Methods for predicting progression free survival and overall survival in subjects having cancer using tumor macrophage hybrid cells

Abstract

Methods for predicting overall survival (OS) and progression free survival (PFS) of subjects having cancer, based on the presence of Tumor Macrophage Hybrid Cells (TMHCs), a unique group of cell types that include fusions and / or clusters of different cells found in the blood of subjects having solid tumors, are provided.

Description

METHODS FOR PREDICTING PROGRESSION FREE SURVIVAL AND OVERALL SURVIVAL IN SUBJECTS HAVING CANCER USING TUMOR MACROPHAGE HYBRID CELLSBACKGROUNDField of the Invention

[0001] The present invention generally relates to the use of biomarkers in the blood and other bodily fluids to make predictions regarding overall survival and progression free survival in subjects having cancer, such as solid tumors.Related Art

[0002] When tumor cells break away from primary solid tumors, they penetrate into the blood or lymphatic circulation, and ultimately leave the blood stream and enter either organs or tissue to form metastasis. 90% of cancer-related deaths are caused by the metastatic process. The most common metastatic sites are the lung, liver, bone and brain. Tumor cells found in the circulation are called circulating tumor cells (CTCs). Many research publications and clinical trials show that CTCs have clinical utility in (i) providing prognostic survival and cancer recurrence information through the enumeration of CTCs in the blood stream, and (ii) providing treatment information through examination of protein expression levels, and the occurrence of gene mutations and translocations in the CTCs. However, CTCs are not consistently associated with the development and / or presence of cancer in a subject, even in stage IV cancer patients. While CTCs are found most often in stage IV of breast, prostate and colorectal cancers, they are rare in early stages of the same cancer. CTCs are also rare in other cancers.

[0003] Circulating Cancer Associated Macrophage-Like cells (CAMLs) are another cancer- related cell type that is found in the blood of subjects having cancer. CAMLs are associated with all solid tumors tested and all stages of cancer. CAMLs are polyploid and very large in size, ~20 pm to -300 pm in size. These polyploid cells can be either CD45(-) or CD45(+) and express CD11c, CD 14 and CD31, which confirms their origin as a myeloid lineage. They are often found in the process of engulfing CTCs and cell debris I14-19!. The examination of protein expression levels in CAMLs can also assist clinicians in making informed treatment decisions.

[0004] Assays associated with additional types of cancer-associated circulating cells, in blood and other body fluids, could be used to provide further important prognostic and treatment information for a subject having cancer. The present invention is directed to discovery of such cells and other important goals.SUMMARY

[0005] The present invention is directed prognostic methods that make use of Tumor Macrophage Hybrid Cells (TMHCs), a unique group of cell types that include fusions and / or clusters of different cells that are found in the blood of subjects having solid tumors, including carcinoma, sarcoma, neuroblastoma and melanoma. These circulating cells have been shown to be associated with the presence of solid tumors in a subject having cancer. TMHCs have been found in peripheral blood of subjects having solid tumors via microfdtration using precision microfilters.

[0006] As discussed in detail below, the present inventors have discovered that the presence of TMHCs in the blood of a subject having cancer is an indicator of more aggressive disease with worse clinical outcomes. Thus, medical applications associated with TMHCs include, but are not limited to, use of the cells themselves as biomarkers to provide early detection of cancer and diagnosis of cancer, in particular, in the early detection and diagnosis of cancer relapse or recurrence, and in the determination of cancer mutation.

[0007] More specifically, and in a first embodiment, the present invention is directed to a method for predicting overall survival (OS) and / or progression free survival (PFS) of a subject having cancer. The method comprises determining the presence of TMHCs in a biological sample from a subject having cancer, wherein the presence of TMHCs predicts lower OS and / or PFS than a subject having the same cancer without the presence of TMHCs.

[0008] In a second embodiment, the invention is directed to a method for predicting presence of metastatic spread and / or metastatic progression in a subject having cancer. The method comprises determining the presence of TMHCs in a biological sample from a subject having cancer, wherein the presence of TMHCs predicts presence of metastatic spread and / or metastatic progression in the subject.

[0009] In a third embodiment, the invention is directed to a method for predicting cancer progression in a subject having cancer. The method comprises determining the presence TMHCs in a first biological sample and a second biological sample at a later date, and optional additional biological samples, obtained from a subject having cancer, wherein when TMHCs are present in the second and / or additional biological sample but not present in the first biological sample, the cancer is predicted to progress in the subject.

[0010] In a fourth embodiment, the invention is directed to a method for predicting response to treatment in a subject having cancer. The method comprises determining the presence TMHCs in a first biological sample and a second biological sample at a later date, and optional additional biological samples, obtained from a subject having cancer, wherein the first sample is obtained from the subject prior to or during cancer treatment, wherein the second sample and optional additional samples are obtained from the subject after at least one cancer treatment, wherein when TMHCs are present in the first biological sample but not present in the second and / or additional biological sample, the subject is predicted to respond to the treatment.

[0011] In each of the embodiments and aspects of the invention, the TMHCs comprise one or more of the following cell types:(a) partial tumor macrophage fusion cells;(b) homodimeric tumor macrophage fusion cells;(c) cannibalistic tumor macrophage fusion cells;(d) binucleated tumor macrophage fusion cells;(e) heterotypic circulating tumor cell (CTC) clusters;(f) homotypic circulating tumor cell (CTC) clusters; and(g) epithelial-mesenchymal transition circulating tumor cell (EMT CTCs) clusters.

