Use of extracellular vesicles and micronuclei from circulating stromal cells as pan-cancer biomarkers to predict clinical outcome

Abstract

Provided are methods for predicting overall survival (OS) and progression-free survival (PFS) in subjects with cancer based on the presence of certain structures associated with circulating cancer-associated macrophage-like cells (CAML), including micronuclei (MN), extracellular vesicles (EV), multinucleated nuclei (EPN), internalized intact cells, and large internal cellular debris.

Description

[Technical Field]

[0001] The present invention relates generally to the use of biomarkers in blood and other body fluids to make predictions regarding overall survival and progression-free survival in subjects with cancer, such as solid tumors. [Background technology]

[0002] When tumor cells break away from primary solid tumors, they infiltrate the blood and lymphatic circulation and eventually leave the bloodstream to enter organs and tissues, metastasizing. 90% of cancer-related deaths are due to the metastatic process. The most common sites of metastasis are the lungs, liver, bone, and brain. Tumor cells found in the circulation are called circulating tumor cells (CTCs). Numerous research publications and clinical trials have demonstrated the clinical utility of CTCs in the following ways: (i) enumeration of CTCs in the bloodstream can provide information about prognosis, survival, and cancer recurrence, and (ii) examining protein expression levels and the occurrence of genetic mutations and translocations in CTCs can provide information about treatment. However, even in patients with stage IV cancer, the correlation between CTCs and the onset and / or presence of the target cancer is inconsistent. CTCs are most frequently found in stage IV breast, prostate, and colorectal cancers, but are rare in earlier stages of these cancers. CTCs are also rare in other cancers.

[0003] Circulating cancer-associated macrophage-like cells (CAML) are another type of cancer-associated cell found in the blood of subjects with cancer. CAML have been associated with all solid tumors and all stages of cancer examined. CAML are polyploid and very large, ranging in size from approximately 20 μm to approximately 300 μm. These polyploid cells are CD45(-) or CD45(+) and express CD11c, CD14, and CD31, confirming their myeloid origin. CAML are often found engulfing CTCs and cellular debris. [14,22] Examination of protein expression levels in CAML can also help clinicians make informed treatment decisions.

[0004] Assays related to the identification and characterization of biomarkers in CTCs and CAMLs in blood and other body fluids can be used to provide important information regarding the prognosis and treatment of subjects with cancer. The present invention provides clinicians with such tools and solves other important problems. Summary of the Invention

[0005] The present invention relates to a prognostic method utilizing a type of cell with specific characteristics found in the blood of subjects with solid tumors, including carcinoma, sarcoma, neuroblastoma, and melanoma. These circulating cells, called "circulating cancer associated macrophage-like cells" (CAML), have been found to be associated with the presence of solid tumors in cancer-bearing subjects. CAML have been consistently detected in the peripheral blood of subjects with stage I to stage IV solid tumors by microfiltration using precision microfilters.

[0006] Both an increase in the number of CAMLs in a subject's blood and the expansion of CAMLs have been shown to indicate more aggressive disease with worse clinical outcomes. Therefore, medical applications related to CAMLs include, but are not limited to, the early detection and diagnosis of cancer, particularly the early detection and diagnosis of cancer recurrence or relapse, and the use of the cells themselves as biomarkers for identifying cancer mutations.

[0007] CAMLs were also observed to possess irregular cellular characteristics not typically seen in less aggressive cancer states, such as (a) micronuclei (MNs), (b) extracellular vesicles (EVs), (c) enlarged polynuclearization (EPNs), (d) the presence of internalized intact cells, and (e) large internal cellular debris. The data presented here demonstrate that these five CAML-associated structures have clinical utility in predicting disease progression and patient survival, which may aid in informed treatment decisions.

[0008] More specifically, in a first embodiment, the present invention relates to a method for predicting overall survival (OS) and / or progression-free survival (PFS) of a subject with cancer, comprising measuring the presence of one or more of the following in circulating cells or produced by circulating cells from a biological sample from the subject with cancer, wherein the presence of one or more of (a) micronuclei (MN), (b) extracellular vesicles (EV), (c) multinucleated spread (EPN), (d) one or more internalized intact cells, and (e) internal cell debris is predictive of shorter OS and / or PFS than a subject with the same cancer in the absence of the presence of one or more of (a), (b), (c), (d), or (e).

[0009] In a second embodiment, the present invention relates to a method for predicting the presence of metastatic spread and / or metastatic progression in a subject with cancer, the method comprising measuring the presence of one or more of (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells from a biological sample from the subject with cancer, wherein the presence of one or more of (a), (b), (c), (d), and (e) predicts the presence of metastatic spread and / or metastatic progression in the subject.

[0010] In a third embodiment, the present invention relates to a method for predicting cancer progression in a subject with cancer. The method includes measuring the presence of one or more of (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a later second biological sample, and any additional biological samples obtained from a subject with cancer; and predicting that the cancer will progress in the subject if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and / or additional biological samples but not present in or produced by circulating cells of the first biological sample; and predicting that the cancer will not progress in the subject if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the first biological sample but not present in or produced by circulating cells of the second and / or additional biological samples.

[0011] In a fourth embodiment, the present invention relates to a method of predicting cancer progression in a subject with cancer, the method comprising measuring the presence of one or more of: (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject with cancer, wherein 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, and wherein one or more of (a), (b), (c), (d), or (e) are If (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and / or additional biological samples but not present in or produced by circulating cells of the first biological sample, a prediction is made that the cancer will not progress in the subject; and if (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample, a prediction is made that the cancer will progress in the subject.

