Method for Monitoring Treatment Response and Disease Progression in a Subject Using Circulating Cells

Abstract

To provide a method of monitoring treatment response and disease progression in subjects having cancer, such as a solid tumor, using circulating cell biomarkers in the blood and other bodily fluids.SOLUTION: Means for monitoring treatment response and disease progression in subjects are disclosed, where the predictions are based on the change of the number and / or size of circulating cancer associated macrophage-like cells (CAMLs) found in a biological sample, such as blood, from the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention generally relates to methods for monitoring treatment response and disease progression in subjects having cancer, such as solid tumors, using circulating cell biomarkers in blood and other body fluids. Related Art

[0002] When tumor cells detach from the primary solid tumor, they penetrate the blood or lymph circulation and ultimately leave the bloodstream to enter organs or tissues, forming metastases. Ninety percent of cancer-related deaths are caused by the metastatic process. The most common sites of metastasis are the lung, liver, bone, and brain. Tumor cells found in the circulation are referred to as circulating tumor cells (CTCs). Numerous research publications and clinical trials have shown the clinical utility of CTCs in (i) providing information on prognosis survival and cancer recurrence through enumeration of CTCs in the bloodstream, and (ii) providing treatment information through examination of protein expression levels and occurrence of gene mutations and translocations in CTCs. However, CTCs are not consistently associated with the progression and / or presence of cancer in the subject, even in stage IV cancer patients. CTCs are most frequently seen in stage IV breast, prostate, and colorectal cancers, and they are rare in the early 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 having cancer. They are circulating giant stromal cells. CAMLs are associated with all solid tumors and all stages of cancer that have been tested. CAMLs are polyploid and very large in size, with sizes ranging from ~25 to ~300 μm. These polyploid cells can be either CD45(-) or CD45(+), and in most cases, express CD11c, CD14, and CD31, which confirms their origin as myeloid lineage. They are often found in the process of phagocytosing CTCs and cell debris [1,6].

[0004] Assays related to the identification and characterization of biomarkers such as CAMLs in blood and other body fluids can be used to provide diverse information, including whether the disease is progressing in a subject with cancer, whether a subject with cancer is responding to treatment, whether a subject with cancer is likely to respond to future treatment, and information regarding overall survival (OS) and progression-free survival (PFS) in such subjects. The present invention is directed to providing clinicians with such tools for predicting disease progression, treatment response, and other important endpoints.

[0005] The importance of being able to rapidly predict disease progression and treatment response by blood tests is related to providing an opportunity to change treatment if the initial treatment is not effective. The ability to make changes quickly helps to minimize cancer progression prior to another treatment. In immunotherapy, rapid switching can also reduce costs as it can cost over $150,000 to $350,000 per year. SUMMARY OF THE INVENTION

[0006] Abstract The present invention is directed to methods of using cell types having unique characteristics found in the blood of subjects having solid tumors, including carcinomas, sarcomas, neuroblastomas, and melanomas. These circulating cells, termed "circulating cancer-associated macrophage-like cells" (CAMLs), have been shown to be associated with the presence of solid tumors in subjects with cancer. Five morphologies associated with CAMLs have been characterized and described [1,2]. CAMLs have been found in the peripheral blood of cancer patients in a variety of ways. Size exclusion methods such as precision filtration using a precision microfilter provide reliable separation of CAMLs from the peripheral blood of subjects with solid tumors from stage I to stage IV.

[0007] Medical applications related to CAMLs include, but are not limited to, early detection and diagnosis of cancer, particularly early detection and diagnosis of cancer recurrence or relapse, and use of these cells as biomarkers for determining cancer mutations. CAMLs have been shown to have clinical utility as biomarkers in knowing the prognosis regarding a patient's survival. CAMLs have also been shown through the data presented herein to have clinical utility as biomarkers in predicting treatment response and disease progression.

[0008] Accordingly, the present invention is directed to a method for predicting disease progression and / or treatment response in a subject having cancer, such as solid tumor cancer.

[0009] In a first embodiment, the method of the present invention includes determining the size of circulating cells, such as CAMLs, in two or more biological samples obtained from a subject having cancer over time, and when a decrease in the size of the circulating cells between samples is seen over time, it is predicted that the cancer is not progressing and / or it is found that the subject is responding to treatment. In a related embodiment, the method of the present invention includes determining the number of circulating cells, such as CAMLs, in two or more biological samples obtained from a subject having cancer over time, and when a decrease in the number of circulating cells between samples is seen over time, it is predicted that the cancer is not progressing and / or it is found that the subject is responding to treatment.

[0010] In a related aspect, the method includes determining the size of circulating cells, such as CAMLs, in a first and a second biological sample, and any additional biological samples, obtained from a subject having cancer, wherein the first sample is obtained from the subject before cancer treatment or during cancer treatment, the second and any additional samples are obtained from the subject at least one time after cancer treatment, and when the size of at least one cell in the first sample is about 50 μm or more and the size of each cell in the second and any additional samples is less than about 50 μm, it is predicted that the cancer is not progressing and / or it is found that the subject is responding to treatment.

[0011] In another related aspect, the method includes determining the average size of circulating cells, such as CAMLs, in first and second biological samples, and any additional biological samples, obtained from a subject having cancer, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and the subject is identified as responding to treatment if the average size of the circulating cells in the second and any additional samples is decreased compared to the average size of the cells in the first sample. In one aspect, the average size of the circulating cells in the first sample is about 50 μm or greater.

[0012] In a second embodiment, the invention is directed to a method for predicting cancer progression in a subject having cancer, including determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject having cancer and making a prediction based thereon, where if the size of each circulating cell in the sample is less than about 50 μm, cancer is predicted not to progress, and if the size of at least one circulating cell in the sample is about 50 μm or greater, cancer is predicted to progress.

[0013] In one aspect of this embodiment, the invention is directed to cancer progression in a subject having cancer, including determining the size of circulating cells, such as CAMLs, in first and second biological samples, and any additional biological samples, obtained from the subject having cancer, where the first sample is obtained from the subject before or during cancer treatment, and the second sample and any additional samples are obtained from the subject after at least one cancer treatment. If the average size of the CAMLs in the second and any additional samples is decreased compared to the average size of the CAMLs in the first sample, cancer is predicted not to progress in the subject, and the subject is optionally identified as responding to treatment; or, If the average size of the CAMLs in the second and any additional samples is maintained or increased compared to the average size of the CAMLs in the first sample, cancer is predicted to progress in the subject, and the subject is optionally identified as not responding to treatment; or If at least one CAML of the first sample has a size of about 50 μm or more and the size of each cell of the second and any additional samples is less than about 50 μm, the cancer is predicted not to progress and the subject is optionally identified as responding to treatment; or, If the size of each CAML of the first sample is less than about 50 μm and the size of at least one CAML of the second and any additional samples is greater than about 50 μm, the cancer is predicted to progress and the subject is optionally identified as not responding to treatment. In one aspect, the average size of the circulating cells in the first sample is about 50 μm or more.

[0014] In another aspect of this embodiment, the present invention is directed to a method for predicting the progression of cancer in a subject having cancer, including measuring the number of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject having cancer, and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second sample and any additional samples are obtained from the subject at least one time after cancer treatment, and if the number of circulating cells in the second and any additional samples is decreased compared to the number of circulating cells in the first sample, the cancer is predicted not to progress and the subject is optionally identified as responding to treatment, and if the number of circulating cells in the second and any additional samples is maintained or increased compared to the number of circulating cells in the first sample, the cancer is predicted to progress and the subject is identified as not responding to treatment.

[0015] In a third embodiment, the present invention is directed to a method for predicting a subject having cancer's response to treatment, including determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject and predicting based thereon, wherein if the size of each circulating cell in the sample is less than about 50 μm, the subject is predicted to respond to treatment, and if the size of at least one circulating cell in the sample is about 50 μm or more, the subject is predicted not to respond to treatment.

[0016] In one aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, including determining the size of circulating cells, such as CAMLs, in first and second biological samples obtained from a subject having cancer, and any additional biological samples, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the average size of CAMLs in the second and any additional samples is decreased compared to the average size of CAMLs in the first sample, the subject is predicted to respond to the treatment; wherein, if the average size of CAMLs in the second and any additional samples is maintained or increased compared to the average size of CAMLs in the first sample, the subject is predicted not to respond to the treatment; if the number of circulating cells with a size exceeding about 50 μm is maintained or increased in the first to second and any additional samples, the subject is predicted not to respond to the treatment; or, if the number of circulating cells with a size exceeding about 50 μm decreases in the first to second and any additional samples, the subject is predicted to respond to the treatment; or if the size of each circulating cell in the second or subsequent sample is decreased compared to the first sample and the size of each circulating cell in the second or any additional sample is less than about 50 μm, the subject is predicted to respond to the treatment and there is a possibility of cancer cure.

[0017] In another aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, including determining that there are no circulating cells, such as CAMLs, larger than about 50 μm in a biological sample from the subject, and predicting that this subject is responding to the treatment.

[0018] In a further aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, comprising determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject having cancer, wherein the sample is obtained from the subject after at least one cancer treatment, and when the size of each circulating cell is about 50 μm or less, the subject is predicted to respond to the treatment.

[0019] In an additional aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, comprising determining the number of circulating cells, such as CAMLs, in a first and a second biological sample, and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and when the number of circulating cells decreases from the first to the second and any additional samples, the subject is predicted to respond to the treatment, and when the number of circulating cells is maintained or increased from the first to the second and any additional samples, the subject is predicted not to respond to the treatment.

[0020] In a fourth embodiment, the present invention is directed to a method for predicting resistance to treatment in a subject having lung cancer, comprising determining the size of circulating cells, such as CAMLs, in a biological sample from the subject having cancer, wherein when the size of at least one cell in the sample is about 50 μm or more, the subject is predicted to be more resistant to cancer treatment than a subject having cancer without cells having a size greater than about 50 μm. In one aspect of this embodiment, the cancer is lung cancer.

