Representative diagnostic method

Abstract

To provide the preparation of representative samples from clinical samples, e.g., tumors (whole or in part), lymph nodes, metastases, cysts, polyps, or a combination or portion thereof, using mechanical and / or biochemical dissociation methods to homogenize intact samples or large portions thereof.SOLUTION: The disclosure generally relates to the preparation of representative samples from clinical samples, e.g., tumors (whole or in part), lymph nodes, metastases, cysts, polyps, or a combination or portion thereof, using mechanical and / or biochemical dissociation methods to homogenize intact samples or large portions thereof. The resulting homogenate provides the ability to obtain a correct representative sample despite spatial heterogeneity within the sample, increasing detection likelihood of low prevalence subclones, and is suitable for use in various diagnostic assays as well as the production of therapeutics, especially "personalized" anti-tumor vaccines or immune cell based therapies.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of the filing dates of U.S. Provisional Patent Application No. 62 / 418,146, filed on November 4, 2016; U.S. Provisional Patent Application No. 62 / 354,622, filed on June 24, 2016; U.S. Provisional Patent Application No. 62 / 279,405, filed on January 15, 2016, and U.S. Provisional Patent Application No. 62 / 252,153, filed on November 6, 2015, the disclosures of which are hereby incorporated by reference in their entireties.

[0002] The present disclosure generally relates to the development of methods for generating representative tissue samples, such as whole organs, tumors, lymph nodes, metastases, or combinations thereof, to address the problem of tissue heterogeneity in clinical samples, particularly samples for use in clinical oncology. More specifically, the present disclosure provides the ability to obtain accurate representative samples and increase the likelihood of detection for small sub - clone populations and / or low - prevalence events, despite spatial heterogeneity within tissues, such as tumors, by applying mechanical, chemical, and / or biochemical, e.g., enzymatic dissociation methods, to intact fixed (or preserved) tissue samples, such as whole organs, tumors, lymph nodes, metastatic tissue, or any combination thereof. Representative tissue samples, such as tumor samples, are suitable for use in various diagnostic assays. In particular, these representative tumor samples and portions thereof are useful in assay methods for evaluating cancer prognosis (e.g., tumor stage classification) and in the selection and design of appropriate treatment regimens. Representative samples contain diverse antigens expressed by a given tumor (one or more), and thus representative tissue samples, such as tumor samples and portions thereof, can be used as therapeutic agents or to generate therapeutic agents, e.g., as vaccines, or in the manufacture of cancer vaccines or immune cell - based therapies.

Background Art

[0003] The tumor sampling techniques used for all diagnostic tests in medical oncology are rooted in the co - evolution of anatomical pathology and surgical oncology in the late 1800s. Before the discovery of ether - based anesthesia in 1842 and the invention of aseptic surgical techniques in 1867, pathological samples were typically obtained from autopsies. Before these two significant advancements in surgical techniques, the tissue samples obtained by pathologists typically originated from patients who died on the operating table due to shock or soon after due to infection. Thus, due to the high mortality rate of surgery, pathologists were unable to correlate the anatomical and microscopic characterization of tumor structure with patient survival statistics. The impact of anesthesia and aseptic techniques led to an immediate and dramatic increase in the number of patients surviving surgery and a substantial increase in the number of complex surgical procedures.

[0004] The above - mentioned advancements in the operating room coincided with the emergence of paraffin embedding of tissues in 1869 and the widespread use of formalin fixation in the early 1890s. Along with these two innovations, anatomical pathologists obtained the first clarity in tissue structure and microscopic morphology in history. In the early 1900s, insights were made into correlating different patterns of tissue structure and morphology with specific tumor types. Over time, anatomical pathologists and surgical oncologists were able to correlate these specific anatomical and microscopic features within the same tumor type with differences in overall survival. The correlation between the histological features of tumors and patient prognosis was ultimately systematized into the TNM staging classification system in the early 1950s.

[0005] The TNM staging system aims to determine the prognosis of cancer patients by evaluating the morphological aspects of the tumor (T), the extent to which the tumor cells have spread to the lymph nodes (N), and whether the tumor has metastasized to distant organs (M). For the "T" part of the TNM staging analysis, it is necessary to obtain a very small tumor sample (typically 3-5) at the interface between the tumor and the surrounding normal tissue. The sample should be of a consistent size (about 20×20×3 millimeters) to enable proper formalin fixation and paraffin embedding. 4-μm sections of the formalin-fixed paraffin-embedded (FFPE) sample are cut by a microtome, placed on a slide glass, and examined by a pathologist. Many pathology laboratories are expected to have the basic equipment to enable TNM staging of tumors: plastic tissue cassettes, formalin fixation buffers, a tissue processor to dehydrate the tissue and embed it in paraffin, a microtome to cut thin sections (generally 4 μm thick), and slide glasses to place the tissue sections.

[0006] The TNM staging system became an internationally recognized method for cancer staging in 1987 and is currently administered by the American Joint Commission on Cancer (AJCC) and the Union Internationale Contre le Cancer (UICC). The AJCC, UICC, and the College of American Pathologists (CAP) review and regularly update the guidelines for TNM staging criteria worldwide. The histological and anatomical features that are input into the TNM annotations are conditional on the surgical pathologist obtaining consistent tissue samples worldwide. Thus, the sampling techniques required for the TNM staging system, based on the techniques and methods developed in the late 1800s, have become fixed in medical oncology.

[0007] Obtaining an "accurate" tissue sample for tumor TNM stage classification is a major goal of surgical pathology, as evidenced by the fact that multiple textbooks describe and illustrate how surgical pathologists should handle surgical specimens. Generally, sections are taken that best demonstrate the features observed on gross examination. Many of these textbooks describe procedures approved by the AJCC, UICC, and CAP from recognized medical institutions and include examples that detail the exact portions of the tumor from which samples are taken (see FIGS. 1A-1C). The purpose of these textbooks is to train surgical pathologists worldwide to take specific regions of resected solid tumors in a consistent, reproducible manner to avoid random sampling. For example, see Surgical Pathology Dissection: An Illustrated Guide, 2nd Edition, p. 29, which states, "The key to an economical and thorough approach is selective sampling. Selective sampling is a strategic approach that attempts to maximize the information that can be obtained from a given tissue section. In contrast to random and haphazard sampling of specimens, selective tissue sampling is selective, increases the information that can be obtained histologically, and requires fewer sections to do so." These references state that to facilitate TNM stage classification, specific regions of solid tumors should be collected in a consistent, reproducible manner. Generally, sections with the highest grade are used for all diagnostic tests, and the remaining samples are discarded.

[0008] Since there is no additional information to be gained from increasing tumor sampling, surgical pathologists are taught to refrain from increasing the total number of samples taken from surgical specimens. There is no recognition that more diagnostic information may be available within the remaining tumor that has not been sampled.

[0009] Tumor metastasis to regional draining lymph nodes is a significant prognostic indicator (i.e., "N" in the prognostic TNM staging system). The presence of tumor cells within the draining lymph nodes can be a decisive factor in determining whether a patient receives adjuvant chemotherapy. Conventionally, tumor cells are detected by microscopic examination of single thin sections of lymph nodes and FFPE tissues after surgical removal. Metastatic growths in lymph nodes may be microscopic, filling the entire organ or containing only a few cancer cells. Since only a single thin section of the excised lymph node is examined for the presence of metastatic tumors, small metastatic growths can be missed within the lymph node. Depending on the size of the lymph node, a single FFPE tissue section can contain only 0.2 - 0.3 percent of the total amount of the excised node. Metastatic tumors growing in areas of the lymph node that are not sampled are not identified, resulting in false-negative analysis. As a result, patients receive a treatment regimen that is less invasive than what is necessary for individuals with locally advanced cancer (i.e., lymph node metastasis, N-positive).

[0010] Case reports are compiled by the pathology department, and after tumor samples are cut from the excised tumor material and the TNM stage classification is calculated, they are submitted to the oncologist. The remaining surgical material containing the remaining tumor tissue not used for the TNM stage classification is then typically destroyed by incineration as required under the Health Insurance Portability and Accountability Act (HIPAA) of 1996. However, CAP recommends retaining all paraffin blocks and cut slides for 10 years. There is a worldwide system in which all diagnostic information from solid tumors is based on a few tumors themselves. See, for example, De Petris, Proteome Science, 8:9 (2010) ("De Petris"). In De Petris, only "biopsies from areas representative of the tumor" were taken for further testing, and hemolysis and non-fixation requirements were made such that the samples were unsuitable for follow-up histological testing.

[0011] The discovery of tumor heterogeneity runs counter to the conventional theory of tumorigenesis. In the 1950s, the common belief was that the final, clinically detectable tumor was the product of successive selection of a specific subpopulation of the tumor "stem line." In this theory, most of the tumor is dominated by a single "stem line" that is superior to other "stem lines" due to natural selection. As the concept of the tumor "stem line," the TNM staging system, in which most tumors are composed of a single "type" of tumor, was developed during the same period. See Peter Nowel, Science (1976) 194:23-28.

[0012] Rather than being homogeneous in composition, solid tumors are in fact heterogeneous. Some solid tumors have been reported to be composed of multiple genetically distinct, spatially separated populations of cancer cells. See Gerlinger et al., NEJM (2012) 366:883-92; and Yachida et al., nature (2010) 467(7319):1114-1117. As further described herein, the inventors have shown that conventional sampling methods for histological analysis of tumors provide an inadequate sample of the tumor (or tissue potentially containing cancer cells such as lymph nodes) and can be improved.

[0013] Physicians and scientists guided by modern pathology usually do not assign any meaningful medical value to the remaining tumor tissue once a sample has been taken for the TNM staging system and thus destroy it. Morphological analysis of tissue sections and biomarker-based analysis are also limited by the phenotypic, morphological, and genetic heterogeneity exhibited by malignant populations within or between tumors. Small populations of tumor cells may include the major sources of malignant tumors and metastases and may constitute a non-significant amount of the genetic makeup of the entire tumor. As a result, biomarkers developed for large tumor masses often cannot capture the very reason for cancer mortality caused by a small percentage of tumors.

[0014] The heterogeneous gene structure within a given tumor poses a major challenge for therapy decisions in diagnostic oncology that use information taken from a small number of tumors under the assumption that tumors are composed of cells with a uniform composition. For example, the spatial location of gene subtypes within a sample can greatly affect clinical outcomes. Traditional routine tissue section procedures cannot sample the entire surgical specimen, even if they are based on selective sampling guided by recognized literature. Furthermore, current sampling procedures can only examine a small number of tumors, leaving much of the tumor unexamined and ultimately destroyed.

[0015] Under procedures approved by AJCC, UICC, and CAP, pathologists use only a small surgically excised tissue specimen and discard the remaining sample. Since tumor samples can carry multiple genetically and spatially distinct populations of tumor cells, even the most experienced pathologists cannot have the 3 - 5 small samples they collect represent all the genetically diverse groups of malignancies or metastases within the entire specimen. Furthermore, since the DNA sequences of tumor cells are not revealed upon gross examination, the location of any genomically distinct population of cancer cells cannot be known presumptively. In fact, it is the remaining tumor material that is discarded, which contains most of the cellular, genomic, and proteomic heterogeneity within the primary tumor, yet there is no time - and cost - effective method or instrument that enables whole - tumor sampling.

[0016] Current pathological practice requires more thorough sampling and processing methods for the whole tumor that can help ensure that cellular, genomic, and proteomic heterogeneity within the entire primary tumor is captured in the diagnostic sample. SUMMARY OF THE INVENTION

[0017] The present disclosure provides a processed homogenate composition derived from a heterogeneous tissue sample that includes a substantially uniformly distributed cellular structure, wherein the ratio of the cellular structure in each subset of the homogenate is substantially similar to the ratio of the cellular structure in the original heterogeneous tissue sample. The homogenate composition is a new, unique tissue sample that represents an important feature of the original heterogeneous tissue sample. The disclosed compositions address and overcome the limitations of prior art methods that cannot account for tissue heterogeneity in clinical samples, particularly samples for use in clinical oncology.

[0018] The homogenate composition is derived from or originates from various tissues, organs or samples thereof, such as lymph nodes, metastases, polyps, cysts, excisions, organs, or fractions thereof, or spatially separated cells. In one aspect, the homogenate comprises from about 25% to about 100% of the cellular structure of the tissue sample. The homogenate may contain protein fractions, lipids, nucleic acids or other moieties present in the starting tissue, such as the whole tumor, lymph node or metastasis from which the homogenate is derived, and the relative proportions of such replacement components are representative of the starting tissue. The substantially uniform cellular structure may include a single cell or a plurality of cell clusters isolated from normal or abnormal tissue. In certain embodiments, the homogenate may include liquid or non-liquid tissue samples obtained by various methods such as fine needle aspiration for cytodiagnosis, effusion samples or cervical cytology.

[0019] In other aspects, the homogenate can be isolated from preserved tissue, such as formalin-fixed tissue. In other aspects, the tissue sample is not preserved or fixed and / or contains live cells. The heterogeneous tissue sample can be isolated from one or more tissues obtained from one or more patients.

[0020] The homogenate is suitable for use in a variety of diagnostic, prognostic, and clinical applications, including but not limited to the generation of representative data, including representative oncology data, cancer stage classification, identification and evaluation of disease prognosis (e.g., tumor stage classification), selection and design of appropriate treatment regimens, clinical trial matching, identification and characterization of markers, tissue profiling, and preservation of the homogenate composition. Since the representative sample contains a variety of antigens expressed by a given tumor (one or more), the homogenate composition is also useful for screening therapeutic agents or for generating therapeutic agents in the treatment of patients, for example, as a vaccine, or in the manufacture of cancer vaccines or immune cell-based therapeutic agents.

[0021] The present disclosure also provides a homogenate composition representative of a heterogeneous tissue sample, for example, a method for generating a representative sample. More specifically, the present disclosure increases the likelihood of detection for small subclone populations and / or low incidence events by providing an accurate representative sample, despite the spatial heterogeneity within the original tissue or organ, for example, within a tumor. This is achieved by applying mechanical, chemical, and / or biochemical, for example, enzymatic dissociation methods to intact tissue-containing samples, such as whole organs, tumors, lymph nodes, metastatic tissue, or any combination thereof (from the same or different patients).

[0022] In one aspect, the present disclosure generally relates to the development of methods for generating representative tissue samples of, for example, whole organs, tumors, lymph nodes, metastases, or combinations thereof, to address the problem of heterogeneity, such as tumor heterogeneity, in clinical specimens, particularly clinical specimens for use in clinical oncology, and to the use of such representative samples or portions thereof in various diagnostic and therapeutic methods, as well as to compositions containing such representative samples for use in diagnosis and treatment, particularly in oncology.

[0023] Furthermore, representative samples derived from different patients or different tissues of a single or different patients can be labeled respectively with unique identification labels, such as haptens, and the labeled samples or tissues of different patients can be combined and used in a desired assay method.

[0024] Using representative samples derived from exemplary embodiments of the present disclosure, the accuracy of detecting, diagnosing, and / or staging different tumor types can be improved regardless of tumor histotype, location, size, or volume. The methods disclosed herein are useful for the production of representative samples from normal tissue or putative pre-cancerous tissue (e.g., obtained from subjects at high risk of developing cancer due to genetic risk or previous cancer) for the identification of rare cells.

[0025] In one aspect, the present disclosure provides a method for producing a biological sample suitable for evaluating the heterogeneity of cells within a tumor, lymph node, or metastasis, and / or for evaluating the prognosis of a particular cancerous state in a subject, and / or for determining an appropriate treatment protocol for a subject having a cancerous state. The method includes (i) obtaining a tissue (such as a tumor sample, lymph node, or metastasis) that includes spatially distinct regions of the tissue or the entire tumor or a substantial portion thereof, and (ii) homogenizing the tissue such that the cell heterogeneity is substantially evenly distributed within the resulting homogenate or a portion or fraction thereof. The sample(s) can be optionally fixed and / or preserved, e.g., formalin-fixed, ethanol-fixed, frozen, or freeze-dried, stored in wax (such as paraffin), before or after homogenization of the entire or substantially the entire tumor, lymph node, metastasis, or even an entire organ such as the kidney.

[0026] In another aspect, the present disclosure provides a method for producing a biological sample suitable for assessing the heterogeneity of cells within a sample (such as a tumor sample, lymph node, metastasis, or a combination thereof) and / or for assessing the prognosis of a specific cancer condition in a subject, the method comprising: (i) obtaining one or more intact samples, preferably each intact sample containing at least about 100-200; 200-1,000; 1,000-5,000; 10,000-100,000; 100,000-1,000,000; 1,000,000-5,000,000; 5,000,000-1,000,000,000; 1,000,000,000-5,000,000,0000 cells, or alternatively, at least 1,000; 10,000; 100,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000; 1,000,000,000; 5,000,000,000; 10,000,000,000; 50,000,000,000; 100,000,000,000; 500,000,000,000; 1,000,000,000,000; 5,000,000,000,000; 10,000,000,000,000; 50,000,000,000,000; 100,000,000,000,000 cells, optionally fixed or preserved (such as a sample fixed or preserved with formalin, paraffin, or ethanol), from a solid tumor or lymph node, and (ii) homogenizing one or more samples, separately or together, such that each homogenate substantially represents the heterogeneity of the corresponding sample(s). In one embodiment, the intact sample(s) derived from the solid tumor or lymph node comprise, consist essentially of, or consist of a part of the solid tumor or lymph node. In another embodiment, the intact sample(s) derived from the solid tumor or lymph node comprise, consist essentially of, or consist of substantially a part of the solid tumor or lymph node.In a further embodiment, the intact sample(s) derived from the solid tumor or lymph node comprises, consists essentially of, or consists of the whole solid tumor or whole lymph node.

[0027] Optionally, the representative sample can be further dissociated and / or processed to remove or isolate specific types of molecules, such as specific cell types, proteins, nucleic acids, or lipids, using, for example, CAVA computer analysis of Illumina sequencing output used in diagnostic and therapeutic methods.

[0028] In yet another aspect, the present disclosure provides a method for producing a biological sample suitable for evaluating the heterogeneity of cells within a tumor or lymph node or metastasis or a combination thereof, the method comprising: (i) obtaining one or more biopsy samples from a solid tumor or lymph node or metastasis, preferably wherein each biopsy sample contains at least about 100 - 200; 200 - 1,000; 1,000 - 5,000; 10,000 - 100,000; 100,000 - 1,000,000; 1,000,000 - 5,000,000; 5,000,000 - 1,000,000,000; 1,000,000,000 - 5,000,000,0000 cells, or alternatively, at least 1,000; 10,000; 100,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000; 1,000,000,000; 5,000,000,000; 10,000,000,000; 50,000,000,000; 100,000,000,000; 500,000,000,000; 1,000,000,000,000; 5,000,000,000,000; 10,000,000,000,000; 50,000,000,000,000; 100,000,000,000,000 cells, and optionally, the sample is fixed or preserved (such as a sample fixed or preserved with formalin, paraffin, or ethanol), and (ii) homogenizing the one or more biopsy samples separately or together under conditions such that the resulting homogenate(s) is substantially dissociated into individual cells and the resulting homogenate(s) is substantially homogeneous.

[0029] In another aspect, the present disclosure provides a method for producing a biological sample suitable for assessing whether a subject has or is at risk of developing a particular cancer of a pathogenic type and / or whether the subject has a cancer of a pathogenic type, the method comprising: (i) obtaining, preferably, one or more intact biopsy samples from a solid tumor or lymph node or metastasis or pre-cancerous cyst, wherein each biopsy sample contains at least about 100-200; 200-1,000; 1,000-5,000; 10,000-100,000; 100,000-1,000,000; 1,000,000-5,000,000; 5,000,000-1,000,000,000; 1,000,000,000-5,000,000,0000 cells, or alternatively, at least 1,000; 10,000; 100,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000; 1,000,000,000; 5,000,000,000; 10,000,000,000; 50,000,000,000; 100,000,000,000; 500,000,000,000; 1,000,000,000,000; 5,000,000,000,000; 10,000,000,000,000; 50,000,000,000,000; 100,000,000,000,000 cells, and optionally, fixed or preserved (such as a sample fixed or preserved with formalin, paraffin, or ethanol), and (ii) homogenizing the one or more biopsy samples, separately or together, such that each resulting homogenate contains substantially uniformly the heterogeneity of the corresponding biopsy sample(s), and optionally, isolating or detecting the presence of at least one biomarker. In this aspect, the presence or absence of the biomarker indicates a cancer of a pathogenic type, or alternatively, upregulation or downregulation of the biomarker is associated with a particular cancer of a pathogenic type.

[0030] In yet another aspect, the present disclosure provides a method for characterizing the situation within a heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst and / or detecting genetically distinct subclones within a heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst and / or identifying low-incidence events within a tumor, lymph node, metastasis, or pre-cancerous cyst and / or determining the incidence rate of a target within a tumor, lymph node, metastasis, or pre-cancerous cyst, the method comprising: (i) obtaining a sample (s) of a tumor, lymph node, metastasis, or pre-cancerous cyst that includes spatially distinct regions of the tumor, lymph node, metastasis, or pre-cancerous cyst, optionally fixed or preserved prior to homogenization, e.g., using formalin, paraffin, and / or ethanol; and (ii) producing a set of homogenates that are representative of the heterogeneous situation within the tumor, lymph node, metastasis, or pre-cancerous cyst and are suitable for characterizing the situation of the tumor and / or detecting genetically distinct subclones within a heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst and / or identifying low-incidence events within a tumor, lymph node, metastasis, or pre-cancerous cyst and / or determining the incidence rate of a target within a tumor, lymph node, metastasis, or pre-cancerous cyst by separately homogenizing the sample (s) of the tumor, lymph node, metastasis, or pre-cancerous cyst. These situations are related to genomic diversity (e.g., the number of point mutations, insertions, and deletions within the tumor), tumor phenotypic diversity (e.g., the amount of tumor that has undergone epithelial-to-mesenchymal transition), host immune response diversity (e.g., the diversity of expression of immune checkpoint regulators in tumors and immune cells), diversity of any potential resistance mechanisms (e.g., the number and diversity of tumor mutations conferring resistance to targeted therapy), tissue structure diversity (e.g., the amount of squamous cell carcinoma and adenocarcinoma tumors in lung cancer), diversity of neoantigens expressed by the tumor, and / or other complex phenotypic, morphological, histological, genomic, proteomic, metabolomic situations among all diseased or potentially diseased tissues excised from the subject.

[0031] In yet another aspect, the present disclosure provides a method for detecting pre-cancerous or cancerous cells in putative normal or putative pre-cancerous tissue in a patient, such as a patient at risk of developing cancer due to a genetic mutation or previous cancer, or a patient having a pre-cancerous cyst or polyp, the method comprising: (i) obtaining a sample(s) of putative normal or putative pre-cancerous tissue, such as a pre-cancerous cyst or polyp, comprising spatially distinct regions of the patient's putative normal or putative pre-cancerous tissue, optionally fixed or preserved prior to homogenization; and (ii) producing a homogenate representative of the putative normal or putative pre-cancerous tissue by homogenizing the sample(s), the homogenate being suitable for detecting rare cancerous cells or cancer stem cells, for example, even before the signs of the disease appear in the patient.

[0032] In another aspect, the present disclosure provides a method of using a representative sample and a portion thereof produced by any of the methods described above in different assay formats, which assays can be performed in high-throughput, simultaneously, or at different times or different locations, and / or by automation (fully automated or semi-automated).

[0033] In yet another aspect, the present disclosure provides a representative sample or a portion thereof produced by any of the methods described above, preserved for future use, non-limiting examples of such preservation including, for example, freezing, formalin fixation, paraffin embedding, treatment with ethanol, or lyophilization.

[0034] In yet another aspect, the present disclosure provides a representative sample or a portion thereof produced by any of the methods described above, which is used to induce (optionally, purify) an antibody or antigen specific for a particular antigen derived from a cancer cell or cell type in a patient sample, the antibody or antigen potentially being useful in the production of personalized medicine, i.e., a therapeutic or prophylactic cancer vaccine.

[0035] The homogenization step in all of the above methods can be carried out by a method that preserves the integrity of the cells in the sample, i.e., the majority of the cells in the homogenized sample(s) are not lysed, whereby the resulting homogenate and portions thereof "represent" the sample(s). Thus, the cells in the sample or a portion thereof reflect the percentages of the different cell types in the whole tissue sample(s), e.g., in a solid tumor or lymph node. This can be achieved, for example, by mechanical dissociation of the tumor sample or a portion thereof (such as mechanical dissociation carried out with or without 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 (such as treatment with an enzyme that acts selectively or preferentially or mainly on extracellular matrix proteins as compared to membrane-bound proteins). Alternatively, the homogenization method can result in dissociation of cells while still generating a sample that represents the starting tissue, such as an entire tumor. The homogenized representative sample can optionally be further dissociated and / or processed to remove or isolate specific types of molecules, such as specific cell types, proteins, nucleic acids, or lipids, to generate other representative samples that can be used in diagnostic and therapeutic methods.

[0036] Any of the above methods may further comprise detecting the expression of at least one biomarker, e.g., at least one lipid, protein, or nucleic acid biomarker, in the homogenate or a portion or fraction thereof. Further, the method may further comprise detecting the percentage of tumor cells in the homogenate or a portion or fraction thereof that express a specific biomarker or combination of biomarkers. Optionally, the relative frequency or percentage of tumor stem cells and / or tumor subclones in the homogenate or a portion or fraction thereof is detected and / or isolated. Further, the method may comprise detecting genetic targets (such as point mutations, deletions, insertions, translocations, gene fusions, or gene amplifications).

[0037] Using any of the above methods, it is also possible to detect, isolate, and / or quantify specific immune cells (such as B lymphocytes, T lymphocytes, macrophages, NK cells, monocytes, or combinations thereof, etc.) present in a homogenate or a part or fraction thereof that provides useful clinical information, such as immune status and disease status, for selecting a suitable treatment protocol, such as a checkpoint inhibitor, cytokine, or other immunomodulator.

[0038] The resulting homogenate or representative sample can consist essentially of, or further consist of, about 100 - 200; 200 - 1,000; 1,000 - 5,000; 10,000 - 100,000; 100,000 - 1,000,000; 1,000,000 - 5,000,000; 5,000,000 - 1,000,000,000; 1,000,000,000 - 5,000,000,0000 cells, or alternatively, at least 1,000; 10,000; 100,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000; 1,000,000,000; 5,000,000,000; 10,000,000,000; 50,000,000,000; 100,000,000,000; 500,000,000,000; 1,000,000,000,000; 5,000,000,000,000; 10,000,000,000,000; 50,000,000,000,000; 100,000,000,000,000 cells.