[0012] In a fifth embodiment, the present invention relates to a method of predicting therapeutic response in a subject with cancer, the method comprising measuring the presence of one or more of (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject with cancer, wherein 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, and wherein one or more of (a), (b), (c), (d), or (e) are present. is present in or produced by circulating cells of the first biological sample but not present in or produced by circulating cells of the second and / or additional biological samples, the subject is predicted to respond to treatment; and one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample is predicted to not respond to treatment.

[0013] In some aspects of the first embodiment, OS and / or PFS is for a period of at least 12 months. In other aspects of the first embodiment, OS and / or PFS is for a period of at least 24 months.

[0014] The size of the biological sample in each embodiment of the present invention is 5 to 15 mL.

[0015] In various embodiments of the present invention, the circulating cells have the following characteristics: (a) a plurality of individual nuclei and / or one or more fused nuclei having a size of about 14 to 64 μm; (b) cell size of approximately 20 to 300 μm in size, and (c) a morphological shape selected from the group consisting of spindle-shaped, tadpole-shaped, round, oval, two-legged, two or more-legged, thin-legged, and irregular; It has.

[0016] In embodiments of the present invention, the circulating cells may have the following additional characteristics: (d) CD14 expression; (e) CD45 expression; (f) EpCAM expression, (g) vimentin expression; (h) PD-L1 expression, (i) CD11c marker expression on monocytes and macrophages; (j) endothelial CD146 marker expression; (k) Endothelial and macrophage CD202b marker expression; (l) endothelial, macrophage, and leukocyte CD31 marker expression; and (m) epithelial cancer cell CK8, 18, and / or 19 marker expression; The device may have one or more or all of:

[0017] In each embodiment of the present invention, the source of the biological sample is one or more of peripheral blood, blood, lymph nodes, bone marrow, cerebrospinal fluid, and urine. For example, the biological sample may be antecubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, or jugular vein blood.

[0018] In embodiments of the present invention, the cancer is stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colon cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal cancer, brain and ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, cervix, uterine cancer or other solid tumor cancer.

[0019] Circulating cells may be isolated from the biological sample for the measurement using one or more techniques selected from the group consisting of size exclusion, immunocapture, red blood cell lysis, leukocyte depletion, polymeric polysaccharides such as FICOLL®, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, enrichment of large cells, removal of small cells, or combinations thereof.

[0020] Circulating cells may be isolated from a biological sample using a size exclusion method using a microfilter. The microfilter may have a pore size ranging from about 5 μm to about 20 μm. Furthermore, the pores of the microfilter may have a circular, racetrack, elliptical, square, or rectangular pore shape. The microfilter may also have a precise pore shape and a uniform pore distribution.

[0021] Circulating cells may be isolated by either physical size-based sorting using microfluidic chips, hydrodynamic size-based sorting, grouping, trapping, immunocapture, enrichment for large cells, or size-based removal of small cells.

[0022] A microfiltration assay may be used to separate circulating cells from the biological sample for the measurement.

[0023] In related embodiments, the treatment is one or more of chemotherapy, monotherapy, combination therapy, immunotherapy, radiation therapy, chemoradiotherapy, radiation combined with one or more other drugs, chemoradiotherapy combined with one or more other drugs, cancer vaccine, and cell therapy. As a non-limiting example, the treatment is a cancer vaccine and the subject expresses at least one HLA allele.

[0024] In related embodiments, the subject may have been treated with one or more of chemotherapy, monotherapy, combination drug therapy, immunotherapy, radiation therapy, chemoradiotherapy, radiation combined with one or more other drugs, chemoradiotherapy combined with one or more other drugs, cancer vaccines, and cell therapy. As a non-limiting example, the immunotherapy may be PD-L1 immunotherapy.

[0025] CAML and the aforementioned CAML-related structures may be used alone as cancer markers or in combination with biomarker expression by other circulating cells, such as circulating tumor cells (CTCs). Suitable CTC subtypes include, but are not limited to, pathologically definable CTCs (PDCTCs), apoptotic CTCs, and CTC subtypes that undergo epithelial-mesenchymal transition (EMTCs). Other suitable circulating cell types include circulating cancer-associated vascular endothelial cells (CAVEs). The presence of these additional cell types themselves, as well as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomics, metabolomics, lipidomics, and other biomarkers, may provide a more complete understanding of a patient's disease.

[0026] In each aspect and embodiment of the invention, the method further comprises administering a therapeutically effective amount of a cancer treatment to the subject. The subject may be a subject predicted to have a shorter OS and PFS than another subject. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows cell differentiation markers used to identify and subtype CAML.

[0028] [Figure 2] This figure shows three examples of CAML with micronuclei (MN). The white dashed lines encircle the MN. The white dashed boxes can be enlarged and recolored to better observe the MN within the cells.

[0029] [Figure 3A] Images of a CAML with micronuclei and nuclear enlargement. Nuclear enlargement (a, green-blue), CD45 staining (b, purple), diffusible cytokeratin (c, green), and DAPI (d, blue) are shown to reveal cellular structure (box = 85 μm). The enlarged section of the cell reveals a single micronucleus (e, white arrow) emerging from the main nucleoplasmic mass, as well as two distinct micronuclei within the cytoplasm (box = 20 μm).

[0030] [Figure 3B] Micronucleus-positive CAML (a, 66 μm diameter) stained with PD-L1 (red) and DAPI (blue). The size of the micronuclei varies from 4 μm (b) to 2 μm (c).