[0021] The present invention is also directed to a method of treating cancer, wherein the treatment decision can be based on the prediction of treatment response using circulating cells in the methods described above.

[0022] For example, in a fifth embodiment, the present invention is directed to a method of treating a subject having cancer, comprising administering a therapeutically effective amount of cancer treatment to the subject having cancer, and determining the size of circulating cells, such as CAMLs, in two or more biological samples obtained from the subject having cancer, wherein a first biological sample is obtained from the subject before or during cancer treatment, a second biological sample is obtained from the subject during or after cancer treatment, and if a decrease in the size of circulating cells is seen over time between the samples, it is understood that the subject is responsive to the treatment and the treatment is continued, and if no decrease in the size of circulating cells is seen over time between the samples, it is understood that the subject is non-responsive to the treatment and the treatment is not continued.

[0023] In related embodiments, the method of the invention comprises administering a therapeutically effective amount of cancer treatment to a subject having cancer, and determining the number of circulating cells, such as CAMLs, in two or more biological samples obtained from the subject having cancer, wherein a first biological sample is obtained from the subject before or during cancer treatment, a second biological sample is obtained from the subject during or after cancer treatment, and if a decrease in the number of circulating cells is found over time between the samples, it is understood that the subject is responsive to the treatment and the treatment is continued, and if no decrease in the number of circulating cells is seen over time between the samples, it is understood that the subject is non-responsive to the treatment and the treatment is not continued.

[0024] In one aspect of this embodiment, the method comprises administering a therapeutically effective amount of cancer treatment to a subject having cancer, and determining the size of circulating cells, such as CAMLs, in first and second biological samples obtained from the subject, and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the size of at least one cell in the first sample is about 50 μm or greater and the size of each cell in the second sample and any additional samples is less than about 50 μm, it is understood that the subject is responsive to the treatment and the treatment is continued.

[0025] In another related aspect, the method includes administering a therapeutically effective amount of cancer treatment to a subject having cancer and determining the average size of circulating cells, such as CAMLs, in first and second biological samples obtained from the subject and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and the subject is identified as responsive to the treatment if the average size of the circulating cells in the second and any additional samples is decreased compared to the average size of the cells in the first sample, the treatment is continued, and the subject is identified as non-responsive to the treatment if the average size of the circulating cells in the second and any additional samples is not decreased compared to the average size of the cells in the first sample, and the treatment is not continued. In one aspect, the average size of the circulating cells in the first sample is about 50 μm or greater.

[0026] In a sixth embodiment, the invention is directed to a method of treating a subject having cancer, the method including administering a therapeutically effective amount of cancer treatment to the subject having cancer and determining the size of circulating cells, such as CAMLs, in first and second biological samples obtained from the subject and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the average size of the CAMLs in the second and any additional samples is decreased compared to the average size of the CAMLs in the first sample, the subject is predicted to be responsive to the treatment and the treatment is continued; if the average size of the CAMLs in the second and any additional samples is maintained or increased compared to the average size of the CAMLs in the first sample, the subject is predicted to be non-responsive to the treatment and the treatment is not continued; if the number of circulating cells having a size greater than about 50 μm increases from the first sample to the second and any additional samples, the subject is predicted to be non-responsive to the treatment and the treatment is not continued; If the number of circulating cells greater than about 50 μm decreases in the first through second and any additional samples, the subject is predicted to respond to treatment and the treatment is continued; or If the size of each circulating cell in the second or subsequent sample decreases compared to the first sample and the size of each circulating cell in the second or any additional sample is less than about 50 μm, the subject is predicted to respond to treatment and the treatment is continued.

[0027] In one aspect of this embodiment, the invention is directed to a method of treating a subject having cancer, comprising administering a therapeutically effective amount of a cancer treatment to the subject having cancer and determining that there are no circulating cells, such as CAMLs, greater than about 50 μm in a biological sample obtained from the subject, wherein the sample is obtained from the subject after at least one cancer treatment, and if there are no circulating cells greater than about 50 μm in the biological sample, the subject is predicted to respond to treatment and the treatment is continued.

[0028] In a further aspect of this embodiment, the invention is directed to a method of treating a subject having cancer, comprising administering a therapeutically effective amount of a cancer treatment to the subject having cancer and determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject, wherein the sample is obtained from the subject after at least one cancer treatment, and if the size of each circulating cell is about 50 μm or less, the subject is predicted to respond to treatment and the treatment is continued.

[0029] In an additional aspect of this embodiment, the present invention is directed to a method of treating a subject having cancer, comprising administering a therapeutically effective amount of a cancer treatment to the subject having cancer, and determining the number of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject, and any additional biological samples, wherein the first sample is obtained from the subject before or during the cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the number of circulating cells decreases from the first to the second and any additional samples, the subject is predicted to respond to the treatment and the treatment is continued, and if the number of circulating cells is maintained or increased from the first to the second and any additional samples, the subject is predicted not to respond to the treatment and the treatment is not continued.

[0030] In each of these embodiments and aspects of the present invention, if no circulating cells are seen in the biological sample at the end of the treatment, it can be concluded that the cancer has been removed. If circulating cells are still found in the biological sample at the end of the treatment, it can be concluded that the cancer has not been removed in the patient and that there is residual disease in the patient.

[0031] The pathogenicity of the residual disease can be predicted by the size of the circulating cells. For example, if the size of at least one CAML in the biological sample is 50 μm or more, the cancer progresses faster.

[0032] In each of the embodiments and aspects of the present invention, the circulating cells can be CAMLs. CAMLs are defined as having each of the following characteristics. (a) A large, non-diploid nucleus with a size of about 14-64 μm, multiple individual nuclei, and / or one or more fused nuclei with a size of about 14-64 μm; (b) A cell size with a size of about 20-300 μm; and (c) A morphological shape selected from the group consisting of spindle, otamajakushi, circular, oval, two legs, three or more legs, thin legs, and amorphous.

[0033] In certain aspects of embodiments of the present invention, circulating cells can be further defined as having one or more of the following additional characteristics. (d) CD14 positive phenotype; (e) CD45 expression; (f) EpCAM expression; (g) Vimentin expression; (h) PD-L1 expression; (i) Expression of the CD11C marker; (j) Expression of the CD146 marker; (k) Expression of the CD202b marker; (l) Expression of the CD31 marker; and (m) CK8, 18, 19 epithelial phenotype.

[0034] In each of the embodiments and aspects of the present invention, the size of the biological sample is from 0.5 to 50 mL. The size of the biological sample may be from 5 to 15 mL. In certain aspects of the present invention, the size of the biological sample is about 7.5 mL.

[0035] In each of the embodiments and aspects of the present invention, the source of the biological sample can be one or more of blood, lymph node, bone marrow, cerebrospinal fluid, tissue, urine, peripheral blood mononuclear cells (PBMCs), and cryopreserved PBMCs. When the biological sample is blood, the blood can be, for example, peripheral blood, median cubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, or jugular vein blood. The sample can be a fresh sample or a thawed cryopreserved sample appropriately prepared.

[0036] In certain aspects of embodiments of the present invention, the cancer is a solid tumor, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, colorectal cancer, liver cancer, head and neck cancer, kidney cancer, ovarian cancer, esophageal cancer, or other solid tumor cancer.

[0037] In certain aspects of embodiments of the present invention, circulating cells are separated from a biological sample using one or more means selected from size exclusion methods, immunocapture, multivalent cell capture via dendrimers, affinity-based surface capture, biomimetic surface coating supplementation, selectin-coated surface capture, other functionalized surface capture, inertial focusing chips, erythrocyte lysis, leukocyte depletion, Ficoll separation, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, or combinations thereof.

[0038] In one aspect of the present invention, circulating cells are separated from a biological sample using a size exclusion method that includes using a microfilter. The microfilter can have a pore size in the range of about 5 microns to about 20 microns. Suitable ranges of pore size include, but are not limited to, pore sizes in the ranges of about 5-7 microns, about 7-8 microns, 8-10 microns, 11-13 microns, 14-16 microns, 17-20 microns, 5-10 microns, 11-15 microns, 15-20 microns, 9-15 microns, 16-20 microns, and 9-20 microns. Another suitable range includes pore sizes in the range of about 5-20 microns, excluding pores of 7-8 microns. The pores of the microfilter can have a circular, racetrack, elliptical, slit, square, rectangular, and / or other shape. The microfilter can have a precise pore shape, a uniform pore distribution, multiple pore shapes, and / or a non-uniform pore distribution. The microfilter can be a single layer or a multilayer having different shapes in different layers. A microfilter having circular pores with a size of about 7-8 microns is particularly optimal when a polymeric microfilter is used. In a preferred aspect, the microfilter has a precise pore shape and a uniform pore distribution.

[0039] In another aspect of the present invention, circulating cells are separated from a biological sample using a CellSieve™ low pressure precision filtration assay.

[0040] In another aspect of the present invention, circulating cells are separated from a biological sample using a microfluidic chip via sorting based on physical size, slits, channels, sorting based on hydrodynamic size, grouping, trapping, immunocapture, enrichment of large cells, or exclusion of small cells based on size.

[0041] In certain aspects of embodiments of the present invention, a subject is undergoing treatment. The treatment can be one or more of chemotherapy, a single drug, a combination of drugs, immunotherapy, radiation therapy, chemoradiation therapy, radiation in combination with one or more drugs, chemoradiation therapy in combination with one or more drugs, a cancer vaccine, and cell therapy. If the treatment is a cancer vaccine, the subject may express at least one HLA allele.

[0042] In each of the embodiments and aspects of the present invention, CAMLs can be used as cancer markers independently or in combination with other circulating cells such as circulating tumor cells (CTCs), epithelial-mesenchymal transition cells (EMTs), circulating cancer-associated vascular endothelial cells (CAVEs), and also in combination with free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomic, metabolomic, lipidomic, and other biomarkers, thereby providing a more complete understanding of the patient's disease. CAMLs are typically the only cell type larger than 30 μm in size among the groups of circulating cells mentioned herein.

