A method for predicting progression-free survival and overall survival in cancer patients using tumor macrophage hybrid cells.

Abstract

A method is provided for predicting overall survival (OS) and progression-free survival (PFS) of subjects with cancer based on the presence of tumor macrophage hybrid cells (TMHC(s)), which are a specific group of cell types containing fusions and / or clusters of different cells, found in the blood of subjects with solid tumors.

Description

[Technical Field]

[0001] In summary, this invention relates to the use of biomarkers in blood and other bodily fluids for predicting overall survival and progression-free survival in subjects with cancer, such as solid tumors. [Background technology]

[0002] When tumor cells detach from a primary solid tumor, they infiltrate the blood or lymphatic circulation, eventually leaving the bloodstream and entering any organ or tissue, forming metastases. 90% of cancer-related deaths are caused by the metastatic process. The most common sites of metastasis are the lungs, liver, bones, and brain. Tumor cells found in circulation are called circulating tumor cells (CTCs). Numerous research publications and clinical trials have shown that CTCs have clinical utility in (i) providing prognostic survival and cancer recurrence information by measuring the number of CTCs present in the bloodstream, and (ii) providing therapeutic information through examination of protein expression levels in CTCs and the occurrence of gene mutations and translocations. However, CTCs are not consistently associated with the development and / or presence of cancer in subjects, even in stage IV cancer patients. CTCs are most frequently found in stage IV breast, prostate, and colorectal cancers, but they are rare in the earlier stages of the same cancers. CTCs are also rare in other cancers.

[0003] Circulating cancer-associated macrophage-like cells (CAMLs) are another cancer-associated cell type found in the blood of subjects with cancer. CAMLs are associated with all solid tumors and all stages of cancer tested. CAMLs are polyploid and very large, ranging in size from approximately 20 μm to 300 μm. These polyploid cells can be either CD45(-) or CD45(+) and express CD11c, CD14, and CD31, confirming their myeloid origin. They are often found in the process of taking up CTCs and cellular debris. [14,19] Testing protein expression levels in CAMLs can also help clinicians make informed treatment decisions.

[0004] Assays related to additional types of cancer-associated circulating cells in blood and other bodily fluids can be used to provide further important prognostic and therapeutic information for subjects with cancer. This invention is directed toward the discovery of such cells and other important objectives. [Overview of the project]

[0005] This invention relates to a prognostic method utilizing tumor macrophage hybrid cells (TMHCs), a specific group of cell types containing fusions and / or clusters of different cells, 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 subjects with cancer. TMHCs have been found in the peripheral blood of subjects with solid tumors by microfiltration using precision microfilters.

[0006] As detailed below, the inventors have discovered that the presence of TMHCs in the blood of subjects with cancer is an indicator of a more aggressive disease with a worse clinical outcome. Therefore, medical applications related to TMHCs include, but are not limited to, the use of the cells themselves as biomarkers for early detection and diagnosis of cancer, particularly early detection and diagnosis of cancer recurrence or relapse, and identification of cancer mutations.

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

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

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

[0010] In a fourth embodiment, the present invention relates to a method for predicting a response to treatment in a subject having cancer. The method comprises measuring the presence of TMHCs in a first biological sample and a second biological sample obtained at a later date from a subject having cancer, and in any further biological samples, the first biological sample being obtained from the subject before or during cancer treatment, and the second biological sample and optionally additional biological samples being obtained from the subject after at least one cancer treatment. Here, if TMHCs are present in the first biological sample but not in the second and / or additional biological samples, the subject is predicted to respond to treatment.

[0011] In each embodiment and aspect of the present invention, TMHCs are the following cell types: (a) Partial tumor macrophage fusion cells; (b) Homodimeric tumor macrophage fusion cells; (c) Cannibalistic tumor macrophage fusion cells; (d) Dinuclear tumor macrophage fusion cells; (e) heterotype circulating tumor cell (CTC) clusters; (f) Homotyped circulating tumor cell (CTC) clusters; and (g) Epithelial-Mesenchymal Transition (EMT) circulating tumor cell (CTC(s)) clusters, Includes one or more of the following.

[0012] In an aspect of the first embodiment, the OS and / or PFS are for a period of at least 12 months. In another aspect of the first embodiment, the OS and / or PFS are for a period of at least 24 months.

[0013] In each embodiment and aspect of the present invention, the biological sample is one or more of the following: blood (such as peripheral blood), lymph nodes, bone marrow, cerebrospinal fluid, and urine. For example, the biological sample may be cubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, or jugular vein blood.

[0014] In each embodiment and aspect of the present invention, the size of the biological sample is 5 to 15 mL.

[0015] In each embodiment of the present invention, cancer is 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 cancer, brain and ONS cancer, laryngeal cancer, bronchial cancer, oral and pharyngeal cancer, stomach cancer, testicular cancer, thyroid cancer, cervical cancer, uterine cancer, or other solid tumor cancer, or hematological cancer.