[0012] In aspects of the first embodiment, OS and / or PFS is over a period of at least 12 months. In other aspects of the first embodiment, OS and / or PFS is over a period of at least 24 months.

[0013] In each of the embodiments and aspects of the invention, the biological sample is one or more of blood (such as peripheral blood), lymph node, bone marrow, cerebral spinal fluid, and urine. For example, the biological sample may be antecubital-vein blood, inferior-vena-cava blood, femoral vein blood, portal vein blood, or jugular-vein blood.

[0014] In each of the embodiments and aspects of the invention, the size of the biological sample is between 5 and 15 mb.

[0015] The cancer in each of the embodiments of the invention is a Stage I cancer, Stage II cancer, Stage III cancer, Stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal, brain & ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, uterine cervix, uterine corpus cancer or other solid tumor cancers, or a blood cancer.

[0016] The TMHCs may be isolated from the biological samples for the determining steps using one or more means selected from the group consisting of size exclusion methodology, immunocapture, red blood cell lysis, white blood cell depletion, a high-molecular weight polysaccharide such as FICOLL®, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, concentrating large cells, eliminating small cells, or a combination thereof.

[0017] The TMHCs may be isolated from the biological samples using size exclusion methodology that comprises using a microfilter. The microfdter may have a pore size ranging from about 5 microns to about 20 microns. Additionally, the pores of the microfdter may have a round, race-track shape, oval, square and rectangular pore shape. The microfdter may also have precision pore geometry and uniform pore distribution.

[0018] The TMHCs may be isolated using a microfluidic chip via physical size-based sorting, hydrodynamic size-based sorting, grouping, trapping, immunocapture, concentrating large cells, or eliminating small cells based on size.

[0019] The TMHCs may be isolated from the biological samples for the determining steps using a microfdtration assay.

[0020] In relevant embodiments, the treatment is one or more of chemotherapy, single drug, combination of drugs, immunotherapy, radiation therapy, chemoradiation, radiation combined with single or multiple drugs, chemoradiation combined with single or multiple drugs, cancer vaccine, and cell therapy. As a non-limiting example, the treatment may be a cancer vaccine and the subject expresses at least one HLA allele.

[0021] In relevant embodiments, the subject may be treated with one or more of chemotherapy, single drug, combination of drugs, immunotherapy, radiation therapy, chemoradiation, radiation combined with single or multiple drugs, chemoradiation combined with single or multiple drugs, cancer vaccine, and cell therapy. As a non-limiting example, the immunotherapy may be PD-L1 immunotherapy.

[0022] TMHCs may be used independently as cancer markers, or in combination with biomarker expression by other circulating cells, such as CAMLs and other circulating tumor cells (CTCs). Suitable CTC subtypes include, but are not limited to, pathologically definable CTCs (PDCTCs), apoptotic CTCs, circulating cancer associated vascular endothelial cells (CAVEs). The presence of these additional cell types themselves and also cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomic, metabolomic, lipidomic and other biomarkers may provide a more complete understanding of a patient’s disease.

[0023] In each aspect and embodiment of the invention, the methods further comprise administering a therapeutically effective amount of a cancer treatment to the subject. The subject may be a subject in which the OS and PFS is predicted to be lower or shorter than the OS and PFS of another subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. l is a diagram of the different TMFC subtypes and images of TMFCs from metastatic breast cancer patient blood.

[0025] FIG. 2 is a diagram of the different CTC cluster subtypes and images of CTC clusters from metastatic breast cancer patient blood.

[0026] FIG. 3 shows survival times. A. shows progression free survival (PFS) for mBC patients with any TMFCs (types (1 )-(4)) detected in blood. B. shows overall survival (OS) for mBC patients with any TMFCs (types (l)-(4)) detected in blood.

[0027] FIG. 4 shows Cox Proportional Survival Analysis of PFS (A.) and OS (B.) for no CTCs (Green Line), with CTCs (Black Line) or with TMFCs (not including hyperploidy fusion cells (CAMLs)) (Red Line) at BL.

[0028] FIG. 5 shows PFS of Homotypic vs Heterotypic CTC Clusters.

[0029] FIG. 6 shows PFS of EMT Clusters vs > 50 pm CAMLs.DETAILED DESCRIPTION

[0030] The matters defined in the description such as a detailed construction and elements are nothing but the ones provided to assist in a comprehensive understanding of the invention. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the invention.

[0031] Cancer is one of the most feared illness in the world, affecting all populations and ethnicities in all countries. Approximately 40% of both men and women will develop cancer in their lifetime. In the United States alone, at any given time there are more than 12 million cancer patients, with 1.7 million new cancer cases and more than 0.6 million deaths estimated yearly. Cancer death worldwide is estimated to be about 8 million annually, of which 3 million occur in developed countries where patients have access to treatment.

[0032] Liquid biopsies provide real-time, sequential tracking of diagnostically -important circulating cells isolated from a subject having cancer. Cells such as circulating tumor cells (CTCs) are found in the peripheral blood of cancer patients and previous work has shown that CTC-based assays can be used as a substitute to tissue biopsies[1'4].