[0043] When the method of the present invention is related to predicting a treatment response in a subject, the time scale for obtaining treatment response information can be as short as 2 treatment cycles or 60 days, depending on the type of treatment.

[0044] In related aspects of the present invention, tests for predicting a cancer treatment response can be one or more of the following: · Independent of the type of solid tumor, · Independent of the stage of cancer, · Independent of the type of treatment, · At the start of treatment, during ongoing treatment, or at the end of treatment, · Based on a blood sample, · Not requiring tissue, and · Conducted through a determination obtained prior to imaging diagnosis.

[0045] The subject referred to in the method of the present invention is a human, a non - human primate, a bird, a horse, a cow, a goat, a sheep, a companion animal such as a dog, a cat or a rodent, or other mammal.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0057] DETAILED DESCRIPTION As used herein, "a" or "an" can mean one or more. When used in conjunction with the term "comprising" herein, the term "a" or "an" can mean one or more. As used herein, "another" can mean at least a second or more. Further, unless the context requires otherwise, singular terms include pluralities and plural terms include singulars.

[0058] As used herein, "about" refers to a numerical value, including integers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values that one of ordinary skill in the art would consider to be equivalent to the recited value (e.g., having the same function or result), such as a range of + / - 5, 6, 7, 8, 9, or 10% of the recited value. In some cases, the term "about" may include a value rounded to the nearest significant digit. For example, "about 50 μm" should be interpreted to mean 50 μm + / - 10%. Thus, unless otherwise stated, "about 50 μm" encompasses sizes from 45 to 55 μm.

[0059] The matters defined in the description of detailed configurations and elements are provided only to assist in the overall understanding of the present invention. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0060] Cancer is one of the most feared diseases in the world, affecting all populations and ethnic groups in all countries. Approximately 40% of men and women will develop cancer during their lifetime. In the United States alone, there are always more than 12 million cancer patients, and in 2018, an estimated 1.7 million new cancer patients and more than 600,000 deaths were reported. The estimated annual number of cancer deaths worldwide is about 8 million, of which 3 million occur in developed countries where patients can receive treatment.

[0061] Ideally, there would be a diagnostic method that can quickly determine whether the selected treatment is working and provide a prognosis for the progression of the disease.

[0062] This disclosure shows a cell type that is more consistently seen in the blood of solid tumor patients in stages I - IV than any other cancer-related cells. These circulating cells are macrophage-like cells that contain the same tumor markers as the primary tumor, and here they are referred to as circulating cancer-associated macrophage-like cells (CAMLs).

[0063] Along with circulating tumor cells (CTCs), CAMLs present in a biological sample from a patient having cancer can be separated and characterized, for example, by use of a size exclusion method including a precision filtration method. The microfilter can be formed with pores large enough to allow red blood cells and most white blood cells to pass through while retaining larger circulating cells such as CTCs and CAMLs. The collected cells can then be characterized directly on the filter or via other means.

[0064] CAMLs are useful alone for many clinical applications. However, the characterization of CAMLs in a biological sample using the methods of the present invention can be combined with the assay of other markers for the same characteristics (e.g., size and number) or other characteristics. These other markers include CTCs, epithelial-mesenchymal transition cells (EMTs), circulating cancer-associated vascular endothelial cells (CAVEs), as well as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomic, metabolomic, lipidomic, and other biomarkers, and free proteins in the blood, further improving the sensitivity and specificity of the methods defined herein. This is particularly true for CAMLs and CTCs, as they can be simultaneously separated and identified using the same methods disclosed herein. Circulating cancer-associated macrophage-like cells (CAMLs)

[0065] As defined herein, CAMLs have one or more of the following characteristics: · Despite having enlarged and fused nucleoli being common, CAMLs have large, often highly irregular ploidy nuclei or multiple discrete nuclei, often scattered within the cell. CAML nuclei generally range in size from about 10 μm to about 70 μm in diameter, more commonly from about 14 μm to about 64 μm in diameter. ·In many cancers, CAMLs express cancer markers of the disease. For example, CAMLs associated with epithelial cancer may express CK8, 18 or 19, vimentin, etc. This marker is typically diffuse or associated with vacuoles and / or ingested substances. The staining pattern of any marker is almost uniformly diffused throughout the cell. In sarcomas, neuroblastomas, and melanomas, other markers associated with cancer can be used instead of CK8, 18, 19. ·CAMLs can be CD45 positive or CD45 negative, and the present invention encompasses the use of both types of CAMLs. ·CAMLs are large, with a longest diameter size ranging from about 20 microns to about 300 microns. ·CAML is seen in many different morphological shapes, including spindle-shaped, amoeboid, circular, oval, two legs, three or more legs, thin legs, or amorphous. ·CAMLs of cell tumors typically have diffuse cytokeratin. ·When CAMLs express EpCAM, EpCAM is typically diffuse throughout the cell or associated with vacuoles and / or ingested substances and is almost uniform throughout the cell. However, in some tumors, the expression of EpCAM is extremely weak or absent, so not all CAMLs express EpCAM. ·When CAMLs express a marker, the marker often diffuses throughout the cell or is associated with vacuoles and / or ingested substances and is almost uniform throughout the cell. However, not all CAMLs express the same marker at the same intensity in a limited number, and the marker is not uniformly distributed throughout the cell. ·CAMLs often express markers associated with markers of tumor origin. For example, if the tumor is of prostate cancer origin and expresses PSMA, CAMLs from such patients also express PSMA. As another example, if the primary tumor is of pancreatic origin and expresses PDX-1, CAMLs from such patients also express PDX-1. As a further example, if the primary tumor or CTC of cancer origin expresses CXCR-4, CAMLs from such patients also express CXCR-4. · When the primary tumor of cancer origin or CTCs express the biomarker of the drug target, the CAMLs from such patients also express the biomarker of the drug target. An example of such a biomarker for immunotherapy is PD-L1. · CAMLs express monocyte markers (e.g., CD11c, CD14) and endothelial markers (e.g., CD146, CD202b, CD31). · CAML has the ability to bind to the Fc fragment.

[0066] A broad set of markers was evaluated for their expression on CAMLs, and the results are shown in Figure 9. In one aspect of the invention, the CAMLs of the present invention express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all 21 of the markers shown in Figure 9. The markers were screened against 1118 CAMLs from 93 different patients with different cancers.

[0067] The markers, and thus the CAMLs themselves, can be detected and / or characterized using various means known to those skilled in the art. For example, antibodies having binding specificity for a particular marker can be used to select, detect and / or identify cells expressing one or more markers. CAMLs were initially separated and identified with DAPI, cytokeratin, and CD45; then sequentially re-stained with a total of 27 markers including markers of myeloid / macrophage, leukocyte, megakaryocyte, epithelial, endothelial, progenitor / stem, and motility. As can be seen from Figure 9, the marker expression range is from 0% to 96%. It was found that almost all CAMLs express levels of CD31 and generally co-expressed cytokeratin, CD14, CXCR4, vimentin and other markers. However, although CAMLs contained the distinct myeloid lineage marker (CD14), the CD31 marker was expressed more frequently at 96%.

[0068] CAMLs also present a number of phenotypes that do not appear to be consistent with the understanding of classical cell differentiation (i.e., co-expression of CD45 [leukocytes] and cytokeratin [epithelium], CD11c / CD14 [macrophages] and CD41 [macrophages / megakaryocytes], CD146 [endothelial cells] and CD6 [macrophages / endothelial cells / megakaryocytes], CD31 [leukocytes / macrophages / endothelial cells / megakaryocytes / stem cells] and CD68 / CD163 [macrophages]). Many of the markers appear on multiple cell types. The combination of these data indicates that CAMLs are bone marrow-derived cells at an early stage of the differentiation process and have many phenotypic characteristics related to stem cells and the ability to promote angiogenesis.

[0069] As shown in Figure 9, CAMLs can be visualized by colorimetric staining such as H&E or fluorescence staining of specific markers. In the cytoplasm, CD31 is the most positive phenotype. CD31 alone, or in combination with other positive markers in Figure 9, or cancer markers associated with tumors are recommended.

[0070] Thus, in various embodiments and aspects of the present invention, CAMLs can be defined as having each of the following characteristics and can be identified in biological samples based solely on these characteristics. (a) A large, non-regularly shaped, polyploid nucleus with a size of about 14 - 64 μm, or multiple nuclei within the same cell; (b) A cell size with a size of about 20 - 300 μm; and (c) A morphological shape selected from the group consisting of spindle-shaped, amoeboid, circular, oval, two-legged, three or more legs, thin-legged, and amorphous.

[0071] In certain aspects of embodiments of the present invention, CAMLs can be further defined as having one or more of the following additional features: (d) A CD14 positive phenotype; (e) CD45 expression; (f) EpCAM expression; (g) Vimentin expression; (h) PD-L1 expression; (i) Expression of CD11C marker; (j) Expression of CD146 marker; (k) CD202b marker expression; (l) Expression of CD31 marker; and (m) CK8, 18, 19 epithelial phenotypes.

[0072] In metastatic cancer, diagnostic information obtained from a patient is useful in (i) ruling out cancer at the metastatic site and (ii) methods of treating and / or curing cancer, i.e., ruling out cancer anywhere in the subject. A decrease in the number of CAMLs is an indicator of tumor regression and clearance at the metastatic site. This concept is applicable to all therapies associated with a large number of CAMLs and all cancers. For example, in a subject with breast cancer, there are usually a large number of CAMLs, and in stage IV patients, there are approximately 30 - 50 CAMLs per 7.5 mL of blood. In contrast, in a subject with lung cancer, there are typically only about 5 CAMLs per 7.5 mL of blood in stage IV patients. The presence of CAMLs is an indicator of the presence of cancer. The size of a CAML is also an indicator of the pathogenicity of the disease. Circulating tumor cells

[0073] CTCs express a number of cytokeratins (CKs). CK8, 18, and 19 are most commonly used for diagnosis and can be used in the methods of the present invention, but the assay is not limited to these three. The surface of solid tumor CTCs usually expresses epithelial cell adhesion molecule (EpCAM). However, this expression is neither uniform nor consistent. Since CTCs are white blood cell markers, they should not express any CD45. In assays for identifying tumor-related cells such as CTCs and CAMLs, antibodies against CK8, 18, or 19, or antibodies against CD45 or DAPI are sufficient. By combining the presence of staining and morphology, pathologically definable CTCs, apoptotic CTCs, and CAMLs can be identified.