[0016] Size exclusion, immunocapture, red blood cell lysis, leukapheresis, FICOLL (R) TMHCs can be isolated from biological samples for the measurement using one or more means selected from the group consisting of high molecular weight polysaccharides such as electrophoresis, dielectrophoresis, flow cell method, magnetic levitation, and sorting, grouping, trapping, enrichment of large cells, removal of small cells, or combinations thereof based on various microfluidic chips, slits, channels, and hydrodynamic size.

[0017] TMHCs can be isolated from biological samples using size exclusion methods, including the use of microfilters. Microfilters may have pore sizes ranging from approximately 5 microns to approximately 20 microns. Furthermore, the pores of microfilters may have circular, racetrack-shaped, elliptical, square, and rectangular pore shapes. Microfilters may also have precise pore shapes and uniform pore distribution.

[0018] TMHCs can be isolated using a microfluidic chip via sorting based on physical size, sorting based on hydrodynamic size, grouping, trapping, immunocapture, enrichment of large cells, or removal of small cells based on size.

[0019] TMHCs can be isolated from a biological sample for measurement using a microfiltration assay.

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

[0021] In related embodiments, the subject can be treated with one or more of chemotherapy, a single agent, a combination of drugs, immunotherapy, radiation therapy, chemoradiation therapy, radiation therapy combined with one or more drugs, chemoradiation therapy combined with one or more drugs, a cancer vaccine, and cell therapy. By way of non-limiting example, the immunotherapy can be PD-L1 immunotherapy.

[0022] TMHCs can be used independently as cancer markers or in combination with biomarker expression by other circulating cells such as CAMLs and other circulating tumor cells (CTC(s)). Preferred CTC subtypes include, but are not limited to, pathologically definable circulating tumor cells (PDCTC(s)), apoptotic CTCs, and circulating cancer-associated vascular endothelial cell(s) (CAVE(s)). The presence of these additional cell types themselves, as well as the presence of cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomics, metabolomics, lipidomics, and other biomarkers, can provide a more complete understanding of the patient's disease.

[0023] In each aspect and embodiment of the present invention, the method further includes administering a therapeutically effective dose of cancer treatment to a subject. The subject may be one in which OS and PFS are predicted to be lower or shorter than those of another subject. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows images of TMFCs from the blood of patients with different TMFC subtypes and metastatic breast cancer.

[0025] [Figure 2] Figure 2 shows images of CTC clusters from different CTC cluster subtypes and from the blood of patients with metastatic breast cancer.

[0026] [Figure 3] Figure 3 shows survival times. A. Progression-free survival for mBC patients with any TMFCs (types (1) to (4)) detected in the blood. B. Overall survival for mBC patients with any TMFCs (types (1) to (4)) detected in the blood.

[0027] [Figure 4] Figure 4 shows the Cox proportional survival analysis (PFS) (A.) and OS (OS) for BL in the following cases: without CTCs (green line), with CTCs (black line), or with TMFCs (excluding highly polyploid fusion cells (CAML(s))) (red line).

[0028] [Figure 5] Figure 5 shows the progression-free survival period for homozygous versus heterozygous CTC clusters.

[0029] [Figure 6] Figure 6 shows the PFS of EMT clusters versus CAMLs larger than 50 μm. [Modes for carrying out the invention]

[0030] Detailed explanation The detailed configurations and elements defined in this detailed description are provided solely to aid in a comprehensive understanding of the present invention. Therefore, those skilled in the art will recognize that various modifications and alterations 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 diseases in the world, affecting all populations and ethnicities in every country. Approximately 40% of both men and women will develop cancer in their lifetime. In the United States alone, there are at any given time more than 12 million people with cancer, with an estimated 1.7 million new cases and over 600,000 deaths each year. Globally, an estimated 8 million people die from cancer each year, 3 million of which occur in developed countries where patients have access to treatment.

[0032] Liquid biopsy provides real-time, sequential tracking of diagnostically important circulating cells isolated from subjects with cancer. Cells such as circulating tumor cells (CTCs) are found in the peripheral blood of cancer patients, and previous studies have shown that CTC-based tests can be used as a substitute for tissue biopsy. [1-4]