[0039] The resulting homogenate or its fraction or part can optionally be frozen or lyophilized, embedded in wax (such as paraffin), or used in further steps without such freezing or lyophilization or wax. For example, a representative paraffin block, i.e., one produced from a homogenate or its fraction or part embedded in paraffin, is suitable for use in current anatomical pathology workflows, such as sectioning, slide preparation, staining, microscopic observation, antigen retrieval, etc.

[0040] The homogenate may be derived from two or more tumors taken from one or more subjects (e.g., the same subject before and after treatment or multiple subjects before and after the same or different treatments), and the resulting homogenate or fraction thereof from each tumor is used to assess the similarity and / or difference between two or more tumors or disease states of different patients. In a further aspect, homogenates from one or more subjects can be combined for a representative sample of multiple subjects.

[0041] The homogenate may be derived from two or more putative normal or pre-cancerous tissues, e.g., breast, cervical, colorectal, or pre-cancerous cysts or polyps, obtained from a subject (one or more), e.g., a person having a BRCA mutation, and the resulting homogenate or fraction thereof is used to assess whether abnormal cells or disease biomarkers are present.

[0042] Furthermore, non-human cells (such as insect cells and / or mouse cells) or other foreign proteins, nucleic acids, or small molecules can be added to the homogenate to create an internal control for positive protein or nucleic acid detection.

[0043] Small molecules (such as haptens, peptide tags, protein tags, fluorescent tags, and / or nucleic acid tags) can be added to the sample and used to provide spatial information in the representative sample. For example, the sample (tumor or lymph node) can be sectioned, e.g., cut into quadrants, and different haptens (or other suitable small molecules) are "doped" into each section, after which the sections are homogenized to generate a representative sample. The number of sections that can be generated from each sample for "doping" prior to homogenization is not limited, but rather appears to be selected to be commensurate with the size of the sample, i.e., the larger the sample, the greater the number of sections that can be "tagged" with small molecules prior to homogenization. In this way, spatial information can be maintained in the resulting homogenate or fraction thereof.

[0044] In one embodiment, after adding a small molecule to a sample, it is also possible to provide a means for distinguishing the sample when the sample is combined with another sample from a different patient or the same patient and thus run in a multiplex assay format.

[0045] The sample to be homogenized can be stored before or after homogenization, for example, fixed with formalin or treated with ethanol. For safety reasons, tissue samples are generally fixed with formalin or otherwise prior to the processing step of using CAVA computer analysis of Illumina sequencing output in a pathology laboratory prior to use in many diagnostic methods. Formalin or other fixation methods can be achieved by techniques generally known in the art. In such cases, the formalin-fixed tumor sample may be immersed in water or buffered saline solution (such as PBS) prior to homogenization in step (ii).

[0046] Alternatively, or additionally, the tumor sample used in the disclosed method may be stored in ethanol prior to homogenization. However, formalin fixation, methanol or ethanol fixation, or other storage procedures are not essential for the method in question and can be eliminated without compromising the suitability of the resulting homogenized representative sample.

[0047] Representative biological samples can be produced using homogenization of non-fixed tissues. The biological representative samples can be cultured to generate representative tissue culture samples from individual patients. Such representative samples can be divided multiple times to generate multiple representative culture samples, which can be used to determine the effectiveness of chemotherapy (such as antibodies, nucleic acids, small molecules, or polypeptides that antagonize, inhibit, or block the expression or functional activity of at least one known or unknown biomarker). Additionally, specific cell types (such as immune cells or tumor cells) can be selected using FACS analysis. For example, immune cells infiltrating a tumor can be selected and cultured to determine tumor-specific antibodies secreted by the immune system.

[0048] Also, as disclosed herein, the disclosed methods for producing representative samples and their use in diagnostic and therapeutic methods are suitable for both fixed and non-fixed tissue samples.

[0049] Any of the disclosed methods for preparing representative samples may include the addition of at least one collagenase (or other suitable enzyme or combination of enzymes or other chemical substances such as salts that autolyze or facilitate the breakdown of the extracellular matrix) before, during, or after homogenization; the use of elevated temperature and / or buffer conditions (such as cell conditioning buffers, e.g., CC1 or CC2 that disrupt cell crosslinking); and / or the use of devices for mechanical shearing (such as an IKA blender, gentleMACs Disassociator, or functional equivalent). Again, these methods may or may not be performed under conditions that maintain the viability and integrity of the cells within the sample, such as some homogenization conditions under which the cells are not substantially lysed.

[0050] In one aspect, the homogenization process includes the use of mechanical processes, non-limiting examples of such being a mortar and pestle, a Dounce-type homogenizer or tissue grinder, a hand-held electric rotary blade tissue homogenizer (such as the Omni-TH available from Thomas Scientific), a bead-type homogenizer (such as the Bullet Blender available from OMNI or the Burton Precellys 24 Tissue Homogenizer or Bead Ruptor), and optionally, for a rotary homogenizer, at a speed of about 100 - about 75,000 RPM or for a bead beater at a speed of about 0.5 m / s - about 2.5 m / s, for a length of about 30 seconds - about 5 minutes, about 5 minutes - about 10 minutes, about 10 minutes - about 30 minutes, or about 30 minutes - about 60 minutes. As described herein, the mechanical homogenization process can be used alone or in combination with other processes.

[0051] In another embodiment, homogenization involves the use of enzyme preparations, alone or in combination with other processes, non-limiting examples of which include, for example, optionally, 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, pyroglutamic acid aminopeptidase, or any combination thereof, at a concentration of about 0.001 μg / ml to about 1000 mg / ml and for a length of about 1 minute to about 120 minutes.

[0052] The tumor or other sample used in the disclosed methods that encompasses spatially distinct regions of a tumor or other tissue may include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or preferably, all of a surgically removed tumor or tissue sample from a patient. The tumor sample may have a diameter of at least 1, 5, 10, 20, 50, 100 millimeters (mm) or centimeters (cm) or more.

[0053] The samples used in the methods of interest are generally from any suitable tissue sample, e.g., solid tumor(s) (including primary and metastatic tumors), lymph node, metastasis, or pre-cancerous tissue such as cyst or polyp. Alternatively, or additionally, the methods may be effective for non-solid tumors, e.g., blood cancers. For example, the tissue sample or solid tumor sample to be homogenized may optionally be combined with a liquid patient sample, e.g., blood, lymph, effusion specimen, cerebrospinal fluid, bile, mucus, and / or urine sample derived from the patient. The sample to be homogenized may further, or alternatively, include a complete or partial sample, e.g., biopsied "normal" or pre-cancerous tissue, for example, to detect disease cells prior to disease symptoms.

[0054] Such tumor(s) or other tissue sample(s) used in the disclosed methods may be from any source, e.g., breast, colon, lung, pancreas, gallbladder, skin, bone, muscle, liver, kidney, neck, ovary, prostate, esophagus, stomach, or other organ, e.g., breast cancer tumor, lung cancer tumor, liver cancer tumor, prostate cancer tumor, colon cancer tumor, bladder cancer tumor, or kidney cancer tumor. In one embodiment, the tumor sample or other tissue used in the disclosed methods is of human origin, but may be from any suitable tissue source.

[0055] The tumor or other tissue sample used in the disclosed methods is at least 1 cm 3 , at least 2 cm 3 , at least 3 cm 3 , at least 4 cm 3 , at least 5 cm 3 , at least 6 cm 3 , at least 7 cm 3 , at least 8 cm 3 , at least 9 cm 3 , at least 10 cm 3 , at least 15 cm 3 , at least 20 cm 3 , at least 25 cm 3 , at least 50 cm 3 , at least 100 cm3 、 at least 250 cm 3 、 at least 500 cm 3 、 at least 1,000 cm 3 、 at least 2,500 cm 3 、 at least 5,000 cm 3 、 at least 7,500 cm 3 、 at least 10,000 cm 3 may have a volume of 15 or more.

[0056] The tumor or other tissue sample used in the disclosed method may have a maximum width of at least 0.5 cm, at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, at least 5 cm, at least 6 cm, at least 7 cm, at least 10 cm, at least 25 cm, at least 50 cm or more.

[0057] In a further embodiment, the representative sample may be made from tissue that has been pre-fixed in formalin and embedded in paraffin wax. In particular, after melting the wax, recovering the tissue, and hydrating it, the methods described herein, i.e., homogenization, can be applied to a sample suitable for use in any number of assays. Thus, by using the disclosed method to appropriately melt the wax, recover the sample, rehydrate the tissue, and homogenize it, a representative sample can be generated using the sample(s) already prepared for TNM stage classification.

[0058] Each of the above methods may further include (iii) dispensing the homogenate or a part or fraction thereof onto one or more slides or other solid supports, and optionally, staining one or more slides or other solid supports containing the homogenate or a part or fraction thereof with hematoxylin and eosin dyes; performing immunohistochemical staining on the slide or other solid support containing the homogenate or a part or fraction thereof; or performing in situ hybridization on the slide or other solid support containing the homogenate or a part or fraction thereof, that is, any one of these is considered to be step (iv) in the above method. For example, the homogenate or a part thereof can be analyzed on an automated platform for analysis. Such platforms are known in the art and are commercially available from Ventana Medical Systems, Inc. (see Ventana.com for exemplary automated platforms).

[0059] Furthermore, each of the above methods may further include (iii) purifying nucleic acids (such as DNA or mRNA) from the homogenate or a part or fraction thereof. The purified nucleic acids can be subjected to Northern blot, DNA sequencing, PCR, RT-PCR, microarray profiling, differential display, or in situ hybridization. Alternatively, the purified nucleic acids can be conjugated to nanoparticles (quantum dots, paramagnetic nanoparticles, superparamagnetic nanoparticles, and metal nanoparticles, preferably, by way of example and not limitation, alloy quantum dots including, for example, CdSe, ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN, etc.).

[0060] It is also contemplated that any of the above methods may further include purifying lipids or exosomes or other organelles from the homogenate or a part or fraction thereof. The purified lipids can be subjected to mass spectrometry or histochemical analysis.

[0061] Furthermore, it is also contemplated that any of the above methods may further include purifying proteins from the homogenate or a part or fraction thereof. The purified proteins can be subjected to Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, chromatography, mass spectrometry, microarray profiling, interferometric spectroscopy, electrophoretic staining, or immunohistochemical staining. Alternatively, or in addition to the above, the purified proteins can be used to produce antiserum specific to a tumor or tissue sample.

[0062] Furthermore, it is contemplated that any of the above methods may further include performing genomic, epigenomic, transcriptomic, proteomic, and / or metabolomic analysis on (iii) the homogenate or a part or fraction thereof.

[0063] Furthermore, each of the above methods further comprises (iii) affinity purifying a specific cell type from the homogenate or a part or fraction thereof. The specific cell type may contain the biomarker of interest.Exemplary biomarkers of interest include Her2, bRaf, ERBB2 amplification, P13KCA mutation, FGFR2 amplification, p53 mutation, BRCA mutation, CCND1 amplification, MAP2K4 mutation, ATR mutation, or any other biomarker whose expression correlates with a particular cancer; AFP, ALK, BCR-ABL, BRCA1 / BRCA2, BRAF, V600E, Ca-125, CA19.9, EGFR, Her-2, KIT, PSA, S100, KRAS, ER / Pr, UGT1A1, CD30, CD20, F at least one of 1P1L1-PDGRFa, PDGFR, TMPT, and TMPRSS2; or ABCB5, AFP-L3, alpha-fetoprotein, alpha-methylacyl-CoA racemase, BRCA1, BRCA2, CA15-3, CA242, CA27-29, CA-125, CA15-3, CA19-9, calcitonin, carcinoembryonic antigen, carcinoembryonic antigen peptide-1, des-gamma carboxyprothrombin, desmin, early prostate cancer antigen-2, estrogen receptor, fibrinogen degradation products, glucose-6-phosphate isomerase, HPV antigens such as E6, E7, L1, L2 or p16INK4a human chorionic gonadotropin, IL-6, keratin 19, lactate dehydrogenase, leucyl aminopeptidase, lipotropin, methamphetamine, neprilysin, NMP22, normethamphetamine, PCA3, prostate-specific antigen, prostatic acid phosphatase, synaptophysin, thyroglobulin, TNF, ERG, ETV1 (ER81), FLI1, ETS1, ETS2, ELK1, ETV6 (TE L1), ETV7 (TEL2), GABPa, ELF1, ETV4 (E1AF; PEA3), ETV5 (ERM), ERF, PEA3 / E1AF, PU.1, ESE1 / ESX, SAP1 (ELK4), ETV3 (METS), EWS / FLI1, ESE1, ESE2 (ELF5), ESE3, PDEF, NET (ELK3; SAP2), NERF (ELF2), or FEV, a transcription factor selected from tumor-associated glycoprotein 72, c-kit, SCF, pAKT, pc-kit, and vimentin.

[0064] Alternatively, or further, the biomarker of interest may be, but is not limited to, an immune checkpoint inhibitor such as CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, KIR, TIM3, GAL9, GITR, LAG3, VISTA, KIR, 2B4, TRPO2, CD160, CGEN-15049, CHK1, CHK2, A2aR, TL1A, and B-7 family ligands or combinations thereof, or a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof.

[0065] The method of the present disclosure may also include, or may contain, the detection of at least one biomarker associated with acute lymphoblastic leukemia (etv6, am11, cyclophilin b), B-cell lymphoma (Ig idiotype), glioma (E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn), bladder cancer (p21ras), cholangiocarcinoma (p21ras), breast cancer (MUC family, HER2 / neu, c-erbB-2), cervical cancer (p53, p21ras), colon cancer (p21ras, HER2 / neu, c-erbB-2, MUC family), colorectal cancer (colorectal-associated antigen (CRC)-C017-1A / GA733, APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastric cancer (HER2 / neu, c-erbB-2, ga733 glycoprotein), hepatocellular carcinoma (alpha-fetoprotein), Hodgkin lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), melanoma (p5 protein, gp75, tumor fetal antigen, GM2 and GD2 gangliosides, Melan-A / MART-1, cdc27, MAGE-3, p21ras, gp100.sup.Pmel117), myeloma (MUC family, p21ras), non-small cell lung cancer (HER2 / neu, c-erbB-2), nasopharyngeal cancer (Imp-1, EBNA-1), ovarian cancer (MUC family, HER2 / neu, c-erbB-2), prostate cancer (prostate-specific antigen (PSA) and its antigen epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2 / neu, c-erbB-2, ga733 glycoprotein), kidney cancer (HER2 / neu, c-erbB-2), squamous cell carcinoma of the cervix and esophagus (viral products such as human papillomavirus proteins), testicular cancer (NY-ESO-1), and / or T-cell leukemia (HTLV-1 epitope).

[0066] The methods of the present disclosure further include (iii) treating the homogenate or a part or fraction thereof with a collagenase or other enzyme or chemical or a combination thereof that degrades the extracellular matrix, incubating the homogenate or a part or fraction thereof under high temperature conditions, and / or mechanically agitating the homogenate or a part or fraction thereof to dissociate the cells therein. Generally, these methods are expected to generate a population of individual cells or small cell clusters from a representative sample that can be used in the disclosed analytical or therapeutic methods or a combination thereof.

[0067] Furthermore, any of the above methods may further include (iii) filtering or sizing the homogenate or a part or fraction thereof, which can result in the acquisition of single cells or small cell clusters such as doublets or triplets.

[0068] The cellular components of the representative sample can be separated by one or more filtration steps. For example, after homogenization and dissociation of the homogenate by physical and / or biochemical means, the dissociated sample can be filtered through a 1 μm filter paper to remove all intact cell material. The acellular representative sample is expected to contain secreted factors derived from tumors that are clinically useful and normal stroma derived within the tumor, i.e., antibodies, growth factors, immunomodulators, and other unknown factors. The acellular representative sample can be analyzed by ELISA, mass spectrometry, next-generation sequencing, and other diagnostic methods. To the extent that single cells derived from the representative sample are obtained after filtration, such cells can be analyzed using fluorescence-activated cell sorting (FACS) and flow cytometry analysis.

[0069] Considering the representative properties of the homogenate generated by the disclosed method, the homogenate or a part or fraction thereof can be used to detect low-occurrence genetic events (genetic events occurring at an occurrence rate of 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.001%, 0.0001%, 0.00001%, an occurrence rate of 0.000001% or less, etc.). Exemplary genetic events include point mutations, deletions, insertions, translocations, gene fusions, or gene amplifications. Similarly, the method may also include detecting genetic or epigenetic heterogeneity in a tumor sample or a part thereof, and / or detecting cells containing rare genetic or epigenetic changes. Such cells may be present in the tumor sample at a frequency of less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01%.

[0070] The detected rare cells may contain one or more genetic or epigenetic differences that confer resistance to therapy, sensitivity to a particular therapy over another anticancer therapy, and / or promote metastasis. Thus, in one aspect, the detection of such cells facilitates the prognostic diagnosis of cancer and the selection of an appropriate treatment regimen such as chemotherapy, combinatorial targeted therapy, etc., and / or the use of biologics.

[0071] The method also includes acridines and derivatives such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin, coumarin and derivatives such as 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanosin; 4',6-diamidin-2-phenylindole (DAPI); 5',5''-dibromopyrogallol-sulfonphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaminepentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin B and derivatives such as erythrosin and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); 2',7'-difluorofluorescein (OREGON GREEN (registered trademark)); fluorescamine; IR144; IR1446; malachite green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline;Phenol Red; B-Phycoerythrin; o-Phthalaldehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron.RTM. Brilliant Red 3B-A); 6-Carboxy-X-Rhodamine (ROX), 6-Carboxyrhodamine (R6G), Lissamine Rhodamine B Sulfonyl Chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Isothiocyanate, Rhodamine Green, Sulfhorhodamine B, Sulfhorhodamine 101 and sulfonyl chloride derivatives of sulfhorhodamine 101 (Texas Red) and other rhodamine and derivatives; N,N,N’,N’-Tetramethyl-6-carboxyrhodamine (TAMRA); Tetramethylrhodamine; Tetramethylrhodamine Isothiocyanate (TRITC); Riboflavin; Rose bengal and terbium chelate derivatives, and may include the use of at least one detectable label selected from fluorescent molecules or fluorescent dyes such as a thiol-reactive europium chelate that emits at about 617 nm.;

[0072] The disclosed method can be automated in whole or in part. For example, steps (i) and (ii) can be automated, but subsequent steps, such as steps (iii) and (iv), are manual. Alternatively, by way of example, steps (i) and (ii) may be manual, but subsequent steps, such as steps (iii) and (iv), are automated. Further, all steps encompassed by the method can be automated such that the method is fully automated.

[0073] For tumor stage classification, the disclosed method can be used alone or in combination with other known methods (such as TNM). In one aspect, the method further comprises evaluating the degree to which tumor cells have spread to the lymph nodes to which they belong by analyzing one or more aspects of a representative sample of the tumor and a representative sample of the excised lymph nodes, to predict the likelihood of disease recurrence and / or progression.

[0074] The disclosed method may further include using an algorithm to calculate the percentage of sampled cells, such as tumor cells, with or without using specific biomarkers. The relative risk of metastatic (or malignant subclone) progression can be determined based on the percentage of cells in a representative tumor sample and / or a representative lymph node sample using specific detectable biomarkers or combinations of biomarkers.

[0075] The disclosed method may further include the development of individualized dosages or treatment regimens based on biomarker profiles, antigen profiles, mutation profiles, lipid profiles, protein profiles, and / or exosome profiles contained in representative samples. For example, based on the information contained in the representative sample or in combination with information obtained from a representative lymph node sample, the selection of one or more drugs to be administered to a patient and / or the dosage (amount, length of administration, etc.) of such drugs can be modified to individualize treatment based on the patient's individual tissue or cancer profile.

[0076] The disclosed method may further include comparing the genomic profile of a representative sample with the genomic profile of a representative tissue sample from the sample patient or another patient, such as a lymph node sample, and further optionally comparing these profiles with circulating tumor DNA from a distant metastasis or a representative metastatic tumor sample.

[0077] The disclosed method may further include the development of inclusion criteria for clinical trials based on biomarker profiles, antigen profiles, mutation profiles, lipid profiles, protein profiles, and / or exosome profiles contained in representative samples.

[0078] The present disclosure also encompasses representative homogenate compositions produced by any of the foregoing methods, alone or in combination with other compositions and carriers.

[0079] Furthermore, the results of said method (such as the detection of rare genetic and / or epigenetic events, rare cells, etc.) or the compositions produced by any of said methods, including the homogenization of samples such as tumor samples, to prepare representative samples suitable for further analysis using any number of standard diagnostic assays, can be used in the selection of an appropriate treatment regimen for a cancer patient. The treatment regimen may include gene therapy, chemotherapy, targeted small molecules, other targeted therapies, immunomodulator administration, radiation, cytokine administration, surgery, or combinations thereof.

[0080] Furthermore, using the disclosed method, at least one therapeutic agent (such as gene therapy (e.g., CRISPR), T cell therapy (e.g., CAR T cells), antibodies, nucleic acids, small molecules, or polypeptides that antagonize, inhibit, or block the expression or functional activity of at least one detected biomarker) can be selected that is suitable for use in a subject that was the source for a representative sample generated by a method by which a sample or tumor was provided.

[0081] In a further aspect, the present disclosure relates to a method for preparing a representative sample for analysis, the method comprising: (1) obtaining a surgically excised tissue sample from at least one subject; and (2) homogenizing the surgically excised tissue sample to obtain a homogenized sample. In one embodiment, at least a portion of the surgically excised tissue sample is fixed. In a further embodiment, the method further comprises processing a first portion of the surgically excised sample to generate one or more fixed, embedded tissue blocks, and further homogenizing a second portion of the remaining surgically excised tissue sample. A portion of the one or more fixed, embedded tissue blocks can be processed by microtomy to produce one or more tissue sections for morphological analysis. Further, at least one of the one or more fixed, embedded tissue blocks can be homogenized. In one aspect, the surgically excised tissue sample comprises one or more separate tissue pieces. In a further embodiment, the one or more separate tissue pieces comprise at least a portion of one or more primary solid tumor tissue masses excised from the subject to obtain the surgically excised sample. In another aspect, the one or more separate tissue pieces comprise at least a portion of one or more lymph nodes excised from the subject.

[0082] In a further embodiment, the method further comprises separately homogenizing at least a portion of the separate tissue pieces to obtain separately homogenized samples. In a further embodiment, the surgically excised tissue sample comprises a single tissue mass that can be further divided into two or more small pieces of the single tissue mass. Further, at least one of the two or more small pieces of the single tissue mass can be homogenized and stored. In one aspect, homogenization may include physical separation such as cutting, chopping, or mincing. In another aspect, homogenization may include mechanical dissociation such as blending or juicing. In yet another aspect, homogenization is achieved by biochemical dissociation, for example, using a protease.

[0083] In a further aspect, one or more biomolecules can be purified from at least a portion of the homogenate, and such biomolecules may include DNA, RNA, proteins, lipids, and metabolites. The biomolecules can then be analyzed, for example, by PCR, mass spectrometry, next-generation sequencing, or ELISA. Such analysis produces at least one dataset.

[0084] In a further embodiment, at least a portion of the homogenized sample can be embedded in paraffin. In a further aspect, the method further includes preparing one or more thin sections of the paraffin-embedded homogenized sample and performing histological analysis on the sample. Such histological analysis may include H&E staining, IHC staining, ISH staining, and FISH staining. The histological analysis can be interpreted by a human or quantified on an automated device. In a further embodiment, the interpretation or quantification produces at least one dataset.

[0085] In one aspect, the present disclosure also relates to further processing at least a portion of the homogenate to generate cell fragments. Such processing steps may include physical, mechanical, chemical, or enzymatic methods. Such cell fragments may include nuclei, cell membranes, and cell organelles. In another aspect, at least a portion of the cell fragments is fixed to at least one slide glass and optionally subjected to histological analysis. Such histological analysis may include H&E staining, IHC staining, ISH staining, or FISH staining. The analysis can be interpreted by a human or quantified by an automated device. The interpretation or quantification results in the generation of at least one dataset.

[0086] In a further aspect, at least a portion of the cell fragments is analyzed by flow cytometry, FACS, or a particle analyzer, and such analysis produces a data set. In one aspect, at least one cell fragment is purified from at least a portion of the cell fragments. Such purification may be performed, for example, by FACS, affinity purification, size exclusion fractionation centrifugation, filtration, or electrophoresis. In another embodiment, a biomolecule can be isolated from at least one cell fragment purified from at least a portion of the cell fragments. The biomolecule can be analyzed by PCR, mass spectrometry, next-generation sequencing, or ELISA. In one aspect, the analysis produces at least one data set.

[0087] In yet another embodiment, the method of the present disclosure further comprises further processing at least a portion of the homogenate, for example, physically, mechanically, chemically, or enzymatically, to generate at least one dissociated cell. The dissociated cells are normal cells, cancer cells, or bacterial cells. In one aspect, the dissociated cells are fixed on at least one slide glass and subjected to histological analysis. Such analysis may include, for example, H&E staining, IHC staining, ISH staining, or FISH staining. In a further embodiment, the analysis is interpreted by a human or quantified by an automated device. In a further embodiment, the interpretation or quantification produces at least one data set. In a further aspect, at least one cell is purified from at least one dissociated cell by means such as FACS, affinity purification, size exclusion fractionation centrifugation, filtration, or electrophoresis. In a further embodiment, a biomolecule can be isolated from at least one cell purified from at least one dissociated cell. In a further aspect, the biomolecule can be analyzed, for example, by PCR, mass spectrometry, next-generation sequencing, or ELISA. In a further embodiment, such analysis produces at least one data set.

[0088] In a further aspect, at least one purified cell from at least one dissociated cell is fixed to at least one slide glass and optionally subjected to histological analysis. In a further embodiment, the histological analysis is H&E staining, IHC staining, ISH staining, or FISH staining. Such analysis can be interpreted by a human or quantified by an automated device. In a further aspect, the analysis or interpretation produces at least one dataset.

[0089] In a further embodiment, the dataset produced by the method disclosed above is further analyzed. In one aspect, the analysis includes determination of biomarker diversity or phenotypic diversity. In a further embodiment, the analysis includes determination of at least one clinical judgment. Such clinical judgment, in one aspect, includes determination of disease prognosis, prediction of disease recurrence, prediction of disease therapy targets, inclusion of subjects in clinical trials, or treatment strategy for at least one subject.

[0090] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication that include the colored drawings will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fees.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0092] It should be understood that the present disclosure is not limited to the specific embodiments described and, as such, may of course vary. Also, since the scope of the present disclosure is limited only by the appended claims, it should be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting.