[0033] Recently, another circulating cell associated with cancer has been identified in the peripheral blood of cancer patients. This cancer stromal cell subtype has been termed a cancer associated macrophage-like cell or CAML. CAMLs have been identified in the blood using a non-affinity microfiltration based method which captures both CTCs and CAMLs, and allows for singular or parallel analysis of these cancer specific circulating cell subtypes[1, 6'16]. CAMLs are a recently defined circulating myeloid derived stromal cell, found in all the stages of invasive malignancy and in various solid malignancies (e.g. breast, prostate, non-small cell lung carcinoma (NSCLC), and pancreatic)[lt 13, 14, 17]. CAMLs are specialized myeloid polyploid cells in the blood in all stages of solid tumors. They are easy to identify by their large size (greater than 20 pm), polyploid nucleus and morphologies: round, rod shaped, with one tail or two tails 180 degrees apart. CAMLs typically express CD31, CD 14, CD45 and cytokeratin, and can also express EpCAM, CD 146, CD11c and tie2[11, 13, 14, 17].

[0034] As defined herein, a third group of cancer-associated circulating cells has recently been characterized and comprises Tumor Macrophage Hybrid Cells (TMHCs). These cells are a unique group of cell types that include fusions and / or clusters of different cells that are found in the blood of subjects having solid tumors, including carcinoma, sarcoma, neuroblastoma and melanoma. These circulating cells have been shown to be associated with the presence of solid tumors in a subject having cancer. TMHCs have been found in peripheral blood of subjects having solid tumors via microfdtration using precision microfilters.

[0035] The presence of TMHCs in the blood of a subject having cancer is an indicator of more aggressive disease with worse clinical outcomes. Thus, medical applications associated with TMHCs include, but are not limited to, use of the cells themselves as biomarkers to provide early detection of cancer and diagnosis of cancer, in particular, in the early detection and diagnosis of cancer relapse or recurrence, and in the determination of cancer mutation. The simple presence of one or more of these circulating cell types in a blood sample obtained from a subject having cancer provides diagnostic information.Tumor Macrophage Hybrid Cells (TMHCs).

[0036] Recently, it was described that macrophages and tumor cells can fuse to form tumor macrophage fusion cells (TMFCs), which are detectable within primary tumors and a patient’s blood. Similarly, circulating tumor cell clusters (CTCCs) have been described which are aggregated groups of tumor cells that detached from primary tumors and circulate in the bloodstream. While circulating tumor cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined.

[0037] As defined herein, these circulating fusion cells and clustered cells are collectively termed “Tumor Macrophage Hybrid Cells” or TMHCs. Each reference to “circulating cells” is synonymous with TMHCs, and each reference to “TMHCs” is synonymous with circulating cells. The TMHCs of the present invention comprise one or more of the following cell types:(a) partial tumor macrophage fusion cells;(b) homodimeric tumor macrophage fusion cells;(c) cannibalistic tumor macrophage fusion cells;(d) binucleated tumor macrophage fusion cells;(e) heterotypic circulating tumor cell (CTC) clusters;(f) homotypic circulating tumor cell (CTC) clusters; and(g) epithelial-mesenchymal transition circulating tumor cell (EMT CTCs) clusters.

[0038] Partial tumor macrophage fusion cells are cellular fusions between tumor cells and macrophages where there is some membrane interaction between the cells and both cells retaining their original phenotypes (FIG. 1).

[0039] Homodimeric tumor macrophage fusion cells are cellular fusions between tumor cells and macrophages where the two cells have a fused membrane and shared cytoplasm (FIG. 1).

[0040] Cannibalistic tumor macrophage fusion cells are cellular fusions between tumor cells and macrophages where the tumor cells has been taken up by the macrophage and both cells retaining their original phenotypes (FIG. 1).

[0041] Binucleated tumor macrophage fusion cells are cellular fusions between tumor cells and macrophages where both cells completely merge to become one cell with dual expression of phenotypes (FIG. 1).

[0042] Heterotypic CTC clusters are clusters of CTCs and immune / stromal white blood cells (WBCs) (FIG. 2).

[0043] Homotypic CTC clusters are clusters of only CTCs (FIG. 2).

[0044] Epithelial-mesenchymal transition circulating tumor cell (EMT CTCs) clusters are clusters of CTCs undergoing epithelial-mesenchymal transition (EMT), a process whereby tumor cells downregulate epithelial traits and upregulate mesenchymal traits (FIG. 2).

[0045] TMHCs can be visualized by colorimetric stains, such as H&E, or fluorescent staining of specific markers as shown in FIG. 1 and FIG. 2.Circulating Tumor Cells

[0046] As defined herein, CTCs associated with carcinomas express a number of cytokeratins (CKs). CK 8, 18, & 19 are the cytokeratins most commonly expressed and used in diagnostics, but surveying need not be limited to these markers alone. The surface of solid tumor CTCs usually express epithelial cell adhesion molecule (EpCAM). However, this expression is not uniform or consistent. CTCs do not express any CD45 because it is a white blood cell marker. Inassays to identify tumor-associated cells, such as CTCs and CAMLs, it is sufficient to use antibodies against markers associated with the solid tumor such as CK 8, 18 and 19, or antibodies against CD45 or DAPI.

[0047] Different subgroups of CTCs upregulate and / or down regulate phenotypes and marker expression in relation to tumor progression, tumor spread, and in response to tumor treatments. Therefore, assessing CTCs and the markers expressed by such cells in the peripheral blood can provide important information regarding the status of the cancer in the subject.

[0048] CTCs that can be used as cancer diagnostics, as well as means for screening and monitoring treatment and determining the susceptibility of a tumor in a particular subject to a particular treatment, can be divided into three subgroups. The first subgroup is pathologically definable CTCs (PDCTCs)[6’101. PDCTCs can be characterized by: a “cancer-like” nucleus stained by DAPI; cytokeratin having a filamentous pattern; expression of one or more of CK 8, 18 and 19 (CTCs from epithelial cancers usually express at least CK 8, 18 and 19); lack of CD45 expression.