[0033] Recently, another circulating cell type associated with cancer has been identified in the peripheral blood of cancer patients. This cancer stromal cell subtype is called cancer-associated macrophage-like cells, or CAMLs. CAMLs have been identified in the blood using a non-affinity-based microfiltration method that captures both CTCs and CAMLs, allowing for the individual or parallel analysis of these cancer-specific circulating cell subtypes. [1、6~16] CAMLs are recently defined circulating bone marrow-derived stromal cells found in all stages of invasive malignancies and various solid malignancies (e.g., breast cancer, prostate cancer, non-small cell lung carcinoma (NSCLC), and pancreatic cancer). [11,13,14,17] CAMLs are specialized myeloid polyploid cells in the blood at all stages of solid tumors. They are easily identified by their large size (greater than 20 μm), polyploid nucleus, and morphology: round, rod-shaped, with one tail or two tails separated by 180 degrees. CAMLs typically express CD31, CD14, CD45 and cytokeratin, and can also express EpCAM, CD146, CD11c and tie2. [11,13,14,17]

[0034] As defined herein, a third group of cancer-associated circulating cells, recently characterized, includes tumor macrophage hybrid cells (TMHC(s)). These cells are a distinct group of cell types, comprising fusions and / or clusters of different cells, found in the blood of subjects with solid tumors, including carcinomas, sarcomas, neuroblastomas, and melanomas. These circulating cells have been shown to be associated with the presence of solid tumors in subjects with cancer. TMHCs have been found in the peripheral blood of subjects with solid tumors by microfiltration using precision microfilters.

[0035] The presence of TMHCs in the blood of subjects with cancer is an indicator of a more progressive disease with a worse clinical outcome. Therefore, medical applications related to TMHCs include, but are not limited to, the use of the cells themselves as biomarkers to provide early detection and diagnosis of cancer. They are particularly useful in the early detection and diagnosis of cancer relapse or recurrence, as well as in the identification of cancer mutations. The mere fact that one or more of these circulating cell types are present in a blood sample obtained from a subject with cancer provides diagnostic information.

[0036] Tumor macrophage hybrid cells (TMHC(s)) Recently, it has been described that macrophages and tumor cells can fuse to form tumor macrophage fusion cells (TMFCs), which can be detected in primary tumors and the patient's blood. Similarly, circulating tumor cell clusters (CTCCs), which are aggregated groups of tumor cells circulating in the bloodstream away from the primary tumor, have been described. While circulating tumor cells (CTCs) are a well-studied phenomenon, CTCCs remain relatively unexplored and unclear.

[0037] As defined herein, these circulating fusion cells and clustered cells are collectively referred to as “tumor macrophage hybrid cells” or TMHC(s). Each reference to “circulating cell(s)” is synonymous with TMHC(s), and each reference to “TMHC(s)” is synonymous with circulating cell. The TMHCs of this invention are the following cell types: (a) Partial tumor macrophage fusion cells; (b) Homodimeric tumor macrophage fusion cells; (c) Cannibalistic tumor macrophage fusion cells; (d) Dinuclear tumor macrophage fusion cells; (e) heterotype circulating tumor cell (CTC) clusters; (f) Homotyped circulating tumor cell (CTC) clusters; and (g) Cluster of epithelial-mesenchymal transition circulating tumor cells (EMT CTC(s)). Includes one or more of the following.

[0038] Partial tumor macrophage fusion cells are cell fusions between tumor cells and macrophages, with some membrane interactions between the cells, and both cells retaining their original phenotypes (Figure 1).

[0039] Homodimeric tumor macrophage fusion cells are cell fusions between tumor cells and macrophages, where the two cells share a fused membrane and cytoplasm (Figure 1).

[0040] Cannibalistic tumor macrophage fusion cells are cell fusions between tumor cells and macrophages, in which tumor cells are taken up by macrophages, and both cells retain their original phenotypes (Figure 1).

[0041] Binultanate tumor macrophage fusion cells are cell fusions between tumor cells and macrophages, where both cells completely fuse to form a single cell with dual phenotypic expression (Figure 1).

[0042] A heterotyped CTC cluster is a cluster of CTCs and immune / interstitial leukocytes (WBCs) (Figure 2).

[0043] A homotyped CTC cluster is a cluster consisting only of CTCs (Figure 2).

[0044] Epithelial-mesenchymal transition circulating tumor cell (EMT CTC(s)) clusters are clusters of circulating tumor cells undergoing epithelial-mesenchymal transition (EMT), a process in which tumor cells downregulate epithelial traits and upregulate mesenchymal traits (Figure 2).

[0045] As shown in Figures 1 and 2, TMHCs can be visualized by colorimetric staining such as H&E, or by fluorescent staining of specific markers.

[0046] Circulating tumor cells As defined herein, carcinoma-associated CTCs express several cytokeratins (CK(s)). CK 8, 18, and 19 are the most commonly expressed and used cytokeratins in diagnosis, but investigations do not need to be limited to these markers alone. The surface of solid tumor CTCs typically expresses epithelial cell adhesion molecules (EpCAM). However, this expression is not uniform and inconsistent. CTCs do not express any CD45, as it is a leukocyte marker. Assays for identifying tumor-associated cells such as CTCs and CAMLs can be adequately performed using antibodies against solid tumor-associated markers such as CK 8, 18, and 19, or antibodies against CD45 or DAPI.