[0093] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present technology, but the preferred methods, devices, and materials are described herein. All technical and patent publications cited herein are hereby incorporated by reference in their entirety. Nothing in this specification should be construed as an admission that the present technology is not entitled to antedate such disclosure by virtue of prior invention.

[0094] The practice of the present technology uses conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art unless otherwise indicated. For example, Sambrook and Russell (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., N.Y.Series of ); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane (eds.) (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th Edition; Gait (ed.) (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins (eds.) (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins (eds.) (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos (eds.) (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides (ed.) (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker (eds.) (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. (eds.) (1996) Weir’s Handbook of Experimental Immunology. Please refer to.

[0095] All numerical values, including, for example, ranges, pH, temperature, time, concentration, and molecular weight, are (+) or (-) approximations that vary, as appropriate, in increments of 1.0 or 0.1, or alternatively, by fluctuations of ±15%, or alternatively, 10%, or alternatively 5%, or alternatively 2%. Although not always explicitly stated, it should be understood that the term "about" precedes all numerical values. Also, although not always explicitly stated, the reagents described herein are merely exemplary, and it should be understood that equivalents of such are known in the art.

[0096] In the absence of an explicit citation and unless otherwise intended, when the technology relates to polypeptides, proteins, polynucleotides or antibodies, it is presumed that equivalents or biological equivalents of such are intended to be within the scope of the technology.

[0097] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0098] As used herein, the term "animal" refers to living, multicellular vertebrates, including, for example, the categories of mammals and birds. The term "mammal" includes both humans and non-human mammals.

[0099] The terms "subject", "host", "individual", and "patient" are used interchangeably herein and refer to human and veterinary subjects, such as humans, animals, non-human primates, dogs, cats, sheep, mice, horses, and cows. In some embodiments, the subject is a human.

[0100] A "composition" typically contemplates a combination of an active agent, e.g., a compound or composition, with a natural or non-natural, inert (e.g., a detectable agent or label) or active carrier, e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and includes a pharmaceutically acceptable carrier. The carrier also includes proteins, peptides, amino acids, lipids, and carbohydrates (e.g., saccharides including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized saccharides such as alditols, aldonic acids, esterified saccharides, etc.; and polysaccharides or sugar polymers) that are pharmaceutical excipients and additives, which may be present alone or in combination and include 1-99.99% by weight or volume alone or in combination. Exemplary protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / antibody components that can also function in buffering capacity include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. Carbohydrate excipients are also intended to be within the scope of the present technology, and examples thereof include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0102] As used herein, the term "representative sample" refers to a sample (or subset thereof) that accurately reflects the components of an entity, and thus the sample is an unbiased indicator of the entire population. The sample is originally composed of spatially separated cell structures and further derived from solid organs, tissues, and tumors ("OTT") that are organized into spatially separated cell types. The techniques and methods of representative sampling are such that the components of the original spatially stratified OTT (cell structures, cells, peptides, nucleic acids, lipids, metabolites, etc.) are spatially stratified into sub-samples (also known as analytical samples) in the proportions in which they existed in the original organ, tissue, or tumor. It is to sufficiently homogenize, mix, or otherwise disrupt the three-dimensional structure of the OTT. In some embodiments, a representative sample is one that approaches or binds to the entire OTT and represents the diversity of the OTT at the level of clusters of cells, individual cells, cell fragments, organelles, peptides, nucleic acids, lipids, metabolites, etc. It refers to a sample of the OTT that comprises a significantly sufficient portion of the OTT and constitutes as many OTTs as possible. A representative sample may contain the minimum amount of intact OTT required to encompass the diversity of the OTT. In a further embodiment, a representative sample may comprise a plurality of segments or particles, at least some of the particles being embedded in paraffin and at least some of the remaining particles being homogenized.

[0103] Multiple representative samples may be made from a single OTT. In this embodiment, the surgically removed or excised OTT is first processed or otherwise manipulated into separate sub-units such that each sub-unit contains spatially stratified cell structures, cells, peptides, nucleic acids, etc. Each sub-unit is then sufficiently homogenized, mixed, or otherwise disrupted to produce a representative sample of the OTT sub-unit.

[0104] The representative sample can be homogenized, or otherwise mixed or disrupted, to the point where any analytical sample, or a portion of the representative sample, contains a random sampling of the substances present in the representative sample. The analytical sample is a sufficiently large fraction of the representative sample such that it encompasses the diversity of the representative sample (i.e., cells and clusters of associated cells) compared to the intended output of the analytical test to which it is applied. Any analytical sample used for a specific assay produces data that, within experimental error, is consistent with that produced by another analytical sample used for the same assay. Further, any analytical sample selected for a specific assay provides information that can be cross-referenced with data generated using different assays, using analytical samples taken from the same representative sample, or from other representative samples made from OTTs derived from the same patient, different patients, or combinations of patients or subjects. Since the proportions of the original biological components are present in all analytical samples taken from the representative sample, data produced from the analytical samples regarding the proportions of the biological components of the OTT can be compared between patients or combinations of patients.

[0105] Other samples with less diversity than the analytical sample can be taken from the representative sample for analysis, for example, from single cells. However, millions of single cells taken from the representative sample of the OTT generate a "representative dataset" that includes "representative oncological data".

[0106] In one embodiment, the cells or cell components are dissociated within the representative sample such that their relative proportion or percentage within the representative sample or a portion thereof accurately reflects or mimics the relative proportion or percentage of these cell types or components within the whole intact tissue specimen. The specimen may, in one embodiment, be a solid tumor, lymph node, metastasis, polyp, cyst, or a portion thereof or any combination of the foregoing.

[0107] In one embodiment, the representative samples disclosed herein are obtained by homogenizing a large amount of intact tissue or tumor samples (such as clinical tumor samples) obtained from a subject, or lymph nodes or metastases or a combination thereof. For example, an entire tumor or a substantial portion thereof, such as at least 50%, at least 75%, or at least 95%, or preferably all of a tumor or lymph node, can be used as the input material from which the representative sample is generated. Representative samples can be generated from intact tumor biopsy samples derived from solid tumors. In one embodiment, the sample optionally contains at least about 100 - 200; 200 - 1,000; 1,000 - 5,000; 10,000 - 100,000; 100,000 - 1,000,000; 1,000,000 - 5,000,000; 5,000,000 - 1,000,000,000; 1,000,000,000 - 5,000,000,0000 or more cells derived from spatially distinct regions of the tumor. Generally, there are about 1 billion cells in a tumor or a part thereof having a diameter of about 1 cm, and for many parts, this relationship progresses on a linear scale. For example, an excised sample such as a biopsy having a diameter of about 2 cm may contain more than 3 - 5 billion cells. In another embodiment, the representative samples disclosed herein are obtained, for example, by homogenizing one or more normal tissue specimens derived from a subject at risk of developing cancer as a result of a genetic mutation or previous cancer, or adjacent normal tissue derived from a surgical excision for use as a control sample.

[0108] In a further embodiment, the term "representative tumor sample" refers to a representative sample prepared from a tumor, such as an excised tumor, or from a sample potentially containing cancer cells, or from a sample such as a lymph node that is tested for the potential presence of cancer cells. Similarly, the phrase "tumor sample" encompasses a sample prepared from a tumor, or from a sample potentially containing cancer cells, or a sample such as a lymph node that is tested for the potential presence of cancer cells.

[0109] In a further embodiment, the term "representative normal sample" refers to a putative normal tissue, e.g., a representative sample prepared from a biopsy, polyp, or cyst obtained from a patient, or a sample that is tested for the potential presence of cancer or pre-cancerous cells or immune cells that suggest immune abnormalities. Similarly, the phrase "normal sample" encompasses a putative normal tissue, e.g., a biopsy, polyp, or cyst that potentially contains cancer cells, or a sample that is tested for the potential presence of cancer cells, such as a lymph node. In a further embodiment, "normal sample" may refer to a tissue that is likely to be disease-free and against which a tumor sample can be compared to identify phenotypic changes due to the disease state.

[0110] As used herein, the term "representative data" refers to a relatively small amount of data that accurately reflects any set of data (e.g., gene expression, percentage of a particular cell type (e.g., immune cells), protein expression, SNP expression or absence thereof, level, amount, or number of histological subtypes of microRNA expression) or the entire dataset, and the source thereof is derived from a representative sample of a tissue, organ, or tumor. In one embodiment, representative data is unbiased data that shows the overall diversity of a tissue, organ, or tumor. As used herein, a "dataset" is a collection of data. In one embodiment, a dataset is composed of separate elements but can be operated on as a unit. In one embodiment, a dataset may include information regarding biomarker diversity or phenotypic diversity.

[0111] As used herein, the term "histological analysis" refers to the microscopic examination of cells and tissues of plants and animals. This analysis helps to gather information regarding the biological components of a sample, e.g., nucleic acids (RNA, DNA), proteins, lipids, or metabolites. Histological techniques include those known to those of skill in the art and some non-limiting examples include PCR, mass spectrometry, next-generation sequencing, and ELISA. Further, histological analysis may include the simultaneous detection of more than one biological component, i.e., multiplexing.

[0112] As used herein, "clinical judgment" refers to drawing diagnostic conclusions, collecting information to determine what treatment to give a patient, and integrating this information. Such diagnostic conclusions may include the disease the patient is suffering from and what tests should be performed on the patient. In one embodiment, clinical judgment may also include determining disease prognosis, predicting disease recurrence, predicting disease therapy targets, enrolling subjects in a clinical trial, or determining a treatment strategy for at least one subject.

[0113] As used herein, the term "homogenate" refers to the biomass obtained after a tissue has been homogenized or processed. A homogenate may contain any cellular components derived from the tissue, including but not limited to cells, peptides, nucleic acids, lipids, metabolites, etc. In one aspect, a homogenate is a representative sample that accurately reflects a portion, ratio, or fraction of the components of the tissue from which it is derived. In some embodiments, the ratio of the cellular structure, cellular components, or any constituent (such as cells, peptides, nucleic acids, lipids, metabolites, etc.) of a homogenate (or some or each subset of the homogenate) is the same as, similar to, or substantially similar to the ratio of the cellular structure, cellular components, or any constituent in the original intact tissue. Like a representative sample, a homogenate may contain the minimum amount of intact organ, tissue, or tumor required to encompass the diversity of the organ, tissue, or tumor.

[0114] The tissue(s) from which the homogenate is derived may be from one tissue, two tissues, or multiple tissues. In some embodiments, the homogenate is derived from one subject, two subjects, or more than two subjects. In one aspect, two or more subjects are genetically homogeneous subjects. In another aspect, two or more subjects are phenotypically homogeneous subjects. In some aspects, two or more subjects are genetically different subjects. In one aspect, two or more subjects are phenotypically different subjects. In another aspect, two or more subjects are from the same sex. In a further aspect, two or more subjects are from different sexes. In yet another aspect, two or more subjects are from different ethnic groups. In one aspect, two or more subjects are from the same ethnic group. In another aspect, the subject is selected from the group consisting of an animal or a human subject.

[0115] As used herein, the term "substantially" means a high degree in quality or quantity, e.g., at least about 60%, or alternatively, at least about 70%, or alternatively, about 80%, or alternatively, about 85%, or alternatively, about 90%, or alternatively, about 95%, or alternatively, about 98% identity.

[0116] "Similar" means less than 100% identical, or alternatively, more than 98% identical, or alternatively, more than 95% identical, or alternatively, more than 90% identical, or alternatively, more than 85% identical, or alternatively, more than 80% identical, or alternatively, more than 75% identical. The term "homogeneous" means at least 80%, or alternatively, at least 85%, or alternatively, at least 90%, or alternatively, at least 95%, or alternatively, at least 98%, or alternatively, at least 100% identity. Non - homogeneous means less than 80% identity.

[0117] As used herein, the term "processed" means that the homogenate composition has been subjected to at least physical, mechanical, or chemical treatment. In one aspect, the resulting homogenate composition is subjected to more than two types of processing steps. In other aspects, the homogenate composition is subjected to three types of processing steps. In yet another aspect, the homogenate is subjected to four or more types of processing steps.

[0118] The term "derived from" means that the sample was obtained or received from a source.

[0119] The term "subset" means a part or component of a larger group. A "ratio" is a value of the relative amount of the part in relation to the component or part of the larger group.

[0120] As used herein, the terms "cellular structure", "cellular component", or "component" can be used interchangeably to refer to any substance or material within a cell, tissue, or organism, or any substance or material produced during and after a cell, tissue, or organism is processed. The substance or material may be natural or foreign to the cell, tissue, or organism. In some aspects, "cellular structure" and "cellular component" may also include any substance or material that has been modified or processed, such as a cell or nucleic acid containing a dye or radioactive substance. Cellular structures or components include, but are not limited to, cells, receptors, proteins, lipids, cell organelles, membranes, chemicals, nucleic acids, small molecules, bacteria, protozoa, viruses, parasites, and / or parts or fractions thereof. As used herein, "cellular fragment" includes parts of the whole cell, such as nuclei, cell membranes, and cell organelles.

[0121] As used herein, the terms "spatially different" or "spatially separated" refer to elements distributed in different regions of three-dimensional space. In one embodiment, a representative sample captures all spatially different sub-populations of cancer cells within a tumor. In another embodiment, the tumor sample used to generate the representative sample is taken from different regions of the tumor sample. For example, the proximal to distal regions of the tumor, different faces of the tumor, different layers of the tumor, etc., where one is attempting to capture the diversity within the entire tumor.

[0122] The term "to homogenize" or "homogenize" refers to a process (mechanical process and / or biochemical process) of bringing a biological sample to a state where all fractions of the sample are equal in the composition. A representative analytical sample can be prepared by removing a portion of the homogenized sample. Generally, a tumor, lymph node, or other sample referred to as "liquefied" in the context of the present disclosure is understood to be sufficiently mixed or blended to be homogenized. A homogenized sample is mixed such that the removal of a portion (aliquot) of the sample does not substantially change the overall composition of the remaining sample, and the components of the removed aliquot are substantially the same as the components of the remaining sample. In the present disclosure, "homogenize" generally refers to a process that retains the integrity of most of the cells within the sample, for example, at least 50, 80, 85, 90, 95, 96, 97, 98, 99, 99.9% or more of the percentage of cells in the sample are not destroyed or lysed as a result of the homogenization process. The homogenate can be substantially dissociated into individual cells (or clusters of cells), and the resulting homogenate(s) is substantially homogeneous (consisting of, or composed of, similar elements or a uniform effluent). In one embodiment, the term "homogenize" refers to a process by which a tissue or biological sample is processed to such an extent that any subset, portion, or fraction of the tissue is similar, substantially similar, or identical in some aspect.

[0123] In one embodiment, the term "mechanical homogenization" refers to homogenization obtained from mechanical means.

[0124] As used herein, the term "biochemical dissociation" means dissociation using an enzyme such as a protease. Biochemical dissociation can be affected by protease concentration, incubation time, and temperature.

[0125] As used herein, the term "physical separation" or "physical dissociation" of a tissue sample refers to homogenization or dissociation of the sample using mechanical means, e.g., cutting, mincing, or chopping with a sharp instrument. "Cutting" generally results in a tissue section of about 1.0 mm - 5.0 mm in size. Mincing generally results in a tissue section of about 0.5 - 2.0 mm in size. Chopping generally results in a tissue section of about 0.1 - 1.0 mm in size.

[0126] As used herein, "mechanical separation" or "mechanical dissociation" of a tissue sample refers to homogenization or dissociation of the sample using a mechanical source such as a traditional blender, juicer, or bead beater, as known to those skilled in the art.

[0127] As used herein, "dissociated cells" are cells that were once part of a tissue or organ but have not been separated from that tissue or organ.

[0128] Depending on the mechanical and / or biochemical dissociation process applied to the sample to generate a homogenate, the cell clusters may contain from more than one cell to thousands of cells. Depending on the subsequent assays performed using representative samples (e.g., IHC which requires cell clusters containing tens to thousands of cells, or FACS or flow cytometry which requires single cells or cell fragments), the clusters can be dissociated (reducing the size and / or number of cells contained therein) by application of further processing methods, e.g., further mechanical and / or biochemical dissociation, and / or size exclusion.

[0129] The methods described herein are flexible with respect to the degree of sample dissociation. The target cell aggregate size can be controlled by further processing the cell clusters obtained after application of a first mechanical means (such as blending or the equivalent) such that the clusters match the dissociation objectives of the sampling method. In one aspect, mechanical shearing and size exclusion, e.g., using a series of sieves with meshes, can be used to remove cell clusters of a particular size or smaller while retaining larger cell clusters for further processing until the target particle size is reached. The resulting distribution of cell cluster sizes can be determined by size exclusion techniques and specific particles can be removed from the dissociation process until the size reaches a plateau rather than the distribution.

[0130] After homogenization, the resulting clusters may contain at least 1 - 2, 2 - 100, 100 - 500, 500 - 1,000, 1,000 - 10,000, 10,000 - 50,000 or more cells. In one aspect, the clusters may contain single cells, about 2 - 10 cells, about 10 - 20 cells, or about 20 - 40 cells. The size of the resulting clusters varies. See, e.g., FIG. 20.

[0131] As a result of homogenizing the sample, the distribution of cells within the sample is substantially uniformly distributed within the resulting homogenate or a portion or fraction thereof such that the homogenate or any fraction thereof represents the heterogeneity of the original sample. The homogenized sample may be referred to as a liquid or liquefied sample based on its ability to flow, even though many or most of the cells remain intact.

[0132] Other moieties can be added to these homogenates or representative samples, e.g., other cells, haptens or labels.

[0133] The term "heterogeneity" refers to diversity or inconsistency, for example, differences in the composition, or morphology, function, and behavior of different, or dissimilar parts. The term "heterogeneous tissue sample" is intended to mean a sample that is not uniform in composition or properties, for example, diverse in morphology, function, or behavior. In the context of cancer, the term "tumor heterogeneity" describes the fact that different tumor cells can exhibit different morphological, phenotypic, and genetic profiles, such as cell morphology, gene expression, gene mutations, metabolism, motility, proliferation, and metastatic potential. Heterogeneity may exist between tumors (inter-tumor heterogeneity) and within tumors (intra-tumor heterogeneity). Tumor heterogeneity has been observed in a variety of cancers, including but not limited to lung cancer, leukemia, breast cancer, kidney cancer, prostate cancer, colon cancer, brain cancer, esophageal cancer, head and neck cancer, bladder cancer, gynecological cancers, liposarcoma, and multiple myeloma. Two models have been proposed to explain heterogeneity in tumor cells: the cancer stem cell model and the clonal evolution model. The cancer stem cell model states that the heterogeneity observed between tumor cells is the result of differences in the cancer stem cells from which the tumor cells originate. The clonal evolution model states that tumors arise from a single mutated cell, but accumulate additional mutations (each giving rise to a further subpopulation of cells that can divide and further mutate) that cause the observed diversity in cancer cells derived from the same tumor. These models are not considered mutually exclusive and thus may contribute to heterogeneity in varying amounts across different tumor types. Tumor heterogeneity includes overall dispersion (population dispersion) and the spatial structure of that dispersion (population spatial stratification), and thus both elements of dispersion should be considered in sample design. Tumor heterogeneity can result from genetic heterogeneity (e.g., resulting from exogenous factors, genomic instability, therapy, etc.), other heterogeneities (e.g., epigenetic ones), and / or the tumor microenvironment (e.g., regional differences in tumors such as oxygen availability or immune surveillance exert different selection pressures on tumor cells).

[0134] The different tumor cell populations resulting from tumor heterogeneity are called "subclones" and are the progeny of mutated cells arising within a clone.

[0135] The prevalence of subclones within a tumor may vary. A particular subclone may comprise a majority of the tumor but decrease over time and / or after a particular treatment. Other subclones may be undetectable initially but become abundant later. Multiple subclones may coexist and their prevalence may vary over the time it takes for the tumor to grow large enough to be detectable. The term “low prevalence event” or “low prevalence genetic event” within a tumor refers to a rare event or rare genetic event (such as a mutation) that occurs at a rate lower than 10-1%, 1-0.1%, 0.1-0.01%, 0.01-0.001%, 0.001-0.0001%, 0.0001-0.00001%, 0.00001-0.000001%, or 0.000001%. Because the samples generated by the disclosed methods are representative (or substantially representative) of the tumor as a whole, it is possible to detect not only all other subclones that are present at higher prevalence, but also low prevalence subclones (such as down to at least 0.000001%) within the tumor or biological sample.

[0136] As used herein, the term "biological sample" or "tissue sample" refers to any sample containing biomolecules (proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (humans; veterinary animals such as cats, dogs, horses, cows, and pigs; and laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, nematodes, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears like Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation or other methods). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (such as that obtained by surgical biopsy or needle biopsy), nipple aspirate fluid, earwax, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any substance containing biomolecules derived from a first biological sample. In certain embodiments, as used herein, the term "biological sample" refers to a sample (such as a homogenized sample or a liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0137] As used herein, "normal tissue" refers to tissue that, in the context of a disease or cancer, has no detectable lesions or abnormalities that are presumably correlated with an increased incidence of malignant tumors. These normal samples may be derived from patients having gene mutations or conditions that are correlated with an increased incidence of a disease (genetic or otherwise), cancer, or malignant tumor. Normal tissue may be of the same type of tissue corresponding to a pathological tissue from the same individual or a different individual; or it may be normal tissue not associated with a pathological tissue from the same individual or another individual (e.g., derived from different locations in the body or having different tissue types).

[0138] As used herein, the term "preneoplastic tissue" refers to tissue that contains some lesion or abnormality that is presumably correlated with an increased incidence of cancer or malignancy.

[0139] The term "tumor" refers to a mass or neoplasm that is defined as an abnormal new growth of cells that typically grow more rapidly than normal cells and, if untreated, continue to grow and may cause damage to adjacent structures. The size of a tumor may vary widely. A tumor may be solid or fluid-filled. A tumor may refer to a benign (not malignant, generally harmless) or malignant (metastasizing) growth. Some tumors may contain neoplastic cells that are benign (such as carcinoma in situ) and at the same time contain malignant cancer cells (such as adenocarcinoma). It should be understood that this includes neoplasms located at multiple positions in the body. Thus, for the purposes of the present disclosure, tumors include primary tumors, lymph nodes, lymphoid tissue, and metastatic tumors. The boundary lines between cancerous, preneoplastic, and cancerous growths are not always clear, but there are general characteristics of each type of growth. Benign tumors are non-malignant tumors. Benign tumors are usually localized and do not spread (metastasize) to other parts of the body. Many benign tumors respond well to treatment. However, if left untreated, some benign tumors may grow large and cause significant harm or damage due to their size. Thus, benign tumors closely resemble malignant tumors and may thus be treated. Malignant tumors are cancerous growths that are often resistant to treatment, spread to other parts of the body, and may recur after removal. "Cancer" is another term for malignant growth (malignant tumor or neoplasm).

[0140] The pathogenicity of a tumor may vary. Certain cancers may be relatively easy to treat and / or cure, while others are more aggressive. Tumor pathogenicity can be determined, at least in part, by characterizing differential gene expression or genomic changes. In cancerous cells, the mechanisms that activate or silence genes in the cell are damaged. As a result, there is often abnormal activation of genes that are specific to other tissues and / or other developmental stages. For example, in lung cancer, tumor cells that express genes specific to sperm production, which should be silent, are extremely pathogenic (high-risk cancers that exhibit increased proliferative capacity and an increased tendency to hide from the body's immune system). Also, in almost all cancers, it has been shown that dozens of specific genes in the germline are abnormally activated. See, e.g., Rousseaux et al., Ectopic Activation of Germline and Placental Genes Identifies Aggressive Metastasis-Prone Lung Cancers. Science Translational Medicine (2013) 5(186): 186. Thus, because upregulation or downregulation of genes can be associated with the pathogenic type of a particular cancer, even if a tumor is treated adequately early in its development, it is possible to predict, after a diagnostic test, which cancers are at high risk of recurrence and have a fatal prognosis.

[0141] The term "lymph node" refers to oval or kidney-shaped organs of the lymphatic system that are widely present in the body, including the armpit and stomach, and are connected by lymphatic vessels. Lymph nodes contain a diverse number of immune cells, including, but not limited to, B cells and T cells. Lymph nodes are important for the proper functioning of the immune system and can act as filters for foreign particles and cancer cells.

[0142] The term "polyp(s)" refers to abnormal biological masses protruding from a mucous membrane. Polyps can be found in several tissues, including, but not limited to, the colon, stomach, nose, ear, sinus(es), bladder, and uterus.

[0143] The terms "metastasis" or "metastatic tumor" refer to tumors and / or their related components, which are not limited but have originated or spread from one organ or part of the body to another, including blood vessels, bone, and meninges.

[0144] The term "cyst" refers to a round or oval sealed sac with distinct membranes and boundaries compared to adjacent tissues. In some embodiments, a cyst is a cluster of cells that group together to form a sac (in a manner different from how clusters of water molecules group together to form air bubbles). In some embodiments, the cells that form such a sac or the "shell" of the cyst are clearly abnormal when compared to all surrounding cells at a given location. A cyst may contain, but is not limited to, air, fluid, or any semi-solid substance. Some tumors may contain cysts or may be described as "cystic".

[0145] The term "excision" refers to all or part of an organ or other body structure that is removed from a subject.

[0146] As used herein, the term "organ(s)" refers to any anatomical part or tissue having a specific function in an animal. This term includes a part or all of an anatomical part or tissue, for example, a lobe of the lung. Such organs include, but are not limited to, the adrenal gland, appendix, bladder, brain, ear, esophagus, eye, gallbladder, heart, kidney, intestine (e.g., large intestine or small intestine), liver, lung, mouth, muscle, nose, pancreas, parathyroid gland, pineal gland, pituitary gland, skin, spleen, stomach, thymus, thyroid gland, trachea, uterus, vermiform appendix, or a part thereof.

[0147] The term "cell cluster(s)" refers to an aggregate(s) of cells, e.g., malignant cells, fibroblasts, immune cells, stem cells, or endothelial cells. In one aspect, a cell cluster comprises 1 to 10, 10 to 100, 100 to 200; 200 to 1,000; 1,000 to 5,000; 10,000 to 100,000; 100,000 to 1,000,000; 1,000,000 to 5,000,000; 5,000,000 to 1,000,000,000; 1,000,000,000 to 5,000,000,0000 or more cells. The term "multiple cell clusters" means more than one cell cluster. Cell clusters aggregate separately or exist separately. The cells within a cell cluster adhere to each other by proteins such as cadherins and adhere to the surrounding extracellular matrix via integrins. Thus, the cells within a cell cluster are most likely related to each other and can be considered subclones or can be derived from subclones.

[0148] As used herein, the term "organelle" refers to structures bound to the cell membrane such as chloroplasts, mitochondria, and nuclei. The term "organelle" includes natural and synthetic organelles.