[0049] The second subgroup is apoptotic CTCs. When a CTC dies, the cytokeratin pattern degrades into dots. Therefore, early apoptotic CTC have some cytokeratin dots and later apoptotic CTCs have all of the cellular cytokeratin degraded into dots. Apoptotic CTCs can also be characterized as expressing CK 8, 18 and 19; a degrading nuclei; lack of CD45 expression.

[0050] In the third subgroup of CTCs, the cells are undergoing epithelial to mesenchymal transition (EMTCTCs)[2, 3’5'91. EMT is a gradual morphogenetic process, and EMTCTCs encompass cells in various stages of transition[6]. EMTCTCs can be generally described by the down regulation of epithelial proteins, e.g. EpCAM and CK, and the upregulation of mesenchymal stem cell proteins, e.g. vimentin and CD34

[0013] . EMTCTC subtyping is typically performed using non-proteomic methods, i.e. mRNA expression or DNA analysis

[0013] .

[0051] A further type of circulating cell associated with cancer that may serve as a diagnostic is the cancer-associated vascular endothelial cell or CAVE. CAVEs are a subtype of circulating endothelial cells. Tumors require blood supply provided by tumor endothelial cells. CAVES are tumor endothelial cells that have broken off from the tumor site into the blood stream. CAVEs are often found in clusters. CAVEs express cytokeratin and various subtypes endothelial cell markers such as CD31, CD146, CD144, CD105, but do not express CD14 or CD45[18b

[0052] Combining staining techniques with morphology, pathologically-definable CTCs (PDCTC), apoptotic CTCs and CAMLs can be identified[6].Predictive Methods

[0053] As suggested above, unique characteristics of TMHCs make them well-suited for use in clinical methodology including methods of screening and diagnosis diseases such as cancer, monitoring treatment, monitoring of disease progression and recurrence. It has been shown that the presence of TMHCs in the blood of a subject is an indicator of more aggressive disease with worse clinical outcomes.

[0054] As suggested in the Summary above, in a first embodiment, the present invention is directed to a method for predicting overall survival (OS) and / or progression free survival (PFS) of a subject having cancer. The method comprises determining the presence of TMHCs in a biological sample from a subject having cancer, wherein the presence of TMHCs predicts lower OS and / or PFS than a subject having the same cancer without the presence of TMHCs.

[0055] In a second embodiment, the invention is directed to a method for predicting presence of metastatic spread and / or metastatic progression in a subject having cancer. The method comprises determining the presence of TMHCs in a biological sample from a subject having cancer, wherein the presence of TMHCs predicts presence of metastatic spread and / or metastatic progression in the subject.

[0056] In a third embodiment, the invention is directed to a method for predicting cancer progression in a subject having cancer. The method comprises determining the presence TMHCs in a first biological sample and a second biological sample at a later date, and optional additional biological samples, obtained from a subject having cancer, wherein when TMHCs are present in the second and / or additional biological sample but not present in the first biological sample, the cancer is predicted to progress in the subject.

[0057] In a fourth embodiment, the invention is directed to a method for predicting response to treatment in a subject having cancer. The method comprises determining the presence TMHCs in a first biological sample and a second biological sample at a later date, and optional additional biological samples, obtained from a subject having cancer, wherein the first sample is obtained from the subject prior to or during cancer treatment, wherein the second sample and optionaladditional samples are obtained from the subject after at least one cancer treatment, wherein when TMHCs are present in the first biological sample but not present in the second and / or additional biological sample, the subject is predicted to respond to the treatment.

[0058] As used herein, overall survival (OS) means the length of time survived by a subject having cancer from a selected date, such as the date of diagnosis, the date on which treatment began, and the date on which blood is drawn to assess cancer progression.

[0059] As used herein, progression free survival (PFS) means the length of time survived by a subject having cancer from a selected date, such as the date on which treatment began or the date on which blood is drawn to assess cancer progression, and where the cancer has not worsened or progressed.

[0060] In each of the methods of the invention, OS or PFS, or both, is over a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 months, or more. In one aspect of the invention, OS or PFS, or both, is over a period of at least about 12 months or at least about 24 months.

[0061] In each of the methods of the invention, when OS and / or PFS is predicted to be “lower”, “shortened” or “worse”, OS and / or PFS is shorter in duration then would be the case for a subject having cancer in which the CAML associated structures had not been found.

[0062] In each of the methods of the invention, it will be apparent that the amount of the biological sample in which the circulating cells (e.g., TMHCs) are assayed can vary. However, the biological sample should generally be at least about 2.5 mL. The amount of biological sample may also be at least about 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 21, 22, 22.5, 23, 24, 25, 26, 27, 27.5, 28, 29, or 30 mL, or more. The amount of biological sample may also be between about 2.5 and 20 mL, between about 5 and 15 mL, or between about 5 and 10 mL. In one aspect of the invention, the biological sample is about 7.5 mL.

[0063] In each of the embodiments and aspects of the invention, the source of the biological sample may be, but is not limited to, one or more of blood (such as peripheral blood), lymph node, bone marrow, cerebral spinal fluid, and urine. When the biological sample is blood, the blood may be antecubital-vein blood, inferior-vena-cava blood femoral vein blood, portal veinblood, or jugular-vein blood, for example. The sample may be a fresh sample or a cryo-preserved sample that is thawed

[0014] .