[0047] Different subgroups of CTCs upregulate and / or downregulate phenotype and marker expression in relation to tumor progression, tumor spread, and in response to tumor treatment. Therefore, evaluating CTCs in peripheral blood and the markers expressed by such cells provides important information about the cancer status in a subject.

[0048] CTCs, which can be used as a means of cancer diagnosis, as well as for screening and monitoring treatment and determining the susceptibility of tumors to specific treatments in specific subjects, can be divided into three subgroups. The first subgroup is pathologically definable circulating tumor cells (PDCTC(s)). [6-10]PDCTCs can be characterized by "cancer-like" nuclei stained by DAPI; cytokeratin with a fibrillar pattern; expression of one or more of CK 8, 18, and 19 (CTCs derived from epithelial cancers usually express at least CK 8, 18, and 19); and lack of CD45 expression.

[0049] [[ID=�]]The second subgroup is apoptotic CTCs. When one CTC dies, the cytokeratin pattern decomposes into dots. Thus, early apoptotic CTCs have some cytokeratin dots, and late apoptotic CTCs have all cellular cytokeratins decomposed into dots. Apoptotic CTCs are also characterized by expression of CK 8, 18, and 19, nuclear decomposition, and lack of CD45 expression.

[0050] In the third subgroup of CTCs, the cells are undergoing epithelial-mesenchymal transition (EMT CTC(s) [Epithelial to Mesenchymal Transition] CTC(s)) [2,3,5-9] EMT is a progressive morphogenetic process, and EMT CTCs include cells at various transitional stages [6] EMT CTCs are generally explained by downregulation of epithelial proteins (such as EpCAM and CK) and upregulation of mesenchymal stem cell proteins (such as vimentin and CD34)

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

[13] 。

[0051] A further type of circulating cell associated with cancer that can be useful for diagnosis is cancer-associated vascular endothelial cells, or CAVEs. CAVEs are a subtype of circulating endothelial cells. Tumors require a blood supply provided by tumor endothelial cells. CAVEs are tumor endothelial cells that have detached from the tumor site into the bloodstream. CAVEs are often found in clusters. CAVEs express cytokeratin and various subtypes of endothelial cell markers such as CD31, CD146, CD144, CD105, but do not express CD14 or CD45.

[18] .

[0052] By combining staining techniques and morphology, pathologically definable CTCs (PDCTCs), apoptotic CTCs, and CAMLs can be identified. [6] .

[0053] Prediction method As suggested above, the unique characteristics of TMHCs make them suitable for use in clinical methodologies, including screening and diagnostic methods for cancer-like diseases, monitoring of treatment, and monitoring of disease progression and recurrence. The presence of TMHCs in the blood of subjects has been shown to be an indicator of more aggressive diseases with worse clinical outcomes.

[0054] As suggested in the above summary, the present invention relates in a first embodiment to a method for predicting overall survival (OS) and / or progression-free survival (PFS) in subjects with cancer. The method comprises measuring the presence of TMHCs in a biological sample from a subject with cancer, the presence of TMHCs predicts lower OS and / or PFS than that of the same subject with cancer without the presence of TMHCs.

[0055] In a second embodiment, the present invention relates to a method for predicting the presence of metastatic spread and / or metastatic progression in a subject having cancer. The method comprises measuring the presence of TMHCs in a biological sample derived from the subject having cancer, the presence of TMHCs predicting the presence of metastatic spread and / or metastatic progression in the subject.

[0056] In a third embodiment, the present invention relates to a method for predicting cancer progression in a subject with cancer. The method includes measuring the presence of TMHCs in a first biological sample obtained from a subject with cancer, a second biological sample obtained at a later date, and any further biological samples, wherein if TMHCs are present in the second biological sample and / or additional biological samples but not in the first biological sample, cancer is predicted to progress in the subject.

[0057] In a fourth embodiment, the present invention relates to a method for predicting a response to treatment in a subject having cancer. The method comprises measuring the presence of TMHCs in a first biological sample and a second biological sample obtained at a later date from a subject having cancer, and any further biological samples, the first sample being obtained from the subject before or during cancer treatment, and the second sample and any further samples being obtained from the subject after at least one cancer treatment. If TMHCs are present in the first biological sample but not in the second biological sample and / or further biological samples, the subject is predicted to respond to treatment.

[0058] As used herein, overall survival (OS) refers to the length of time a subject with cancer survives from a selected date (e.g., the date of diagnosis, the date treatment began, and the date blood was taken to assess cancer progression).

[0059] When used in this context, progression-free survival (PFS) refers to the length of time a subject with cancer survives from a selected date (e.g., the date treatment is initiated or the date blood is taken to assess cancer progression) during which the cancer has not worsened or progressed.