[0031] [Figure 4] Figure 1 shows three subtypes of EVs distinguished based on their formation method and size: exosomes (approximately 30 nm to 100 nm), microvesicles (approximately 100 nm to 1 μm), and apoptotic bodies (approximately 1 to 5 μm). EVs originate from CAML that have left the tumor site and entered the circulation.

[0032] [Figure 5] FIG. 1 shows progression-free survival (PFS) of stromal cells based on the presence or absence of micronuclei (MN) over 24 months.

[0033] [Figure 6] FIG. 1 shows overall survival (OS) of stromal cells based on the presence or absence of micronuclei (MN) over a 24-month period.

[0034] [Figure 7] Figure 1 shows that the mean PD-L1 expression in all MN+ cells (n=185) was 338, while the mean expression in MN- cells (n=347) was 253. Median (red line), upper error bar (maximum), lower error bar (minimum).

[0035] [Figure 8] This figure shows Kaplan-Meier curves for progression-free survival (PFS) and overall survival (OS) in colorectal cancer patients who were micronuclei positive at the time of initial blood collection. Panel a: Kaplan-Meier curves for PFS in patients who were micronuclei positive (red) or negative (blue). Panel b: Kaplan-Meier curves for OS in patients who were micronuclei positive (red) or negative (blue).

[0036] [Figure 9] Figure 1 shows Kaplan-Meier curves for PFS and OS for patients with micronuclei positivity at the time of first blood draw. Panel a. Kaplan-Meier curves for PFS for patients with micronuclei positive (blue) or negative (red). Panel b. Kaplan-Meier curves for OS for patients with micronuclei positive (blue) or negative (red).

[0037] [Figure 10] FIG. 1 shows a single-factor ANOVA comparing PD-L1 expression in MN-positive or MN-negative cells.

[0038] [Figure 11] FIG. 1 shows a T-test comparing PD-L1 expression in MN-negative, MN1, MN2, or MN3+ cells.

[0039] [Figure 12]Figure 1 shows the presence of micronuclei in various clinical parameters. Panel a. Linear regression plot showing the relationship between the number of CAMLs and the number of micronuclei in each patient at baseline sampling. Panel b. Presence of micronuclei based on patients currently receiving chemotherapy (i.e., genotoxic agents) or newly diagnosed and treatment-naive patients. Panel c. Presence of micronuclei based on non-metastatic or metastatic spread. Graph d. Frequency of micronuclei in all CAMLs (n=776). 91.0% (blue) of CAMLs were negative for micronuclei, and only 9% were positive for at least one micronucleus.

[0040] [Figure 13] This figure shows a case study tracking mean micronuclei in subsequent blood draws. Panel a shows patient A. After progression on maintenance therapy (Xeloda, red box), FOLFOX (orange box) was initiated. After four cycles of treatment, MN increased, which correlated with tumor growth (+17%). Treatment was discontinued due to infection. MN increased further, and new lesions were discovered at cycle 6. FOLFOX was then restarted, but MN continued to increase, and the patient withdrew from the study. Panel b shows patient B. With progressive disease on FOLFOX, this patient was initiated on a single-agent CCR5 inhibitor (leronlimab, blue box), with a corresponding decrease in MN, which correlated with a decrease in tumor size (-39%). Subsequently, MN increased slightly, which correlated with a slight increase in tumor size (+11%), at which point FOLFIRI (purple box) was added to leronlimab. After the introduction of FOLFIRI, a decrease in MN was observed, which correlated with stable disease.

[0041] [Figure 14]This figure shows Kaplan-Meier curves of progression-free survival (PFS) and overall survival (OS) for EV-positive CAML patients with metastatic non-small cell lung carcinoma (mNSCLC). The upper panel shows Kaplan-Meier curves of PFS for patients who were EV-positive (red) or EV-negative (black). The lower panel shows Kaplan-Meier curves of OS for patients who were EV-positive (red) or EV-negative (black).

[0042] [Figure 15] PD-L1 staining of EV-positive CAML (45 μm diameter). Multiple PD-L1-positive EVs range in size from <1 μm to 3 μm.

[0043] [Figure 16] Kaplan-Meier curves for PFS and OS for EV positivity in CAML in patients with initial blood draw from 130 patients with various cancer types. Panel a. Kaplan-Meier curves for PFS in patients with EV positive (blue) or negative (red). Panel b. Kaplan-Meier curves for OS in patients with EV positive (blue) or negative (red).

[0044] [Figure 17] This figure shows the results of tracking changes in EV abundance in response to CRT over a 15-month period in one patient. The patient underwent chemoradiotherapy (CRT) at baseline (BL) and had a 33% decrease in EV abundance at T1 blood draw after completion of CRT. EV abundance increased from T2 to T5, at which point the patient's disease progression was confirmed by radiological imaging (PET / CT). The patient began treatment with ipilumab, but subsequently developed further metastases at T7 and disease progression.

[0045] [Figure 18]Kaplan-Meier curves for progression-free survival (PFS) and overall survival (OS) for EV-positive CAML patients with metastatic non-small cell lung carcinoma (mNSCLC). Panel a. Kaplan-Meier curves for PFS in patients with EV-positive (red) or -negative (blue) patients. Panel b. Kaplan-Meier curves for OS in patients with EV-positive (red) or -negative (blue) patients.