[0064] In each of the embodiments and aspects of the invention, the cancer may be a Stage I cancer, Stage II cancer, Stage III cancer, Stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal, brain & ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, uterine cervix, uterine corpus cancer or other solid tumor cancers. The skilled artisan will appreciate that the methods of the invention are not limited to particular forms or types of cancer and that they may be practiced in association with a wide variety of cancers

[0065] In each of the embodiments and aspects of the invention, TMHCs are isolated from the biological samples for the determining steps using one or more means selected from size exclusion methodology, immunocapture, red blood cell lysis, white blood cell depletion, a high- molecular weight polysaccharide such as FICOLL®, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, concentrating large cells, eliminating small cells, or a combination thereof. In a particular aspect, the size exclusion methodology comprises use of a microfilter.

[0066] In one aspect of the invention, TMHCs are isolated from the biological samples using size exclusion methodology that comprises using a microfilter. Suitable microfilters can have a variety of pore sizes and shapes. The microfilter may have a pore size ranging from about 5 microns to about 20 microns. In certain aspects of the invention, the pore size is between about 5 and 10 microns; in other aspects, the pore size is between about 7 and 8 microns. The larger pore sizes will eliminate most of the WBC contamination on the filter. The pores of the microfilter may have any shape, with acceptable shapes including round, race-track shape, oval, slit, square, rectangular and / or other shapes. The microfilter may have precision pore geometry, uniform pore distribution, more than one pore geometry, and / or non-uniform distribution. The microfilter may be single layer, or multi-layers with different shapes on different layers.

[0067] In another aspect of the invention, TMHCs are isolated from the biological samples using a microfluidic chip via physical size-based sorting, slits, channels, hydrodynamic sizebased sorting, grouping, trapping, immunocapture, concentrating large cells, or eliminating smallcells based on size. The circulating cell capture efficiency can vary depending on the collection method. The size of a circulating cell that can be captured on different platforms can also vary depending, for example, on the identity of the cell. Collection of circulating cells using CELLSIEVE1Mmicrofilters provides 100% capture efficiency and high quality cells.

[0068] In another aspect of the invention, when the biological sample is peripheral blood or blood, the sample may be collected from a subject using a blood collection tube. CELLSAVE™ blood collection tubes (Menarini Silicon Biosystems Inc., San Diego, CA), for example, provide stable cell morphology and size.

[0069] In a further aspect of the invention, TMHCs are isolated from the biological samples using a CELLSIEVE™ microfilter low-pressure microfiltration assay.

[0070] TMHCs may be used independently as cancer markers, or in combination with biomarker expression by other circulating cells, such as circulating tumor cells (CTCs). Suitable CTC subtypes include, but are not limited to, pathologically definable CTCs (PDCTCs), and apoptotic CTCs. Other suitable circulating cell types include CAMLs and circulating cancer associated vascular endothelial cells (CAVEs). The presence of these additional cell types themselves and also cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomic, metabolomic, lipidomic and other biomarkers may provide a more complete understanding of a patient’s disease.Methods of Treatment

[0071] In each aspect and embodiment of the invention, the method optionally further comprises administering a therapeutically effective amount of a cancer treatment to the subject. The subject may be a subject in which the OS and PFS is predicted to be lower or shorter than the OS and PFS of another subject.

[0072] The identity of the cancer treatment will correspond to the particular type of cancer being treated. However, suitable cancer treatments include chemotherapy, single drugs, combination of drugs, immunotherapy, radiation therapy, chemoradiation, chemoradiation combined with single or multiple drug, chemoradiation combined with single or multiple drugs (such as immunotherapy drugs), cell therapy, and other therapies.

[0073] Additional cancer treatments include, but are not limited to, immunotherapeutic agents, chemotherapeutic agents, radiotherapeutic agents, existing cancer drugs, CCR5 antagonists and CXCR4 antagonists. Examples of cancer treatments include, but are not limited to, one or more of antibodies or antagonists that block the activity of CCL3, CCL5 (RANTES), CCL7 or CCL8; Leronlimab (PRO 140); T-VEC, AM-0010, CXCR4 antagonist, TGF-beta kinase inhibitor galunisertib, anti-CSF-lR monoclonal antibody, Abemaciclib, Faslodex, necitumumab, AZD9291, Cyramza (ramucirumab), TPIV 200, Galunisertib, cancer vaccines, cytokines, cellbased therapies, bi- and multi-specific antibodies, tumor-targeting mAbs, Rituximab, oncolytic viruses, reovirus, Blinatumomab, Sipuleucel-T, T-Vec, IL-2, IFN-a, Trastuzumab, Celuximab, bevacizumab, Tim-3, BTLA, anti-IL-10, GM-CSF, anti-angiogenesis treatment, VEGF blockade, HMGB1, Nrpl, TAM receptor tyrosine kinases, Axl , MerTK, ALT-803, IL-15, Immunosuppressive Ligand Phosphatidyl serine (PS), bavituximab, bevacizumab (anti-VEGF), coblmetinib (MEK inhibitor), vemurafenib (BRAE inhibitor), erlotinib (EGFR), alectinib (ALK inhibitor), bevacizumab (anti-VEGF), pazopanib (tyrosine kinase inhibitor), dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), durvalumab (anti-PD-Ll), sunitinib (RTK inhibitor), pazopanib (RTK inhibitor), sargramostim, VISTA, TIM-3, LAG-3, PRS-343, CD137 (4- 1BB) / HER2 bispecific, USP7, anti-HER2, SEMA4D, CTLA-4, PD-1, PD-L1, and PD-L2. As a non-limiting example, the treatment may be a cancer vaccine and the subject expresses at least one HLA allele. As a non-limiting example, the immunotherapy may be PD-L1 immunotherapy.