[0149] As used herein, the term "peptide" means a short polymer of amino acids linked by peptide bonds. All amino acids can have an L- or D-configuration.

[0150] As used herein, the term "nucleic acid" refers to a nucleotide in the form of a polymer of any length, either ribonucleotide, deoxyribonucleotide, or peptide nucleic acid (PNA), which includes purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. The backbone of the polynucleotide may typically include a sugar and phosphate group, or a modified or substituted sugar or phosphate group, as found in RNA or DNA. In one aspect, the polynucleotide includes modified nucleotides such as methylated nucleotides and nucleotide analogs.

[0151] The term "lipid" is used in its conventional sense as a general term encompassing fats, lipids, and the alcohol-ether soluble components of protoplasm that are insoluble in water. Lipids constitute fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulfolipids, aminolipids, pigment lipids (fatty pigments), and fatty acids. This term encompasses both naturally occurring lipids and synthetically produced lipids. Preferred lipids associated with the present disclosure are phospholipids including phosphatidylcholine and phosphatidylethanolamine, and sphingomyelin.

[0152] The term "metabolite" refers to a compound, protein, or any substance, byproduct, or material obtained from an enzymatic reaction, i.e., a compound synthesized by a process in which an enzyme participates.

[0153] The term "liquid tissue" refers to any tissue that is in liquid form or may be, including but not limited to blood, plasma, serum, saliva, semen, cervical secretions, saliva, urine, tears, sweat, breast milk, and amniotic fluid. The term "non-liquid tissue" refers to any tissue that is not liquid tissue.

[0154] The term "aspiration needle biopsy cytodiagnosis" refers to the procedure of fine needle aspiration biopsy (FNAB, FNA or NAB), or fine needle aspiration biopsy cytology (FNAC).

[0155] The term "exudation" refers to the procedure of collecting fluid from a subject. In some embodiments, the fluid collected by exudation can be described as having an abnormal medical condition, disorder, sign or symptom. In another embodiment, the fluid derived from exudation may be an excessive accumulation of fluid in a body cavity, or the peritoneal cavity.

[0156] The term "Pap smear" refers to a screening procedure for cervical cancer. It tests for the presence of pre-cancerous or cancerous cells at the cervix, the opening of the uterus.

[0157] As used herein, the term "abnormal tissue" is intended to mean a tissue that exhibits defining characteristics different from those of normal tissue. For example, breast cancer tissue may, in one aspect, be phenotypically non-cancerous, yet still be "abnormal" compared to breast tissue that may be "normal" with respect to other characteristics, such as gene expression of certain biomarkers.

[0158] The term "phenotypically normal tissue" refers to a tissue having physical characteristics, such as histological appearance, that are the same as, similar to, or substantially similar to those considered normal. The term "phenotypically abnormal tissue" refers to a tissue having physical characteristics that are the same as, similar to, or substantially similar to those considered abnormal.

[0159] The term "phenotypically homogeneous" is intended to mean at least one or more physical characteristics, such as histological appearance, that are the same as, similar to, or substantially similar to the identified characteristic(s) of other members of a group or tissue type, such as breast, colon, or lung.

[0160] The term "genotypically normal tissue" refers to a tissue having genomic characteristics, such as chromosomal, mitochondrial, RNA, microRNA, and / or non-coding RNA characteristics, such as gene sequences, that are the same as, similar to, or substantially similar to those considered normal. The term "genotypically abnormal tissue" refers to a tissue having genetic characteristics, such as gene sequences, that are the same as, similar to, or substantially similar to those considered abnormal.

[0161] The term "genetically diverse" as related to a cell, tissue, or subject refers to a population of at least two members of a group that are different from each other in terms of the subject, tissue, or cell population, or at the genomic level, such as chromosomal, mitochondrial, RNA, microRNA, and / or non-coding RNA, of at least another individual, cell, or tissue. The term "genetically homogeneous subject" as related to two or more individuals refers to an individual that exhibits substantially the same specific marker or characteristic at the genomic level, such as chromosomal, mitochondrial, RNA, microRNA, and / or non-coding RNA.

[0162] The term "ethnic group" refers to a social group that has a common country or cultural tradition. In one aspect, this term is intended to mean members of a group that are derived from a common, or closely related, genetic origin.

[0163] The term "small molecule" refers to a low molecular weight organic substance that can be used to regulate biological processes. In one aspect, the molecular weight ranges from 0 - 100 Daltons, 100 - 200 Daltons, 200 - 300 Daltons, 300 - 400 Daltons, 400 - 500 Daltons, 500 - 600 Daltons, 600 - 700 Daltons, 700 - 800 Daltons, 800 - 900 Daltons, or 900 - 1000 Daltons. In another aspect, the molecular weight is less than 1000 Daltons. Small molecules include, but are not limited to, organic compounds, peptides, metabolites, and lipids.

[0164] The term "pigment" refers to a substance that can impart color to an object by selective absorption of light. In some embodiments, the pigment is soluble or solid. In another embodiment, the pigment is retained in the substrate by absorption, solution, and mechanical retention, or by ionic or covalent chemical bonds. In a further embodiment, the pigment is any organic or inorganic molecule or moiety that absorbs electromagnetic radiation, for example, at a selected wavelength.

[0165] [[ID=!4]] As used herein, the term "quantitative data" means data that represents a particular quantity, amount, or range associated with a unit of measurement. For example, quantitative data regarding a tumor includes, but is not limited to, the size of the tumor, or the expression level of a biomarker.

[0166] As used herein, the term "qualitative data" refers to information that describes the characteristics, features, or other properties of an object. For example, qualitative data regarding cancer includes, but is not limited to, the stage of the tumor, appearance, and other physical characteristics.

[0167] As used herein in connection with measured values, the term "normalized" is intended to refer to the adjustment of values measured on different scales to a nominally common scale.

[0168] A gene is just one type of cancer biomarker. As used herein, the term "biomarker" or "marker" refers to a biological molecule found in blood, other body fluids, or tissues that is an indication of a normal or abnormal process, or of a condition or disease (such as cancer). Biomarkers can be used to determine how well the body will respond to treatment for a disease or condition, or whether a subject has a predisposition to a disease or condition. In the context of cancer, a biomarker refers to a biological substance that indicates the presence of cancer in the body. A biomarker may be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Genetic, epigenetic, proteomic, glycomic, and imaging biomarkers can be used for cancer diagnosis, prognosis, and epidemiology. Such biomarkers can be assayed in non-invasively collected biological fluids such as blood or serum. Some gene- and protein-based biomarkers, such as, but not limited to, AFP (liver cancer), BCR-ABL (chronic myeloid leukemia), BRCA1 / BRCA2 (breast / ovarian cancer), BRAF V600E (myeloma / colorectal cancer), CA-125 (ovarian cancer), CA19.9 (pancreatic cancer), CEA (colorectal cancer), EGFR (non-small cell lung cancer), HER-2 (breast cancer), KIT (gastrointestinal stromal tumor), PSA (prostate-specific antigen), S100 (myeloma), and many others, are already being used in patient management. Biomarkers can be useful as diagnostic agents (to identify early-stage cancer) and / or prognostic agents (to predict how aggressive the cancer is and / or how well a subject will respond to a particular treatment and / or how likely the cancer is to recur).

[0169] As used herein, the term "clinically relevant marker" is intended to mean a marker or biomarker that is associated with a clinical outcome or condition, such as the presence or absence of a disease or condition, such as cancer.

[0170] As used herein, the term "cancerous tissue" refers to any tissue that bears tumor cells. Examples of cancerous tissue include, but are not limited to, muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph node, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, or connective tissue.

[0171] "Prognosis" is intended to mean a prediction of the current state of a subject or a likely outcome, including the likelihood of cancer recurrence and / or cancer metastasis in a patient.

[0172] As used herein, the term "preserve" or "fix" refers to a process in the preparation of a biological sample, such as a tissue section. Methods of preservation or fixation include, but are not limited to, chemical fixation, heat fixation, immersion, perfusion, and / or lyophilization. Thus, a "fixed sample" is a sample that has been processed as described above. <##

[0173] The term "live cell" refers to any cell that has not been fixed or preserved. In some embodiments, the term "live cell" includes cells that are functioning. One of ordinary skill in the art can readily distinguish between live cells and dead cells for the purposes of the present disclosure.

[0174] As used herein, the term "wax embedding" or "paraffin embedding" is used for a process that involves injecting paraffin wax into a tissue specimen and has the added benefit of retaining its cellular structure when sectioned using a microtome and being suitable for long-term storage.

[0175] As used herein, the term "one or more tissues" refers to tissues derived from one or more subjects, or one or more tissues derived from the same subject. The term "two or more tissues" is used with respect to tissues derived from two or more subjects, or two or more tissues derived from the same subject or patient.

[0176] As used herein, the term "premalignant or malignant cells" is used to describe any cell that has undergone malignant transformation or is predisposed to undergo malignant transformation. Characteristics of premalignant or malignant cells include, but are not limited to, uncontrolled growth, metastasis, abnormal cell metabolism, avoidance of apoptosis, self-sufficiency in growth signals, and sustained angiogenesis. For example, a colonic polyp can be premalignant for colon cancer.

[0177] As used herein, the term "circulating tumor cells" refers to tumor or cancer cells that are circulating in blood vessels, lymphatic vessels, or other fluids. Examples of circulating tumor cells include, but are not limited to, leukemia cells.

[0178] As used herein, the term "normal adjacent tissue" refers to normal tissue adjacent to tumor cells or a tumor tissue.

[0179] The term "reconstituting" with respect to reconstitution of a homogenized sample is intended to mean mixing or combining.

[0180] The term "extracting components" with respect to a homogenate is intended to mean isolating or purifying components of the cell structure.

[0181] As used herein, the term "FFPE sample" means a formalin-fixed paraffin-embedded sample. FFPE samples can be used for several medical studies, including, but not limited to, diagnosis, IHC, and profiling of gene expression or origin of diseases.

[0182] The term "predetermined value" in this specification generally represents a value used to calculate data fluctuations. In one aspect, the predetermined value is calculated based on historical data, or different sample groups, or sample groups similar to the data being tested.

[0183] The term "risk value" refers to a quantitative or qualitative value associated with risk. For example, the risk value of cancer is a value that quantifies or qualifies the risk of developing cancer.

[0184] The term "chromosomal translocation" refers to a chromosomal abnormality caused by the rearrangement of parts between non-homologous chromosomes.

[0185] The term "intrachromosomal inversion" refers to a chromosomal rearrangement in which a fragment of a chromosome is reversed end to end. In one aspect, an inversion occurs when a single chromosome undergoes breakage and rearrangement within itself.

[0186] The term "treatment regimen" is used according to its well-known meaning in the art. For example, this term refers to a treatment plan for an individual suffering from a medical condition (e.g., chronic hepatitis C infection or cancer) that identifies factors such as the agent(s) administered to the patient, the dosage of such agent(s), and the schedule and duration of treatment. An individualized dosage or treatment regimen is a therapy or dosage regimen based on the concept of precision medicine that takes into account the individual characteristics of the patient or subject, such as genetic structure, pharmacogenetics, ethnicity, treatment history, family history, clinical chemistry, or other relevant characteristics or measurements.

[0187] The term "chemotherapy" refers to treatment using chemical agents. Chemotherapy can be defined as the use of pharmaceuticals specifically designed to target, fight against, and / or destroy diseased cells. Non-limiting examples of diseases that can be treated by chemotherapy include cancer, systemic sclerosis, lupus erythematosus, rheumatoid arthritis, autoimmune diseases such as vasculitis, and viral infections. In one aspect, chemotherapeutic agents destroy cancer cells by targeting cells that divide rapidly in the body. Due to the lack of specificity for cancer cells, the toxic effects of chemotherapy are also seen in other rapidly dividing non-cancerous cells. Blood cells, cells in the mouth, the digestive tract, the nose, nails, and hair are some of the cells that divide rapidly in the body. The destruction of normal cells in the body causes side effects such as alopecia, cachexia, anemia, leukopenia, and neutropenia. These side effects limit the effectiveness of chemotherapy and increase the risk of dose reduction, which directly affects the survival of patients.

[0188] The term "immunotherapy" refers to treatment that involves activation or inactivation of specific immune responses and / or one or more immune effector functions. The term "radiation" or "radiation therapy" relates to treatment, including but not limited to, the use of high-energy particles or waves such as x-rays, gamma rays, electron beams, or protons to treat a disease (e.g., cancer) or medical condition.

[0189] The term "surgery" relates to any tissue activity, with or without the use of instruments, on a patient to bring about a curative or therapeutic effect.

[0190] The term "gene therapy" preferably refers to the use of a gene delivery process or a gene editing process (e.g., CRISPR) to cause a therapeutic effect in a subject or patient.

[0191] As used herein, the term "hormone therapy" is defined as a treatment related to regulating hormones. Hormone therapy may include, but is not limited to, removing glands that synthesize hormones or prohormones, blocking or inhibiting hormone synthesis, preventing the binding of a hormone to its receptor, downregulating or degrading a hormone receptor.

[0192] As used herein, the term "stem cell therapy" is a treatment by using stem cells to treat or prevent a disease or medical condition.

[0193] The term "blood transfusion" relates to a procedure of receiving blood through a venous line.

[0194] As used herein, the term "physical therapy" refers to the treatment of physical dysfunction or injury by using therapeutic exercises and the application of modalities, which are intended to restore or facilitate normal function or development.

[0195] As used herein, the term "photodynamic therapy" refers to the process of directing light of a specific wavelength at a tissue or cell that is to receive a treatment or investigation made photosensitive by the administration of a photosensitive agent or photosensitizer. In one embodiment, the goal may be diagnosis, in which case the wavelength of the light is selected to cause the photosensitive agent to fluoresce and thus obtain information about the tissue without damaging the tissue. Also, the goal may be treatment, in which case the wavelength of the light delivered to the target tissue being treated is selected to cause a photochemical interaction of the photosensitive agent with oxygen, resulting in high-energy species such as singlet oxygen, causing local tissue lysis or destruction, or inducing an immune response in the photosensitized tissue or cells.

[0196] As used herein, the term "differential expression" refers to the difference in gene or protein expression levels between two or more samples. In one aspect, the results of differential expression can be used to identify any pattern that may be associated with a disease, biomarker, or medical condition.

[0197] As used herein, the term "first profile" refers to a dataset of a subject or group of subjects. In one embodiment, the first profile can be used as a baseline to determine changes in subsequent profile(s). In another embodiment, the data in the first profile can be incorporated into subsequent analyses or processes that generate subsequent profiles.

[0198] As used herein, the term "predetermined profile" refers to a dataset or panel of datasets associated with a physiological condition. A predetermined profile may be derived from a subject or a group of subjects. In one aspect, a predetermined profile may include a histological or known dataset of a physiological condition. In another aspect, a predetermined profile is used as a baseline for determining changes in a physiological condition (e.g., tumor progression).

[0199] As used herein, the term "quantitative score" refers to a numerical representation of a physiological state (eg, disease risk).

[0200] As used herein, the term "proliferation" refers to the growth and division of cells. In some embodiments, the term "proliferation" as used herein with reference to cells refers to a population of cells that are capable of increasing in number over a period of time.

[0201] The term "apoptosis" refers to the process of programmed cell death. In some aspects, apoptosis involves morphological changes in cells and loss of cell viability.

[0202] The term "necrosis" encompasses necrotic states of cells, as well as intermediate states that exhibit characteristics of necrosis and apoptosis.

[0203] As used herein, the term "cell migration" refers to the movement of cells induced by a physiologically active substance or caused by a physiological change (eg, transformation).

[0204] The term "epithelial-mesenchymal transition" or "EMT" refers to the process by which epithelial cells, which are normally non-proliferative and non-motile, undergo a transition to mesenchymal cells characterized by a proliferative and migratory phenotype. EMT is a central mechanism for diversifying the cells found in complex tissues and is thus a process involved in organizing the construction of the body plan (Kalluri and Nelson J Clin Invest 112(12):1776-1784, 2003). Epithelial cells were once thought to ultimately differentiate, but the epithelium is recognized to have an element of plasticity that allows for the transition to migratory mesenchymal cells (Boyer et al., Biochem Pharmacol 60:1099, 2000; Nieto Nat Rev Mol Cell Biol 3:155-166, 2002). Thus, EMT is necessary in adult tissues to allow for the formation of fibroblasts in injured tissues (Strutz et al., J Cell Biol 130:393-405, 1995; Iwano et al., J Clin Invest 110:341-350, 2002) and is necessary to initiate metastasis in epithelial cancers (Kiermer et al., Oncogene 20:6679-6688, 2001; Janda et al., J Cell Biol 156:299-313, 2002; Xue et al., Cancer Res 63:3386-3394, 2003).

[0205] In one aspect, EMT is the process of disassembling the epithelial unit and reforming the epithelium for migration in the formation of mesenchymal cells. The transition generally requires molecular reprogramming of the epithelium, which is thought to be by various cytokines, metalloproteinases, and membrane assembly inhibitors (Kalluri and Neilson 2003, supra; Yang and Liu Am J Pathol 159:1465-1475, 2001; Zeisberg et al., Am J Pathol 159:1313-1321, 2001; Fan Kindney Int 56:1455-1467, 1999).

[0206] As used herein, the term "mitosis" can be used interchangeably with the term "cell division". In some embodiments, mitosis refers to just one stage of the cell division process, a process in which sister chromatids are equally distributed between two daughter cells. In eukaryotic cells, after mitosis, cytokinesis occurs, a process in which the cytoplasm of the cell is cleaved into two distinct but genetically identical daughter cells.

[0207] At the onset of mitosis, small intracellular filamentous structures known as cytoplasmic microtubules, whose major component is a protein called tubulin, disassemble into tubulin molecules. The tubulin then reassembles into microtubules, forming an intracellular structure known as the "mitotic spindle". The mitotic spindle plays an important role in accurately partitioning the chromosomes within the dividing cell between the two daughter nuclei. Cancer cells are characterized by more rapid cell division and proliferation than is observed in many healthy cells, and many anticancer agents function by inhibiting cell division. Because cancer cells divide more rapidly than healthy cells, cancer cells are preferentially killed by anticancer agents that inhibit mitosis. Such compounds are called "antimitotic drugs".

[0208] The term "cell cycle arrest" refers to a point of arrest in the cell cycle where the cell is not in the processes surrounding replication and division. The natural cell cycle includes several checkpoints that determine whether the cell proceeds to divide or to arrest. These arrests can also be induced by external factors such as exposure to radiation or pharmaceuticals used to control cell growth.

[0209] The first stage of the cell cycle is G1, during which the cell prepares for replication. The cell genetic material is replicated during the S phase. Cell damage is repaired during the M phase and the G2 phase before moving into mitosis. After mitosis, the cell either re-enters G1 or moves into G0, a resting phase. Checkpoints temporarily halt the cell cycle at each stage, allowing the cell to determine whether it should continue. Some cells are programmed to replicate rarely, while damaged cells may take time for repair or destruction.

[0210] In some embodiments, cell cycle arrest precedes apoptosis, or cell death. This occurs when the cell can no longer function due to DNA damage. The cell becomes a target for destruction. Cell cycle arrest allows the cell to regularly check for signs of DNA damage that could cause functional problems or lead to tumor development.

[0211] The term "S phase" refers to the period during the cell cycle in which DNA is replicated. The S phase typically occurs between the G1 and G2 phases. Precise and accurate DNA replication during the S phase is necessary to prevent genetic abnormalities.

[0212] As used herein, the term "senescence" refers to the permanent cessation of DNA replication and cell proliferation that cannot be reversed by growth factors. This phenomenon can occur at the end of the proliferative lifespan of normal cells or in normal or tumor cells in response to cytotoxic agents or DNA damage. Senescence is characterized by certain morphological properties such as, but not limited to, an increase in cell size, a flattened cell morphology, an increase in granularity, and the presence of senescence-associated β-galactosidase activity (SA-β-gal).

[0213] As used herein, the term "differentiation" refers to the process by which the structure or function of a cell becomes specialized during its division, proliferation, and growth. Generally, differentiation refers to the phenomenon by which a relatively simple system is divided into two or more qualitatively different subsystems.

[0214] As used herein, the term "detection" refers to the act or process of identifying the presence of a particular molecule. In one embodiment, detection of a biomarker may refer to identifying the expression of the biomarker.

[0215] As used herein, the term "fixative" refers to an agent used for fixation of tissues for several purposes, such as delivery, preservation, histological studies, and processing, although not limited thereto.

[0216] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymer form of nucleotides of any length that are ribonucleotides or deoxyribonucleotides. Thus, the term includes, although not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0217] The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that can be transcribed and / or translated to produce mRNA, either in its native state or when manipulated by methods well known to those skilled in the art. The antisense strand is the complement of such a nucleic acid and the coding sequence can be deduced therefrom.

[0218] As used herein, the term "vector" refers to a nucleic acid construct designed for introduction between different hosts, such as, although not limited to, plasmids, viruses, cosmids, phages, BACs, YACs, etc. In some embodiments, plasmid vectors can be prepared from commercially available vectors. In other embodiments, viral vectors can be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector.

[0219] As used herein, the term "promoter" refers to any sequence, such as a gene, that regulates the expression of a coding sequence. A promoter may be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence that is a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. It may contain gene elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors.

[0220] As used herein, the term "isolated cell" generally refers to a cell that is substantially separated from other cells of a tissue.

[0221] "Effective amount" or "therapeutically effective amount" refers to the amount of a drug, or the combined amount of two or more drugs, that is sufficient to effect such treatment for a disease when administered for the treatment of a patient, mammal, or other subject. The "effective amount" is expected to vary depending on the drug(s), the disease and its severity, and the age, weight, etc. of the subject being treated.

[0222] As used herein, the term "detectable marker or label" refers to at least one marker that can directly or indirectly produce a detectable signal. A non-exhaustive list of markers includes, for example, enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase that produce signals detectable by colorimetry, fluorescence, luminescence; chromophores such as fluorescent, luminescent dyes; groups having an electron density detectable by an electron microscope or by its electrical properties such as conductivity, current measurement, voltammetry, impedance; for example, groups of a size sufficient to induce a detectable modification of the physical and / or chemical properties of a molecule, and such detection can be achieved by optical methods such as diffraction, surface plasmon resonance, surface changes; physical methods such as contact angle changes or atomic force spectroscopy, tunneling effect; or 32 P, 35 S or 125Examples of radioactive molecules include I and the like.

[0223] As used herein, the term "purification marker" refers to at least one marker useful for purification or identification. A non-exhaustive list of markers includes His, lacZ, GST, maltose binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YEP, cherry, thioredoxin, poly(NANP), V5, Snap, HA, chitin binding protein, Softag1, Softag3, Strep, or S protein. Suitable direct or indirect fluorescent markers include FLAG, GFP, YEP, RFP, dTomato, cherry, Cy3, Cy5, Cy5.5, Cy7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluor, FITC, TRITC, or any other fluorescent dye or hapten.

[0224] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. When the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from a sample can be directly compared to the expression level of that gene from a control sample or reference sample. In another aspect, the expression level of a gene from a sample can be directly compared to the expression level of that gene from the same sample after administration of a compound.

[0225] When used in the context of two or more nucleic acid or polypeptide sequences, as used herein, "homology" or "identical", "percent identity" or "similarity" means the same or, over a particular region (e.g., the nucleotide sequence encoding an antibody described herein or the amino acid sequence of an antibody described herein), the same, e.g., having at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, of a particular percentage of nucleotide or amino acid residues, of two or more sequences or subsequences. Homology can be determined by comparing the positions in each of the sequences that can be aligned for comparison. If a position in the sequences being compared is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. Alignment and percent homology or sequence identity can be determined using software programs known in the art, e.g., those described in Current Protocols in Molecular Biology (Ausubel et al. (eds.), 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. In particular, preferred programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; description = 50 sequences; sort by HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+Genbank CDS translations+SwissProtein+SP updates+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.The terms "identity" or "identical", "percent identity" or "similarity" also refer to, or can apply to, the complement of a test sequence. This term may also include sequences with deletions and / or additions, as well as those with substitutions. As described herein, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably, over a region of at least 50 - 100 amino acids or nucleotides in length. "Unrelated" or "non - homologous" sequences have less than 40% identity, or alternatively, less than 25% identity with one of the sequences disclosed herein.

[0226] In one aspect, the terms "equivalent" or "biological equivalent" of an antibody mean the ability of the antibody to selectively bind to its epitope protein or a fragment thereof when measured by ELISA or other suitable methods. Biologically equivalent antibodies include, but are not limited to, antibodies, peptides, antibody fragments, antibody variants, antibody derivatives, and antibody mimetics that bind to the same epitope as the reference antibody.

[0227] In the absence of explicit description and unless otherwise specifically intended, where the present disclosure relates to a polypeptide, protein, polynucleotide or antibody, it should be presumed that equivalents or biological equivalents of such are intended to be within the scope of the present disclosure. As used herein, the term "its biological equivalent" is intended to be synonymous with "its equivalent" which, when referring to a reference protein, antibody, polypeptide or nucleic acid, refers to something having minimal homology while still maintaining the desired structure or function. Unless specifically described herein otherwise, any polynucleotide, polypeptide or protein described herein is intended to include its equivalents. For example, the equivalents are intended to have at least about 70% homology or identity, or at least 80% homology or identity, or or at least about 85%, or or at least about 90%, or or at least about 95%, or or 98% homology or identity percentage and exhibit biological activity substantially equivalent to the reference protein, polypeptide or nucleic acid. Alternatively, when referring to a polynucleotide, its equivalent is a polynucleotide that hybridizes to the reference polynucleotide or its complement under stringent conditions.