[0046] [Figure 19] Figure 1 shows Kaplan-Meier curves of PFS and OS for CAML EV positivity in patients with metastatic non-small cell lung cancer (mNSCLC) treated with or without immunotherapy (IMT).

[0047] [Figure 20] Figure 1 shows Kaplan-Meier curves of PFS and OS for patients with DAPI nuclear increase in CAML at the time of first blood collection. Panel a. Kaplan-Meier curves of PFS in patients with nuclear area >465 μm2 (red) or <465 μm2 (blue). Panel b. Kaplan-Meier curves of OS in patients with nuclear area >460 μm2 (red) or <465 μm2 (blue).

[0048] [Figure 21] Figure 1 shows Kaplan-Meier curves for PFS and OS for patients with WBC CAML positivity at first blood draw. Panel a. Kaplan-Meier curves for PFS in patients with CAML WBC positive (blue) or negative (red). Panel b. Kaplan-Meier curves for OS in patients with CAML WBC positive (blue) or negative (red).

[0049] [Figure 22] Figure 1 shows Kaplan-Meier curves for PFS and OS for patients with positive debris at first blood draw. Panel a. Kaplan-Meier curves for PFS in patients with positive (blue) or negative (red) debris. Panel b. Kaplan-Meier curves for OS in patients with positive (blue) or negative (red) debris.

[0050] [Figure 23]

[0033] Figure 1 shows Kaplan-Meier curves for PFS and OS in patients with MN, EV, and / or CAML with a nucleus larger than 465 μm2 at the time of first blood collection. Panel a. Kaplan-Meier curves for PFS in patients who were positive (blue) or negative (red) for MN, EV, or CAML with a nucleus larger than 465 μm2. Panel b. Kaplan-Meier curves for OS in patients who were positive (blue) or negative (red) for MN, EV, or CAML with a nucleus larger than 465 μm2. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description The details of the configuration and elements defined in this description are presented merely to aid in a comprehensive understanding of the present invention, and therefore, it will be apparent to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0052] Cancer is one of the world's most feared diseases, affecting every country and every demographic. Approximately 40% of both men and women will develop cancer in their lifetime. In the United States alone, it is estimated that there are more than 12 million cancer patients at any given time, with 1.7 million new cases diagnosed each year and more than 600,000 deaths. Globally, the number of cancer deaths is estimated to be approximately 8 million per year, with 3 million of these occurring in developed countries where patients have access to treatment.

[0053] Liquid biopsies provide real-time, continuous tracking of diagnostically important circulating cells isolated from cancer-bearing subjects. Cells such as circulating tumor cells (CTCs) are present in the peripheral blood of cancer patients, and previous studies have shown that CTC-based assays can be used as an alternative to tissue biopsies. [1-4] .

[0054] Recently, another cancer-associated circulating cell type has been identified in the peripheral blood of cancer patients. This cancer stromal cell subtype has been termed cancer-associated macrophage-like cells or CAML. CAMLs were identified in blood using a non-affinity microfiltration-based method that captures both CTCs and CAMLs, allowing for the separate or parallel analysis of these cancer-specific circulating cell subtypes. [1,6-16] CAML are recently defined circulating bone marrow-derived stromal cells that have been found in all stages of aggressive malignancies and in various solid malignancies, including breast cancer, prostate cancer, non-small cell lung cancer (NSCLC), and pancreatic cancer. [11,13,14,17] CAML are a type of specialized myeloid polyploid cells present in the blood of solid tumors of all stages. They are easily identified by their large size (greater than 20 μm), polyploid nuclei, and morphology, i.e., round, rod-like, single tail, or two tails separated by 180 degrees. CAML typically expresses CD31, CD14, CD45, and cytokeratin, and may also express EpCAM, CD146, CD11c, and tie2. [11,13,14,17] .

[0055] While the mere presence of one or more of these circulating cells in a blood sample obtained from a subject with cancer provides diagnostic information, the presence of specific cellular structures or biomarkers provides additional information. For example, as described herein, the present invention is based on the inventors' discovery that CAML possesses irregular cellular characteristics not typically seen in less aggressive cancer states, such as (a) micronuclei (MN), (b) extracellular vesicles (EV), (c) enlarged polynuclearized megakaryocytes (EPN), (d) the presence of internalized intact cells, and (e) large internal cellular debris. These five CAML-associated structures have been shown by the data presented herein to have clinical utility in predicting disease progression and patient survival, which may aid in informed treatment decisions. A detailed discussion of these CAML-associated structures is provided below, following detailed information on the associated cell types.