[0074] The subjects mentioned in the methods of the present invention will be a human, a nonhuman primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal. The subject having cancer may be undergoing treatment for the cancer. Such treatments include, but are not limited to targeted agents, chemotherapy, and radiation therapy.Companion Diagnostics

[0075] The information obtained using the methods of the invention can be used as a companion or complementary diagnostic. A companion or complementary diagnostic is a diagnostic test that can be used in combination with a therapeutic drug for a selected disease or condition. The companion diagnostic determines the suitability of the drug for treatment of the disease or condition in a particular patient, i.e. the companion diagnostic can help to predictwhether the subject will be a responder or non-responder to the effects of the drug on the disease or condition in the subject. Thus, the companion diagnostic provides information that is used in treatment decisions and that is essential for the safe and effective use of a corresponding drug or biological product.ExamplesA. Tumor Macrophage Fusion Cells (TMFCs)

[0076] Four types of Tumor Macrophage Fusion Cells (TMFCs) were categorized and enumerated, namely (1) Partial, (2) Homodimeric, (3) Cannibalistic, and (4) Binucleated, along with (5) Hyperploidy CAMLs and (6) Hyper-engorged CAMLs, as shown in FIG. 1, from a prospective pilot study using n = 122 metastatic breast cancer (mBC) patients that were starting new lines of treatment. Whole peripheral blood (7.5 mL) was procured, filtered and stained using cytokeratin CD45 / CD14 to identify TMFCs. The presence of the various types of TMFCs & CTCs were compared to a patient’s progression free survival (PFS) and overall survival (OS) hazard ratios (HRs), analyzed by censored univariate analysis based on RECIST vl.l over 24 months.

[0077] CTCs were found in 39% of patients, Partial TMFCs in 25% of patients, Homodimeric TMFCs in 6% of patients, Cannibalistic TMFCs in 0% of patients, Binucleated TMFCs in 2% of patients, Hyperploidy CAMLs in 96% of patients.

[0078] Neither CTCs alone, Binucleated TMFCs, nor Hyperploidy CAMLs were prognostic for PFS or OS (Table 1).

[0079] TMFCs with Partial or Homodimeric fusions were prognostic for worse PFS and OS (Table 1)

[0080] Combining patients with any TMFCs into one group (minus Hyperploidy TMFCs) was highly significant for worse PFS and OS (Fig. 3A and Fig. 3B).

[0081] Thus, TMFCs were detected and described in the blood of mBC patients, demonstrating an association with poor clinical outcomes. These data suggest a TMFC involvement in the pathogenesis of cancer.B. Tumor Macrophage Fusion Cells (TMFCs)

[0082] In a prospective study, 7.5 mL blood samples were collected from n=137 metastatic breast cancer (mBC) patients (Table 2) enrolled in prospective trials from multiple institutions before starting new treatment lines for newly progressive mBC. If possible, an optional followup (FU) sample was collected (n=73) after baseline (BL) (median=4.9 weeks). TMFCs in this analysis did not include Hyperploidy Fusion cells (i.e. CAMLs). TMFCs & CTCs were isolated using a CellSieve™ microfilter and differentiated by staining for CK, CD45, CD14, and DAPI. Cox proportional regression hazard ratios (HRs) with 95% Confidence Intervals (95%CI) for progression free survival (PFS) and overall survival (OS) by univariate & multivariate analyses were based on RECIST vl .1, determined by local institutional pathologist over 24 months (M).

[0083] TMFCs were detected in 26% of patient samples at BL and 27% of patient samples at FU. TMFCs were significantly prognostic for worse PFS and OS at both BL and FU. Any type of CTCs were detected in 39% at BL and 38% at FU. Any type of CTCs were significantly prognostic for worse PFS but not OS at BL. At FU, CTCs were significantly prognostic for worse PFS and OS. At BL, patients without CTCs or TMFCs had the best survival outcomes (Fig. 4A and 4B), followed by patients with just CTC, and patients with TMFCs having the worst outcomes. TMFCs at BL and FU were the only significant independent parameters for PFS and OS (Table 3).

[0084] TMFCs appears to represent a subgroup of CTCs with additional prognostic value not previously analyzed. Presence of TMFCs correlates with significantly worse PFS and OS versus patients without CTCs, or versus patients with normal CTCs. Patients with drops in TMFC populations after induction of new treatments had better outcomes, indicating responses to specific treatment types.C. Circulating Tumor Cell Clusters (CTCCs)

[0085] Circulating tumor cell clusters (CTCCs) are aggregated groups of tumor cells that detached from primary tumors and circulate in the bloodstream. However, while Circulating Tumor Cells (CTCs) are a well studied phenomenon, CTCCs remain relatively unexplored and ill-defined, with only initial studies evaluating their clinical utility. Adding to the CTCC complexity is that various subtypes exist (Fig. 2), including homotypic clusters made of only tumor cells and heterotypic CTCCs made of CTCs attached to immune / stromal white blood cells (WBCs). Furthermore, CTCs can undergo Epithelial-Mesenchymal Transition (EMT), a process where tumor cells downregulate epithelial traits and upregulate mesenchymal traits, and also form clustered EMTs (CEMTs). Further, CTCs can fuse with macrophages forming Cancer- Associated Macrophage-Like cells (CAMLs) when in circulation. Single CTCs, EMTs, and CAMLs, were enumerated, as well as homotypic CTCCs, heterotypic CTCCs and EMT CTCCs from the blood of metastatic breast cancer (mBC) patients to quantify these CTC populations and assess their clinical utility by median progression free survival (mPFS) and median overall survival (mOS) over 24 months.