[0228] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another array, when aligned, means that that percentage of bases (or amino acids) is the same in the comparison of the two sequences. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al. (eds.), 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. In particular, preferred programs are BLASTN and BLASTP with the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation value = 10; matrix = BLOSUM62; description = 50 sequences; selection by = HIGH SCORE; database = non-redundant, GenBank+EMBL+DDBJ+PDB+Genbank CDS translation+SwissProtein+SP update+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0229] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of nucleotide residues. The hydrogen bonds may occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex may include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction may constitute a step in a broader process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0230] Examples of stringent hybridization conditions include an incubation temperature of about 25°C - about 37°C; a hybridization buffer concentration of about 6×SSC - about 10×SSC; a formamide concentration of about 0% - about 25%; and a washing solution of about 4×SSC - about 8×SSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C - about 50°C; a buffer concentration of about 9×SSC - about 2×SSC; a formamide concentration of about 30% - about 50%; and a washing solution of about 5×SSC - about 2×SSC. Examples of high stringency conditions include an incubation temperature of about 55°C - about 68°C; a buffer concentration of about 1×SSC - about 0.1×SSC; a formamide concentration of about 55% - about 75%; and a washing solution of about 1×SSC, 0.1×SSC, or deionized water. Generally, the incubation time for hybridization is 5 minutes - 24 hours, including one, two or more washing steps, and the incubation time for washing is about 1, 2, or 15 minutes. SSC is 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0231] As used herein, the term "isolated" refers to a molecule, cell or biologic agent or cell material substantially free of other materials. In one aspect, the term "isolated" refers to a nucleic acid such as DNA or RNA, or a protein or polypeptide (e.g., an antibody or derivative thereof), or a cell or cell organelle, or a tissue or organ that has been separated from other DNA or RNA, or protein or polypeptide, or cell or cell organelle, or tissue or organ present in its natural source. The term "isolated" also refers to a nucleic acid or peptide substantially free of cell material, viral material, or culture medium if produced by recombinant DNA techniques, or chemical precursors or other chemicals if chemically synthesized. Further, "isolated nucleic acid" means including nucleic acid fragments that do not exist as fragments in nature and are not found in their natural state. The term "isolated" is also used herein to refer to a polypeptide isolated from other cellular proteins and is meant to encompass both purified polypeptides and recombinant polypeptides. Also, the term "isolated" is used herein to refer to a cell or tissue isolated from other cells or tissues and is meant to encompass both cultured and engineered ones.

[0232] The term "individual cell" or "single cell" means the structural and / or functional unit of an organism. In some aspects, the individual cell includes, but is not limited to, cytoplasm, nucleus, or cell membrane.

[0233] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by generating hybrid antibody-forming cells from a fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0234] The terms "protein", "peptide", and "polypeptide" are used interchangeably and, in their broadest sense, refer to compounds of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. In another aspect, the subunits can be linked by other bonds, such as ester, ether bonds, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may be included in the sequence of the protein or peptide. As used herein, the term "amino acid" refers to natural and / or non-natural, or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0235] As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein, biological complex, or other active compound is one that has been isolated in whole or in part from proteins or other contaminants. Generally, a substantially purified peptide, protein, biological complex, or other active compound for use in the present disclosure comprises more than 80% of all macromolecular species present in a preparation prior to mixing or formulation with a pharmaceutical carrier, excipient, buffer, absorption enhancer, stabilizer, preservative, adjuvant, or other co-components in a complete pharmaceutical formulation for therapeutic administration. More typically, a peptide, protein, biological complex, or other active compound is purified to comprise more than 90%, often more than 95% of all macromolecular species present in a purified preparation prior to mixing with other formulation components. In other cases, the purified preparation may be essentially homogeneous and other macromolecular species are not detectable by conventional techniques.

[0236] As used herein, the term "recombinant protein" generally refers to a polypeptide produced by recombinant DNA technology in which DNA encoding the polypeptide is inserted into a suitable expression vector and then used to transform a host cell to produce a heterologous protein.

[0237] As used herein, the term "sonication" refers to the application of sound waves (acoustic energy) transmitted through a liquid medium. The sound waves can cause particles (e.g., cells or cell clusters) to vibrate around their average positions. In one aspect, sonication results in the dissociation of cell clusters into a single cell suspension.

[0238] As used herein, "treating" a disease in a subject or its "treatment" means: (1) preventing a symptom or disease from occurring in a subject who has a predisposition to the disease or who has not yet manifested symptoms of the disease; and / or (2) inhibiting the disease or arresting its development; and / or (3) ameliorating the disease or its symptoms or causing regression thereof. As understood in the art, "treatment" is a procedure for obtaining a beneficial or desired result, including clinical outcomes. For the purposes of the present technology, beneficial or desired results include, but are not limited to, the reduction or improvement of one or more detectable or undetectable symptoms, a decrease in the degree of a condition (including a disease), stabilization of the condition (including a disease) (i.e., not getting worse), delay or deceleration of the condition (including a disease), progression, improvement or remission (partial or total) of the condition (including a disease), condition and situation.

[0239] substantially homogeneous representative sample The present disclosure addresses the limitations of prior art clinical sampling methods that cannot provide representative samples. A representative sample is equivalent to the important variables and characteristics of a larger entity or sample. Current practices in selective sampling aim to collect tissue samples to meet the requirements of the TNM staging classification system. Samples for the TNM staging classification system are specifically taken to reflect the normal anatomy of the removed organ containing the tumor. Although important for the prognostic stage classification of the TNM system, this selective sampling method produces biased tumor samples or samples that do not contain the genetic and phenotypic diversity found throughout the tumor mass.

[0240] The present disclosure provides a processed homogenate composition derived from a heterogeneous tissue sample that includes a substantially uniformly distributed cellular structure, wherein the ratio of the cellular structure in each and / or any subset of the representative sample is substantially similar to the ratio of the cellular structure in the originating heterogeneous tissue sample. The homogenate composition is a new, unique tissue sample that also represents the important characteristics of the original, originating heterogeneous tissue sample. The composition and the method for preparing the composition described herein overcome the failure of prior art methods that cause problems with tissue heterogeneity in clinical samples, particularly samples for use in clinical fields such as clinical oncology.

[0241] The representative samples of the present disclosure are illustrated in FIGS. 2A and 2B, which show schematic diagrams of the homogenates of the present disclosure. FIG. 2A shows a tumor containing three subclones present in different proportions. The disclosed homogenization method produces a representative sample containing the subclones in the proportions in which they exist within the solid tumor. Any sample taken from the homogenate contains each subclone in the same proportion as present in the original tumor. FIG. 2B is an illustration of how the homogenate facilitates the detection of subclones with low prevalence.

[0242] Representative samples of the present disclosure overcome the sampling challenges imposed by the spatially stratified three-dimensional structure of tissues. In a representative sample, the components (cell structures, cells, peptides, nucleic acids, lipids, metabolites, etc.) of the original spatially stratified organ, tumor, or tissue (“OTT”) are present in a sub-sample or subset of the sample in the proportions in which they were present in the original OTT. In some embodiments, a representative sample refers to a sample of OTTs that comprises as many OTTs as possible that approach, or come close to, representing the diversity of the OTT at the level of clusters of cells, individual cells, cell fragments, organelles, peptides, nucleic acids, lipids, metabolites, etc., that are intact and sufficient in number to approach the goal of representing the diversity of the OTT. A representative sample may contain the minimum amount of intact OTT required to encompass the diversity of the OTT.

[0243] Multiple representative samples can be made from a single OTT. In this embodiment, the surgically removed OTT is first processed or otherwise manipulated into separate sub-units such that each sub-unit is composed of spatially stratified cell structures, cells, peptides, nucleic acids, etc. Each sub-unit is then sufficiently homogenized, mixed, or otherwise disrupted to produce a representative sample of the OTT sub-unit.

[0244] The representative sample can be homogenized, or otherwise mixed or disrupted, to the point where any analytical sample, or a portion of the representative sample, contains a random sampling of the materials present in the representative sample. It is a characteristic of the analytical sample that it is a sufficiently large fraction of the representative sample to encompass the diversity of the representative sample, as compared to the intended output of the analytical test being applied (i.e., cell vs. large number of cells). In the representative sample, any analytical sample used for a particular assay produces data that, within experimental error, agrees with that of another analytical sample used for the same assay. Further, any subset of the representative sample selected for a particular assay provides information that can be cross-referenced with data generated using different assays with analytical samples taken from the same representative sample or from other representative samples made from OTTs derived from the same patient. It is also contemplated that data produced from analytical sub-samples regarding the proportion of the biological components of the OTT can be compared between patients, since the original proportions of the original biological components are present in all analytical sub-samples.

[0245] In one embodiment, the representative sample is a processed homogenate composition derived from a heterogeneous tissue sample. The homogenate composition comprises, consists essentially of, or consists of a substantially uniformly distributed cell structure, and the ratio of the cell structure in each subset of the homogenate is substantially similar to the ratio of the cell structure in the tissue sample. In one embodiment, the tissue sample is selected from the group consisting of a tumor, lymph node, metastasis, polyp, cyst, resection, organ, or a fraction thereof. In another embodiment, the tissue sample comprises, consists essentially of, or consists of spatially separated cell structures. In another aspect, the cell structure comprises, consists essentially of, or consists of cell clusters, individual cells, cell fragments, organelles, peptides, nucleic acids, lipids, metabolites, or a combination thereof. In one aspect, the homogenate comprises, consists essentially of, or consists of cell structures derived from up to 25% of the tissue sample. In one aspect, the homogenate comprises, consists essentially of, or consists of cell structures derived from up to 50% of the tissue sample. In another aspect, the homogenate comprises, consists essentially of, or consists of cell structures derived from up to 75% of the tissue sample. In different aspects, the homogenate comprises, consists essentially of, or consists of cell structures derived from up to 100% of the tissue sample. In another aspect, the homogenate comprises, consists essentially of, or consists of cell structures derived from 100% of the tissue sample.

[0246] In another embodiment, the homogenate comprises, consists essentially of, or consists of cell structures derived from 100% of the tissue sample. In one aspect, the tissue sample comprises, consists essentially of, or consists of a non-liquid tissue sample. In another aspect, the tissue sample comprises, consists essentially of, or consists of a liquid tissue sample. In one aspect, the liquid tissue sample comprises, consists essentially of, or consists of tissue isolated by one or more of surgical resection, fine needle aspiration biopsy, effusion sample, or Pap smear.

[0247] In yet another embodiment, the substantially homogeneous cell structure comprises, consists essentially of, or consists of a plurality of single cells or a plurality of cell clusters. In one aspect, the cell structure is isolated from normal tissue. In another aspect, the cell structure is isolated from phenotypically or genetically normal tissue. In yet another aspect, the cell structure is isolated from abnormal tissue. In one aspect, the cell structure is isolated from phenotypically or genetically abnormal tissue.

[0248] In one embodiment, the tissue sample comprises, consists essentially of, or consists of stem cells, epithelial cells, blood cells, adipocytes, skin cells, endothelial cells, tumor cells, or immune cells. In one aspect, the tumor cells are derived from cancerous tissue selected from the group consisting of lung cancer, leukemia, breast cancer, prostate cancer, colon cancer, brain cancer, esophageal cancer, head and neck cancer, bladder cancer, gynecological cancer, ovarian cancer, cervical cancer, liposarcoma, melanoma, lymphoma, plasmacytoma, sarcoma, glioma, thymoma, liver cancer, and myeloma. In another aspect, the immune cells are cells selected from the group consisting of neutrophils, monocytes, dendritic cells, macrophages, lymphocytes, T cells, B cells, or natural killer cells.

[0249] In another embodiment, the tissue sample is not preserved or fixed. In one aspect, the tissue sample comprises live cells or cells freshly isolated from a subject. In another embodiment, the tissue sample is preserved or fixed. In one aspect, the preserved or fixed tissue sample comprises a sample frozen or fixed by a method of the group consisting of freezing, freeze-drying, and wax embedding.

[0250] In one embodiment, the heterogeneous tissue sample is isolated from one or more tissues from the same or different subjects. In one aspect, the tissue sample is isolated from 1 subject. In another aspect, the tissue sample comprises or consists essentially of or consists of tissues isolated from 2 or more subjects and from the same or similar tissue types or different tissue types. In a further aspect, the 2 or more subjects are genetically homogeneous subjects. In another aspect, the 2 or more subjects are phenotypically homogeneous subjects. In a further aspect, the 2 or more subjects are genetically diverse subjects. In one aspect, the 2 or more subjects are phenotypically diverse subjects. In another aspect, the 2 or more subjects are from the same sex or different sexes.

[0251] In a further aspect, the 2 or more subjects are from different sexes. In yet another aspect, the 2 or more subjects are from different ethnic groups. In one aspect, the 2 or more subjects are from the same ethnic group. In another aspect, the subject is selected from the group consisting of animals, livestock, pets, and human subjects.

[0252] In one embodiment, the homogenate further comprises or consists essentially of or consists of one or more of non-human cells, human cells, a detectable label, a purified label, a non-natural protein, nucleic acid or polynucleotide, a small molecule, a dye, a virus, a bacterium, a parasite, a protozoan, or a chemical substance. In one aspect, the small molecule comprises or consists essentially of or consists of a hapten, a peptide tag, a protein, a fluorescent tag, a nucleic acid tag, and combinations thereof.

[0253] Method for generating representative data The present disclosure also relates to generating representative data from the representative samples or homogenate compositions described herein. In one aspect, a method for generating representative data includes analyzing a homogenate composition described herein. In a further aspect, the analysis includes generating quantitative and / or qualitative data regarding markers in the homogenate composition. Any suitable method for obtaining data associated with the markers can be used, and non-limiting examples of such include measurements by single cell sequencing, single nucleus sequencing, flow cytometry, immunohistochemical staining, hematoxylin and eosin staining, whole genome sequencing, high throughput sequencing, mass spectrometry, DNA microarray, or a combination thereof.

[0254] The methods described herein are, in part, a powerful means for generating a data set due to the unique sampling techniques and compositions of the present disclosure. The cellular structure and / or individual components of the representative sample essentially accurately reflect or mimic the relative proportions or percentages of these cellular structures (such as, but not limited to, type, constitution, and variations, etc.) in the entire tissue specimen, generally in solid tumors, lymph nodes, metastases, polyps, cysts, or any part or combination of the foregoing. Thus, a set of data derived from the analysis of a representative sample (or homogenate composition) or a subset thereof accurately reflects the corresponding information of the entire tissue or biological sample from which the representative sample is derived. In some embodiments, the representative data exhibits all the characteristics of the entire population of the original organ, tissue, or tumor from which the representative sample was derived.

[0255] As described above, a method for generating representative data includes, consists essentially of, or consists of generating quantitative and / or qualitative data regarding markers in the homogenate composition.

[0256] In the context of the methods and compositions of the present invention, a marker comprises, consists essentially of, or consists of a polynucleotide, DNA, protein, RNA, lipid, cell organelle, metabolite, or cell. In one aspect, the protein comprises a modification selected from the group consisting of acetylation, ADP-ribosylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination. In another aspect, a marker comprises, consists essentially of, or consists of a genomic polymorphism, a pharmacogenomic single nucleotide polymorphism (SNP), a genomic SNP, a somatic polymorphism, and differential expression of a protein, lipid, and / or cell organelle. In a further aspect, a marker comprises, consists essentially of, or consists of a single nucleotide position; an intronic or intergenic region; an exon or intron, or a fragment thereof; a coding or non-coding region; a promoter, enhancer, 5' untranslated region (5'UTR), or 3' untranslated region (3'UTR), or a fragment thereof; cDNA or a fragment thereof; an SNP; a somatic mutation, a germline mutation, or both; a point mutation or a single mutation; a deletion mutation; an in-frame deletion, an intragenic deletion, a whole gene deletion; an insertion mutation; an intragenic insertion; an inversion mutation; an intrachromosomal inversion; a ligation mutation; a ligation insertion mutation; an inverted duplication mutation; a tandem duplication; an intrachromosomal tandem duplication; a translocation; a chromosomal translocation, a non-reciprocal translocation; a rearrangement; a genomic rearrangement; a rearrangement of one or more introns, or a fragment thereof; a rearranged intron; a 5' or 3'UTR, or a combination thereof.In different embodiments, the marker comprises, consists essentially of, or consists of a changed nucleotide sequence encoding a changed amino acid sequence, a chromosomal translocation, an intrachromosomal inversion, a change in copy number, a change in expression level, a change in protein level, a change in protein activity, or a change in methylation state in cancer tissue or cancer cells as compared to normal, healthy tissue or cells.

[0257] In another aspect, the marker is a tumor marker selected from the group consisting of a protein, an antigen, an enzyme, a hormone, DNA, RNA, microRNA, or a carbohydrate. In a further aspect, the marker is Her2, bRaf, ERBB2, Pl3KCA, FGFR2, p53, BRCA, CCND1, MAP2K4, ATR, AFP, ALK, BCR-ABL, BRCA1 / BRCA2, BRAF, V600E, Ca-125, CA19.9, EGFR, Her-2, KIT, PSA, S100, KRAS, ER / Pr, UGT1A1, CD30, CD20, F1P1L1-PDGRFα, PDGFR, TMPT, TMPRSS2; ABCB5, AFP-L3, alpha-fetoprotein, alpha-methylacyl-CoA racemase, BRCA1, BRCA2, CA15-3, CA242, Ca27-29, CA-125, CA15-3, CA19-9, calcitonin, carcinoembryonic antigen, carcinoembryonic antigen peptide-1, des-gamma-carboxyprothrombin, desmin, early prostate cancer antigen-2, estrogen receptor, fibrin degradation product, glucose-6-phosphate isomerase, vE6, E7, L1, L2 or p16INK4a, human chorionic gonadotropin, IL-6, keratin 19, lactate dehydrogenase, leucyl aminopeptidase, lipotropin, metanephrine, neprylisin, NMP22, normetanephrine, PCA3, prostate specific antigen, prostate acid phosphatase, synaptophysin, thyroglobulin, TNF, ERG, ETV1(ER81), FLI1, EST1, EST2, ELK1, ETV6, ETV7, GABPα, ELF1, ETV4, ETV5, ERF, PEA3 / E1AF, PU.1, ESE1 / ESX, SAP1(ELK4), ETV3(METS), EWS / FLI1, ESE1, ESE2(ELF5), ESE3, PDEF, NET(ELK3;A tumor marker selected from the group consisting of a transcription factor selected from SAP2), NERF (ELF2), or FEV, XXX, tumor-associated glycoprotein 72, c-kit, SCF, pAKT, pc-kit, vimentin, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, KIR, TIM3, GAL9, GITR, LAG3, VISTA, KIR, 2B4, TRPO2, CD160, CGEN-15049, CHK1, CHK2, A2aR, TL1A, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, the B-7 family, or a combination thereof. In one aspect, the marker is a tumor marker comprising, consisting essentially of, or further consisting of genomic polymorphisms, pharmacogenomic single nucleotide polymorphisms (SNPs), genomic SNPs, somatic polymorphisms, and differential expression of proteins, lipids, and cell organelles. In another aspect, the tumor marker is a single nucleotide position; an intronic or intergenic region; an exon or intron, or a fragment thereof; a coding or non-coding region; a promoter, enhancer, 5'untranslated region (5'UTR), or 3'untranslated region (3'UTR), or a fragment thereof; cDNA or a fragment thereof; SNP; somatic mutations, germline mutations, or both; point mutations or single mutations; deletion mutations; in-frame deletions, intragenic deletions, whole gene deletions; insertion mutations; intragenic insertions; inversion mutations; intrachromosomal inversions; ligation mutations; ligation insertion mutations; inverted duplication mutations; tandem duplications; intrachromosomal tandem duplications; translocations; chromosomal translocations, non-reciprocal translocations; rearrangements; genomic rearrangements; rearrangement of one or more introns, or a fragment thereof; rearranged introns;It is selected from the group consisting of 5' or 3' UTR, or a combination thereof. In different embodiments, the marker is a "changed" amino acid sequence, chromosomal translocation, intrachromosomal inversion, copy number change, expression level change, protein level change, protein activity change, and methylation state change in cancer tissue or cancer cells, respectively, compared to normal, healthy tissue or cells, and includes, or alternatively consists essentially of, or further consists of, a marker derived from the group of changed nucleotide sequences encoding the same. ;

[0258] Data is generated from analyzing the homogenate compositions described herein. Non-limiting examples of tissues for analysis are samples selected from the group of one or more premalignant or malignant cells, solid tumors, soft tissue tumors or cells derived from metastatic lesions, tissues or cells derived from resection margins, histologically normal tissues, one or more circulating tumor cells (CTCs), normal adjacent tissues (NAT), blood samples from the same subject with or at risk of having a tumor, or FFPE samples.

[0259] In one embodiment, the representative data includes qualitative and quantitative data generated from a single marker. In one aspect, the representative data includes qualitative and / or quantitative data generated from two or more different markers. In a further aspect, the representative data is generated by measuring the same or multiple markers at different time points, for example, before and after therapy, and in one aspect, this can be used to monitor the therapy or the patient's condition over the course of treatment.

[0260] In one embodiment, a method for generating representative data further comprises, consists essentially of, or consists of, assigning internal values to qualitative and / or quantitative data. In another embodiment, a method for generating representative data further comprises, consists essentially of, or consists of, comparing the representative data with a predetermined value for the data. In a further aspect, the measured value of a marker is normalized and a composite score is obtained based on the normalized measured value of the marker. The composite score can be further compared with a predetermined score. In the context of cancer, in yet another embodiment, a method for generating representative data further comprises, consists essentially of, or consists of: (a) measuring a tumor marker in a first biological sample, wherein the measured value of the tumor marker is normalized; (b) obtaining a composite score based on the normalized measured value; and (c) comparing the composite score with a predetermined score to determine a cancer risk value for the subject.

[0261] In another embodiment, the predetermined value is selected from clinical trial data, data regarding a subject, data derived from scientific literature, and data regarding a biologic or small molecule in clinical development. In one aspect, the representative data comprises, consists essentially of, or consists of representative oncology data, and the representative oncology data comprises, consists essentially of, or consists of quantitative and / or qualitative data of at least one tumor marker derived from a first biological sample, wherein the tumor marker is associated with the presence of a tumor.

[0262] In one embodiment, the predetermined score is derived from clinical trial data, representative oncology data derived from a second biological sample, representative oncology data derived from a group of biological samples, and a group of data for the clinical development of a biologic or small molecule.

[0263] Method for determining a phenotypic profile In one embodiment, the present disclosure relates to a method of determining a phenotypic profile of a tissue sample, the method comprising, consisting essentially of, or consisting of, analyzing the cellular structure of a homogenate composition. In one aspect, the cellular structure that can be analyzed for the phenotypic profile includes, consists essentially of, or consists of cell clusters, individual cells, cell fragments, organelles, peptides, nucleic acids, lipids, metabolites, or combinations thereof. In another aspect, the cellular structure includes a single cell or nucleus, and the single cell or nucleus is intact. In some embodiments, the analysis includes, consists essentially of, or consists of analyzing the number, type, state, percentage, and / or expression of the cellular structure. In another aspect, the analysis includes, consists essentially of, or consists of single cell analysis, single nucleus analysis, single organelle analysis, or combinations thereof. In one aspect, the state of the cellular structure includes, consists essentially of, or consists of proliferation, apoptosis, necrosis, migration, epithelial-mesenchymal transition (“EMT”), mitosis, cell cycle arrest, S phase, senescence, and / or differentiation. In another aspect, the analysis includes, consists essentially of, or consists of analyzing markers derived from the homogenate.

[0264] In one embodiment, the marker is selected from the group consisting of DNA, protein, RNA, lipids, cell organelles, metabolites, or cells. In one aspect, the analysis of the marker includes, consists essentially of, or consists of detecting the marker. In another aspect, the analysis of the marker includes analyzing the marker derived from a single cell or a single nucleus.

[0265] Method of treating a disease In another aspect, the present disclosure relates to a method of treating a disease by selecting an effective treatment regimen based on representative data generated using the methods of the present disclosure. Using accurate amounts or types of information derived from a patient, the treatment regimen can be adjusted for the best response and highest safety margin to achieve an outcome regarding the patient. Further, with this information, the patient can also significantly improve the quality of health management by receiving many other benefits, such as earlier diagnosis, risk assessment, and effective treatment.

[0266] The selection of an effective treatment regimen depends on several factors. First, it is necessary to achieve a reliable diagnosis of the disease or medical condition. In the case of infectious diseases, cancer, or other acute life-threatening diseases, this diagnosis must be rapid and efficient because time plays a decisive role in the survival rate of patients suffering from these diseases. Second, the treatment of an individual patient is more effective when the diagnosis is accurate. For example, when cancer is sometimes treated with a standard "cocktail" of anticancer drugs, the cocktail often shows severe side effects for the patient. Unless the type of cancer (or other disease) is accurately determined, an individual treatment regimen for this type of disease is not necessarily more effective than any other treatment regimen. Thus, effectiveness directly depends on data or information obtained from the patient being treated. A more effective treatment is expected to be possible when the treatment regimen cross-checks with a regimen that has already been successfully applied to this or other patients based on the physiological response to the initial regimen. Further, an accurate diagnosis of the disease results in a reduction in the cost of an individual treatment regimen because unnecessary and ineffective drugs are avoided. However, currently, even those with up-to-date treatment information require time to absorb that information and understand how it relates to other treatment information to provide the best available treatment for the patient.

[0267] Accordingly, the present disclosure provides a method of treating a disease in a subject, the method comprising, consisting essentially of, or consisting of selecting an appropriate treatment regimen based on representative data including a first profile of the subject.

[0268] In one aspect, non-limiting examples of the first profile include, consist essentially of, or consist of a profile derived from the group of marker profile, antigen profile, protein profile, mutation profile, lipid profile, exosome profile, or a combination thereof. In another aspect, the markers are Her2, bRaf, ERBB2, Pl3KCA, FGFR2, p53, BRCA, CCND1, MAP2K4, ATR, AFP, ALK, BCR-ABL, BRCA1 / BRCA2, BRAF, V600E, Ca-125, CA19.9, EGFR, Her-2, KIT, PSA, S100, KRAS, ER / Pr, UGT1A1, CD30, CD20, F1P1L1-PDGRFα, PDGFR, TMPT, TMPRSS2; ABCB5, AFP-L3, alpha-fetoprotein, alpha-methylacyl-CoA racemase, BRCA1, BRCA2, CA15-3, CA242, Ca27-29, CA-125, CA15-3, CA19-9, calcitonin, carcinoembryonic antigen, carcinoembryonic antigen peptide-1, des-gamma-carboxyprothrombin, desmin, early prostate cancer antigen-2, estrogen receptor, fibrin degradation products, glucose-6-phosphate isomerase, vE6, E7, L1, L2 or p16INK4a, human chorionic gonadotropin, IL-6, keratin 19, lactate dehydrogenase, leucyl aminopeptidase, lipotropin, metanephrine, neprilysin, NMP22, normetanephrine, PCA3, prostate specific antigen, prostate acid phosphatase, synaptophysin, thyroglobulin, TNF, ERG, ETV1(ER81), FLI1, EST1, EST2, ELK1, ETV6, ETV7, GABPα, ELF1, ETV4, ETV5, ERF, PEA3 / E1AF, PU.1. A transcription factor selected from ESE1 / ESX, SAP1 (ELK4), ETV3 (METS), EWS / FLI1, ESE1, ESE2 (ELF5), ESE3, PDEF, NET (ELK3; SAP2), NERF (ELF2), or FEV, XXX, tumor-associated glycoprotein 72, c-kit, SCF, pAKT, pc-kit, vimentin, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, KIR, TIM3, GAL9, GITR, LAG3, VISTA, KIR, 2B4, TRPO2, CD160, CGEN-15049, CHK1, CHK2, A2aR, TL1A, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, the B-7 family, and combinations thereof, comprising one or more derived from the group consisting of, or alternatively consisting essentially of, or even consisting of. In some embodiments, the first profile comprises a profile generated from a group of one or more markers, one or more antigens, one or more proteins, one or more mutations, one or more lipids, one or more exosomes, or combinations thereof..