[0060] In each method of the present invention, the OS or PFS, or both, lasts for 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 longer. In one aspect of the present invention, the OS or PFS, or both, lasts for a period of at least about 12 months or at least about 24 months.

[0061] In each method of the present invention, if OS and / or PFS are predicted to be “lower,” “shorter,” or “worse,” the OS and / or PFS will be shorter in duration than in subjects with cancer in which no CAML-related structures were found.

[0062] It is clear that in each method of the present invention, the amount of biological sample into which circulating cells (e.g., TMHC(s)) are assayed may 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 about 2.5–20 mL, about 5–15 mL, or about 5–10 mL. In one embodiment of the present invention, the biological sample is about 7.5 mL.

[0063] In each embodiment and aspect of the present invention, the source of the biological sample may be, but is not limited to, one or more of the following: blood (e.g., peripheral blood), lymph nodes, bone marrow, cerebrospinal fluid, and urine. When the biological sample is blood, examples of blood include cubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, and jugular vein blood. The sample may be a fresh sample or a thawed, frozen sample.

[14] .

[0064] In each embodiment and aspect of the present invention, cancer may be stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell carcinoma, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal cancer, brain and ONS cancer, laryngeal cancer, bronchial cancer, oral and pharyngeal cancer, gastric cancer, testicular cancer, thyroid cancer, cervical cancer, uterine cancer, or other solid tumor cancers. Those skilled in the art will understand that the methods of the present invention are not limited to any particular form or type of cancer and that they may be applied in relation to a wide variety of cancers.

[0065] In each embodiment and aspect of the present invention, TMHCs are subjected to size exclusion, immune capture, erythrolysis, leukocyte removal, and FICOLL. (R) High molecular weight polysaccharides are isolated from the biological sample for the measurement step using one or more means selected from various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, enrichment of large cells, removal of small cells, or a combination thereof. In certain embodiments, the size exclusion method includes the use of microfilters.

[0066] In one embodiment of the present invention, TMHCs are isolated from a biological sample using a size exclusion method that includes the use of a microfilter. Suitable microfilters can have a variety of pore sizes and shapes. Microfilters can have pore sizes ranging from about 5 microns to about 20 microns. In some embodiments of the present invention, the pore size is about 5 to 10 microns. In other embodiments, the pore size is about 7 to 8 microns. Larger pore sizes eliminate most of the WBC contamination on the filter. The pores of the microfilter may have any shape, with acceptable shapes including circular, racetrack, elliptical, slit, square, rectangular, and / or other shapes. Microfilters may have precise pore shapes, uniform pore distribution, two or more pore shapes, and / or non-uniform distributions. Microfilters may be single-layer or multi-layer with different shapes on different layers.

[0067] In another aspect of the present invention, TMHCs are isolated from a biological sample using a microfluidic chip via physical size-based sorting, slit, channel, hydrodynamic size-based sorting, grouping, trapping, immunocapture, enrichment of large cells, or removal of small cells based on size. Circulating cell capture efficiency may vary depending on the collection method. The size of circulating cells that can be captured on different platforms may also vary, for example, depending on the cell type. CELLSIEVE TM Collecting circulating cells using microfilters provides 100% capture efficiency and high-quality cells.

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

[0069] In a further embodiment of the present invention, TMHCs are CELLSIEVE TMIt is isolated from biological samples using a 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 (CTC(s)). Preferred CTC subtypes include, but are not limited to, pathologically definable CTCs (PDCTCs) and apoptotic CTCs. Other preferred circulating cell types include CAMLs and circulating cancer-associated vascular endothelial cells (CAVE(s)). The presence of these additional cell types themselves, as well as the presence of cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomics, metabolomics, lipidomics, and other biomarkers, can provide a more complete understanding of the patient's disease.

[0071] Treatment method In each aspect and embodiment of the present invention, the method optionally further includes the step of administering a therapeutically effective dose of cancer treatment to a subject. The subject may be one in which the OS and PFS are predicted to be lower or shorter than those of another subject.

[0072] The type of cancer treatment corresponds to the specific type of cancer being treated. However, appropriate cancer treatments include chemotherapy, monotherapy, combination therapy, immunotherapy, radiation therapy, chemoradiotherapy, chemoradiotherapy with one or more drugs, chemoradiotherapy with one or more drugs (e.g., immunotherapy drugs), cell therapy, and other therapies.