[0056] Circulating cancer-associated macrophage-like cells (CAML) As defined herein, circulating cells used in the methods of the invention can be referred to as "CAML." The term "circulating cells" is synonymous with CAML, and the term "CAML" is synonymous with circulating cells. Whether referred to as CAML or "circulating cells," these cells are characterized by having one or more of the following characteristics: CAML has large, atypical polyploid nuclei or multiple individual nuclei, often scattered throughout the cell, although enlarged, fused nucleoli are also common. The diameter of the nuclei in CAML typically ranges from about 10 μm to about 70 μm, more commonly from about 14 μm to about 64 μm. In many cancers, CAML expresses cancer markers of the disease. For example, CAML associated with epithelial cancers may express CK8, 18, or 19, EpCAM, vimentin, etc. These markers are usually diffuse or associated with vacuoles and / or endocytic substances. The staining pattern of either marker is generally uniform and diffuse throughout the cell. For sarcoma, neuroblastoma, and melanoma, other cancer-associated markers can be used instead of CK8, 18, or 19. CAML can be CD45 positive or CD45 negative, and the present invention encompasses the use of both types of CAML. CAMLs are large, measuring from about 20 μm to about 300 μm in their longest dimension. CAMLs are found in many different morphological forms, including spindle-shaped, tadpole-shaped, round, oblong, two-legged, more than two-legged, thin-legged, or amorphous. 1,2 . Carcinoma-derived CAML usually has diffuse cytokeratin. When CAML expresses EpCAM, it is usually diffuse throughout the cell or associated with vacuoles and / or ingested material, with a fairly uniform distribution throughout the cell; however, some tumors have very low or no EpCAM expression, and not all CAML express EpCAM. When CAMLs express markers, the markers are often diffuse throughout the cell or associated with vacuoles and / or ingested material, and are distributed fairly uniformly throughout the cell; however, not all CAMLs express the same markers with the same intensity, and the limited number of markers they express may not be distributed uniformly throughout the cell. CAML often expresses markers related to tumor-derived markers. For example, if the tumor is of prostate cancer origin and expresses PSMA, CAML from such a patient will also express PSMA. As another example, if the primary tumor is of pancreatic origin and expresses PDX-1, CAML from such a patient will also express PDX-1. As yet another example, if the primary cancer tumor or CTCs express CXCR-4, CAML from such a patient will also express CXCR-4. If the primary tumor or CTCs from the cancer express a biomarker for the drug target, CAML from such patients will also express the biomarker for the drug target. An example of such an immunotherapy biomarker is PD-L1. CAML expresses monocyte markers (e.g., CD11c, CD14) and endothelial markers (e.g., CD146, CD202b, CD31). · CAML has the ability to bind to Fc fragments.

[0057] The results of evaluating a broad set of markers for CAML expression are shown in Figure 1. In one aspect of the present invention, CAML 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 1. The markers were screened for 1,118 CAMLs obtained from 93 different patients with different cancers. CAMLs were initially isolated and identified using DAPI, cytokeratin, and CD45, and then sequentially re-stained with a total of 27 markers, including myeloid / macrophage, leukocyte, megakaryocyte, epithelial, endothelial, progenitor / stem, and motility markers. As can be seen in Figure 1, marker expression ranged from 0% to 96%. Most CAMLs expressed CD31, and commonly co-expressed cytokeratin, CD14, CXCR4, vimentin, and other markers. CAML contained a clear myeloid marker (CD14), but the CD31 marker was more commonly expressed in 96%.

[0058] CAML also display multiple phenotypes that appear inconsistent with traditional understandings of cell differentiation (i.e., coexpression of CD45 (leukocytes) and cytokeratin (epithelial), CD11c / CD14 (macrophages) and CD41 (macrophages / megakaryocytes), CD146 (endothelial) and CD61 (macrophages / endothelial / megakaryocytes), and CD31 (leukocytes / macrophages / endothelial / megakaryocytes / stem cells) and CD68 / CD163 (macrophages)). Many markers are expressed on multiple cell types. Taken together, these data suggest that CAML are bone marrow-derived cells at an early stage of differentiation that possess many phenotypic characteristics associated with stem cells and angiogenic potential.

[0059] CAML can be visualized by colorimetric staining, such as H&E, or by fluorescent staining for specific markers, as shown in Figure 1. Regarding the cytoplasm, CD31 is the most positive phenotype. CD31 alone or in combination with other positive markers shown in Figure 1 or tumor-associated cancer markers is recommended.

[0060] Thus, and in various embodiments and aspects of the present invention, circulating cells (CAML) have each of the following characteristics: (a) a plurality of individual nuclei and / or one or more fused nuclei having a size of about 14 to 64 μm; (b) cell size of approximately 20 to 300 μm, and (c) a morphological shape selected from the group consisting of spindle-shaped, tadpole-shaped, round, oval, two-legged, more than two-legged, thin-legged, and irregular; can be defined as having

[0061] In some aspects of embodiments of the present invention, the circulating cells (CAML) have the following additional characteristics: (d) CD14 expression; (e) CD45 expression; (f) EpCAM expression, (g) vimentin expression; (h) PD-L1 expression, (i) CD11c marker expression on monocytes and macrophages; (j) endothelial CD146 marker expression; (k) Endothelial and macrophage CD202b marker expression; (l) endothelial, macrophage, and leukocyte CD31 marker expression; and (m) epithelial cancer cell CK8, 18, and / or 19 marker expression; The compound may be further identified as having one or more of:

[0062] Currently, there is no officially designated term for CAML in the literature. Other terms that appear in the literature to describe the same cell type include: ·Circulating stromal cells ·Immune stromal cells Circulating cancer-related cells Circulating tumor-macrophage hybrid cells ·Cell-cell fusion cells Tumor-associated macrophage hybrid cells Double-positive circulating cells (see the presence of CK and CD45 markers) Fusion of tumor cells and macrophages Circulating giant tumor macrophage fusion cells Tumor cell-macrophage fusion cells Fusion hybrid cells Circulating hybrid cells Tumor hybrid cells

[0063] Circulating tumor cells As defined herein, carcinoma-associated CTCs express multiple cytokeratins (CKs). CK8, 18, and 19 are the cytokeratins most commonly expressed by CTCs and used in diagnosis, but investigations need not be limited to these markers. The surface of solid tumor CTCs typically expresses epithelial cell adhesion molecule (EpCAM). However, this expression is not uniform or consistent. CTCs do not express CD45, as CD45 is a leukocyte marker. Assays to identify tumor-associated cells, such as CTCs and CAML, can be performed using antibodies against solid tumor-associated markers such as CK8, 18, and 19, or antibodies against CD45 or DAPI.