[0269] In some embodiments, the markers of the method are selected from the group consisting of genomic polymorphisms, pharmacogenomic single nucleotide polymorphisms (SNPs), genomic SNPs, somatic polymorphisms, and differential expression of proteins, lipids, and cell organelles. In one aspect, the marker is a single nucleotide position; an intragenic or intergenic region; an exon or intron, or a fragment thereof; a coding or non-coding region; a promoter, enhancer, 5' untranslated region (5'UTR), or 3' untranslated region (3'UTR), or a fragment thereof; cDNA or a fragment thereof; SNP; somatic mutation, germline mutation or both; point mutation or single mutation; deletion mutation; in-frame deletion, intragenic deletion, whole gene deletion; insertion mutation; intragenic insertion; inversion mutation; intrachromosomal inversion; ligation mutation; ligation insertion mutation; inversion duplication mutation; tandem duplication; intrachromosomal tandem duplication; translocation; chromosomal translocation, non-reciprocal translocation; rearrangement; genomic rearrangement; rearrangement of one or more introns, or a fragment thereof; rearranged intron; 5' or 3'UTR, and combinations thereof.

[0270] The method can be used to determine the use of a treatment regimen that includes, consists essentially of, or further consists of an individualized dosage regimen. In one aspect, the treatment regimen is selected from the group consisting of chemotherapy, immunotherapy, radiation, surgery, gene therapy, hormone therapy, stem cell therapy, blood transfusion, physical therapy, photodynamic therapy, and combinations thereof.

[0271] In another embodiment, a method of treating a disease in a subject further includes, consists essentially of, or further consists of comparing a first profile of the subject with a predetermined profile to determine whether a treatment regimen is appropriate for the subject. In one aspect, the predetermined profile is determined based on data selected from the group consisting of clinical trial data, a second profile of the subject, profiles of different biological samples or groups of biological samples, profiles of different subjects or groups of subjects, data regarding biologics or small molecules, and combinations thereof.

[0272] In a further aspect, the treatment involves the selection of one or more drugs and / or the dosage (amount, length of administration, etc.) of such drugs to be administered to the patient to individualize the treatment based on the patient's individual tissue or cancer profile. For example, if two biomarkers in a representative sample predict response to a specific drug X and a different drug Y found within a single representative sample, both drugs can be administered to the patient. If the biomarker for drug X is present within 75% and the biomarker for drug Y is present at 25%, drug X can be prioritized and delivered first, followed by drug Y. Alternatively, drug Y may precede drug X.

[0273] The method can be repeated at different time points of the therapy and modified based on changes in the marker expression of the profile. In this aspect, the method is useful for monitoring therapy and disease progression in a patient or among different patients having the same or similar diseases undergoing the same or different therapies.

[0274] Method for identifying clinically relevant markers Data or information regarding clinically relevant markers or biomarkers can provide an indication of the likelihood of a medical condition. Using the representative data in the present disclosure, clinically relevant markers, particularly those not previously associated with any medical condition, can be identified.

[0275] Thus, another aspect of the present disclosure relates to a method of identifying a clinically relevant marker, which includes comparing representative data with predetermined data. Non-limiting examples of markers are those described above. In one aspect, the marker is selected from the group consisting of a protein, an antigen, an enzyme, a hormone, DNA, RNA, microRNA, or a carbohydrate. In another aspect, the marker comprises, consists essentially of, or consists of genomic polymorphisms, pharmacogenomic single nucleotide polymorphisms (SNPs), genomic SNPs, somatic polymorphisms, and differential expression of proteins, lipids, protein modifications, and cell organelles. In some aspects, the marker is a single nucleotide position; an intronic or intergenic region; an exon or intron, or a fragment thereof; a coding or non-coding region; a promoter, enhancer, 5'untranslated region (5'UTR), or 3'untranslated region (3'UTR), or a fragment thereof; cDNA or a fragment thereof; an SNP; a somatic mutation, a germline mutation, or both; a point mutation or a single mutation; a deletion mutation; an in-frame deletion, an intragenic deletion, a whole gene deletion; an insertion mutation; an intragenic insertion; an inversion mutation; an intrachromosomal inversion; a ligation mutation; a ligation insertion mutation; an inversion duplication mutation; a tandem duplication; an intrachromosomal tandem duplication; a translocation; a chromosomal translocation, a non-reciprocal translocation; a rearrangement; a genomic rearrangement; a rearrangement of one or more introns, or a fragment thereof; a rearranged intron; a 5'or 3'UTR, and combinations thereof, selected from the group consisting of. In one aspect, the marker is selected from the group consisting of a changed nucleotide sequence encoding a changed amino acid sequence, a chromosomal translocation, an intrachromosomal inversion, a change in copy number, a change in expression level, a change in protein level, a change in protein activity, and a change in methylation state in cancer tissue or cancer cells as compared to normal, healthy tissue or cells, respectively.

[0276] In one embodiment, the predetermined data is generated from data selected from the group consisting of clinical trial data, data of a subject or a group of subjects, data of a tissue sample or a group of tissue samples, data of a biologic or small molecule in clinical development, or a combination thereof.

[0277] In one aspect, a method for determining the prognosis of cancer in a subject is provided, the method including evaluating representative data derived from the subject. In one embodiment, the method for determining the prognosis of cancer further includes, consists essentially of, or consists of calculating quantitative data regarding the prognosis of cancer, and the prognosis is classified based on a quantitative score.

[0278] In one embodiment, the representative data includes information regarding the number, type, state, and / or percentage of cell structures in the homogenate. In another embodiment, the cell structures include, consist essentially of, or consist of stem cells, epithelial cells, blood cells, adipocytes, skin cells, endothelial cells, cancer cells, or immune cells. In one embodiment, the immune cells include, consist essentially of, or consist of neutrophils, monocytes, macrophages, dendritic cells, natural killer cells, T cells, and / or B cells. In some embodiments, the T cells include, consist essentially of, or consist of killer T cells, helper T cells, regulatory T cells, pan T cells, naive T cells, activated T cells, and / or gamma delta T cells.

[0279] In one embodiment, the state of the cell structure comprises, consists essentially of, or consists of proliferation, apoptosis, necrosis, migration, epithelial-mesenchymal transition ("EMT"), mitosis, cell cycle arrest, S-phase, senescence, and / or differentiation. In one embodiment, the representative data includes information regarding markers in the homogenate. In another embodiment, the marker is selected from DNA, protein, RNA, lipid, cell organelles, metabolites, or groups of cells. In some embodiments, the marker is Her2, bRaf, ERBB2, Pl3KCA, FGFR2, p53, BRCA, CCND1, MAP2K4, ATR, AFP, ALK, BCR-ABL, BRCA1 / BRCA2, BRAF, V600E, Ca-125, CA19.9, EGFR, Her-2, KIT, PSA, S100, KRAS, ER / Pr, UGT1A1, CD30, CD20, F1P1L1-PDGRFα, PDGFR, TMPT, TMPRSS2; ABCB5, AFP-L3, alpha-fetoprotein, alpha-methylacyl-CoA racemase, BRCA1, BRCA2, CA15-3, CA242, Ca27-29, CA-125, CA15-3, CA19-9, calcitonin, carcinoembryonic antigen, carcinoembryonic antigen peptide-1, des-gamma-carboxyprothrombin, desmin, early prostate cancer antigen-2, estrogen receptor, fibrin degradation products, glucose-6-phosphate isomerase, vE6, E7, L1, L2 or p16INK4a, human chorionic gonadotropin, IL-6, keratin 19, lactate dehydrogenase, leucyl aminopeptidase, lipotropin, metanephrine, neprylisin, NMP22, normetanephrine, PCA3, prostate specific antigen, prostate acid phosphatase, synaptophysin, thyroglobulin, TNF, ERG, ETV1(ER81), FLI1, EST1, EST2, ELK1, ETV6, ETV7, GABPα, ELF1, ETV4, ETV5, ERF, PEA3 / E1AF, PU.1, ESE1 / ESX, SAP1(ELK4), ETV3(METS), EWS / FLI1, ESE1, ESE2(ELF5), ESE3, PDEF, NET(ELK3;A transcription factor selected from SAP2), NERF (ELF2), or FEV, XXX, tumor-associated glycoprotein 72, c-kit, SCF, pAKT, pc-kit, vimentin, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, KIR, TIM3, GAL9, GITR, LAG3, VISTA, KIR, 2B4, TRPO2, CD160, CGEN-15049, CHK1, CHK2, A2aR, TL1A, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, the B-7 family, and one or more of combinations thereof, or alternatively consists essentially of or further consists of the foregoing. In another embodiment, the marker is selected from the group consisting of protein modifications, said modifications being selected from the group consisting of acetylation, ADP-ribosylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme, covalent attachment of nucleotide or nucleotide derivative, covalent attachment of lipid or lipid derivative, covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslink, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination. In some embodiments, the marker is a genomic polymorphism, a pharmacogenomic single nucleotide polymorphism (SNP), a genomic SNP, a somatic polymorphism, and differential expression of proteins, lipids, and / or cell organelles, a single nucleotide position; an intragenic region or an intergenic region; an exon or an intron, or a fragment thereof; a coding region or a non-coding region; a promoter, an enhancer, a 5' untranslated region (5'UTR), or a 3' untranslated region (3'UTR), or a fragment thereof; cDNA or a fragment thereof; SNP; somatic mutation, germline mutation or both; point mutation or single mutation; deletion mutation; in-frame deletion, intragenic deletion, whole gene deletion; insertion mutation; intragenic insertion; inversion mutation;Intrachromosomal inversion; linked mutation; linked insertion mutation; inversion duplication mutation; tandem duplication; intrachromosomal tandem duplication; translocation; chromosomal translocation, non-reciprocal translocation; rearrangement; genomic rearrangement; rearrangement of one or more introns, or fragments thereof; rearranged intron; a changed nucleotide sequence encoding a 5' or 3' UTR, a changed amino acid sequence, a chromosomal translocation, an intrachromosomal inversion, a change in copy number, a change in expression level, a change in protein level, a change in protein activity, or a change in methylation state in a cancer tissue or cancer cell as compared to a normal, healthy tissue or cell, selected from the group consisting of.;

[0280] In another aspect, the present disclosure relates to a method of monitoring a disease in a patient, comprising, or alternatively consisting essentially of, or further consisting of, analyzing a clinically relevant marker, wherein the clinically relevant marker is identified based on representative data. In one aspect, the marker is selected from the group consisting of a protein, an antigen, an enzyme, a hormone, DNA, RNA, microRNA, or a carbohydrate. In another aspect, the marker is DNA or RNA isolated from a sample selected from the group consisting of one or more premalignant or malignant cells, solid tumors, soft tissue tumors or metastatic lesions, tissue or cells derived from a resection margin, histologically normal tissue, one or more circulating tumor cells (CTCs), normal adjacent tissue (NAT), a blood sample from the same subject with or at risk of having a tumor, or an FFPE sample. In one aspect, the disease is cancer.

[0281] Method for preserving a representative sample (or homogenate composition) Once a representative sample has been constructed, the sample can be transported for further processing and / or analysis. Thus, the present disclosure also relates, in some embodiments, to a method of preserving a homogenate composition, comprising, or alternatively consisting essentially of, or further consisting of, mixing the composition with an effective amount of a preservation reagent, non-limiting examples of which are provided herein.

[0282] In one embodiment, the storage reagent comprises, consists essentially of, or consists of a preservative, a chaotropic agent, a surfactant, a reducing agent, a chelating agent, a buffer, or a combination thereof. In one aspect, the mixed composition retains the phenotypic and genotypic characteristics of the composition prior to mixing with the storage reagent. In another aspect, the mixed composition comprises, consists essentially of, or consists of a denatured protein, an inactivated nuclease, an inactivated protease, an inactivated pathogen, a non-degraded nucleic acid, or a combination thereof. In some aspects, the chaotropic agent comprises, consists essentially of, or consists of guanidine thiocyanate, guanidine isothiocyanate, guanidine hydrochloride, or a combination thereof. In one aspect, the surfactant comprises, consists essentially of, or consists of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or a combination thereof. In another aspect, the reducing agent comprises, consists essentially of, or consists of mercaptoethanol, tris(2-carboxyethyl)phosphine, dithiothreitol, dimethyl sulfoxide, tris(2-carboxyethyl)phosphine, or a combination thereof. In a further aspect, the chelating agent comprises, consists essentially of, or consists of ethylene glycol tetraacetic acid, hydroxyethylethylenediamine triacetic acid, diethylenetriamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediamine tetraacetic acid, citric anhydride, sodium citrate, calcium citrate, ammonium citrate, diammonium citrate, citric acid, diammonium citrate, ammonium iron citrate, lithium citrate, or a combination thereof.In different embodiments, the buffer comprises, consists essentially of, or consists of tris(hydroxymethyl)aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1,3-bis(tris(hydroxymethyl)methylamino)propane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 3-(N-morpholine)propanesulfonic acid, bicarbonate, phosphate, or combinations thereof.

[0283] Method for generating representative samples The present disclosure generally relates to the development of methods for generating representative tissue samples of, for example, whole organs, tumors, lymph nodes, metastases, or combinations thereof, for addressing issues of heterogeneity, such as tumor heterogeneity, in clinical specimens, particularly clinical specimens for use in clinical oncology, and the use of such representative samples or portions thereof in various diagnostic and therapeutic methods, as well as compositions comprising such representative samples for use in diagnostics and therapies, particularly in oncology.

[0284] This application shows that acceptable sampling methods for cancer diagnosis, which use small samples of tumors for diagnostic testing, can introduce significant sampling bias in diagnostic pathology and oncology. Decisions regarding patient care, both prognostic (e.g., expected survival time of a patient) and predictive (e.g., whether a patient will respond to a particular therapy), are often made using a single FFPE tissue section in tumors considered "small," i.e., tumors that are 2 centimeter masses. Using conventional methods, all diagnostic data are typically taken from less than 0.03% of the tumor volume (i.e., a single section from an FFPE block). Further, these tissue samples from tumors are conventionally taken from very distinct areas of the tumor, and diagnostic oncology becomes independent of the heterogeneity present in the remaining tumor. As a result, the dataset is small (compared to the population) and, consequently, biased.

[0285] Similarly, just as the probability of detecting a small subpopulation of genetically distinct cancer cells within a solid tumor is low, small metastatic tumors within lymph nodes surrounding the primary tumor site cannot be detected using conventional histological examinations. Lymph nodes range in size from directly 1 millimeter to several centimeters. The presence of tumor cells within a lymph node depends on DNA mutations that result in the motility and invasion of tumor cells, as well as mutations that confer the ability to survive in a new environment (i.e., breast versus lymph node). The size of a metastatic tumor within a lymph node depends on the growth rate of the tumor and the length of time the metastatic tumor has been growing within the lymph node. Diagnostic tests for the presence of tumor cells within a lymph node use one or two tissue sections (typically 4 μm thick) derived from FFPE blocks. An important factor for detection using such methods is the size of the tumor relative to the size of the lymph node. A metastatic tumor that is 0.1 mm in diameter fills 10% of the volume of a small lymph node and may have a reasonable probability of detection, whereas a tumor of the same size occupies only 0.005% of a lymph node that is 2 centimeters in diameter, and the probability of detection is very low when using current histological techniques. A significant number of patients mislabeled as node-negative using conventional techniques can be detected and more appropriately treated using the methods disclosed herein. For example, more sensitive detection of node-positive patients can better inform the decision of whether to administer adjuvant chemotherapy.

[0286] IHC analysis of representative samples derived from lymph node tissue (e.g., prepared from surgically removed lymph nodes) can detect very small tumor micrometastases by staining epithelial markers in combination with proliferation markers (e.g., cytokeratin 8 / 18 dual IHC using Ki67). This can be achieved by using other markers of metastatic cells or markers that were positive in the primary tumor using other diagnostic markers. Also, metastatic tumor cells can be detected by identifying nucleic acids, for example, by using next-generation sequencing panels for identifying cancer-related mutations, including mutations present in the primary tumor. These methods identify metastatic tumor cells. Furthermore, the course of the natural evolution of the disease for each patient can be determined and correlated with specific mutations that can be targeted for therapy.

[0287] Current clinical practice for evaluating tumors involves obtaining only small portions of tumor tissue for embedding and sectioning. In a basic scenario, the location of the region within the tumor containing the subclone of interest is assumed to be a random event. Thus, current practice carefully instructs on which regions to sample and to obtain the most appropriate information for TNM stage classification, which is not necessarily useful for localizing subclone regions of the tumor. Also, it is not possible to determine from gross inspection whether subclones are present.

[0288] Embodiments of the present method can address tumor heterogeneity in a clinical oncology setting by providing a method for the efficient and reproducible production of cell samples representative of a patient's entire lymph nodes, tumor(s). As shown in FIG. 2A, a "representative" sample according to the present disclosure includes different partial subpopulations of cancer cells contained within the tumor, regardless of its size. A "representative" sample according to the present disclosure may alternatively include different partial subpopulations within a normal or control population or a mixed sample of tumor cells and normal cells.

[0289] Alternatively, representative samples according to the present disclosure may comprise representative or homogeneous biomolecules derived from a whole tumor, lymph node, or metastasis, which are fractions containing proteins, lipids, nucleic acids, or other moieties present in the starting tissue, e.g., whole tumor, lymph node, or metastasis, used to derive the sample or a fraction thereof, and the relative proportions of such substitutions again represent the starting tissue. For example, such biomolecules can be derived by further dissociation or chemical or enzymatic treatment of the sample and / or by use of methods to isolate or remove specific portions of the sample, e.g., size exclusion, e.g., sieving, to isolate or remove molecules of a specific size or molecular weight, affinity purification methods to isolate or remove specific types of molecules from the representative sample, etc. Thus, such methods essentially result in other types of representative samples according to the present disclosure, e.g., homogeneous or representative samples containing all proteins, nucleic acids, or lipids of the starting sample, e.g., whole tumor, lymph node, metastasis, or organ, and such representative samples can be used for protein, nucleic acid, and / or lipid analysis methods, which reflect the whole tumor sample.

[0290] Thus, regardless of origin, the relative percentage of the cell subpopulation of the starting tissue, e.g., a tumor (s) present in a tumor or lymph node metastasis or organ, or other specific portion, in such representative samples is accurately reflected in the sample. Further, these representative samples, unlike samples obtained by conventional diagnostic methods, can be used in multiple assay methods without impairing the ability to use the specimen in traditional diagnostic assays. Further, representative samples produced according to the present disclosure can be used separately or simultaneously (potentially reused) in several different assay formats to detect the presence of further minor subclone populations or other moieties such as tumor antigens or nucleic acids within the sample, e.g., tumor, lymph node, or metastasis.

[0291] Furthermore, as discussed below, representative samples derived from different patients or from different tissues of a single or different patients can be labeled with unique identification labels, such as haptens, respectively, and the labeled samples or tissues of different patients can be combined and used in a desired assay method. In essence, this provides multiplexing of different patient samples.

[0292] Based thereon, representative samples derived by way of exemplary embodiments of the methods described herein should facilitate the detection, diagnosis, and / or staging of different types of tumors, i.e., different solid tumors, regardless of the histotype, location, size, or volume of the tumor, and substantially improve the accuracy thereof. Also, using the methods, representative samples can be produced from the proposed normal tissue samples or putative pre-cancerous tissues (e.g., obtained from subjects at high risk of developing cancer due to genetic risk or previous cancer) to identify rare cell types, such as cancer stem cell lines, that may be present therein even before the symptoms of the disease appear.

[0293] In a further embodiment, the disclosure relates to a method for preparing a tissue or biological sample containing a heterogeneous cell structure, comprising: (a) homogenizing the sample; and (b) reconstructing the homogenized sample into a homogenate containing a substantially uniform cell structure, wherein the ratio of the cell structures in a subset of the homogenate is substantially similar to the ratio of the cell structures in the tissue sample. In one aspect, the homogenate comprises, consists essentially of, or consists of a plurality of single cells or a plurality of cell clusters.

[0294] In one embodiment, the method for preparing a tissue sample comprises, consists essentially of, or consists of fixing the homogenate with a fixative. In another aspect, the fixative comprises, consists essentially of, or consists of formalin, calcium, acetic acid, saline, alcohol, urea, bronopol, water, or a combination thereof. In different aspects, the fixed homogenate is mounted on a slide.

[0295] In some embodiments, a method for preparing a tissue sample further comprises, consists essentially of, or consists of extracting components from the homogenate. In one aspect, the components are DNA, RNA, protein, lipid, cell organelles, exosomes, cells, or combinations thereof. In another embodiment, a method for preparing a tissue sample further comprises, consists essentially of, or consists of isolating cell structures or components from the homogenate. In one aspect, the cell structures or components include, consist essentially of, or consist of a single cell or a single nucleus. In one aspect, the isolation includes, consists essentially of, or consists of isolating a single cell or a single nucleus. In another aspect, the isolation of a single cell is performed by flow cytometry, laser microdissection, manual cell selection, random seeding and dilution, microfluidics devices, lab-on-a-chip devices, or combinations thereof. In one aspect, the isolation of a single nucleus is performed by flow cytometry.

[0296] In one embodiment, homogenization comprises, consists essentially of, or consists of chemical and / or biochemical dissociation and / or optionally mechanical homogenization. In one aspect, the homogenization process does not lyse cells. In another aspect, the chemical treatment of the sample comprises, consists essentially of, or consists of enzymatic digestion of the sample, said enzymatic digestion comprising the use of an enzyme selected from the group 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 pyroglutamic acid aminopeptidase. In one aspect, mechanical homogenization is performed by a device selected from the group consisting of a blender, a disassociator, an extractor, a mortar, a pestle, a Dounce-type homogenizer, a tissue grinder, a rotary blade tissue homogenizer, and a bead-type homogenizer. In another aspect, the homogenate is created by hand mincing using a surgical scalpel or knife. In one embodiment, homogenization further comprises, consists essentially of, or consists of conditioning of the cells, said conditioning of the cells comprising adjusting the pH and heat or treating the sample with a cell conditioning buffer.

[0297] In some embodiments, the sample is treated with hormones, proteins, enzymes, lipids, surfactants, sonication, physical agitation, or combinations thereof, before or after homogenizing the sample.

[0298] In a further embodiment, the homogenized sample comprises, consists essentially of, or consists of cells and / or cell clusters. In some embodiments, the homogenized sample comprises, consists essentially of, or consists of cell clusters of uniform size. In another embodiment, the homogenized sample comprises, consists essentially of, or consists of cell clusters of non-uniform cells. In some embodiments, the cell clusters comprise, consist essentially of, or consist of 1-100 cells, or 100-1,000 cells, 1,000-10,000 cells, or 10,000-100,000 cells. In a further embodiment, the cell clusters comprise, consist essentially of, or consist of more than 100,000 cells.

[0299] In some embodiments, a method for preparing a tissue sample comprises passing the homogenized sample through a mesh, filter, or series of meshes or filters. In one embodiment, the mesh or filter has a pore size in the range of about 1 μm to about 500 μm. In another embodiment, the mesh or filter has a pore size of less than 1 μm. In some embodiments, the mesh or filter has a pore size in the range of about 1 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 300 μm, about 300 μm to about 400 μm, or about 400 μm to about 500 μm. In a further embodiment, the mesh or filter has a pore size of greater than 500 μm.

[0300] In a further embodiment, the tissue sample is collected from a tissue selected from the group consisting of tumor, lymph node, metastasis, polyp, cyst, biopsy, whole organ, and combinations thereof. In one aspect, the tissue sample is a solid sample or a liquid sample. In another aspect, the liquid sample comprises, consists essentially of, or consists of aspirated needle biopsy cytology, effusion sample, or Pap smear. In one aspect, the cell structure of the homogenate comprises, consists essentially of, or consists of at least one cell. In different aspects, the cell structure of the homogenate comprises, consists essentially of, or consists of at least 100 cells. In another aspect, the cell structure of the homogenate comprises about 100 - about 200 cells, about 200 - about 1,000 cells, about 1,000 - about 5,000 cells, or about 10,000 - about 100,000 cells. In different aspects, the cell structure of the homogenate comprises, consists essentially of, or consists of about 100,000 - about 1,000,000 cells, about 1,000,000 - about 5,000,000 cells, about 5,000,000 - about 1,000,000,000 cells, or about 1,000,000,000 - about 5,000,000,000 cells. In a further aspect, the cell structure of the homogenate comprises, consists essentially of, or consists of more than about 5,000,000,000 cells.

[0301] In one embodiment, the homogenate is not preserved or fixed. In one aspect, the homogenate comprises, consists essentially of, or consists of live cells. In another aspect, the homogenate is preserved or fixed. In one aspect, the homogenate is frozen, lyophilized, or embedded in an embedding medium. In a further aspect, the homogenate comprises cells from one or more tissues and / or one or more subjects.

[0302] In one embodiment, the tissue sample is isolated from a tumor, lymph node, metastasis, polyp, cyst, resection, whole organ, or a combination thereof. In one aspect, the homogenate further comprises, consists essentially of, or consists of non-human cells, human cells, non-natural proteins, nucleic acids, or small molecules. In one embodiment, the small molecule is selected from the group consisting of a hapten, peptide tag, protein tag, fluorescent tag, nucleic acid tag, and combinations thereof. In a further aspect, the small molecule comprises, consists essentially of, or consists of a hapten, peptide tag, protein tag, fluorescent tag, nucleic acid tag, luminescent tag, biotin, and combinations thereof.

[0303] In a further embodiment, the method for preparing the tissue sample further comprises, consists essentially of, or consists of evaluating a substantially uniform cell structure within the homogenate. In one aspect, the substantially uniform cell structure is evaluated by measuring the distribution of an internal control within the homogenate. In another aspect, the internal control is selected from the group consisting of non-human cells, human cells, non-natural proteins, nucleic acids, small molecules, dyes, chemicals, and combinations thereof.