[0074] Pathologically definable CTCs are identified by the following characteristics: · They have "cancer-like" nuclei stained by DAPI. The exception is when the cell is dividing; the nucleus is condensed. · They express at least CK 18 and 19. Cytokeratin has a fibrous pattern. · They lack CD45 expression. Long exposure is used for imaging to avoid missing low-expressing CD45 cells.

[0075] Thus, the pathologically definable CTCs of the present invention include CTCs having one, two, or three of the following characteristics: (a) cancer-like nuclei; (b) expressing one or more of cytokeratins 8, 18, and 19, and the cytokeratin has a fibrous pattern; and (c) a CD45-negative phenotype.

[0076] Apoptotic CTCs are identified by the following characteristics: · They have cancer nuclei. · They express at least CK8, 18, 19; the cytokeratin is not filamentous but appears fragmented in spots. · They do not express CD45.

[0077] Thus, the apoptotic CTCs of the present invention include CTCs having one, two, or three of the following characteristics: (a) cancer-like nuclei; (b) expressing one or more of cytokeratins 8, 18, and 19, and the cytokeratin is fragmented in spots; and (c) a CD45-negative phenotype. Capture of CAMLs and CTCs

[0078] As suggested above, the unique characteristics of the CAMLs and CTCs described herein make them highly suitable for use in clinical methodologies including screening and diagnosis of diseases such as cancer, monitoring of treatment, and monitoring of disease progression and recurrence.

[0079] Cells that are larger and / or less flexible than other cells present in a body fluid, such as CMALs and CTCs, can be collected by filtering the body fluid. For example, target cells indicative of a condition can be collected by filtering the body fluid through a filter having an aperture that is too small for the target cells to pass through but large enough for other cells to pass through. Once collected, any number of analyses of the target cells can be performed. Such analyses can include, for example, identification, counting, characterization of marker expression, obtaining molecular analysis, and / or culturing of the collected cells.

[0080] In each of the embodiments and aspects of the present invention, circulating cells (e.g., CAMLs and CTCs) can be separated from a biological sample in the methods of the present invention using any suitable related means known to those of skill in the art. Suitable means include, but are not limited to, size exclusion methods, immunocapture, erythrocyte lysis, leukocyte depletion, FICOLL separation, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and physical size-based sorting, slits, channels, hydrodynamic size-based sorting, grouping, trapping, enrichment of large cells, exclusion of small cells, or one or more means selected from various microfluidic chips through combinations thereof. In certain aspects, size exclusion methods include the use of microfilters.

[0081] As an example, circulating cells (e.g., CAMLs and CTCs) can be separated from a biological sample using a size exclusion method that includes using a microfilter. Suitable microfilters can have various pore sizes and shapes. For example, the microfilter can have a pore size in the range of about 5 microns to about 20 microns. Suitable ranges of pore sizes include, but are not limited to, pore sizes in the ranges of about 5-7 microns, 7-8 microns, 8-10 microns, 11-13 microns, 14-16 microns, 17-20 microns, 5-10 microns, 11-15 microns, 15-20 microns, 9-15 microns, 16-20 microns, and 9-20 microns. Another suitable range includes pore sizes in the range of about 5-20 microns, excluding pores of 7-8 microns. In one aspect of the invention, the pore size is about 5-10 microns; in other aspects, the pore size is about 7-8 microns. Larger pore sizes eliminate most white blood cell (WBC) contamination on the filter. The pores of the microfilter can have any shape, and shapes including circular, racetrack, oval, slit, square, rectangular, and / or other shapes are acceptable. The microfilter can have a precise pore shape, a uniform pore distribution, multiple pore shapes, and / or a non-uniform distribution. The microfilter can be a single layer or a multilayer with different shapes in different layers.

[0082] Circulating cells (e.g., CAMLs and CTCs) can also be separated from a biological sample by a microfluidic chip via sorting based on physical size, slits, channels, sorting based on hydrodynamic size, grouping, trapping, immunocapture, concentration of large cells, or exclusion of small cells based on size. The capture efficiency of circulating cells varies depending on the collection method. The size of circulating cells that can be captured on different platforms can also vary. The principle of using circulating cell size to determine disease progression and treatment response is the same, but the statistical values will vary. Collection of circulating cells using the CellSieve™ microfilter provides 100% capture efficiency and high-quality cells.

[0083] A biological sample may be collected in a blood collection tube, especially when the sample is blood. The CellSave blood collection tube (Menarini Silicon Biosystems Inc., San Diego, California) provides stable cell morphology and size. Other available blood collection tubes may not provide cell stability. Cells can expand and may even rupture in most other blood collection tubes.

[0084] The size of the biological sample assayed by the method of the present invention can vary without changing the theory and characteristics underlying the present invention. Suitable sample sizes generally range from about 0.5 mL to about 50 mL. Suitable sizes include samples in the range of about 5 to about 15 mL, about 5 to about 10 mL, about 10 to about 15 mL, about 15 to about 20 mL, about 20 to about 25 mL, about 25 to about 30 mL, about 30 to about 35 mL, about 35 to about 40 mL, about 40 to about 45 mL, and about 45 to about 50 mL. In one aspect of the present invention, the size of the biological sample is about 7.5 mL.

[0085] The source of the biological sample used in the method of the present invention is limited only in that it must contain one of the types of circulating cells referred to herein, such as CAMLs and / or CTCs. Suitable sources of biological samples include blood, lymph nodes, bone marrow, cerebrospinal fluid, tissue, urine, peripheral blood mononuclear cells (PBMCs), and cryopreserved PBMCs. When the biological sample is blood, the blood can be, for example, peripheral blood, median cubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, or jugular vein blood. The sample may be a fresh sample or a thawed appropriately prepared cryopreserved sample.

[0086] In a further aspect of the present invention, circulating cells (CAMLs) are separated from a biological sample using the CellSieve™ low pressure precision filtration assay.

[0087] When comparing the size of circulating cells between two subjects with cancer, it is preferred that the subjects have the same type of cancer. However, among other factors, it may be difficult to perfectly match the two subjects with respect to cancer type, cancer stage, cancer progression rate, treatment history, and history of cancer remission and / or recurrence. Thus, it should be understood that there may be some variation in the cancer characteristics of the two subjects being compared by the relevant methods of the present invention. Identification of CAMLs

[0088] As indicated above, various embodiments and aspects of the present invention are based on determining the size or number of CMALs in a sample obtained from a subject with cancer. Specific means for identifying CAMLs in a sample are as previously described and include the following: (i) determination of the size, shape, and number of nuclei within the cell; (ii) determination of the overall cell size; (iii) determination of the morphological shape of the cell; and (iv) use of one or more of the markers shown in FIG. 9.

[0089] However, it should be made clear that each of the embodiments and aspects of the present invention can be implemented without explicitly identifying circulating cells in a biological sample as CAMLs per se. Instead, for example, simply identifying cells based on cell size may be used. Examples of other techniques include the use of colorimetric staining such as H&E staining, or the simple identification of CK(+) cells collected within or from a biological sample. Subject

[0090] The subject referred to in the inventive method will be a human, a non-human primate, a bird, a horse, a cow, a goat, a sheep, a companion animal such as a dog, a cat, a rodent, or other mammal. Animals can develop cancer. Cancer accounts for nearly 50% of the causes of death in pets over 10 years old. Some common types of cancer found in pets include skin cancer, breast cancer, head and neck cancer, lymphoma, leukemia, testicular cancer, abdominal cancer, and bone cancer. Examples of cancers that are commonly found in pets and also commonly found in humans are lymphoma, melanoma, and osteosarcoma. CAMLs are found in animals other than humans. Therefore, the clinical utility of CAMLs in humans can also be applied to other animals.

[0091] In various aspects and embodiments of the present invention, the subject is a subject having cancer. The cancer can be a solid tumor, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, colorectal cancer, liver cancer, head and neck cancer, kidney cancer, ovarian cancer, esophageal cancer, or other solid tumor cancers. Those skilled in the art will fully understand that the method of the present invention is not limited to a specific form or type of cancer and can be implemented in relation to a wide variety of cancers. Treatment

[0092] In certain aspects and embodiments of the present invention, the subject is undergoing treatment. The treatment can be one or more of chemotherapy, a single drug, a combination of drugs, immunotherapy, radiation therapy, chemoradiation therapy, radiation combined with one or more drugs, chemoradiation therapy combined with one or more drugs, a cancer vaccine, and cell therapy.

[0093] A cancer vaccine can be administered to a subject in the form of cells that express a marker intended as a vaccine target. The cells can be in the form of cancer cells, modified viruses, and other types of modified cells. After the subject receives the vaccine, the body's immune system produces T cells that recognize and attack the antigen expressed by the vaccine, along with the cancer cells. If the treatment is a cancer vaccine, the subject may express at least one HLA allele.

[0094] The CAML size still predicts cancer pathogenicity. Additional information can be obtained from the number of CAMLs. The immune system may be able to eliminate cancer more quickly from smaller metastatic sites than from the largest cancer sites. The killer T cells of the immune system may be more likely to infiltrate tumors in some organs than in others. For example, immunotherapy typically works best for lung cancer and melanoma, but less well for breast cancer and prostate cancer. Often, as immune cells infiltrate the tumor and kill tumor cells, pseudoprogression is seen as the tumor grows. This is pseudoprogression in the indicator of a good immunotherapy treatment response.