[0064] Different subgroups of CTCs up- and / or down-regulate phenotypes and marker expression in association with tumor progression, tumor spread, and in response to tumor treatment. Therefore, assessment of CTCs in peripheral blood and the markers expressed by such cells can provide important information regarding the state of cancer in a subject.

[0065] CTCs, which can be used for cancer diagnosis, as well as for screening and monitoring treatment and for determining tumor sensitivity to a particular treatment in a particular subject, can be divided into three subgroups: the first subgroup is pathologically definable CTCs (PDCTCs); [6-10] PDCTCs can be characterized by "cancer-like" nuclei stained with DAPI, expression of cytokeratins showing a fibrous pattern, and expression of one or more of CK8, 18, and 19 (CTCs derived from epithelial cancers usually express at least CK8, 18, and 19), and lack of expression of CD45.

[0066] The second subgroup is apoptotic CTCs. Upon CTC death, the cytokeratin pattern is degraded into punctate patterns. Thus, early apoptotic CTCs have some punctate cytokeratin, while late apoptotic CTCs have all intracellular cytokeratin degraded into punctate patterns. Apoptotic CTCs can also be characterized by the expression of CK8, 18, and 19, degraded nuclei, and the absence of CD45 expression.

[0067] In a third subgroup of CTCs, cells are undergoing epithelial to mesenchymal transition (EMTCTCs). [2,3,5-9] EMT is a gradual morphogenetic process, and EMT encompasses cells at various stages of transformation. [6] EMTCTCs are typically characterized by downregulation of epithelial proteins, such as EpCAM and CK, and upregulation of mesenchymal stem cell proteins, such as vimentin and CD34.

[13] Subtype classification of EMTCTCs is usually performed using methods other than proteomics, i.e., mRNA expression or DNA analysis.

[13] .

[0068] Yet another type of cancer-associated circulating cell that may be diagnostically useful is the cancer-associated vascular endothelial cell, or CAVE. CAVE is a subtype of circulating endothelial cell. Tumors require a blood supply from tumor endothelial cells. CAVE are tumor endothelial cells that have been released into the bloodstream from the tumor site. CAVE are often found in clusters. CAVE express cytokeratins and various subtypes of endothelial cell markers, including CD31, CD146, CD144, and CD105, but not CD14 or CD45.

[20] .

[0069] Combining morphology and staining techniques allows the identification of pathologically definable CTCs (PDCTCs), apoptotic CTCs, and CAML. [6] .

[0070] Forecasting Method As mentioned above, the unique characteristics of CAML make it suitable for clinical applications such as screening and diagnosis of diseases such as cancer, monitoring treatment, and monitoring disease progression and recurrence.

[0071] Both increased numbers of CAMLs in a subject's blood and increased CAML enlargement have been shown to be indicators of more aggressive disease associated with worse clinical outcomes. However, in less aggressive cancer states, CAMLs have also been observed to possess other unusual and irregular cellular features, such as (a) micronuclei (MNs), (b) extracellular vesicle (EV) formation, (c) multinucleated enlargement (EPN), (d) the presence of internalized intact cells, and (e) large internal cellular debris. These five "CAML-associated structures" form the basis of the present invention.

[0072] Micronuclei (MN) are the result of the body's DNA repair mechanisms. 33,34 MNs are formed by intrachromosomal abnormalities that represent subclonal cancer populations with high cell viability and resistance to drug therapy. MNs are often observed as small fragments of nucleic acid excised from the primary nucleus in both tumor cells and surrounding tumor-immune stromal cells (Figures 2 and 3). Cells in which DNA has been damaged and undergoing repair mechanisms, such as those forming MNs, appear to have upregulated expression of programmed cell death ligand (PD-L1). MNs are defined as small, DAPI-positive, round structures within the cell cytoplasm, separate from the primary nucleus. MN sizes vary from 10 nm to 10 μm, but are typically between 0.5 μm and 4 μm.

[0073] Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are involved in cell-to-cell communication, tumor growth, and metastasis in cancer (Figure 4). EVs are broadly defined as membrane-bound vesicles secreted by cells into the extracellular space. EVs play an important role in intercellular communication and cancer recognition, and are intricately involved in tumor growth, progression, and metastasis in cancer. EV budding can be observed as small spherical projections protruding from cells such as CAML. EV budding in CAML is characterized as small spherical projections ranging in size from 1 to 10 μm, but typically ranges from 0.5 to 5 μm.

[0074] Multinucleated cytoplasmic papillae (EPN), or abnormally high amounts of nucleic acid in cells, are an indicator of abnormal cell function resulting from many disease states, including viral infections and cancer. 2 These are nuclear masses with an area of ​​more than 460 μm 2 Ultra, 1000μm 2 Ultra, 2000μm 2 Over 3000 μm 2 It can also be defined as super.

[0075] The presence of internalized intact cells or large internal cellular debris can result from many biological mechanisms, such as phagocytosis, intracellular repair, and cell extrusion, and can lead to downregulation of the immune response. As used herein, the term internalized intact cells refers to whole or part of a white blood cell (WBC), whole or part of a CTC, or WBC bound to a CAML. As used herein, large internal cellular debris refers to the inclusion of undisintegrated cellular debris or other internal structures within a CAML.