[0304] In another aspect, the present disclosure provides a method for producing a biological sample suitable for assessing the heterogeneity of cells within a sample (such as a tumor sample or lymph node or metastasis or combination thereof) and / or for assessing the prognosis of a particular cancer condition in a subject, the method comprising: (i) obtaining one or more intact biopsy samples from a solid tumor or lymph node, preferably each biopsy sample containing at least about 100 - 200; 200 - 1,000; 1,000 - 5,000; 10,000 - 100,000; 100,000 - 1,000,000; 1,000,000 - 5,000,000; 5,000,000 - 1,000,000,000; 1,000,000,000 - 5,000,000,000 cells and optionally being fixed or preserved (a sample fixed or preserved with formalin, paraffin, or ethanol), and (ii) homogenizing one or more biopsy samples separately or in combination such that each of the one or more homogenates substantially uniformly represents the heterogeneity of the corresponding biopsy sample(s).

[0305] As described, optionally, these representative samples can be further dissociated and / or processed to remove or isolate specific types of molecules, such as specific cell types, proteins, nucleic acids, or lipids, to generate other representative samples that can be used in diagnostic and therapeutic methods.

[0306] In yet another aspect, the present disclosure provides a method of producing a biological sample suitable for evaluating the heterogeneity of cells in a tumor or lymph node or metastasis or a combination thereof, the method comprising: (i) obtaining one or more biopsy samples from a solid tumor or lymph node or metastasis, wherein preferably each biopsy sample contains at least about 100-200; 200-1,000; 1,000-5,000; 10,000-100,000; 100,000-1,000,000; 1,000,000-5,000,000; 5,000,000-1,000,000,000; 1,000,000,000-5,000,000,000 cells and is optionally fixed or preserved (samples fixed or preserved with formalin, paraffin, or ethanol); and (ii) homogenizing the one or more biopsy samples separately or in combination under conditions such that the resulting homogenate(s) is substantially dissociated into individual cells and the resulting homogenate(s) is substantially homogeneous.

[0307] Again, optionally, these representative samples can be further dissociated and / or processed to remove or isolate specific types of molecules, such as specific cell types, proteins, nucleic acids, or lipids, to generate other representative samples that can be used in diagnostic and therapeutic methods.

[0308] In another aspect, the present disclosure provides a method for producing a biological sample suitable for evaluating whether a subject has or is at risk of developing a particular cancer of a pathogenic type and / or whether a subject with cancer has that particular cancer of the pathogenic type, the method comprising: (i) obtaining one or more intact biopsy samples from a solid tumor or lymph node or metastasis or pre-cancerous cyst, preferably wherein each biopsy sample contains at least about 100 - 200; 200 - 1,000; 1,000 - 5,000; 10,000 - 100,000; 100,000 - 1,000,000; 1,000,000 - 5,000,000; 5,000,000 - 1,000,000,000; 1,000,000,000 - 5,000,000,000 cells and is optionally fixed or preserved (formalin, paraffin, or ethanol-fixed or preserved samples); and (ii) separately or in combination homogenizing the one or more biopsy samples such that the resulting homogenate(s) is substantially dissociated into individual cells and the resulting one or more homogenates each substantially uniformly represent the heterogeneity of the corresponding biopsy sample(s), and optionally isolating or detecting the expression of at least one biomarker. Upregulation or downregulation of the biomarker is associated with the particular cancer of the pathogenic type.

[0309] In yet another aspect, the present disclosure provides a method for characterizing phenotypic diversity within a heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst, and / or for detecting genetically distinct subclones within a heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst, and / or for identifying low-frequency events within a tumor, lymph node, metastasis, or pre-cancerous cyst, and / or for determining the prevalence of a target within a tumor, lymph node, metastasis, or pre-cancerous cyst, the method comprising: (i) optionally, obtaining a sample(s) of a tumor, lymph node, metastasis, or pre-cancerous cyst that has been fixed or preserved, e.g., using formalin, paraffin, and / or ethanol, prior to homogenization, the sample(s) including spatially distinct regions of the tumor, lymph node, metastasis, or pre-cancerous cyst; and (ii) producing a homogenate by homogenizing the sample(s) of the tumor, lymph node, metastasis, or pre-cancerous cyst, the homogenate being representative of the phenotypic diversity of the heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst, characterizing the tumor context, and / or detecting genetically distinct subclones within the heterogeneous tumor, lymph node, metastasis, or pre-cancerous cyst, and / or identifying low-frequency events within the tumor, lymph node, metastasis, or pre-cancerous cyst, and / or determining the prevalence of a target within the tumor, lymph node, metastasis, or pre-cancerous cyst.

[0310] In yet another aspect, the present disclosure provides a method for detecting precancerous or cancerous cells in a proposed normal tissue or putative precancerous tissue in a patient who is at risk of developing cancer, for example due to a genetic mutation or previous cancer, or who has a precancerous cyst or polyp, the method comprising: (i) optionally, prior to homogenization, obtaining a sample(s) of the proposed normal tissue or putative precancerous tissue of the patient, which may include spatially distinct regions of the proposed normal tissue or putative precancerous tissue such as a precancerous cyst or polyp that is optionally fixed or preserved using, for example, formalin, paraffin, and / or ethanol; and (ii) producing a homogenate that is representative of the proposed normal tissue or putative precancerous tissue by homogenizing the sample(s), which is suitable for detecting rare cancerous cells or cancer stem cells, for example even before the signs of the disease appear in the patient.

[0311] In another aspect, the present disclosure provides a method of using a representative sample and a portion thereof produced by any of the methods described above in different assay formats, which assays can be performed in high throughput, simultaneously, or at different times or positions, and / or by automation (fully or semi-automation).

[0312] In another aspect, a representative sample of the present disclosure or a portion thereof produced by any of the methods described above is preserved, for example frozen or lyophilized, for future use.

[0313] In another aspect, a representative sample of the present disclosure or a portion thereof produced by any of the methods described above is used to induce (optionally purify) an antibody or antigen specific for a particular antigen derived from a cancer cell or cell type in a patient sample, which antibody or antigen can potentially be used in personalized medicine, i.e., in the production of a therapeutic or prophylactic cancer vaccine.

[0314] The homogenization step in all of the above methods can be carried out by a method that preserves the integrity of the cells in the sample, i.e., a large number of cells in the sample(s) to be homogenized are not lysed, whereby the resulting homogenate and parts thereof "represent" the sample(s). Again, this means that the cells in the sample or part thereof reflect the percentage of different cell types throughout the tissue sample(s), e.g., the whole solid tumor or lymph node. This can be done, for example, by mechanical dissociation of the tumor sample or part thereof (such as mechanical dissociation carried out with or without addition of liquid to the tumor sample or part thereof) and / or chemical or enzymatic dissociation of the tumor sample or part thereof (such as treatment with an enzyme that acts selectively, preferentially, or mainly on extracellular matrix proteins as compared to membrane-bound proteins). Alternatively, the homogenization method can result in dissociation of cells while still generating a sample that represents the starting tissue, e.g., the whole tumor. Optionally, the homogenized representative sample can be dissociated and / or treated to remove or isolate specific types of molecules such as specific cell types, proteins, nucleic acids, or lipids, to generate other representative samples that can be used in diagnostic and therapeutic methods.

[0315] Any of the above methods may also include detecting the expression of at least one biomarker, e.g., at least one lipid, protein, or nucleic acid biomarker, in the homogenate or a part or fraction thereof. Further, the method may further include detecting the percentage of tumor cells in the homogenate or a part or fraction thereof that express a specific biomarker or combination of biomarkers. Optionally, detect and / or isolate the relative frequency or percentage of tumor stem cells and / or tumor subclones in the homogenate or a part or fraction thereof. Further, the method may also include detecting genetic targets (such as point mutations, deletions, additions, translocations, gene fusions, or gene amplifications).

[0316] Using any of the above methods, specific immune cells (such as B lymphocytes, T lymphocytes, macrophages, NK cells, monocytes, or combinations thereof, etc.) present in a homogenate or a part or fraction thereof that provide useful clinical information, such as immune status and disease state, can be detected, isolated, and / or quantified, and also a suitable treatment protocol such as a checkpoint inhibitor, cytokine, or other immunomodulator can be selected.

[0317] The resulting homogenate or representative sample may contain at least 1,000; 10,000; 100,000; 1,000,000; 5,000,000; 10,000,000; 50,000,000; 100,000,000; 500,000,000; 1,000,000,000; 5,000,000,000; 10,000,000,000; 50,000,000,000; 100,000,000,000; 1,000,000,000,000 or more cells.

[0318] The resulting homogenate or a fraction or part thereof can optionally be frozen, freeze-dried, embedded in wax (such as paraffin), or used for further steps (some of which are considered below) without using such freezing or freeze-drying or wax. For example, a representative paraffin block, i.e., the resulting homogenate or a fraction or part thereof embedded in paraffin, is suitable for use in current anatomical pathology workflows, such as sectioning, slide preparation, staining, microscopy, antigen retrieval, etc.

[0319] The homogenate may be derived from two or more tumors collected from one or more subjects, and the resulting homogenate or a fraction thereof from each tumor is used to evaluate the similarity and / or difference of two or more tumors or disease states of different patients.

[0320] Furthermore, the homogenate may be derived from two or more putative normal or pre-cancerous tissues, such as breast, cervical, colorectal, or pre-cancerous cysts or polyps obtained from a subject, such as those having a BRCA mutation, and the resulting homogenate or fractions thereof are used to assess whether abnormal cells or biomarkers of disease are present.

[0321] Furthermore, non-human cells (such as insect cells and / or mouse cells) or other foreign proteins, nucleic acids, or small molecules can be added to the homogenate to create an internal control for positive protein or nucleic acid detection.

[0322] Also, small molecules (such as haptens, peptide tags, protein tags, fluorescent tags, and / or nucleic acid tags) can be added to the sample and used to provide spatial information in the representative sample. For example, a sample (such as a tumor or lymph node) can be sectioned, e.g., cut into quadrants, and different haptens (or other suitable small molecules) "doped" into each section, after which the sections are homogenized to generate a representative sample. The number of sections that can be generated from each sample for "doping" prior to homogenization is not limited, but rather is selected according to the size of the sample, i.e., the larger the sample, the more sections can be "tagged" with small molecules prior to homogenization, as should be understood. In this way, spatial information can be maintained in the resulting homogenate or fractions thereof.

[0323] Also, after adding small molecules to the sample, the sample can be combined with another patient or a different sample from the same patient, thus providing a means for identifying the sample when run in a multiplex assay format.

[0324] The sample to be homogenized can optionally be fixed with formalin or stored in ethanol before or after homogenization. Due to safety concerns, generally, tissue samples are fixed with formalin or other means and then used in pathological or diagnostic methods. Formalin or other fixation methods are generally known in the art. Exemplary methods are disclosed below. In such cases, the formalin-fixed tumor sample may be immersed in water or buffered physiological saline solution (such as PBS) before homogenization in step (ii).

[0325] Alternatively, or additionally, the tumor samples used in the disclosed methods can be stored in ethanol before homogenization. However, it should be emphasized that formalin fixation or ethanol or other storage procedures are not essential for the methods in question and can be eliminated without compromising the suitability of the resulting homogenized representative samples.

[0326] Homogenization of unfixed tissue can be used to produce representative live samples. Representative live samples can be cultured to generate representative tissue culture samples derived from individual patients. Such representative samples can be divided several times to produce multiple representative culture samples, which can be used to determine the effectiveness of chemotherapy (such as antibodies, nucleic acids, small molecules, or polypeptides that antagonize, inhibit, or block the expression or functional activity of at least one known or unknown biomarker). Additionally, specific cell types (such as immune cells or tumor cells) can be selected using FACS analysis. For example, immune cells infiltrating a tumor can be selected and cultured to determine tumor-specific antibodies secreted by the immune system.

[0327] Also, as shown herein, the disclosed methods for deriving representative samples and their use in diagnostic and therapeutic methods are suitable for both fixed and unfixed tissue samples.

[0328] Any of the disclosed methods for preparing representative samples (such as homogenates prepared from tumor biopsy samples) may include the addition of at least one collagenase or other suitable enzyme or combination enzyme or other chemical such as a salt that degrades itself before, during, or after homogenization or facilitates degradation of the extracellular matrix; the use of high temperature and / or buffer conditions, such as buffers CC1 or CC2, that disrupt cell cross-linking; and / or the use of a device for mechanical shearing (such as an IKA blender, gentleMAC Disassociator, or functional equivalent). These methods may or may not be performed under conditions that maintain the viability and integrity of the cells within the sample, for example, under some homogenization conditions that do not substantially lyse the cells.

[0329] In one aspect, homogenization includes the use of a mortar and pestle, a Dounce-type homogenizer or tissue grinder, a hand-held electric rotary blade tissue homogenizer (such as the Omni-TH available from Thomas Scientific), a bead-type homogenizer (such as the Bullet Blender available from OMNI or the Burton Precellys 24 Tissue Homogenizer or Bead Ruptor), and optionally, for rotary homogenizers, at a speed of about 100 - about 75,000 RPM or for bead beaters at a speed of about 0.5 m / s - about 2.5 m / s, for a length of about 30 seconds - about 5 minutes, about 5 minutes - about 10 minutes, about 10 minutes - about 30 minutes, or about 30 minutes - about 60 minutes.

[0330] In another embodiment, the homogenization optionally includes the use 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, endoprotease, trypsin, chymotrypsin, endoprotease Asp-N, endoprotease Arg-C, endoprotease Glu-C (V8 protease), endoprotease 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, pyroglutamate aminopeptidase, or any combination thereof, at a concentration of from about 0.001 μg / ml to about 1000 mg / ml and for a length of from about 1 minute to about 120 minutes.

[0331] The tumor sample used in the disclosed method that encompasses spatially distinct regions of a tumor or other neoplasm may include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, at least 85%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or preferably, all of the tumor surgically removed from a patient. The tumor sample may have a diameter of at least 1, 5, 10, 20, 50, 100 millimeters (mm) or centimeters (cm) or more.

[0332] In the methods described, the samples used are generally derived from solid tumors (primary and metastatic), lymph nodes, metastases, or pre-cancerous tissues such as cysts or polyps. Alternatively, or additionally, the methods can potentially be performed on non-solid tumors, such as blood cancers. For example, homogenized solid tumor samples can optionally be combined with liquid patient samples such as blood, lymph, effusion specimens, cerebrospinal fluid, bile, mucus, and / or urine samples derived from the patient. The homogenized samples can further, or alternatively, include biopsied "normal" or pre-cancerous tissues, for example, to detect disease cells prior to disease symptoms.

[0333] Such tumors or other tumor samples (s) used in the disclosed methods can be from any source, such as those derived from the breast, colon, lung, pancreas, gallbladder, skin, bone, muscle, liver, kidney, neck, ovary, prostate, esophagus, stomach, or other organs, such as those derived from breast cancer tumors, lung cancer tumors, liver cancer tumors, prostate cancer tumors, colon cancer tumors, bladder cancer tumors, or kidney cancer tumors. Generally, the tumor samples or other tissues used in the disclosed methods are of human origin.

[0334] The tumor or other tissue sample used in the disclosed method is at least 1 cm 3 at least 2 cm 3 at least 3 cm 3 at least 4 cm 3 at least 5 cm 3 at least 6 cm 3 at least 7 cm 3 at least 8 cm 3 at least 9 cm 3 at least 10 cm 3 at least 15 cm 3 at least 20 cm 3 at least 25 cm 3 at least 50 cm 3 at least 100 cm 3 at least 250 cm 3 at least 500 cm3 、 at least 1,000 cm 3 、 at least 2,500 cm 3 、 at least 5,000 cm 3 、 at least 7,500 cm 3 、 at least 10,000 cm 3 may have a volume of 10,000 cm or more.

[0335] The tumor or other tissue sample used in the disclosed method may have a maximum width of at least 0.5 cm, at least 1 cm, at least 1.5 cm, at least 2 cm, at least 2.5 cm, at least 3 cm, at least 3.5 cm, at least 4 cm, at least 4.5 cm, at least 5 cm, at least 6 cm, at least 7 cm, at least 10 cm, at least 25 cm, at least 50 cm or more.

[0336] Further, in some embodiments, representative samples can be made from tissues previously fixed in formalin and embedded in paraffin wax. In particular, after melting the wax, removing the tissue, and hydrating it, the methods described herein, i.e., homogenization, can be applied to samples suitable for use in some assays (see, e.g., FIG. 24). FIG. 24 shows the staining of HPV16 ISH on Caski cells in a representative sample prepared from tissue recovered from a paraffin block. Tissue previously embedded in paraffin wax was recovered and homogenized in IKA to generate a representative sample. Thus, by dissolving the wax, recovering the sample, rehydrating the tissue, and appropriately homogenizing, the disclosed method can be used to generate representative samples using samples (one or more) already prepared for TNM staining.

[0337] Each of the above methods may further include (iii) distributing the homogenate or a part or fraction thereof on one or more slides or other solid supports, and optionally, staining one or more slides or other solid supports containing the homogenate or a part or fraction thereof with hematoxylin and eosin dyes; performing immunohistochemical staining on the slide or other solid support containing the homogenate or a part or fraction thereof; or performing in situ hybridization on the slide or other solid support containing the homogenate or a part or fraction thereof, that is, any one of these may be considered as step (iv) in the method.

[0338] Furthermore, each of the above methods may further include (iii) purifying nucleic acid (such as DNA or mRNA) from the homogenate or a part or fraction thereof. The purified nucleic acid can be subjected to Northern blot, DNA sequencing, PCR, RT-PCR, microarray profiling, differential display, or in situ hybridization. Alternatively, the purified nucleic acid can be conjugated to nanoparticles (such as alloy quantum dots including quantum dots, paramagnetic nanoparticles, superparamagnetic nanoparticles, and metal nanoparticles, preferably, by way of example and not limitation, such as CdSe, ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN).

[0339] It is also contemplated that each of the above methods may further include purifying lipids or exosomes or other organelles from the homogenate or a part or fraction thereof. The purified lipids can be subjected to mass spectrometry or histochemical analysis.

[0340] Furthermore, it is also contemplated that any of the above methods may further include purifying the protein from the homogenate or a part or fraction thereof. The purified protein can be subjected to Western blot, ELISA, immunoprecipitation, chromatography, mass spectrometry, microarray profiling, interferometric spectrometry, electrophoretic staining, or immunohistochemical staining. Alternatively, or in addition to the above, the purified protein can be used to produce antiserum specific for the tumor.

[0341] Furthermore, it is also contemplated that any of the above methods may further include performing genomic, transcriptomic, proteomic, and / or metabolomic analysis on the (iii) homogenate or a part or fraction thereof.

[0342] Furthermore, each of the above methods is contemplated to further include (iii) affinity purifying a specific cell type from the homogenate or a part or fraction thereof. The specific cell type may contain the biomarker of interest.Exemplary biomarkers of interest include Her2, bRaf, ERBB2 amplification, Pl3KCA mutation, FGFR2 amplification, p53 mutation, BRCA mutation, CCND1 amplification, MAP2K4 mutation, ATR mutation, or any other biomarker whose expression correlates with a specific cancer; at least one of AFP, ALK, BCR-ABL, BRCA1 / BRCA2, BRAF, V600E, Ca-125, CA19.9, EGFR, Her-2, KIT, PSA, S100, KRAS, ER / Pr, UGT1A1, CD30, CD20, F1P1L1-PDGRFa, PDGFR, TMPT, and TMPRSS2; or at least one biomarker selected from ABCB5, AFP-L3, alpha-fetoprotein, alpha-methylacyl-CoA racemase, BRCA1, BRCA2, CA15-3, CA242, CA27-29, CA-125, CA15-3, CA19-9, calcitonin, carcinoembryonic antigen, carcinoembryonic antigen peptide-1, des-gamma-carboxyprothrombin, desmin, early prostate cancer antigen-2, estrogen receptor, fibrin degradation products, glucose-6-phosphate isomerase, vE6, E7, L1, L2 or p16INK4a HPV antigens such as human chorionic gonadotropin, IL-6, keratin 19, lactate dehydrogenase, leucyl aminopeptidase, lipotropin, methanfurin, neprylisin, NMP22, normetanfurin, PCA3, prostate specific antigen, prostate acid phosphatase, synaptophysin, thyroglobulin, TNF, ERG, ETV1 (ER81), FLI1, ETS1, ETS2, ELK1, ETV6 (TEL1), ETV7 (TEL2), GABPa, ELF1, ETV4 (E1AF; PEA3), ETV5 (ERM), ERF, PEA3 / E1AF, PU.1, ESE1 / ESX, SAP1 (ELK4), ETV3 (METS), EWS / FLI1, ESE1, ESE2 (ELF5), ESE3, PDEF, NET (ELK3; SAP2), NERF (ELF2), transcription factors selected therefrom, or at least one biomarker selected from tumor-associated glycoprotein 72, c-kit, SCF, pAKT, pc-kit, and vimentin may be included.

[0343] Alternatively, or further, the biomarker of interest may be, but is not limited to, an immune checkpoint inhibitor such as CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, KIR, TIM3, GAL9, GITR, LAG3, VISTA, KIR, 2B4, TRPO2, CD160, CGEN-15049, CHK1, CHK2, A2aR, TL1A, and B-7 family ligands or combinations thereof, or a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof.

[0344] The method of any of the preceding claims includes the detection of at least one biomarker associated with acute lymphoblastic leukemia (etv6, am11, cyclophilin b), B-cell lymphoma (Ig idiotype), glioma (E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn), bladder cancer (p21ras), cholangiocarcinoma (p21ras), breast cancer (MUC family, HER2 / neu, c-erbB-2), cervical cancer (p53, p21ras), colon cancer (p21ras, HER2 / neu, c-erbB-2, MUC family), colorectal cancer (colorectal-related antigen (CRC)-C017-1A / GA733, APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastric cancer (HER2 / neu, c-erbB-2, ga733 glycoprotein), hepatocellular carcinoma (alpha-fetoprotein), Hodgkin lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), melanoma (p5 protein, gp75, tumor fetal antigen, GM2 and GD2 gangliosides, Melan-A / MART-1, cdc27, MAGE-3, p21ras, gp100.sup.Pmel117), myeloma (MUC family, p21ras), non-small cell lung cancer (HER2 / neu, c-erbB-2), nasopharyngeal cancer (Imp-1, EBNA-1), ovarian cancer (MUC family, HER2 / neu, c-erbB-2), prostate cancer (prostate-specific antigen (PSA) and its antigen epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2 / neu, c-erbB-2, ga733 glycoprotein), kidney cancer (HER2 / neu, c-erbB-2), squamous cell carcinoma of the cervix and esophagus (viral products such as human papillomavirus protein), testicular cancer (NY-ESO-1), and / or T-cell leukemia (HTLV-1 epitope).

[0345] Also, each of the above methods further includes (iii) treating the homogenate or a part or fraction thereof with collagenase or other enzyme or chemical substance or a combination thereof that destroys the extracellular matrix, incubating the homogenate or a part or fraction thereof under high temperature conditions, and / or mechanically agitating the homogenate or a part or fraction thereof to dissociate the cells within the homogenate or a part or fraction thereof. Generally, these methods produce another representative sample that can be used in the disclosed analytical or therapeutic methods or a combination thereof.

[0346] Furthermore, each of the above methods further includes (iii) filtering or sizing the homogenate or a part or fraction thereof, and it is contemplated that this can result in the obtaining of single cells or small cell clusters such as doublets or triplets.

[0347] The cellular components of the representative sample can be separated by one or more filtration steps. For example, after homogenization and dissociation of the homogenate by physical and / or biochemical means, the dissociated sample can be filtered through a 1 μm filter to remove all intact cell material. The acellular representative sample is expected to contain secreted factors derived from tumors and normal stroma derived within the tumor that are clinically useful, namely, antibodies, growth factors, immunomodulators, and other unknown factors. The acellular representative sample can be analyzed by ELISA, mass spectrometry, next-generation sequencing, and other diagnostic methods. To the extent that single cells are obtained from the representative sample after filtration, such cells can be analyzed using fluorescence-activated cell sorting (FACS) and flow cytometry analysis.

[0348] Considering the representative properties of the homogenate generated by the disclosed method, the homogenate or a part or fraction thereof can be used to detect genetic events with low occurrence rates (such as genetic events occurring at occurrence rates of 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.001%, 0.0001%, 0.00001%, and occurrence rates of 0.000001% or less). Exemplary genetic events include point mutations, deletions, insertions, translocations, gene fusions, or gene amplifications. Similarly, the method may also include detecting the genetic or epigenetic heterogeneity of cells in a tumor sample or a part thereof, and / or detecting cells containing rare genetic or epigenetic changes. Such cells may be present in the tumor sample at a frequency of less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01%.

[0349] The rare cells detected may contain one or more genetic or epigenetic differences that confer resistance to anti-cancer therapy and / or promote metastasis. Thus, the detection of such cells facilitates the prognosis of cancer and the selection of appropriate treatment regimens such as surgery and chemotherapy, and / or the use of biologics.

[0350] The method also includes acridines and derivatives such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine and acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarins and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanosin; 4',6-diamidino-2-phenylindole (DAPI); 5',5''-dibromopyrogallol-sulfonphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaminepentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin B and derivatives such as erythrosin and erythrosin isothiocyanate; ethidium; fluoresceins and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); 2',7'-difluorofluorescein (OREGON GREEN (registered trademark)); fluorescamine; IR144; IR1446; malachite green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline;Phenol Red; B-Phycoerythrin; o-Phthalaldehyde; Pyrene and derivatives such as pyrene, pyrenebutyrate, and succinimidyl 1-pyrenebutyrate; Reactive Red4 (Cibacron.RTM. Brilliant Red 3B-A); 6-Carboxy-X-Rhodamine (ROX), 6-Carboxyrhodamine (R6G), Lissamine Rhodamine B Sulfonyl Chloride, Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Isothiocyanate, Rhodamine Green, Sulfhorhodamine B, Sulfhorhodamine 101, and sulfonyl chloride derivatives of sulfhorhodamine 101 (Texas Red) and other rhodamine and derivatives; N,N,N’,N’-Tetramethyl-6-carboxyrhodamine (TAMRA); Tetramethylrhodamine; Tetramethylrhodamine Isothiocyanate (TRITC); Riboflavin; Rose bengal and terbium chelate derivatives, thiol-reactive europium chelates that emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997;(J. Biol. Chem. 274:3315-22, 1999), and at least one detectable label selected from fluorescent molecules or fluorescent dyes such as GFP, Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine, and xanthene (as described in U.S. Patent No. 5,800,996 to Lee et al.), sold by Invitrogen (see, for example, The Handbook--A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen Detection Technologies, Molecular Probes, Eugene, Oreg), or as disclosed in U.S. Patent No. 5,866,366 to Nazarenko may be included. Other fluorophores known to those of skill in the art, for example, those available from Invitrogen Detection Technologies, Molecular Probes (Eugene, Oreg.), and dyes of the ALEXA FLUOR™ series (e.g., as described in U.S. Patents Nos. 5,696,157, 6,130,101, and 6,716,979), dyes of the BODIPY series (e.g., dipyrrometheneboron difluoride as described in U.S. Patents Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663, and 5,433,896), Cascade Blue (amine-reactive derivative of sulfonated pyrene as described in U.S. Patent No. 5,132,432) and Marina Blue (U.S. Patent No. 5,830,912), semiconductor nanocrystals such as QUANTUM DOT™ (e.g., available from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg;It is also possible to use fluorescent nanoparticles such as those described in U.S. Patent No. 6,815,064, 6,682,596, and 6,649,138 (see also). Semiconductor nanocrystals, radioisotopes (; 3 such as H), Gd 3+DOTA and DPTA chelates of radioactive or paramagnetic metal ions such as, and liposomes, enzymes such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase or β-lactamase, for example, those sold by Invitrogen Corporation, Eugene Oreg., and chromogens, fluorescent or luminescent compounds that generate a detectable signal in combination with the enzyme. Specific examples of chromogenic compounds include, in particular, diaminobenzidine (DAB), 4-nitrophenyl phosphate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azinodi-[3-ethylbenzothiazolinesulfonic acid] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl-beta-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-beta-galactopyranoside (X-Gal), methylumbelliferyl-beta-D-galactopyranoside (MU-Gal), p-nitrophenyl-alpha-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsine, iodonitrotetrazolium (INT), tetrazolium blue and tetrazolium violet.