[0073] Additional cancer treatments include, but are not limited to, immunotherapies, chemotherapy agents, radiotherapy agents, existing cancer drugs, CCR5 antagonists, and CXCR4 antagonists. Examples of cancer treatments include one or more of the following antibodies or antagonists that block the activity of CCL3, CCL5 (RANTES), CCL7, or CCL8: leronlimab (PRO 140); T-VEC, AM-0010, CXCR4 antagonists, TGF-beta kinase inhibitor garnicertib, anti-CSF-1R monoclonal antibody, abemaciclib, fathlodex, nesitumumab, AZD9291, Cyramza (ramucirumab), TPIV 200, garnicertib, cancer vaccines, cytokines, cell-based therapies, bispecific and multispecific antibodies, tumor-targeted mAbs, rituximab, oncolytic viruses, reoviruses, blinatumomab, Sipuleucel-T, T-Vec, IL-2, IFN-α, trastuzumab, cetuximab, bevacizumab, Tim-3, BTLA, anti-IL-10, GM-CSF, anti-angiogenic therapy, VEGF inhibitors, HMGB1, Nrp1, TAM receptor tyrosine kinase, Axl, MerTK, ALT-803, IL-15, immunosuppressive ligand phosphatidylserine (PS), bavituximab, bevacizumab (anti-VEGF), cobblemetinib This includes, but is not limited to, HLA-1, PD-1, PD-L1, and PD-L2 (MEK inhibitors), vemurafenib (BRAF inhibitor), erlotinib (EGFR), alectinib (ALK inhibitor), bevacizumab (anti-VEGF), pazopanib (tyrosine kinase inhibitor), dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), durvalumab (anti-PD-L1), sunitinib (RTK inhibitor), pazopanib (RTK inhibitor), salglamostim, 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. In 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 referred to in the methods of the present invention are humans, non-human primates, birds, companion animals such as horses, cattle, goats, sheep, dogs, cats, or rodents, or other mammals. Subjects with cancer may be receiving cancer treatment. Such treatments include, but are not limited to, targeted drugs, chemotherapy, and radiotherapy.

[0075] Companion Diagnosis Information obtained using the method of the present invention can be used as a companion or complementary diagnostic test. A companion or complementary diagnostic test is a diagnostic test that can be used in combination with a therapeutic agent for a selected disease or condition. A companion diagnostic test determines the suitability of a drug for the treatment of a disease or condition in a particular patient; that is, a companion diagnostic test can help predict whether a subject is a responder or non-responder to the effect of a drug on the disease or condition in the subject. Thus, a companion diagnostic test provides information that can be used in treatment decisions and is essential for the safe and effective use of the corresponding cultured drug or biological product. [Examples]

[0076] A. Tumor macrophage fusion cells (TMFC(s)) From a prospective pilot study involving n=122 patients with metastatic breast cancer (mBC) who had initiated a new treatment line, four types of tumor macrophage fusion cells (TMFCs), namely (1) partial, (2) homodimeric, (3) cannibalistic, and (4) binuclear, were classified and enumerated, along with (5) hyperploid CAMLs and (6) hyper-engorged CAMLs, as shown in Figure 1. Whole peripheral blood (7.5 mL) was collected, filtered, and stained with cytokeratin CD45 / CD14 to identify TMFCs. The presence of various types of TMFCs and CTCs was compared to the hazard ratios (HR) of patients' progression-free survival (PFS) and overall survival (OS), analyzed by censored univariate analysis based on RECIST v1.1 over 24 months.

[0077] CTCs were found in 39% of patients, partial TMFCs in 25%, homodimeric TMFCs in 6%, cannibalistic TMFCs in 0%, dinuclear TMFCs in 2%, and polyploid CAMLs in 96%.

[0078] Neither CTCs alone, nor binuclear TMFCs, nor high-ploid CAMLs were prognostic factors for PFS or OS (Table 1).

[0079] TMFCs with partial or homodimeric fusions were associated with poor prognosis for PFS and OS (Table 1).

[0080] Combining patients with any type of TMFC (excluding high-ploid TMFCs) into a single group was highly significant in terms of worsening PFS and OS (Figures 3A and 3B).

[0081] Therefore, TMFCs have been detected, described, and demonstrated to be associated with poor clinical outcomes in the blood of mBC patients. These data suggest the involvement of TMFCs in the pathogenesis of cancer. [Table 1]

[0082] B. Tumor macrophage fusion cells (TMFC(s)) In this prospective study, 7.5 mL blood samples were collected from 137 metastatic breast cancer (mBC) patients enrolled in a multicenter prospective trial (Table 2) before initiating a new line of treatment for newly advanced mBC. Where possible, optional follow-up (FU) samples (n=73) were collected after baseline (BL) (median=4.9 weeks). TMFCs in this analysis did not contain highly polyploid fusion cells (i.e., CAML(s)). TMFCs and CTCs were analyzed using CellSieve. TM The cells were isolated using microfilters and distinguished by staining for CK, CD45, CD14, and DAPI. Univariate and multivariate analyses using Cox proportional regression hazard ratios (HR) with 95% confidence intervals (95% CI) for progression-free survival (PFS) and overall survival (OS) were based on RECIST v1.1 identified by local institution pathologists over 24 months (M).