[0086] The 6 populations were enumerated from a prospective pilot study of n=79 mBC patients. Whole peripheral blood (7.5 mL) was filtered and stained with cytokeratin (CK) & CD45 / CD14 to identify CTCs. CTCs were defined as having an intact DAPI nucleus and strong filamentous CK. Homotypic CTCCs were defined as >2 CTCs attached together. Heterotypic CTCCs were defined as >1 CTC attached to >1 WBC. EMTs were defined as having DAPI nuclei and weak non-filamentous CK. EMT CTCCS were defined as >2 EMTs. CAMLs weredefined as having an enlarged polynucleated DAPI, and positive for CD45 / CD14 or non- filamentous CK.

[0087] Single CTCs were found in 57% of patients (n=34 / 79), homotypic CTCCs 15% (n=12 / 79), heterotypic CTCCs 66% (n=27 / 79), EMTs 56% (n=44 / 79), EMT CTCCs 23% (n=18 / 79), any CAML in 97% (n=77 / 79), and Giant > 50 pm CAMLs in 84% (n=66 / 79) (Table 4).

[0088] Over 24 months, patients with heterotypic CTCCs, homotypic CTCCs and Giant > 50 pm CAMLs had the worst PFS, followed by any CTCs, EMTs, and EMT CTCCs (Figs. 5 & 6). Both CTCCs and EMT CTCCs were rare in HER2+ patients at 8.7% (n=2 / 23) and 17.4% (n=4 / 23), respectively.

[0089] Despite no established definition, CTC Clusters appear to represent an array of subtypes with different biological and clinical meanings. The CTC cluster subtypes from the blood of mBC patients were stratified and enumerated, and compared to clinical outcomes. CAML hyperploidy and CTC clustering appears to indicate poor prognosis.CITATIONS1. Adams DL, Zhu P, Makarova OV, Martin SS, Charpentier M, Chumsri S, et al. The systematic study of circulating tumor cell isolation using lithographic microfilters. RSC Advances. 2014;4:4334-42.2. Lianidou ES, Markou A. Circulating tumor cells in breast cancer: detection systems, molecular characterization, and future challenges. Clinical chemistry. 2011;57: 1242-55.3. Pantel K, Brakenhoff RH, Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nature reviews Cancer. 2008;8:329-40.4. Paterlini-Brechot P, Benali NL. Circulating tumor cells (CTC) detection: clinical impact and future directions. Cancer letters. 2007;253: 180-204.5. Adams D, Tsai S, Makarova OV, Zhu P, Li S, Amstutz PT, et al. Low cytokeratin-and low EpCAM-expressing circulating tumor cells in pancreatic cancer. ASCO Annual Meeting Proceedings; 2013. p. 11046.6. Adams DL, Stefansson S, Haudenschild C, Martin SS, Charpentier M, Chumsri S, et al. Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the cellsearch((R)) CTC test. Cytometry Part A : the journal of the International Society for Analytical Cytology. 2015;87: 137-144.7. Krebs MG, Hou JM, Sloane R, Lancashire L, Priest L, Nonaka D, et al. Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches. J Thorac Oncol. 2012;7:306-15.8. Farace F, Massard C, Vimond N, Drusch F, Jacques N, Billiot F, et al. A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas. British journal of cancer. 2011;105:847-53.9. Lecharpentier A, Vielh P, Perez-Moreno P, Planchard D, Soria JC, Farace F. Detection of circulating tumour cells with a hybrid (epithelial / mesenchymal) phenotype in patients with metastatic non-small cell lung cancer. British journal of cancer. 2011;105: 1338-41.10. Adams DL, Adams DK, Stefansson S, Haudenschild C, Martin SS, Charpentier M, et al. Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas. Breast cancer research : BCR. 2016; 18:44.11 . Adams DL, Adams DK, Alpaugh RK, Cristofanilli M, Martin SS, Chumsri S, et al. Circulating Cancer-Associated Macrophage-Like Cells Differentiate Malignant BreastCancer and Benign Breast Conditions. Cancer Epidemiol Biomarkers Prev. 2016;25: 1037-42. Adams DL, Alpaugh RK, Martin SS, Charpentier M, Chumsri S, Cristofanilli M, et al. Precision microfilters as an all in one system for multiplex analysis of circulating tumor cells. RSC Advances. 2016;6:6405-14. Adams DL, Alpaugh RK, Tsai S, Tang CM, Stefansson S. Multi -Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining. Sci Rep. 2016;6:33488. Adams DL, Martin SS, Alpaugh RK, Charpentier M, Tsai S, Bergan RC, et al. Circulating giant macrophages as a potential biomarker of solid tumors. Proceedings of the National Academy of Sciences of the United States of America. 2014; 111:3514-9. Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004; 10:6897-904. Anantharaman A, Friedlander T, Lu D, Krupa R, Premasekharan G, Hough J, et al. Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients. BMC Cancer. 2016; 16:744. Mu Z, Benali-Furet N, Uzan G, Znaty A, Ye Z, Paolillo C, et al. Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer. Int J Mol Sci. 2016;17. Lin SH, He J, Edelman M, Xu T, Gao H, Reuben J, et al. Sequential Assessment of DNA Damage Response and PD-L1 Expression in Circulating Tumor Cells of Lung Cancer Patients during Radiotherapy. JOURNAL OF THORACIC ONCOLOGY; 2015: ELSEVIER SCIENCE INC 360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USA. p. S266-S7. Daniel L. Adams, R. Katherine Alpaugh, Steven H. Lin, Jeffrey R. Marks, Raymond Bergan, Stuart S. Martin, Saranya Chumsri, Massimo Cristofanilli, Cha-Mei Tang, Steingrimur Stefansson, “Multiplex phenotyping of circulating cancer associated macrophage-like cells in patients with solid tumors”, Proceedings of AACR, Vol. 58, April 2017. Abstract #778. International Patent Application Publication No. WO 2016 / 33103, dated March 3, 2016.

