FACS) was then used to specifically isolate the Ki67-expressing cell populations. Finally, genomic DNA was extracted from these highly proliferative, Ki67-sorted cells and subjected to whole exome sequencing (WES) to identify underlying genomic variations.

[0128] When sequencing the targeted Ki67-positive populations, additional driver mutations were successfully identified in five specific cases, designated as HF299, HF414, HF105, HF039, and HF 168. Crucially, in three of these cases, the newly identified driver mutations were clinically actionable. These included a second-hit PIK3CA mutation in HF039, a POLE mutation in HF105, and a MAP3K1 mutation in HF299. Genomic analysis revealed that these actionable variations were subclonal and arose late in the cancer's evolutionary lineage, typically representing at least a second-order branching on the phylogenetic tree. Because of their low overall prevalence in the bulk tumor and their late emergence, achieving the limit of detection necessary to uncover them required the specific phenotypic enrichment for Ki67.

[0129] The clinical utility of this method is heavily underscored by the specific case of patient HF299. This patient exhibited striking regional variability in Ki67 expression across the primary tumor mass. By explicitly sorting and sequencing the Ki67-positive cells, the method successfully captured a metastasizing subclone that was localized exclusively within those highlyAttorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) proliferative regions. Ultimately, identifying these high-risk, metastasis-seeding variations via Ki67 enrichment provides a vital tool for clinical decision-making. It allows clinicians to intentionally select these rare genomic variations for inclusion in a patient-specific molecular residual disease (MRD) panel, thereby informing the escalation of targeted adjuvant therapies proactively before any clinical recurrence can occur.

[0130] Example 2; Utilizing LVI Positivity to Guide FFPE Block Selection for High-Risk Genomic Sequencing

[0131] This example details a method for leveraging Lymphovascular Invasion (LVI) status to strategically select the most informative formalin-fixed paraffin-embedded (FFPE) tissue block for genomic sequencing, thereby ensuring the capture of specific mutations driving metastatic disease. Standard-of-care sequencing typically relies on a single, routinely selected FFPE block from the primary tumor. However, this generalized approach frequently misses critical, metastasis-seeding driver mutations due to the spatial heterogeneity of the tumor mass. To overcome this limitation, primary tumor samples from a cohort of patients with invasive breast cancer are first evaluated histologically to determine the presence or absence of LVI across the various available tissue blocks. Clinical analysis utilizing a generalized multivariate linear model demonstrates that the presence of LVI in a tissue section is significantly and positively correlated with future metastatic disease progression.

[0132] With this histological data, clinicians can intentionally and specifically choose the LVLpositive FFPE blocks for genomic extraction and sequencing. By directing sequencing efforts exclusively toward these high-risk, LVI-positive tissue sections rather than a standard bulk section, the method accurately captures the dangerous genomic variations responsible for metastatic competence. The critical importance of this targeted block selection was highlighted in seven distinct patient cases within the cohort. In these cases, actionable driver mutations, which were later confirmed to be the exact variations seeding the patients' metastatic disease, were successfully identified by sequencing the LVI-positive blocks, despite being completely undetected by standard-of-care single-region FFPE sampling. Ultimately, utilizing LVI positivity as the primary deciding factor for which FFPE block to sequence provides a highly enriched, metastasis-specific genomic profile. Additionally, this workflow allows clinicians to accurately identify the highest-risk variations for integration into a highly sensitive, patient-specific Molecular Residual Disease (MRD) monitoring panel.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application)

[0133] All the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments.

[0134] Although the present disclosure has been described with reference to a number of illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and / or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) CLAIMS1. A method of identifying one or more genomic variations associated with cells harboring metastatic potential in one or more formalin-fixed paraffin-embedded (FFPE) tissue samples, comprising: a. determining a lymphovascular invasion (LVI) status in each of the one or more FFPE tissue samples; b. sequencing genomic material derived from the one or more FFPE tissue samples determined to be positive for LVI to provide one or more sequencing data sets; c. identifying the one or more genomic variations associated with cells harboring metastatic potential in the one or more sequencing data sets; and d. determining at least one clinical decision based on the identification of the one or more genomic variations associated with cells harboring metastatic potential.