[0083] TMFCs were detected in 26% of BL patient samples and 27% of FU patient samples. TMFCs were a significant prognostic factor for worsening PFS and OS in both BL and FU. Both types of CTCs were detected in 39% of BL patients and 38% of FU patients. Both types of CTCs were a significant prognostic factor for poor PFS in BL, but not for OS. In FU, CTCs were a significant prognostic factor for poor PFS and OS. In BL, patients without CTCs or TMFCs had the best survival outcome (Figures 4A and 4B), followed by patients with only CTCs, and patients with TMFCs had the worst outcome. TMFCs were the only significant independent factors for PFS and OS in both BL and FU (Table 3).

[0084] TMFCs are thought to constitute a subgroup of CTCs with additional prognostic value that has not been previously analyzed. The presence of TMFCs is associated with significantly worse PFS and OS compared to patients without CTCs or patients with typical CTCs. Patients whose TMFC population decreased after the introduction of new treatments showed better outcomes and responded to specific types of treatment. [Table 2] [Table 3]

[0085] C. Circulating tumor cell clusters (CTCC(s)) Circulating tumor cell clusters (CTCC(s)) are aggregated groups of tumor cells that separate from primary tumors and circulate in the bloodstream. However, while circulating tumor cells (CTC(s)) are a well-studied phenomenon, CTCCs remain relatively unexplored and unclear, with only early studies evaluating their clinical utility. In addition to the complexity of CTCCs, various subtypes exist (Figure 2), including homotype clusters consisting solely of tumor cells and heterotype CTCCs consisting of CTCs attached to immune / stromal leukocytes (WBC(s)). Furthermore, CTCs can undergo epithelial-mesenchymal transition (EMT), a process in which tumor cells downregulate epithelial traits and upregulate mesenchymal traits, and can form clustered EMTs (CEMTs). Moreover, when circulating, CTCs can fuse with macrophages to form cancer-associated macrophage-like cells (CAML(s)). Single cytotoxic tumor cells (CTCs), empyema-mediated cytoplasmic tumors (EMTs), and carcinoma-mediated cytoplasmic tumors (CAMLs) were counted from the blood of patients with metastatic breast cancer (mBC). Homotyped CTCCs, heterotyped CTCCs, and EMT-CTCCs were also counted. These CTC populations were quantified, and their clinical utility was evaluated based on median progression-free survival (mPFS) and median overall survival (mOS) over a 24-month period.

[0086] Six populations were counted from a prospective pilot study of n=79 mBC patients. Whole peripheral blood (7.5 mL) was filtered and stained with cytokeratin (CK) and CD45 / CD14 to identify cytokeratinocytes (CTCs). CTCs were defined as having intact DAPI nuclei and strong fibrous CK. Homotyped CTCCs were defined as two or more CTCs adhering to each other. Heterotyped CTCCs were defined as one or more CTCs adhering to one or more WBCs. EMTs were defined as having DAPI nuclei and weak non-fibrous CK. EMT CTCCS were defined as two or more EMTs. CAMLs were defined as having hypertrophied multinucleated DAPI and being positive for CD45 / CD14 or non-fibrous CK.

[0087] Single CTCs were found in 57% of patients (n=34 / 79), homotype CTCCs in 15% (n=12 / 79), heterotype CTCCs in 66% (n=27 / 79), EMTs in 56% (n=44 / 79), EMT-CTCCs in 23% (n=18 / 79), any type of CAML in 97% (n=77 / 79), and giant CAMLs ≥50 μm in 84% (n=66 / 79) (Table 4).

[0088] Over a 24-month period, patients with heterotyped CTCCs, homotyped CTCCs, and large CAMLs ≥50 μm showed the worst PFS, followed by any type of CTC, EMTs, and EMT-CTCCs (Figures 5 and 6). Both CTCCs and EMT-CTCCs were rare in HER2+ patients, occurring in 8.7% (n=2 / 23) and 17.4% (n=4 / 23), respectively.

[0089] Despite the lack of an established definition, CTC clusters are thought to constitute groups of subtypes with distinct biological and clinical significance. CTC cluster subtypes from the blood of mBC patients were stratified, counted, and compared to clinical outcomes. High polyploidy of CAML and clustering of CTCs are considered to suggest a poor prognosis.

Table 4

[0090] Cited Literature 1. 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 Breast Cancer and Benign Breast Conditions. Cancer Epidemiol Biomarkers Prev. 2016;25:1037-42. 12. 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. 13. 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. 14. 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. 15. 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. 16. 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. 17. 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. 18. 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. 18. 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. 19. International Patent Application Publication No. WO 2016 / 33103, dated March 3, 2016.