[0351] The disclosed method can be automated in whole or in part. For example, steps (i) and (ii) can be automated, but subsequent steps, for example, steps (iii) and (iv) are manual. Alternatively, for example, steps (i) and (ii) may be manual, but subsequent steps, for example, steps (iii) and (iv) are automated. Further, all steps encompassed by the method can be automated.

[0352] The disclosed method can be used alone or in combination with other known methods (such as TNM) for tumor stage classification. In one aspect, the method further includes evaluating one or more of the morphological aspects of the tumor, the extent to which tumor cells have spread to the lymph nodes and / or lymphatic system to which they belong, and whether the tumor has metastasized to distant organs, based on genomic, proteomic, and / or lipidomic information contained in a representative sample.

[0353] The disclosed method may further include using an algorithm to calculate the percentage of tumor cells that contain or do not contain a specific biomarker. The relative risk of metastatic (or pathogenic subclone) progression can be determined based on the percentage of cells within a representative tumor sample and / or a representative lymph node sample that includes a specific one.

[0354] The disclosed method may further include the development of an individualized dosage regimen based on the biomarker profile, antigen profile, mutation profile, lipid profile, protein profile, and / or exosome profile contained in a representative sample. For example, based on the information contained in a representative sample (or such information compared to a representative lymph node sample and / or a circulating DNA profile), the selection of a drug and / or the dosage (amount, length of administration, etc.) of such a drug administered to a patient can be modified to individualize the treatment based on the individual cancer profile of the patient.

[0355] The disclosed method may further include comparing the genomic profile of a representative sample with the genomic profile of a representative lymph node sample, and further optionally comparing these profiles with circulating tumor DNA derived from any distant metastasis or representative metastatic tumor sample.

[0356] The present disclosure also encompasses compositions produced by any of the methods in the present disclosure.

[0357] Analysis of Representative Samples Furthermore, the present disclosure contemplates a composition produced by any of the methods described above, including using the results of the methods (such as rare genetic and / or epigenetic events, detection of rare cells, etc.) or preparing a representative sample suitable for further analysis using some standard diagnostic assays in the selection of an appropriate treatment regimen for treating a subject. The treatment regimen may include any of chemotherapy, administration of an immune modulator, irradiation, cytokine administration, surgery, or a combination thereof.

[0358] Furthermore, using the disclosed methods, at least one therapeutic agent (such as an antibody, nucleic acid, small molecule, or polypeptide that antagonizes, inhibits, or blocks the expression or functional activity of at least one detected biomarker) can be selected for use in a subject in which the tumor was a source for a representative sample generated by the provided methods.

[0359] Recent advances in the field of cancer biology have weakened long-held beliefs regarding tumor physiology. Previously, the dominant view was that all cells within a tumor were similar to one another regardless of the stage of cancer. However, with the emergence of new technologies such as single-cell sequencing, it is now understood that cells within a tumor can be highly diverse. See Campbell et al., Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing. PNAS 2008; 105:13081-13086; and Navin et al., Tumor evolution inferred by single-cell sequencing. Nature. 2011; 472:90-94. The discovery of intratumoral heterogeneity highlights new complexities that need to be addressed in clinical oncology such as cancer diagnosis.

[0360] Current pathological methods are based on cutting only a small area from the tumor, placing them in paraffin blocks, and cutting small sections from these blocks that are tested for cancer biomarkers. See Westra et al., Surgical Pathology Dissection: An Illustrated Guide. New York: Springer. 2003. This method may be useful in cancer staging, but the reliability in sampling only a small fraction of the whole tumor makes the diagnostic results unrepresentative of the whole. As a result, current methods often oversample small disease features that 1) cannot identify important disease characteristics and 2) do not determine or affect the progression and / or outcome of the disease.

[0361] These problems are amplified as the size of the tumor increases, and as a result, the long-term survival rate of cancer patients is often negatively affected. For example, regardless of the cancer stage, the 5-year average survival rate decreases as the tumor size increases. See Lopez-Encuentra et al., Staging in lung cancer: is 3cm a prognostic threshold in pathological stage 1 non-small cell lung cancer? A multicenter study of 1,020 patients. Chest. 2002; 121(5):1515-1520; Miller and Grigsby. Measurement of tumor volume by PET to evaluate prognosis in patients with advanced cervical cancer treated by radiation therapy. Int J Radiat Oncol Biol Phys. 2002; 53(2): 353-359; Elkin et al., The effect of changes in tumor size on breast carcinoma survival in the U.S.: 1975-1999. Cancer. 2005; 104(6): 1149-1157; Brookman-May et al., Difference between clinical and pathological renal tumor size, correlation with survival, and implications for patient counseling regarding nephron-sparing surgery. AJR. 2011; 197(5): 1137-1145; and Kornprat et al., Value of tumor size as a prognostic variable in colorectal cancer: a critical reappraisal. Am J Clin Oncol. 2011; 34(1): 43-49.

[0362] Accordingly, there is a need for new technologies in clinical oncology to address the problem of tumor heterogeneity, particularly for patients with large solid tumors. There is also a need for improved methods to identify patient samples that contain abnormal cells prior to the manifestation of disease symptoms in order to enhance the likelihood of a favorable treatment outcome.

[0363] To address the problem of tumor heterogeneity, some experts in the field of oncology have proposed increasing the amount of tumor sampled at one time by testing multiple regions. See, e.g., Alizadeh et al., Toward understanding and exploiting tumor heterogeneity. Nature Medicine. 2015; 21: 846-853. As shown by the probability model above, this technique cannot fully address the problem of tumor heterogeneity. The increased workload on the pathology laboratory responsible for processing this number of samples per patient prohibits this proposed method.

[0364] For a sample to be representative, all different fragments of the starting material need to have an equal chance of ending up in the sample, which needs to be consistent across the entire sample. See Petersen et al., 2005. However, when using current sectioning procedures, a large proportion of the tumor is incinerated after the paraffin block is made. See Westra et al., 2003. Thus, not only have representative tumor samples not been made to date, but current pathological practice prevents and even prohibits their creation.

[0365] In a further aspect, mechanical methods, such as shearing, and / or biochemical methods, such as adjustment by heat and pH and enzymatic digestion of the extracellular matrix, can be used to create representative samples from tumors. The coupling of these techniques results in representative samples of tissue sample(s), such as an entire tumor or a substantial portion thereof, without impairing the ability to use the specimens in traditional tissue-based assays, such as hematoxylin and eosin staining, immunohistochemical analysis, and nucleic acid isolation. Indeed, each representative sample can be used simultaneously in multiple different assays. Further, homogenization of an organ, tissue, or tumor renders it suitable for use in further diagnostic tests, such as whole genome sequencing, which can be important for future pharmaceutical and diagnostic discoveries and for personalized medicine. Additionally, the homogenate is suitable for automated methods similar to those used for diagnostic tests from blood. Thus, representative samples can be used for various diagnostic protocols to identify rare tumor subclones and, by extension, improve clinical diagnosis and individualized cancer treatment. Also, the resulting representative samples can be used to induce antibodies or antigens useful in the development of therapeutic or prophylactic tumor vaccines.

[0366] As exemplified herein, the inventors have demonstrated the ability to create representative samples from clinical specimens, such as human tumor clinical specimens, and further have shown that rare phenotypes that may not be recognized when using traditional tumor sectioning can be detected within the representative samples generated by the methods disclosed herein. Further, the inventors have shown that the disclosed methods can be used to generate representative samples from a variety of different tissue types, fixed or unfixed tissues, and that the resulting representative samples can be used in a variety of diagnostic tests, including IHC and nucleic acid isolation.

[0367] Depending on the mechanical and / or biochemical dissociation processes applied to the sample to generate a homogenate, the cell clusters may contain from more than one cell to thousands of cells. Depending on the subsequent assays performed using representative samples (e.g., FACS and flow cytometry require single cells), further application of methods, such as further mechanical and / or biochemical dissociation and / or size exclusion, can dissociate the clumps (reducing the size and / or number of cells contained therein).

[0368] The method is flexible with respect to the degree of sample dissociation. Thus, for example, the mechanical process(es) can be controlled to obtain a target cell aggregate size by further processing the cell clusters obtained after application of a first mechanical means (blending or equivalent) until the clusters match the dissociation target (such as single cells) of the sampling method. In one aspect, mechanical shearing and size exclusion, such as using a series of sieves with meshes, are used to remove cell clusters of a certain size or below while retaining larger cell clusters for further processing to achieve a target particle size. Thus, the size range may appear to be a normal distribution, but the resulting cell cluster particle size distribution is manipulated by size exclusion, such as using sieve sizes, to remove certain particles from the dissociation process and thus achieve a plateau size rather than a distribution.

[0369] After homogenization, the resulting clusters may contain at least 1 - 2, 2 - 100, 100 - 500, 500 - 1,000, 1,000 - 10,000, 10,000 - 50,000 or more cells. In one aspect, the clusters contain single cells, about 2 - 10 cells, about 10 - 20 cells, or about 20 - 40 cells. The size of the resulting clusters varies. See, for example, FIG. 17.

[0370] As a result of homogenizing a tumor and / or lymph node sample (or of homogenizing a tumor sample), the heterogeneity of cells within the sample, e.g., a tumor or lymph node, is substantially uniformly (evenly) distributed within the resulting homogenate or a part or fraction thereof such that the homogenate (or any fraction thereof) substantially uniformly represents the heterogeneity of the input tumor biopsy sample. By homogenizing a tumor to generate a sample (or homogenate) that is representative of the tumor as a whole, the phenotypic diversity of the tumor, such as the percentage of cells having a particular gene mutation, can be characterized. The sample to be homogenized can often, or regardless of whether many or most cells remain intact, be referred to as a liquid or liquefied sample based on its ability to flow or be poured. In some examples, the representative sample may be a liquid sample (such as a fine needle aspiration biopsy cytology, an effusion sample, or a Pap smear).

[0371] As described, other moieties can be added to these homogenates or representative samples such as other cells, haptens or labels.

[0372] Sequencing of Representative Samples Regarding the ultimate goal of personalized medicine, oncologists rely on the detection of important mutations derived from tumors such that diagnosticians can associate targeted therapies with specific changes within the tumor. Since tumor cells can become irreversibly resistant to therapies over time, capturing sequence information from solid tumors by next-generation sequencing (NGS) is an important component of the clinical oncology workflow. One of the most significant obstacles inherent to all current clinical NGS workflows is that physicians are unable to detect the mutations driving tumor growth across all parts of the tumor and are also unable to detect mutations conferring pre-existing therapy resistance. Furthermore, while some mutations may be present at high prevalence within the tumor, other mutations (including driver and resistance mutations) may be present within only a small fraction of the tumor. Typically, the root of this problem is the fact that physicians use formalin-fixed paraffin-embedded (FFPE) tissue sections derived from samples taken from the primary tumor. While the process of representative sampling can fully resolve the sampling problem, there still remain some significant issues that must be addressed in the clinical NGS workflow in order to fully realize personalized medicine.

[0373] Currently, the use of NGS in the clinic can only detect targetable mutations (i.e., mutations associated with targeted therapies) that are present in the majority of the tumor. Physicians and researchers are primarily focused on targetable mutations due mainly to the volume of data produced by NGS technology. For example, in whole-exome analysis of tissue samples, tens of thousands of genes are examined, and each gene is sequenced in thousands of small fragments. Rather than using all of the data for clinical decision-making, many groups blind most of the data and report only those mutations known to be associated with therapies.

[0374] However, the unmet technical and clinical need is to detect all mutations present in solid tumors, whether the mutations are "targetable" or not. Further, it is the physician's obligation to determine the percentage of tumor cells containing a particular mutation. The physician can understand how to treat patients with multiple "targetable or druggable" mutations using only data that captures most of the genomic diversity of the tumor. For example, if a solid tumor contains a mutation in the EGFR gene with a 55% incidence, a bRaf mutation with a 20% incidence, and a KIT mutation with a 5% incidence, the physician would be expected to want to target the tumor mass with an EGFR inhibitor (e.g., cetuximab, panitumumab) until a clinical response is observed by imaging or over a specific time period. Then, the EGFR inhibitor would be stopped and the next most prevalent target of the therapy, in this case, a bRaf inhibitor (e.g., vemurafenib), would be administered. At this point, the physician may or may not look for a clinical response by imaging. The bRaf inhibitor would be stopped and the least frequent target of the therapy, i.e., a KIT inhibitor (e.g., imatinib, sunitinib), would be administered. Thus, this three-drug regimen could be repeated. If the drugs can be tolerated together, combination therapy is another possibility. Alternatively, the drugs could be administered in the reverse order: KIT inhibitor, bRaf inhibitor, then EGFR inhibitor.

[0375] The important component of this drug schedule is not the NGS technology or inhibitor, but rather the determination of both the presence of all potential targets and their relative prevalence within the tumor, using representative sampling techniques. In the example above, both the KIT mutation and the bRaf mutation were lost because they were present at low prevalence. If regions of the tumor containing a high percentage of bRaf mutant cells had been sampled, it would have appeared as if the bRaf mutation was driving the majority of the tumor and as if a bRaf inhibitor had been given as a single agent. Targeting only one of the three mutations in the example above led to treatment resistance because the tumor cells containing the other two mutations did not respond to the single agent.

[0376] Another use of NGS data from primary tumors is an attempt to determine whether tumor cells can be targeted by the immune system, i.e., through the use of immunotherapy. This includes the prediction of neoantigens that can be targeted by the immune system. Tumor exome sequencing can detect mutations that are predicted to result in changes in the expressed proteins.

[0377] An important factor in determining the prevalence of mutations within solid tumors is the enrichment / purification of tumor cells from a mixed population of tumor and normal cells. One technique that can be used to capture a high percentage of tumor cells is fluorescence-activated cell sorting (FACS). When applying this technique to a representative sample, the homogenized solid tumor needs to be first dissociated into single cells or small multicellular tissue fragments. In addition to the fluorescent detection of tumor markers, single cells can be sorted based on cell and nuclear size. Negative selection of normal cells from tumor cells also results in a sample composed mainly of tumor cells. Multicellular tissue fragment sorting (MTFS) can be enriched by fluorescent detection of tumor markers or, in the case of negative selection, normal markers. This technique not only makes the interpretation of NGS data easier but also enables the detection of mutations with extremely low prevalence in tumors.

[0378] Furthermore, using FACS or MTFS, multiple distinct populations of cells derived from a tumor can be identified that can be processed as distinct samples and analyzed independently of each other. This example is the discrimination of tumor cells, normal epithelial cells, endothelial cells, and immune cells.

[0379] Yet another application of NGS from representative samples is the detection of neoantigens that may be detectable by the immune system. To efficiently use neoantigens in an anti-tumor treatment regimen, physicians must detect all potential antigenic mutations derived from the tumor. Similar to targeted therapies, it is important to detect the majority of neoantigens for chimeric antigen receptor therapy (CAR-T).

[0380] Furthermore, flow cytometry is an important component of a diagnostic workflow that allows a physician to use data from flow cytometry to determine the composition of a representative sample compared to the percentage of samples that are tumors, normal, diploid tumors, or various populations of tumor cells that are positive for specific biomarkers. One example of this workflow is calculating the minimum number of cells required to give statistical power to an IHC assay that requires 1,000 positive cells by calculating the percentage of tumor in a representative sample.

[0381] Homogenization of a tumor or part thereof The present disclosure relates to a method for homogenizing a tumor or other tissue sample, such as a pre-cancerous or putative normal tissue, which can optionally be preserved or fixed before or after homogenization, for example, using formalin, paraffin, and / or ethanol, to generate a "representative sample" which is an unbiased indicator of the entirety of an organization, such as a tumor, lymph node, metastasis, polyp, cyst, biopsy, whole organ, or any combination thereof. Again, these methods can retain the integrity and / or viability of the cells within the sample. Such representative samples may include major clones (having an occurrence rate of more than 50%), primary sub-clones (having an occurrence rate of about 20% - about 50%), secondary sub-clones (having an occurrence rate of about 10% - about 20%), and minor sub-clones (an occurrence rate of less than 10%, preferably less than 5%, more preferably less than 1%, and most preferably less than 0.1%). As discussed above, representative samples facilitate the detection of low-occurrence events, i.e., those with an incidence rate of 0.000001% or less. Representative samples enable the detection of mutations proportional to their incidence in tissues, generally tumors or lymph nodes / lymphoid tissues, which cannot be reliably performed using current methods (such as FFPE slide staining) because they reflect the entirety of a tissue sample, such as a solid tumor or lymph node.

[0382] The concept of homogenizing clinical specimens, such as human tumors, is counter to the historical sampling methods in pathology and oncology. There is no historical precedence for homogenizing an entire human tumor or even a majority of a human tumor. In fact, once a sample for TNM staging classification is taken, the remaining tumor material is destroyed (since it is accepted that all medically relevant information is in the sections collected from the TNM staging classification).

[0383] However, the inventors have identified that information derived from a small sub-population of cancer cells can be detected and analyzed when all (or substantially all) available tumor is preserved and homogenized as a single sample, rather than destroying the remaining tumor material.

[0384] In many cases, samples (tumors, lymph nodes, or metastases) are submitted as a whole, either as a single block or multiple blocks, depending on the size of the mass. For example, many breast tumors less than 2 cm in diameter, which are mostly melanomas, are submitted as a whole. Of the roughly 935,000 estimated cancer cases in 2015, at least one-third of the tumor samples are expected to be suitable for homogenization. Tumors that are particularly suitable for homogenization include colon tumors and kidney tumors.

[0385] The liquid nature of the homogenized tumor enables statistical analysis of the tumor cell population. For example, power analysis suggests that the probability of detecting subclones at low frequencies increases with the analysis of an increasing percentage of samples. For example, a sample size of about 95 cells enables the detection of subclones with an appearance rate of about 20%, a sample size of about 200 cells enables the detection of subclones with an appearance rate of about 0.1%, a sample size of about 2,000 cells enables the detection of subclones with an appearance rate of about 0.01%, and a sample size of about 20,000 cells enables the detection of subclones with an appearance rate of about 0.001%. Tumor samples from the clinic may contain at least about 100 - 200; 200 - 1,000; 1,000 - 5,000; 10,000 - 100,000; 100,000 - 1,000,000; 1,000,000 - 5,000,000; 5,000,000 - 1,000,000,000; 1,000,000,000 - 5,000,000,0000, or more cells (i.e., trillions of cells) from spatially different regions of the tumor, and it is reasonable to predict that representative samples generated from these tumors will have a sufficient number of cells to enable sufficient detection of subclones. Therefore, by providing the power of detection to each diagnostic assay using a sufficient number of cells derived from the representative tumor samples obtained according to the disclosed method, rare subclones within the tumor can be detected, and thus, an unbiased determination of the cancer mutation status becomes easier.

[0386] The disclosed method for preparing representative samples does not require cell lysis and, in some embodiments, maintains the cell structure. As a result, representative samples generated according to the disclosed method that maintain cell integrity can be used, for example, in ICC, IHC, and flow cytometry. Alternatively, or additionally, the sample or representative sample or a portion thereof can be optionally processed to disrupt (lyse) the cells, thus enabling analysis of cell components using, for example, PCR, next-generation sequencing (NGS), and mass spectrometry.

[0387] The initial step in sampling or identifying rare cell types in putative normal or pre-cancerous tissue throughout the entire tumor is to obtain a sufficient amount of tissue, e.g., tumor tissue. Generally, the more tumor tissue available for homogenization, the higher the probability of detecting a rare tumor subpopulation. Realistically, the total amount of tumor material available for creating representative samples is less than 100% (as a result of samples already being removed from the tumor for the TNM staging classification system). However, looking to the future, i.e., once the method of the present invention becomes standard of care, the entire tumor (or at least much of the residual tumor) will be made available for homogenization in the clinic. Current practice is to fix the entire surgical specimen in formalin prior to gross examination and sampling by a surgical pathologist, and both fixed and unfixed tissue are suitable for representative sampling. However, it is envisioned that, for example, once the method of the present invention becomes standard of care, formalin fixation may be excluded or phased out. However, it is also possible to use the current TNM staging classification method together with the representative sampling method disclosed herein.

[0388] Once a tumor or other tissue has been obtained, as much of the tumor or other tissue as possible is placed into a blender (or other suitable device) and generally homogenized as a result of mechanical shearing (however, chemical and / or biochemical, e.g., enzymatic dissociation, can also contribute to homogenization).

[0389] Purely mechanical means, e.g., homogenization by blending, can produce a range of tissue fragments each derived from thousands to hundreds of cells that may conform to a normal distribution. However, the application of other homogenization methods, e.g., biochemical / chemical dissociation methods alone or in combination with mechanical means, can distort the distribution of tissue fragments from a normal distribution.

[0390] Furthermore, a biomarker sample can be generated during or after creating a cell sample by dissociating a sample, e.g., a tumor or lymph node, into tissue fragments (such as single cells and cell clusters) and homogenizing using a combination of mechanical means that are used simultaneously or sequentially. For example, a representative sample containing intact cells can be generated by blending the sample as discussed above, and the resulting "cell sample" can be used to analyze the intact cells; however, the cell sample can be further processed by another mechanical means, e.g., sonication, to produce a biomarker sample, i.e., disruption (lysis) of the intact cells enables analysis of protein and / or DNA biomarkers in the sample.

[0391] The median tissue fragment size is inversely correlated with the energy of the blender (or other suitable device), and at high energy, the tissue fragments are very small. Since dermis requires a large amount of energy for complete dissociation, the tissue component most related to the energy of the blender is the collagen content. The blending time is also important; however, for the most effective clinical applications, it is necessary for the entire tumor to be dissociated within a few minutes. Once the blending time is fixed, it is possible to easily determine the energy required to achieve tumor dissociation under the desired time limit.

[0392] After sufficient mechanical shearing (by blending or other suitable forces) to dissociate the whole tumor, all subpopulations of the originally spatially separated tumor cells are distributed across the newly liquefied tumor sample. Test samples can be taken from the homogenized sample and the tumor subpopulations (including rare, or low prevalence, or minor subpopulations) can be detected using different test modalities.

[0393] For example, an aliquot of the liquefied whole tumor sample can be taken, lysed to release the cellular components, and the nucleic acids purified for analysis by PCR or NGS. For example, the cells can be lysed using a microfluidizer and / or by grinding, milling, chemical or enzymatic lysis and / or other techniques known in the art. Alternatively, or additionally, the protein components of the tumor cells can be purified for proteomic analysis such as mass spectrometry (MS). Further, tissue fragments can be embedded in paraffin wax and sampled using current pathology workflows. For long-term storage, representative samples containing the liquefied tumor can be stored in a suitable buffer for storage, refrigerated or frozen, or embedded in wax (such as paraffin).

[0394] Generally, assays such as those described above (PCR, NGS, MS, IHC, ICC, etc.) that can efficiently use the initially blended whole tumor sample (containing clusters of cells) can be performed without biochemical (enzymatic) processing after the application of mechanical force (by a blender or sonicator or other suitable device for inducing shear). However, representative samples can also be analyzed using assays that require smaller cell populations (such as 2 - 20 cells or even single cells), but additional sample processing steps are required to remove protein cross-links induced during formalin fixation of surgically removed tissue. In particular, enzymatic digestion of the blended tumor tissue is required to create a representative sample consisting of single cells and small cell clusters suitable for use in certain assays, for example, cell sorting.

[0395] As discussed above, homogenization can be carried out using only mechanical means (alone or sequentially or simultaneously with other mechanical means such as blending), only biochemical / chemical means (such as enzymatic dige...

Claims

1. (a) obtaining a representative sample, wherein the representative sample is derived by shearing one or more fixed specimens from the tumor of interest, and the one or more fixed specimens from the tumor of interest are not formalin-fixed and paraffin-embedded (FFPE), any subpopulation of cells that were originally spatially separated within the one or more fixed specimens obtained are substantially uniformly distributed within the representative sample obtained, and any aliquot taken from the representative sample obtained contains the subpopulation of cells present in the one or more fixed specimens from the tumor of interest in the proportion in which they were present; and (b) sorting single cells dissociated from a first aliquot of the representative sample obtained into at least a first population of dissociated cells, wherein the sorting is performed using a fluorescence-activated cell sorter (FACS) or a multicellular tissue fragment sorter (MTFS), A method for sorting cells within a tumor from a subject, comprising:

2. The method according to claim 1, wherein the cells are sorted based on fluorescence detection of a tumor marker.

3. The method according to claim 2, wherein the tumor marker is an intermediate filament.

4. The method according to claim 3, wherein the intermediate filament is cytokeratin or vimentin.

5. The method according to claim 1, wherein the fluorescence detection comprises tyramide signal amplification (TSA).

6. The method according to claim 1, wherein obtaining the representative sample comprises enzymatic dissociation using proteinase K and pepsin.

7. The method according to claim 1, wherein 1% Tween 20 is added to the dissociated single cells to reduce aggregation.

8. The method according to claim 1, wherein the cells are sorted based on cell size or nuclear size.

9. The method according to claim 1, further comprising extracting components from the representative sample, wherein the components are DNA, RNA, protein, lipid, cell organelles, exosomes, cells, or a combination thereof.

10. The method according to claim 9, further comprising analyzing the components.

11. The method according to claim 10, wherein analyzing comprises PCR, mass spectrometry, next-generation sequencing, or ELISA.

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