[0095] An example of a cancer vaccine is SV-BR-1-GM. It is a GM-CSF engineered breast cancer cell line that expresses HER2, PRAME, and class I and class II HLA antigens. SV-BR-1-GM cells loaded with yellow fever virus (YFV) peptides directly activated YFV-specific CD4+ T cells.

[0096] Responders to this SV-BR-1-GM vaccine are predicted to be breast cancer patients who express at least one HLA allele. Methods of the Invention

[0097] As described in the above summary, the present invention is directed, inter alia, to methods for predicting treatment response and / or disease progression (e.g., cancer progression) in a subject based on the number and / or size of circulating cells. These methods involve determining the number and / or size of circulating cells in a biological sample from a subject, such as a subject having cancer, and, in some aspects and embodiments, comparing the results from samples taken from the same subject at different time points. Changes in the size or number of circulating cells being assayed are indicators of treatment response and / or disease progression in the subject, and predictions can be based thereon.

[0098] Specific embodiments and aspects of the present invention are presented in the following paragraphs. However, when the methods of the present invention are related to predicting treatment response in a subject, it can first be noted that the time scale for obtaining treatment response information depends on the type of treatment, but is generally as short as 2 treatment cycles or 60 days. Treatment response depends on the type of treatment, but it will be apparent that it can be seen after 1 treatment cycle or even as little as 10 days.

[0099] It can also be noted here that by analyzing the size and number of CAMLs, and changes in the size and number of CAMLs together with other parameters, the probability of making a correct determination of treatment response and / or disease progression can be improved. Such parameters include the following: 1. Good treatment response, for example: · Disappearance of CTCs · Decrease in the number of CTCs when initially very high · Decrease in circulating tumor DNA (ctDNA) 2. Poor treatment response, for example: · Increase in the number of CTCs [3] · Appearance of CTCs in mitosis [7] · Appearance of CTCs within clusters · Increase in the amount of ctDNA · Increase in mutations of ctDNA · Increase in mutations of CAML DNA

[0100] Furthermore, the size and number of CAMLs, as well as changes in the size and number of CAMLs, can provide a prediction of treatment response and / or disease progression before image diagnosis can provide prognostic information. Image diagnosis requires substantial growth of the tumor so that differences in disease progression can be discerned. CT image diagnosis also generates an undesirable radiation dose that can cause cancer and is undesirable at 30 - 45 day intervals. Finally, image diagnosis is expensive.

[0101] An increase in the number of CTCs is known to form the basis for predicting disease progression. However, it is only applicable by itself to stage IV breast, prostate, and colorectal cancers. CTCs are rare in the early stages of cancer and other types of solid tumors. Determination of the size of circulating cells

[0102] In one embodiment, the method of the present invention includes determining the size of circulating cells, such as CAMLs, in two or more biological samples obtained over time from a subject having cancer, where if a decrease in the size of the circulating cells is seen over time between the samples, the cancer is predicted not to progress and / or it is determined that the subject is responsive to treatment. In a related embodiment, the method of the present invention includes determining the number of circulating cells, such as CAMLs, in two or more biological samples obtained over time from a subject having cancer, where if a decrease in the number of the circulating cells is seen over time between the samples, the cancer is predicted not to progress and / or it is determined that the subject is responsive to treatment.

[0103] In a related aspect, the method includes determining the size of circulating cells, such as CAMLs, in a first and a second biological sample, and any additional biological samples, obtained from a subject having cancer, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the size of at least one cell in the first sample is about 50 μm or greater and the size of each cell in the second and any additional samples is less than about 50 μm, it is predicted that the cancer will not progress and / or it is determined that the subject is responsive to treatment.

[0104] In another related aspect, the method includes determining the average size of circulating cells, such as CAMLs, in a first and a second biological sample, and any additional biological samples, obtained from a subject having cancer, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the average size of the circulating cells in the second and any additional samples is decreased compared to the average size of the cells in the first sample, the subject is identified as being responsive to treatment. In one aspect, the average size of the circulating cells in the first sample is about 50 μm or greater. Prediction of Cancer Progression

[0105] In another embodiment, the present invention is directed to a method for predicting cancer progression in a subject having cancer, the method including determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject having cancer and predicting based thereon, where if the size of each circulating cell in the sample is less than about 50 μm, it is predicted that the cancer will not progress, and if the size of at least one circulating cell in the sample is about 50 μm or greater, it is predicted that the cancer will progress.

[0106] In one aspect of this embodiment, the present invention is directed to a method for predicting cancer progression in a subject having cancer, comprising determining the size of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject having cancer, and any additional biological sample, The first sample is obtained from the subject before or during cancer treatment, and the second sample and any additional samples are obtained from the subject after at least one cancer treatment, If the average size of CAMLs in the second and any additional samples is decreased compared to the average size of CAMLs in the first sample, the cancer is predicted not to progress in the subject, and the subject is optionally identified as responding to the treatment; or If the average size of CAMLs in the second and any additional samples is maintained or increased compared to the average size of CAMLs in the first sample, the cancer is predicted to progress in the subject, and the subject is optionally identified as not responding to the treatment; or If the size of at least one CAML in the first sample is about 50 μm or more and the size of each cell in the second and any additional samples is less than about 50 μm, the cancer is predicted not to progress, and the subject is optionally identified as responding to the treatment; or If the size of each CAML in the first sample is less than about 50 μm and the size of at least one CAML in the second and any additional samples is greater than about 50 μm, the cancer is predicted to progress, and the subject is optionally identified as not responding to the treatment. In one aspect, the average size of the circulating cells in the first sample is about 50 μm or more.

[0107] In another aspect of this embodiment, the present invention is directed to a method for predicting cancer progression in a subject having cancer, comprising determining the number of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject having cancer, and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second sample and any additional samples are obtained from the subject after at least one cancer treatment, and if the number of circulating cells in the second and any additional samples is decreased compared to the number of circulating cells in the first sample, the cancer is predicted not to progress and the subject is optionally identified as responding to the treatment, and if the number of circulating cells in the second and any additional samples is maintained or increased compared to the number of circulating cells in the first sample, the cancer is predicted to progress and the subject is optionally identified as not responding to the treatment. Prediction of treatment response

[0108] In a further embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, comprising determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject and predicting based thereon, wherein if the size of each circulating cell in the sample is less than about 50 μm, the subject is predicted to respond to the treatment, and if the size of at least one circulating cell in the sample is about 50 μm or greater, the subject is predicted not to respond to the treatment.

[0109] In one aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, comprising determining the size of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject having cancer, and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second sample and any additional samples are obtained from the subject after at least one cancer treatment, if the average size of CAMLs in the second and any additional samples is decreased compared to the average size of CAMLs in the first sample, the subject is predicted to respond to the treatment; If the average size of CAMLs in the second and any additional samples is maintained or increased compared to the average size of CAMLs in the first sample, the subject is predicted not to respond to treatment; If the number of circulating cells with a size greater than about 50 μm is maintained or increased from the first sample to the second and any additional samples, the subject is predicted not to respond to treatment; or If the number of circulating cells with a size greater than about 50 μm decreases from the first sample to the second and any additional samples, the subject is predicted to respond to treatment; or If the size of each circulating cell in the second or subsequent sample is decreased compared to the first sample and the size of each circulating cell in the second or any additional sample is less than about 50 μm, the subject is predicted to respond to treatment and there is a possibility of cancer cure.

[0110] In another aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, comprising determining that there are no circulating cells, such as CAMLs, greater than about 50 μm in a biological sample obtained from the subject, and predicting that the subject is responding to treatment.

[0111] In a further aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, comprising determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject having cancer, where the sample is obtained from the subject after at least one cancer treatment, and if the size of each circulating cell is about 50 μm or less, the subject is predicted to respond to treatment.

[0112] In an additional aspect of this embodiment, the present invention is directed to a method for predicting the response of a subject having cancer to treatment, including determining the number of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject having cancer, and any additional biological samples, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the number of circulating cells decreases from the first to the second and any additional samples, the subject is predicted to respond to the treatment, and if the number of circulating cells is maintained or increases from the first to the second and any additional samples, the subject is predicted not to respond to the treatment. Prediction of resistance to treatment

[0113] In a final embodiment, the present invention is directed to a method for predicting resistance to treatment in a subject having lung cancer, the method including determining the size of circulating cells, such as CAMLs, in a biological sample from the subject having cancer, where if the size of at least one cell in the sample is about 50 μm or greater, the subject is predicted to be more resistant to cancer treatment than a subject having cancer without cells having a size greater than about 50 μm. In one aspect of this embodiment, the cancer is lung cancer.

[0114] In each of these embodiments and aspects of the present invention, if no circulating cells are seen in the biological sample at the end of treatment, it can be concluded that the cancer has been removed. If circulating cells are still found in the biological sample at the end of treatment, it can be concluded that the patient's cancer has not been removed and that the patient has residual disease.

[0115] In each of the embodiments and aspects of the present invention, CAMLs can be used as cancer markers independently or in combination with other circulating cells such as circulating tumor cells (CTCs), epithelial-mesenchymal transition cells (EMTs), and circulating cancer-associated vascular endothelial cells (CAVEs), as well as free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomic, metabolomic, lipidomic, and other biomarkers, providing a more complete understanding of the patient's disease. CAMLs are the only cell type typically larger than 30 μm in size among the groups of circulating cells mentioned here. Treatment method

[0116] As summarized above, the present invention is also directed to a method of treating cancer, wherein the treatment decision can be based on the prediction of the treatment response using circulating cells in the above method.

[0117] Accordingly, the present invention includes a method of treating a subject having cancer, comprising administering a therapeutically effective amount of cancer treatment to the subject having cancer and determining the size of circulating cells such as CAMLs in two or more biological samples obtained from the subject having cancer, wherein the first biological sample is obtained from the subject before or during cancer treatment, the second biological sample is obtained from the subject during or after cancer treatment, and if a decrease in the size of circulating cells is observed over time between the samples, it is understood that the subject is responding to the treatment and the treatment is continued, and if no decrease in the size of circulating cells is observed over time between the samples, it is understood that the subject is not responding to the treatment and the treatment is not continued.