2. The method of claim 1, wherein the LVI status is determined by staining the one or more FFPE tissue samples with a morphological stain.

3. The method of claim 1, wherein the LVI status is determined by virtually staining the one or more FFPE tissue samples.

4. The method of any one of claims 1-3, wherein the LVI status is determined visually.

5. The method of any one of claims 1-3, wherein the LVI status is determined using a trained classifier.

6. The method of any one of claims 1-5, further comprising adding the one or more genomic variations associated with cells harboring metastatic potential to a molecular residual disease (MRD) panel.

7. The method of any one of claims 1-6, wherein the determined clinical decision comprises disease prognosis, predicting recurrence of disease, inclusion of subjects of clinical trials, or therapeutic treatment strategy.

8. The method of any one of claims 1-6, wherein the determined clinical decision is to escalate therapy.

9. The method of any one of claims 1-6, wherein the determined clinical decision is to deescalate therapy.

10. The method of any one of claims 1-9, wherein the sequencing comprises exome sequencing.

11. The method of any one of claims 1-9, wherein the sequencing comprises next-generation sequencing.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) 12. The method of claim 11, wherein the next-generation sequencing comprises pyrosequencing, nanopore sequencing, sequencing-by-synthesis, sequencing-by-ligation, and single molecule real-time sequencing.

13. The method of claim 1, further comprising detecting, characterizing, or identifying one or more low prevalence genetic events in the generated one or more sequencing data sets.

14. The method of claim 13, wherein the one or more low prevalence genetic events are selected from a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene.

15. The method of claim 1, wherein at least one identified genomic variation from the one or more FFPE tissue samples determined to be positive for LVI is classified as associated with a metastasis-seeding subclone and is incorporated into a tumor-informed MRD panel.

16. The method of claim 1, wherein the identified genomic variation is associated with an actionable subclonal driver.

17. A method of identifying one or more high-risk genomic variations associated with cells harboring metastatic potential in a single cell composition, comprising: a. obtaining one or more surgical resections or residual surgical material; b. homogenizing the one or more surgical resections or the residual surgical material to provide a homogenized sample; c. dissociating the homogenized sample into the single cell composition; d. staining cells in the single cell composition for the expression of the Ki67 biomarker; e. sorting the cells expressing the Ki67 biomarker into a first population of cells; f. sequencing genomic material derived from the cells in the first population of cells to provide one or more sequencing data sets; g. identifying the one or more high-risk genomic variations in the one or more sequencing data sets; and h. determining at least one clinical decision based on the identification of the one or more high-risk genomic variations.

18. The method of claim 17, further comprising adding the identified one or more high-risk genomic variations associated with cells harboring metastatic potential to a molecular residual disease (MRD) panel.

19. The method of any one of claims 17-18, wherein the determined clinical decision comprises disease prognosis, predicting recurrence of disease, inclusion of subjects of clinical trials, or therapeutic treatment strategy.Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) 20. The method of any one of claims 17-18, wherein the determined clinical decision is to escalate therapy.

21. The method of any one of claims 17-18, wherein the determined clinical decision is to deescalate therapy.

22. The method of any one of claims 17-21, wherein the sequencing comprises exome sequencing.

23. The method of any one of claims 17-21, wherein the sequencing comprises next-generation sequencing.

24. The method of claim 23, wherein the next-generation sequencing comprises pyrosequencing, nanopore sequencing, sequencing-by-synthesis, sequencing-by-ligation, and single molecule real-time sequencing.

25. The method of claim 17, further comprising detecting, characterizing, or identifying one or more low prevalence genetic events in the generated one or more sequencing data sets.

26. The method of claim 25, wherein the one or more low prevalence genetic events are selected from a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene.

27. The method of claim 17, further comprising enriching a second population of cells expressing a second biomarker from the homogenized sample and comparing genomic variations identified in the first population and the second population.

28. The method of claim 27, wherein one or more high-risk genomic variations are identified in the first population but not in the second population and / or are present at lower prevalence in the second population.