[0076] Identifying any of these features in the context of tumor biopsies can be difficult, and they have rarely been studied in the context of liquid biopsies in cancer research. However, as reported here, these five observable biologic features were assayed in CAML from various cancer patients and validated as useful clinical predictors, including association with more aggressive disease likely associated with poor clinical outcomes, including faster progression, higher mortality, and a higher risk of metastatic recurrence. MNs, EVs, EPNs, internalized intact cells, and internal cellular debris were all observed in CAML from multiple stages of cancer and various solid tumor subtypes.

[0077] These observations form the basis of the present invention, as defined therein.

[0078] As suggested in the Summary of the Invention above, in a first embodiment, the present invention provides a method for predicting overall survival (OS) and / or progression-free survival (PFS) of a subject with cancer. The method comprises measuring the presence of one or more of the following in circulating cells or produced by circulating cells from a biological sample from the subject with cancer: (a) micronuclei (MNs), (b) extracellular vesicles (EVs), (c) multinucleated cytoplasm (EPNs), (d) internalized intact cells, and (e) internal cell debris, wherein the presence of one or more of (a), (b), (c), (d), and (e) predicts shorter OS and / or PFS than a subject with the same cancer in the absence of the presence of one or more of (a), (b), (c), (d), or (e).

[0079] In a second embodiment, the present invention provides a method for predicting the presence of metastatic spread and / or metastatic progression in a subject with cancer, the method comprising measuring the presence of one or more of (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in or produced by circulating cells from a biological sample from the subject with cancer, wherein the presence of one or more of (a), (b), (c), (d), and (e) predicts the presence of metastatic spread and / or metastatic progression in the subject.

[0080] In a third embodiment, the present invention provides a method for predicting cancer progression in a subject with cancer. The method includes measuring the presence of one or more of (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a later second biological sample, and any additional biological samples obtained from a subject with cancer; and predicting that the cancer will progress in the subject if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and / or additional biological samples but not present in or produced by circulating cells of the first biological sample; and predicting that the cancer will not progress in the subject if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the first biological sample but not present in or produced by circulating cells of the second and / or additional biological samples.

[0081] In a fourth embodiment, the present invention provides a method for predicting cancer progression in a subject having cancer, the method comprising measuring the presence of one or more of: (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject having cancer, wherein 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, and wherein one or more of (a), (b), (c), (d), or (e) are: If (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and / or additional biological samples but not present in or produced by circulating cells of the first biological sample, a prediction is made that the cancer will not progress in the subject; and if (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample, a prediction is made that the cancer will progress in the subject.

[0082] In a fifth embodiment, the present invention provides a method of predicting therapeutic response in a subject with cancer, the method comprising measuring the presence of one or more of (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject with cancer, wherein 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, and wherein one or more of (a), (b), (c), (d), or (e) are present. is present in or produced by circulating cells of the first biological sample but not present in or produced by circulating cells of the second and / or additional biological samples, the subject is predicted to respond to treatment; and one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample is predicted to not respond to treatment.

[0083] In various embodiments of the present invention, the circulating cells have the following characteristics: (a) a plurality of individual nuclei and / or one or more fused nuclei having a size of about 14 to 64 μm; (b) cell size of approximately 20 to 300 μm in size, and (c) a morphological shape selected from the group consisting of spindle-shaped, tadpole-shaped, round, oval, two-legged, more than two-legged, thin-legged, and amorphous; can be defined as having

[0084] The circulating cells in each embodiment of the invention may have the following additional characteristics: (d) CD14 expression; (e) CD45 expression; (f) EpCAM expression, (g) vimentin expression; (h) PD-L1 expression, (i) CD11c marker expression on monocytes and macrophages; (j) endothelial CD146 marker expression; (k) Endothelial and macrophage CD202b marker expression; (l) endothelial, macrophage, and leukocyte CD31 marker expression; and (m) epithelial cancer cell CK8, 18, and / or 19 marker expression; The device may have one or more or all of:

[0085] In each method of the invention, the number of circulating cells assessed for CAML-associated structures can range from 1 to 100 or more, including ranges of 1 cell, 1-2 cells, 1-3 cells, 1-4 cells, 1-5 cells, 1-6 cells, 1-7 cells, 1-8 cells, 1-9 cells, 1-10 cells, 1-20 cells, 1-30 cells, 1-40 cells, 1-50 cells, 1-100 cells, and 1-200 cells.

[0086] As used herein, the term overall survival (OS) refers to the length of time a subject with cancer has survived from a selected date, such as the date of diagnosis, the date treatment was initiated, or the date blood was drawn to assess cancer progression.

[0087] As used herein, the term progression-free survival (PFS) means the length of time a subject with cancer survives without their cancer getting worse or progressing from a selected date, such as the date treatment was started or the date blood was drawn to assess cancer progression.

[0088] In each method of the invention, OS or PFS, or both, is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 months or longer. In one aspect of the invention, PFS or OS, or both, is at least 12 months or at least about 24 months.

[0089] In each method of the present invention, when OS and / or PFS are predicted to be "shortened" or "worsened," the OS and / or PFS will be shorter than in a subject having a cancer in which a CAML-associated structure has not been found.

[0090] It is clear that the volume of the biological sample in which circulating cells (e.g., CAML) are assayed in each method of the present invention can vary. However, the biological sample should typically be at least about 2.5 mL. The volume of the biological sample may be at least about 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 17.5, 18, 19, 20, 21, 22, 22.5, 23, 24, 25, 26, 27, 27.5, 28, 29, 30 mL, or more. The volume of the biological sample may be about 2.5-20 mL, about 5-15 mL, or about 5-10 mL. In one aspect of the present invention, the biological sample is about 7.5 mL.