Claims

1. A method for assisting in predicting overall survival (OS) and / or progression-free survival (PFS) in subjects with cancer, comprising measuring the presence of tumor macrophage hybrid cells (TMHC(s)) in a biological sample taken from a subject with cancer, wherein the presence of TMHC(s) provides information that predicts lower OS and / or PFS than in the same subject with cancer without the presence of TMHC(s).

2. A method for assisting in predicting the presence of metastatic spread and / or metastatic progression in a subject with cancer, comprising measuring the presence of TMHCs in a biological sample taken from the subject with cancer, wherein the presence of TMHCs indicates information predicting the presence of metastatic spread and / or metastatic progression in the subject.

3. A method for supporting the prediction of cancer progression in a subject with cancer, comprising measuring the presence of TMHCs in a first biological sample taken from a subject with cancer, a second biological sample taken at a later date, and optionally additional biological samples, wherein if TMHCs are present in the second and / or additional biological samples but not in the first biological sample, the method provides information that predicts cancer progression in the subject.

4. A method for assisting in predicting a response to treatment in a subject with cancer, comprising measuring the presence of TMHCs in a first biological sample and a second biological sample taken from a subject with cancer at a later date, and optionally additional biological samples, wherein the first biological sample is obtained from the subject before or during cancer treatment, and the second biological sample and optionally additional biological samples are obtained from the subject after at least one cancer treatment, and the presence of TMHCs in the first biological sample but not in the second and / or additional biological samples indicates information that the subject is expected to respond to the treatment.

5. The aforementioned TMHCs are of the following cell types: (a) Partial tumor macrophage fusion cells; (b) Homodimeric tumor macrophage fusion cells; (c) Cannibalistic tumor macrophage fusion cells; (d) Binary tumor macrophage fusion cells; (e) heterotype circulating tumor cell (CTC) clusters; (f) Homotype circulating tumor cell (CTC) clusters; and (g) Epithelial-mesenchymal transition circulating tumor cell (EMT CTC(s)) cluster, The method comprising one or more of the above.

6. The method according to claim 1, wherein the OS and / or PFS are for a period of at least 12 months.

7. The method according to claim 1, wherein the OS and / or PFS are for a period of at least 24 months.

8. The method according to any one of claims 1 to 4, wherein the biological sample is one or more of blood, peripheral blood, lymph nodes, bone marrow, cerebrospinal fluid, and urine.

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

10. The method according to any one of claims 1 to 4, wherein the cancer is a stage I cancer, a stage II cancer, a stage III cancer, a stage IV cancer, a carcinoma, a sarcoma, a neuroblastoma, a melanoma, an epithelial cell carcinoma, a lung cancer, a breast cancer, a prostate cancer, a pancreatic cancer, a bladder cancer, a kidney cancer, a head and neck cancer, a colorectal cancer, a liver cancer, an ovarian cancer, an osteosarcoma, an esophageal cancer, a brain and ONS cancer, a laryngeal cancer, a bronchial cancer, a oral and pharyngeal cancer, a stomach cancer, a testicular cancer, a thyroid cancer, a cervical cancer, a uterine cancer, or another solid tumor cancer, or a hematological cancer.

11. Size exclusion, immunocapture, red blood cell lysis, leukapheresis, FICOLL （Ｒ） The method according to any one of claims 1 to 4, wherein TMHCs are isolated from the biological sample for the step of measurement using one or more means selected from the group consisting of high molecular weight polysaccharides such as electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and sorting, grouping, trapping, enrichment of large cells, removal of small cells, or a combination thereof, based on various microfluidic chips, slits, channels, and hydrodynamic size.

12. The method according to any one of claims 1 to 4, comprising isolating TMHCs from the biological sample using a size exclusion method that includes using a microfilter.

13. The method according to any one of claims 1 to 4, wherein TMHCs are isolated using a microfluidic chip by sorting based on physical size, sorting based on hydrodynamic size, grouping, trapping, immunocapture, enrichment of large cells, or removal of small cells based on size.

14. The method according to any one of claims 1 to 4, wherein TMHCs are isolated from the biological sample using a microfiltering assay.

15. The method according to any one of claims 1 to 4, wherein the treatment is one or more of chemotherapy, a single drug, a combination of drugs, immunotherapy, radiotherapy, chemoradiotherapy, radiotherapy in combination with one or more drugs, chemoradiotherapy in combination with one or more drugs, cancer vaccines, and cell therapy.

16. The method according to any one of claims 1 to 4, wherein the subject is treated with one or more of the following: chemotherapy, a single drug, a combination of drugs, immunotherapy, radiotherapy, chemoradiotherapy, radiotherapy in combination with one or more drugs, chemoradiotherapy in combination with one or more drugs, cancer vaccines, and cell therapy.

17. The method according to any one of claims 1 to 4, further comprising administering a therapeutically effective dose to a target for cancer treatment.

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

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