29. The method of claim 17, wherein the one or more surgical resections or residual surgical material are obtained from a subject after neoadjuvant endocrine therapy.

30. The method of claim 29, wherein the subject received neoadjuvant endocrine therapy for greater than 30 days.

31. The method of claim 17, wherein the one or more high-risk genomic variations comprise one or more actionable subclonal drivers present in proliferating subclones.

32. The method of claim 27, wherein one or more high-risk genomic variations enriched in the first population relative to the second population are selected for inclusion in a subjectspecific tumor-informed MRD assay.Attorney Docket No.: Ventana-0308WO / P40069-WO (PCT Application) 33. A method of identifying one or more high-risk genomic variations associated with cells harboring metastatic potential in a single cell composition, comprising: a. obtaining one or more formalin-fixed paraffin-embedded (FFPE) tissue samples; b. homogenizing the one or more FFPE tissue samples to provide a homogenized sample; c. dissociating the homogenized sample into the single cell composition; d. staining cells in the single cell composition for the expression of the Ki67 biomarker; e. sorting the cells expressing the Ki67 biomarker into a first population of cells; f. sequencing genomic material derived from the cells in the first population of cells to provide one or more sequencing data sets; g. identifying the one or more high-risk genomic variations in the one or more sequencing data sets; and h. determining at least one clinical decision based on the identification of the one or more high-risk genomic variations.

34. The method of claim 33, further comprising adding the identified one or more high-risk genomic variations associated with cells harboring metastatic potential to a molecular residual disease (MRD) panel.

35. The method of any one of claims 33-34, wherein the determined clinical decision comprises disease prognosis, predicting recurrence of disease, inclusion of subjects of clinical trials, or therapeutic treatment strategy.

36. The method of any one of claims 33-34, wherein the determined clinical decision is to escalate therapy.

37. The method of any one of claims 33-34, wherein the determined clinical decision is to deescalate therapy.

38. The method of any one of claims 33-37, wherein the sequencing comprises exome sequencing.

39. The method of any one of claims 33-37, wherein the sequencing comprises next-generation sequencing.

40. The method of claim 39, wherein the next-generation sequencing comprises pyrosequencing, nanopore sequencing, sequencing-by-synthesis, sequencing-by-ligation, and single molecule real-time sequencing.

41. The method of claim 33, further comprising detecting, characterizing, or identifying one or more low prevalence genetic events in the generated one or more sequencing data sets.Attorney Docket No.: Ventaiia-0308WO / P40069-WO (PCT Application) 42. The method of claim 41, wherein the one or more low prevalence genetic events are selected from a point mutation, a deletion, an addition, a translocation, a genetic fusion, or an amplification of a gene.

43. The method of any one of claims 33-42, wherein the dissociation comprises a non- enzymatic dissociation process.

44. The method of any one of claims 33-43, wherein the one or more FFPE samples are tissue sections / tissue curls.

45. The method of any one of claims 33-43, wherein the one or more FFPE samples are tumor blocks, tissue cores, or tissue biopsies.

46. The method of any one of claims 33-45, wherein the sorting comprises fluorescence- activated cell sorting (FACS).

47. The method of claim 33, further comprising enriching a second population of cells expressing a second biomarker from the homogenized sample and comparing genomic variations identified in the first population and the second population.

48. The method of claim 47, wherein one or more high-risk genomic variations are identified in the first population but not in the second population and / or are present at lower prevalence in the second population.

49. The method of claim 33, wherein the one or more FFPE tissue samples are obtained from a subject after neoadjuvant endocrine therapy.

50. The method of claim 49, wherein the subject received neoadjuvant endocrine therapy for greater than 30 days.

51. The method of claim 33, wherein the one or more high-risk genomic variations comprise one or more actionable subclonal drivers present in proliferating subclones.

52. The method of claim 33, further comprising classifying an identified genomic variation as clonal, subclonal, or indeterminate based on cancer cell fraction.

53. The method of claim 47, wherein one or more high-risk genomic variations enriched in the first population relative to the second population are selected for inclusion in a subjectspecific tumor-informed MRD assay.

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