[0118] In related embodiments, the method of the invention comprises administering a therapeutically effective amount of cancer treatment to a subject having cancer and determining the number of circulating cells, such as CAMLs, in two or more biological samples obtained from the subject having cancer, wherein a first biological sample is obtained from the subject before or during cancer treatment and a second biological sample is obtained from the subject during or after cancer treatment, and if a decrease in the number of circulating cells between the samples is seen over time, it is determined that the subject is responsive to the treatment and the treatment is continued, and if no decrease in the circulating cell number between the samples is seen over time, it is determined that the subject is not responsive to the treatment and the treatment is not continued.

[0119] In one aspect of this embodiment, the method comprises administering a therapeutically effective amount of cancer treatment to a subject having cancer and determining the size of circulating cells, such as CAMLs, in first and second biological samples, and any additional biological samples, obtained from the subject, wherein the first sample is obtained from the subject before or during cancer treatment and the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the size of at least one cell in the first sample is about 50 μm or greater and the size of each cell in the second sample and any additional samples is less than about 50 μm, it is determined that the subject is responsive to the treatment and the treatment is continued.

[0120] In another related aspect, the method includes administering a therapeutically effective amount of cancer treatment to a subject having cancer and determining the average size of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject, and any additional biological samples, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the average size of the circulating cells in the second and any additional samples is decreased compared to the average size of the cells in the first sample, the subject is identified as responding to the treatment and the treatment is continued, and if the average size of the circulating cells in the second and any additional samples is not decreased compared to the average size of the cells in the first sample, the subject is identified as not responding to the treatment and the treatment is not continued. In some aspects, the average size of the circulating cells in the first sample is about 50 μm or greater.

[0121] In another embodiment, the present invention is directed to a method of treating a subject having cancer, the method including administering a therapeutically effective amount of cancer treatment to a subject having cancer and determining the size of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject, and any additional biological samples, where the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, if the average size of the CAMLs in the second and any additional samples is decreased compared to the average size of the CAMLs in the first sample, the subject is predicted to respond to the treatment and the treatment is continued; if the average size of the CAMLs in the second and any additional samples is maintained or increased compared to the average size of the CAMLs in the first sample, the subject is predicted not to respond to the treatment and the treatment is not continued; if the number of circulating cells having a size greater than about 50 μm is maintained or increased in the first through second samples and any additional samples, the subject is predicted not to respond to the treatment and the treatment is not continued; If, for the first to second samples and any additional samples, the number of circulating cells greater than about 50 μm in size decreases, the subject is predicted to respond to the treatment and the treatment is continued; or If the size of each circulating cell in the second or subsequent sample is decreased compared to the first sample and the size of each circulating cell in the second or any additional sample is less than about 50 μm, the subject is predicted to respond to the treatment and the treatment is continued.

[0122] In one aspect of this embodiment, the invention is directed to a method of treating a subject having cancer, comprising administering to the subject having cancer a therapeutically effective amount of a cancer treatment and determining that no circulating cells, such as CAMLs, greater than about 50 μm are present in a biological sample obtained from the subject, wherein the sample is obtained from the subject after at least one cancer treatment, and if no circulating cells greater than about 50 μm are present in the biological sample, the subject is predicted to respond to the treatment and the treatment is continued.

[0123] In a further aspect of this embodiment, the invention is directed to a method of treating a subject having cancer, comprising administering to the subject having cancer a therapeutically effective amount of a cancer treatment and determining the size of circulating cells, such as CAMLs, in a biological sample obtained from the subject, wherein the sample is obtained from the subject after at least one cancer treatment, and if the size of each circulating cell is about 50 μm or less, the subject is predicted to respond to the treatment and the treatment is continued.

[0124] In an additional aspect of this embodiment, the invention is directed to a method of treating a subject having cancer, comprising administering a therapeutically effective amount of cancer treatment to the subject, and determining the number of circulating cells, such as CAMLs, in a first and a second biological sample obtained from the subject, and any additional biological samples, wherein the first sample is obtained from the subject before or during cancer treatment, the second and any additional samples are obtained from the subject after at least one cancer treatment, and if the number of circulating cells decreases from the first to the second and any additional samples, the subject is predicted to respond to the treatment and the treatment is continued, and if the number of circulating cells is maintained or increases from the first to the second and any additional samples, the subject is predicted not to respond to the treatment and the treatment is not continued.

Example

[0125] Example 1 Sample Collection and Processing Size exclusion is one method for collecting both CTCs and CAMLs. There are many size exclusion methods. Here, the filtration method will be described as an example. Peripheral blood was collected into CellSave tubes (Menarini Silicon Biosystems Inc., San Diego, California) and processed within 96 hours. All cancer-related cells (CTCs, EMTs, CECs, and CAMLs) in the blood sample were collected using CellSieve™ microfiltration technology. The CellSieve™ microfilter has over 180,000 pores uniformly arranged with a pore diameter of 7 μm within a 9 mm area. The reagents include a pre-fixation buffer, a post-fixation buffer, a permeabilization buffer, and an antibody cocktail. The filtration was performed using either a syringe pump set [5] aspirated at 5 mL / min or a vacuum pump [4]. The filtration process began by pre-fixing 7.5 mL of blood in 7.5 mL of the pre-fixation buffer before aspiration through the filter. Next, the filter and the captured cells were subjected to washing, post-fixation, washing, permeabilization, and washing. Next, the cells captured on the filter were stained with the antibody cocktail and subsequently washed. The filter was placed on a microscope slide and covered with a coverslip using Fluoromount-G / DAPI (Southern Biotech). One possible antibody cocktail for identifying CTCs and CAMLs in epithelial cancer can include cytokeratin and CD45 antibodies. Additional antibodies for the markers of interest can be included. Next, the slide was read on a fluorescence microscope. The number of CTCs was counted. The size of CAMLs was measured. The number of CTCs, the maximum CAML size, and the maximum CAML size < 50 μm or 50 μm and above were recorded. There are also many other diverse methods for collecting CTCs and CAMLs from blood. Study 1: Study of n = 42 NSCLC patients who received radiotherapy

[0126] Figures 1A - 1D show the CAML sizes related to treatment response and the changes in CAML sizes. The solid curves and lines indicate non - responders, and the dashed lines indicate responders. In the data analysis of this figure, treatment response was defined as the response at the 24 - month time point. In this pilot study of n = 42 samples, the subjects had non - small cell lung cancer (NSCLC), most of whom had received radiotherapy and a minority had also received chemotherapy. Progression was based on the standard of therapeutic imaging diagnosis in lung patients before and after the introduction of chemoradiotherapy. The results show a strong trend even within the first 100 days. Figure 1A shows that most patients with CAMLs less than 50 μm in size at baseline remained less than 50 μm. Figure 1B shows that all patients with CAMLs that increased in size from less than 50 μm to more than 50 μm progressed. Figure 1C shows that one patient who initially had CAMLs larger than 50 μm and had CAMLs smaller than 50 μm at the second time point responded to treatment. Figure 1D shows that there was no response to treatment in patients with CAMLs greater than 50 μm at baseline and no decrease in size over time, and in one subject who showed a decrease in the size of CAMLs.

[0127] To perform qualitative measurements, the accuracy of prediction at a given time point was defined as (true positives + true negatives) / (false positives + false negatives). True positives are the number of patients with CAML size > 50 μm and who progressed. False positives are the number of patients with CAML size > 50 μm but who did not progress. True negatives are the number of patients with CAML size < 50 μm and who did not progress. False negatives are the number of patients with CAML size < 50 μm and who progressed. The use of this 50 - μm size is based on the data shown in International Publication WO2018 / 151865 dated August 23, 2018. The accuracy at baseline was 69% and at the final follow - up was 85%.

[0128] The indicator of treatment response can be obtained within 100 days for most patients. In summary, the final CAML size after treatment provides a basis for making predictions and conclusions regarding treatment response. Study 2: A study of n = 52 NSCLC patients treated with radiotherapy

[0129] To achieve 90% power on both sides with an α of 0.05, a test set of 52 NSCLC patients was conducted. All patients had pathologically confirmed lung cancer stages I (n = 7), II (n = 7), III (n = 29), and IV (n = 9), and received standard PET / CT scans. Baseline (BL) blood samples were collected before treatment initiation. The first follow-up (FU1) was conducted during radiotherapy (~30 days). The second follow-up (FU2) was conducted at the end of radiotherapy (~60 days). Several patients also received chemotherapy within 60 days.

[0130] Blood was filtered by CellSieve™ precision filtration, and CAML size was quantified. Analyses by CAML size <50 μm or ≥50 μm were used to evaluate the progression-free survival (PFS) hazard ratio (HR) by cut-off univariate and multivariate analyses at each time point.

[0131] The following analysis predicts disease progression, as opposed to treatment response. CAMLs were identified in 97% of all samples, with an average of 2.9 CAMLs / 7.5 mL sample at BL, and progression-free survival (PFS) decreased with CAMLs ≥50 μm (HR = 2.9, 95% CI 1.3 - 6.2, p = 0.015). At FU1 (Follow-up 1), the CAML size increased in 7 patients and PFS decreased (HR = 5.0 95% CI 2.3 - 10.9, p < 0.001). At FU2 (Follow-up 2), an additional 7 patients increased their CAML size and further decreased their PFS (HR = 7.1, 95% CI 3.4 - 14.8, p < 0.001). In total, CAMLs with a size ≥50 μm at BL were 65% accurate in predicting the final progression within 24 months, while CAMLs with a size ≥50 μm at FU2 were 89% accurate in predicting progression.

[0132] Note that it is applicable to predicting treatment response or progression in all stages of NSCLC. Study 3: n = 52 NSCLC and n = 29 esophageal cancer patients treated with chemoradiotherapy

[0133] Based on the same 52 NSCLC and 21 esophageal cancer (n = 20 stage III and n = 1 stage IV) patients described in Figures 1A - 1D (stage I, n = 7, stage II, n = 7, stage III, n = 29, and stage IV, n = 9), a larger study was conducted to predict the outcome of radiotherapy including esophageal cancer.