Claims

What is claimed is:

1. A method for predicting overall survival (OS) and / or progression free survival (PFS) of a subject having cancer, comprising determining the presence of Tumor Macrophage Hybrid Cells (TMHCs) in a biological sample from a subject having cancer, wherein the presence of TMHCs predicts lower OS and / or PFS than a subject having the same cancer without the presence of TMHCs.

2. A method for predicting presence of metastatic spread and / or metastatic progression in a subject having cancer, comprising determining the presence of TMHCs in a biological sample from a subject having cancer, wherein the presence of TMHCs predicts presence of metastatic spread and / or metastatic progression in the subject.

3. A method for predicting cancer progression in a subject having cancer, comprising determining the presence TMHCs in a first biological sample and a second biological sample at a later date, and optional additional biological samples, obtained from a subject having cancer, wherein when TMHCs are present in the second and / or additional biological sample but not present in the first biological sample, the cancer is predicted to progress in the subject.

4. A method for predicting response to treatment in a subject having cancer, comprising determining the presence TMHCs in a first biological sample and a second biological sample at a later date, and optional additional biological samples, obtained from a subject having cancer, wherein the first sample is obtained from the subject prior to or during cancer treatment, wherein the second sample and optional additional samples are obtained from the subject after at least one cancer treatment, wherein when TMHCs are present in the first biological sample but not present in the second and / or additional biological sample, the subject is predicted to respond to the treatment.

5. The method of any one of claims 1-4, wherein the TMHCs comprise one or more of the following cell types:(a) partial tumor macrophage fusion cells;(b) homodimeric tumor macrophage fusion cells;(c) cannibalistic tumor macrophage fusion cells;(d) binucleated tumor macrophage fusion cells;(e) heterotypic circulating tumor cell (CTC) clusters;(f) homotypic circulating tumor cell (CTC) clusters; and(g) epitheli al -mesenchymal transition circulating tumor cell (EMT CTCs) clusters.

6. The method of claim 1, wherein OS and / or PFS is over a period of at least 12 months.

7. The method of claim 1, wherein OS and / or PFS is over a period of at least 24 months.

8. The method of any one of claims 1-4, wherein the biological sample is one or more of blood, peripheral blood, lymph node, bone marrow, cerebral spinal fluid, and urine. For example, the biological sample may be antecubital-vein blood, inferior-vena-cava blood, femoral vein blood, portal vein blood, or jugular-vein blood.

9. The method of any one of claims 1-4, wherein the size of the biological sample is between 5 and 15 mL.

10. The method of any one of claims 1-4, wherein the cancer is a Stage I cancer, Stage II cancer, Stage III cancer, Stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal, brain & ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, uterine cervix, uterine corpus cancer or other solid tumor cancers, or a blood cancer.

11. The method of any one of claims 1-4, wherein TMHCs are isolated from the biological samples for the determining steps using one or more means selected from the group consisting of size exclusion methodology, immunocapture, red blood cell lysis, white blood cell depletion, a high-molecular weight polysaccharide such as FICOLL®, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, concentrating large cells, eliminating small cells, or a combination thereof.

12. The method of any one of claims 1-4, wherein TMHCs are isolated from the biological samples using size exclusion methodology that comprises using a microfdter.

13. The method of any one of claims 1-4, wherein TMHCs are isolated using a microfluidic chip via physical size-based sorting, hydrodynamic size-based sorting, grouping, trapping, immunocapture, concentrating large cells, or eliminating small cells based on size.

14. The method of any one of claims 1-4, wherein TMHCs are isolated from the biological samples using a microfdtration assay.

15. The method of any one of claims 1-4, wherein the treatment is one or more of chemotherapy, single drug, combination of drugs, immunotherapy, radiation therapy, chemoradiation, radiation combined with single or multiple drugs, chemoradiation combined with single or multiple drugs, cancer vaccine, and cell therapy.

16. The method of any one of claims 1-4, wherein the subject is treated with one or more of chemotherapy, a single drug, a combination of drugs, immunotherapy, radiation therapy, chemoradiation, radiation combined with single or multiple drugs, chemoradiation combined with single or multiple drugs, cancer vaccine, and cell therapy.

17. The method of any one of claims 1-4, further comprising administering a therapeutically effective amount of a cancer treatment to the subject.

18. The method of claim 17, wherein the subject is a subject in which the OS and / or PFS is predicted to be lower or shorter than the OS and / or PFS of another subject.

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