[0134] Figures 2A - 2C show Kaplan - Meier plots of progression - free survival (PFS) at three time points: baseline (BL) (Figure 2A), first follow - up (FU1) (Figure 2B), and second follow - up (FU2) (Figure 2C). The data were analyzed based on the size of CAMLs and were < 50 μm (blue (upper) curve) or 50 μm or more (red (lower) curve). At baseline (Figure 2A and Table 1), the size of CAMLs provides prognostic information. At the first follow - up (Figure 2B and Table 2), a prediction of treatment response based on CAML size is shown. At the second follow - up after radiotherapy (Figure 2C and Table 3), CAML size provided a much better prediction of PFS for responders compared to non - responders. Multivariate analysis was performed. CAML size provided the best results compared to stage, cancer type, age, and gender. [Table 1] [Table 2] [Table 3]

[0135] Figures 3A-3C show Kaplan-Meier plots of overall survival (OS) at three time points: baseline (BL) (Figure 3A), first follow-up (FU1) (Figure 3B), and second follow-up (FU2) (Figure 3C). The data were analyzed based on the size of CAMLs, <50 μm (blue (upper) curve) or ≥50 μm (red (lower) curve). CAML size provides a good prediction of OS in responders compared to non-responders to chemoradiotherapy for NSCLC and esophageal cancer.

[0136] CAMLs were seen in 97% of all BL samples. On average, 2.9 CAMLs / 7.5 mL of blood were seen. Patients with large CAMLs at BL significantly decreased PFS in NSCLC (HR = 2.9, 95% CI 1.3-6.2, p = 0.015), and was marginal in esophageal cancer (HR = 3.0, 95% CI 0.9-9.9, p = 0.14).

[0137] At FU1, patients with detectable large CAMLs further decreased post-treatment PFS (NSCLC, HR = 5.0, 95% CI 2.3-10.9, p<0.001; EC, HR = 4.0, 95% CI 1.2-13.2, p = 0.05), and was more prominent at FU2 (NSCLC, HR = 7.1, 95% CI 3.4-14.8, p<0.001; EC, HR = 5.6, 95% CI 1.6-18.8, p = 0.01). Combining the two disease datasets, large CAMLs at BL were 70% accurate in predicting final progression within 24 months, while large CAMLs at FU2 were 84% accurate in predicting progression. In multivariate analysis, large CAMLs were the most significant in predicting treatment response.

[0138] Figures 4A-4F show details of the largest CAML in each patient over three time points for patients with breast cancer, prostate cancer, or lung cancer. The red and green curves are for patients with progression (red) vs patients with neither progression nor cure (green).

[0139] Figure 4A shows patients with a CAML size of less than 50 μm at baseline and remaining less than 50 μm at FU2. 81% of the patients did not progress within 24 months.

[0140] Figure 4B shows patients with a CAML size of less than 50 μm at baseline but increasing to more than 50 μm at FU2. 79% of the patients progressed within 24 months.

[0141] Figure 4C shows patients with a CAML size greater than 50 μm at baseline and decreasing to less than 50 μm at FU2. 60% of the patients did not progress within 24 months.

[0142] Figure 4D shows patients with a CAML size greater than 50 μm at baseline and remaining greater than 50 μm at FU2. 96% of the patients progressed within 24 months.

[0143] Figure 4E shows patients with a CAML size of less than 50 μm at FU2. 79% of the patients did not progress within 24 months.

[0144] Figure 4F shows patients with a CAML size greater than 50 μm at FU2. 89% of the patients progressed within 24 months. Study 4: Applicable to many solid tumors and treatments

[0145] Blood-based biomarkers (PSA, CEA, CA125) are used to track the real-time progression of diseases in parallel with imaging diagnosis. However, although there are numerous blood biomarkers, they are specific to cancer types (i.e., PSA for prostate, CEA for colon), and do not appear in all affected individuals. CAMLs have been identified in various solid cancer types where an increase in size and ploidy has been observed during progressive diseases. To evaluate whether the expansion of CAMLs is a biomarker of progression / response, anonymized peripheral blood samples from 34 cancer patients with breast cancer (n = 10), lung cancer (n = 16), and prostate cancer (n = 8) [stage I (n = 2), II (n = 3), III (n = 8), and IV (n = 21)] were used to track CAML growth / shrinkage in a multi-center, prospective, collaborative study. Samples were taken before treatment (BL), at the follow-up time of ~1 month after treatment initiation (FU1), and at the follow-up time of ~3 months (FU2).

[0146] Treatment of patients in this group was very diverse. Treatments included: radiotherapy, chemoradiotherapy, chemotherapy, docetaxel, trastuzumab, trastuzumab / lapatinib, fulvestrant / trastuzumab / lapatinib / brain radiotherapy, letrozole / denosumab, trastuzumab / pertuzumab / eribulin, paclitaxel / herceptin / pertuzumab, abraxane, eligard, lupron, eligard / bicalutamide, casodex, lupron / bicalutamide, lupron, surgery, abiraterone / lupron, nivolumab, carboplatin / taxol, carboplatin / gemcitabine, gemcitabine / trastuzumab + lapatinib, eribulin / trastuzumab / lapatinib, herceptin / fulvestrant / palbociclib, eribulin, paclitaxel / herceptin / pertuzumab, abraxane, eligard + enzalutamide, lupron + enzalutamide, casodex + lupron, vinorelbine / trastuzumab / lapatinib, letrozole / fulvestrant, eribulin / herceptin, herceptin / femara.

[0147] Overall clinical data were: · CAML was seen in 97% of the cancer patients in BL, 97% in FU1, and 94% in FU2. · Over two years, 7 patients showed no progression of clinical disease (blue or upper line in Figures 5 and 6), and 29 patients showed observable progression of clinical disease (red or lower line in Figures 5 and 6). · One of the patients without progression (blue or upper line in Figures 5 and 6, n = 7) had CAMLs of 50 μm or more at all time points, while 6 had only small CAMLs at all time points. · Among the 29 patients with progression, 〇 22 patients had CAMLs of 50 μm or more at all time points 〇 5 patients had CAMLs <50 μm in BL and the size increased by FU2. 〇 1 patient had a CAML of 50 μm or more in BL and decreased by FU2. 〇 1 patient had small CAMLs at all time points.

[0148] Independently of the type of solid tumor and the type of treatment, it was evaluated whether the CAML size could predict the treatment response.

[0149] Figures 5A - 5B show Kaplan - Meier plots of progression - free survival (PFS) at two time points: baseline (BL) (Figure 5A) and second follow - up (FU2) (Figure 5B). The data were analyzed based on the size of CAMLs, <50 μm (blue (upper) curve) or 50 μm or more (red (lower) curve).

[0150] Figures 6A - 6B show Kaplan - Meier plots of overall survival (OS) at two time points: baseline (BL) (Figure 6A) and second follow - up (FU2) (Figure 6B). The data were analyzed based on the size of CAMLs, <50 μm (blue (upper) curve) or 50 μm or more (red (lower) curve).

[0151] We show that increased CAML expansion compared to baseline indicates non - responsiveness to treatment and, as a result, shorter PFS in various types of cancer. Study 5: Combining CTC and CAML Data

[0152] Another set of results is related to different cancers and different treatments. In addition to the size of CAMLs before and after the introduction of new therapies, a 2-year prospective double-blind multi-center joint trial was conducted to evaluate the inclusion of CTCs. A total of n = 91 patients were recruited: breast cancer (n = 14), esophageal cancer (n = 23), NSCLC (n = 23), prostate cancer (n = 21), and small cell lung cancer (SCLC) (n = 10) with stage III (n = 53) or stage IV (n = 38) disease. Baseline (BL) blood samples were taken before the introduction of the new treatment, and one follow-up (FU) was conducted after the start of systemic treatment (~30 days). Blood was filtered by CellSieve filtration. The quantity and subtypes of CTCs and CAMLs were analyzed based on the OS hazard ratio (HR) by cut-off univariate and multivariate analysis.

[0153] Figure 7 shows that CTCs were rare in the lung (6%), esophagus (4%), and prostate (24%), but common in the breast (79%). CAMLs were common to all cancers and were seen in 92% of BL samples and 98% of FU samples, both of which are prognostic for OS.

[0154] CTCs were identified in 21% of BL patients, and single CTCs were prognostic for OS (HR = 2.49 95% CI = 1.1 - 5.1, P = 0.048). Furthermore, CTCs were seen in 23% of FU samples and were also prognostic for OS (HR = 3.1 95% CI = 1.4 - 6.9, p = 0.013). However, CTCs were rare in lung cancer (6%), esophageal cancer (4%) and prostate cancer (24%), but common in breast cancer (79%). In contrast, CAMLs were seen in 92% of BL, and CAMLs more than 50μm were prognostic for OS (HR = 3.0, 95% CI = 1.6 - 5.7, p = 0.001). In FU, CAMLs were seen in 98% of FU samples, and the prognostic value of OS for CAMLs more than 50μm increased (HR = 3.5 95% CI = 1.9 - 6.6, p = 0.001). Furthermore, after the introduction of systemic therapy, the presence of both CTCs > 1 or CAMLs more than 50μm was prognostic for OS HR = 3.7 95% CI = 2.0 - 7.0, p = 0.001 and was 75% accurate in predicting patient survival within 24 hours.

[0155] In FU, single CTCs (Figure 8A) were associated with lower OS (lower (red) curve). Single CAMLs more than 50μm CAML (Figure 8B) were also associated with a decrease in OS (lower (red) curve). The OS of simultaneous measurement of both CTCs and CAMLs by blood test is shown in Figure 8C, together with single CAMLs and CTCs shown as the lower (red) curve.

[0156] This data indicates that simultaneous measurement of both CTCs and CAML size enhances the prognostic value of blood-based diagnosis and can anticipate subsequent treatment benefits.

[0157] This data suggests that CAML size and changes in CAML size are applicable to predicting treatment response or disease progression in major solid tumors. Example 2 Cancer vaccine

[0158] Cancer vaccines can be administered to a subject in the form of cells that express a marker intended as a vaccine target. The cells can be in the form of cancer cells, modified viruses, and other types of modified cells. After the subject receives the vaccine, the body's immune system produces T cells that recognize and attack the antigen expressed by the vaccine along with the cancer cells.

[0159] Data supporting the concept of utilizing CAMLs to monitor and / or predict the treatment response of cancer vaccine patients is based on 10 breast cancer patients treated with the SV-BR-1-GM vaccine. 7.5 mL of blood was collected and analyzed at different time points. The data shown in FIGS. 10 and 11 represent CAML data for patients expressing at least one HLA allele (solid line) and patients not expressing any HLA alleles (dashed line).

[0160] FIG. 10 shows the number of CAMLs during treatment. Patients expressing at least one HLA allele had a decreased number or fewer CAMLs during treatment, indicating a positive response. In one patient where the data point was 200 days, lung and soft tissue metastases were completely removed. Patients not expressing HLA alleles did not appear to benefit from the vaccine, and the number of CAMLs was found to be increasing.

[0161] FIG. 11 shows the size of CAMLs during treatment. All patients except one expressing at least one HLA allele showed a decrease in CAML size during treatment. However, the CAML size of most patients was still much larger than 50 μm, indicating the presence of highly pathogenic cancer. Imaging diagnoses of the patients showed that they all still had cancer (data not shown).

[0162] Changes in the number of CAMLs are applicable to other treatments besides vaccines and other cancers, especially cancers with more than five CAMLs typically present in subjects with stage IV cancer. Example 3

[0163] CAMLs are common circulating stromal cells in the peripheral blood of cancer patients that were hypothesized to be a mechanism in cancer pathogenesis. Shown here are the results of a prospective trial for untreated lung cancer patients before induction and immediately after completion of radical radiotherapy to determine whether CAMLs can predict treatment response and cancer progression.

[0164] Methods: A single-arm prospective single-blind trial was conducted over two years to test the relationship of expanded CAMLs (≥50 μm) to progression-free survival (PFS) in lung cancer patients before and after the introduction of radical radiotherapy. To achieve 95% two-sided power (α = 0.05), a training set of 55 patients with all pathologically confirmed lung cancer was employed: stage I (n = 13), stage II (n = 7), stage IIIa (n = 10), stage IIIb (n = 18), and stage IV (n = 7). Baseline (BL) blood samples were taken before treatment initiation. A second blood sample (T1) was taken ~60 days after radiotherapy completion, n = 46 patients, if possible. Blood was filtered by CellSieve™ filtration and CAMLs were quantified. Analysis by CAML size <50 μm or ≥50 μm was used to evaluate the PFS-hazard ratio (HR) by cutoff univariate and multivariate analysis.

[0165] Results (data not shown): CAMLs were seen in 93% of BL samples at an average of 3.2 CAML / 7.5 mL. PFS was decreased in patients with at least one CAML of 50 μm or more (HR = 2.9, 95% CI 1.4 - 6.0, p = 0.010). Forty-six patients consented to serial blood sampling. Among the 46 patients, 13 had an increase in CAML size to 50 μm or more, while 3 patients had a decrease to <50 μm, resulting in an increase in PFS (HR = 7.7, 95% CI 2.6 - 12.5, p < 0.001). Collectively, 90% of patients with CAMLs of 50 μm or more at BL progressed within 2 years, compared with 46% of patients with CAMLs of <50 μm. Enlargement of CAMLs at T1 predicted progression better, with 92% of patients with CAMLs of 50 μm or more progressing, compared with 21% of patients with CAMLs of <50 μm. Notably, 100% of patients with CAMLs of 50 μm or more at both BL and T1 progressed. In comparison, only 11% of patients with CAMLs of <50 μm at both BL and T1 progressed. In multivariate analysis, CAML size was the most important predictor of PFS and OS, independent of all other clinical variables including stage.

[0166] This data suggests that in patients with lung cancer undergoing radical radiotherapy, the presence of enlarged CAMLs appears to predict patients who are resistant to treatment. Furthermore, these circulating stromal cells may be useful for the sequential monitoring of patients who may be the prognosis of patients prone to progression after treatment. Citation TIFF0007692075000004.tif101166

Claims

1. 1. A method for indicating the likelihood of a subject having cancer responding to a treatment, comprising determining the size of CAMLs in a blood sample obtained from the subject and making a prediction based thereon, indicating that the subject will respond to the treatment if the size of each CAML in the sample is less than about 50 μm, and indicating that the subject will not respond to the treatment if the size of at least one CAML in the sample is greater than or equal to about 50 μm.

2. 1. A method of indicating a likelihood of response to treatment in a subject having cancer, comprising determining the size of CAMLs in a first and a second blood sample, and any further blood samples, obtained from a subject having cancer, wherein the first sample is obtained from the subject before or during a cancer treatment, and the second and any further blood samples are obtained from the subject after at least one cancer treatment; a decrease in the average size of the CAMLs in the second and any additional samples compared to the average size of the CAMLs in the first sample, indicating that the subject will respond to treatment; if the average size of the CAMLs in the second and any additional samples is maintained or increased compared to the average size of the CAMLs in the first sample, indicating that the subject will not respond to treatment; if the number of CAMLs greater than about 50 μm in size is maintained or increased from the first to the second and any additional samples, indicating that the subject will not respond to the treatment; or a decrease in the number of CAMLs greater than about 50 μm in size from the first to the second and any additional samples indicates that the subject will respond to the treatment; or A method in which the size of each CAML in the second or subsequent sample is reduced compared to the first sample, and where the size of each CAML in the second or any additional sample is less than about 50 μm, indicating the subject will respond to the treatment and the cancer may be cured.

3. A method for indicating the likelihood of a subject having cancer responding to treatment, comprising determining the absence of any CAMLs larger than about 50 μm in a blood sample from the subject after treatment, indicating that the subject is responding to the treatment.

4. A method for indicating the likelihood of a subject having cancer responding to a treatment, comprising determining the size of CAMLs in a blood sample obtained from the subject having cancer, the sample being obtained from the subject after at least one cancer treatment, wherein if the size of each CAML is about 50 μm or less, indicating that the subject will respond to the treatment.

5. A method for indicating a likelihood of a subject having cancer responding to a treatment, comprising determining the number of CAMLs in first and second blood samples, and any additional blood samples, obtained from a subject having cancer, wherein the first sample is obtained from the subject before or during a cancer treatment, and the second and any additional samples are obtained from the subject after at least one cancer treatment, wherein a decrease in the number of CAMLs from the first to the second and any additional samples indicates that the subject will respond to the treatment, and a decrease in the number of CAMLs from the first to the second and any additional samples indicates that the subject will not respond to the treatment, and a decrease in the number of CAMLs from the first to the second and any additional samples indicates that the subject will not respond to the treatment.

6. The method according to any one of claims 1 to 5, wherein the CAMLs have the following characteristics: (a) a plurality of individual nuclei and / or one or more fused nuclei having a size of about 14-64 μm; (b) cell size of approximately 20-300 μm in size; and (c) a morphological shape selected from the group consisting of spindle-shaped, tadpole-shaped, round, oval, two-legged, three or more-legged, thin-legged, and amorphous.

7. The method of claim 6, wherein the CAMLs have one or more of the following additional properties: (d) CD14 positive phenotype; (e) CD45 expression; (f) EpCAM expression; (g) vimentin expression; (h) PD-L1 expression; (i) expression of monocyte CD11C marker; (j) expression of endothelial CD146 marker; (k) expression of endothelial CD202b marker; (l) expression of the endothelial CD31 marker; and (m) Expression of epithelial cancer cell CK8, 18, and / or 19 markers.

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

9. The method according to any one of claims 1 to 5, wherein the blood sample is peripheral blood.

10. The method according to any one of claims 1 to 5, wherein the blood sample is antecubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, or jugular vein blood.

11. The method according to any one of claims 1 to 5, wherein the cancer is a solid tumor, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, colon cancer, liver cancer, head and neck cancer, kidney cancer, ovarian cancer, esophageal cancer or other solid tumor cancer.

12. 6. The method of any of claims 1 to 5, wherein CAMLs are isolated from blood samples using one or more means selected from the group consisting of size exclusion, immunocapture, dendrimer-mediated multivalent cell capture, affinity-based surface capture, biomimetic surface coated capture, selectin-coated surface capture, other functionalized surface capture, inertial focusing chips, red blood cell lysis, leukocyte depletion, FICOLL separation, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, or combinations thereof.

13. 13. The method of claim 12, wherein the circulating cells are isolated from the blood sample using a size exclusion method involving the use of a microfilter.

14. 14. The method of claim 13, wherein the microfilter has a pore size ranging from about 5 microns to about 20 microns.

15. 15. The method of claim 14, wherein the pores of the microfilter have a circular, racetrack, elliptical, square and / or rectangular pore shape.

16. 15. The method of claim 14, wherein the microfilter has a precise pore shape and uniform pore distribution.

17. 13. The method of claim 12, wherein circulating cells are separated using a microfluidic chip via physical size-based sorting, hydrodynamic size-based sorting, grouping, trapping, immunocapture, enrichment of large cells, or exclusion of small cells based on size.

18. The method according to any of claims 1 to 5, wherein circulating cells are isolated from a blood sample for the determination step using a CellSieve™ low pressure microfiltration assay.

19. 6. The method of any of claims 1 to 5, wherein the treatment is one or more of chemotherapy, a single drug, a combination of drugs, immunotherapy, radiation therapy, chemoradiotherapy, radiation therapy in combination with single or multiple drugs, chemoradiotherapy in combination with single or multiple drugs, cancer vaccine, and cell therapy.

20. 20. The method of claim 19, wherein the treatment is a cancer vaccine and the subject expresses at least one HLA allele.

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