[0091] In each embodiment and aspect of the present invention, the source of the biological sample may be one or more of, but is not limited to, peripheral blood, blood, lymph nodes, bone marrow, cerebrospinal fluid, and urine. When the biological sample is blood, the blood may be, for example, antecubital 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 sample that has been cryopreserved.

[14] .

[0092] In embodiments and aspects of the present invention, the cancer may be stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colon cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal cancer, brain and ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, cervix, uterine corpus cancer or other solid tumor cancer. It will be apparent to those skilled in the art that the methods of the present invention are not limited to a particular form or type of cancer, but may be practiced in connection with a wide variety of cancers.

[0093] In embodiments and aspects of the present invention, circulating cells (e.g., CAML) may be separated from a biological sample for measurement using one or more techniques selected from the group consisting of size exclusion, immunocapture, red blood cell lysis, leukocyte depletion, polymeric polysaccharides such as FICOLL®, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, enrichment of large cells, removal of small cells, or combinations thereof. In certain aspects, size exclusion involves the use of a microfilter.

[0094] In one aspect of the present invention, circulating cells are isolated from a biological sample using a size exclusion method employing the use of a microfilter. Suitable microfilters can have a variety of pore sizes and shapes. The microfilter may have a pore size ranging from about 5 μm to about 20 μm. In some aspects of the present invention, the pore size is about 5 μm to about 10 μm, and in other aspects, the pore size is about 7 μm to about 8 μm. The larger the pore size, the more WBC contamination on the filter is removed. The pores of the microfilter may be of any shape; acceptable shapes include round, racetrack, oval, slit, square, rectangular, and / or other shapes. The microfilter may have a precise pore shape, a uniform pore distribution, multiple pore shapes, and / or a non-uniform distribution. The microfilter may be single-layered or multi-layered, with different shapes in different layers.

[0095] In another aspect of the present invention, circulating cells are separated from a biological sample using a microfluidic chip by physical size-based sorting, slits, channels, hydrodynamic size-based sorting, grouping, trapping, immunocapture, enrichment for large cells, or size-based removal of small cells. The efficiency of circulating cell capture can vary depending on the collection method. The size of circulating cells that can be captured by different platforms can also vary, for example, depending on the identity of the cells. Collecting circulating cells using a CELLSIEVE™ microfilter provides 100% capture efficiency and high-quality cells.

[0096] In another aspect of the invention, when the biological sample is peripheral blood or blood, the sample may be collected from the subject using a blood collection tube, for example, CELLSAVE™ blood collection tubes (Menarini Silicon Biosystems, San Diego, CA), which provide consistent cell morphology and size.

[0097] In another aspect of the present invention, circulating cells can be captured and analyzed without specifically identifying them as CAML cells themselves. Instead, cells can be identified based on the size of their cytoplasm and nucleus. Examples of this include techniques using colorimetric stains such as H&E staining, or simply examining CK(+) cells.

[0098] Furthermore, in one aspect of the present invention, circulating cells are isolated from a biological sample using a CELLSIEVE™ microfilter low-pressure microfiltration assay.

[0099] CAML and the aforementioned CAML-related structures may be used alone as cancer markers or in combination with biomarker expression by other circulating cells, such as circulating tumor cells (CTCs). Suitable CTC subtypes include, but are not limited to, pathologically definable CTCs (PDCTCs), apoptotic CTCs, and CTC subtypes undergoing epithelial-mesenchymal transition (EMTC) cells. Other suitable circulating cell types include circulating cancer-associated vascular endothelial cells (CAVEs). The presence of these additional cell types themselves, as well as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), methylated DNA, proteomics, metabolomics, lipidomics, and other biomarkers, may provide a more complete understanding of a patient's disease.

[0100] When comparing the number of CAML-related structures in circulating cells (e.g., CAML) of two subjects with cancer, it is desirable that the two subjects have the same type of cancer.However, it may be difficult to completely match two subjects in factors such as cancer type, cancer stage, cancer progression rate, treatment history, cancer remission and / or history of recurrence.Therefore, it should be understood that there may be some differences in the cancer characteristics of the two subjects compared by this method.

[0101] It should also be understood that the data to which the measurement results are compared, for example, the number of CAML-related structures obtained from subjects with the same type of cancer, may be data from a single subject or a combined result from data from two or more subjects. As reference values ​​are established based on data from a group of subjects with the same or similar cancers, the predictive value of the method of the present invention improves over time. Thus, the term "subjects with the same type of cancer" as used herein refers to data from a single subject or a group of two or more subjects with the same type of cancer.

[0102] Treatment method In each aspect and embodiment of the present invention, the method optionally further comprises administering a therapeutically effective amount of a cancer treatment to the subject. The subject may be a subject predicted to have a shorter OS and PFS than another subject.

[0103] The specific cancer treatment corresponds to the particular type of cancer being treated, where suitable cancer treatments include chemotherapy, monotherapy, drug combination therapy, immunotherapy, radiation therapy, chemoradiotherapy, chemoradiotherapy with monotherapy or combination therapy, chemoradiotherapy with monotherapy or combination therapy (e.g., immunotherapeutic agents), cell therapy, and other therapies.

[0104] Other cancer treatments include, but are not limited to, immunotherapeutic agents, chemotherapy agents, radiotherapy agents, existing anticancer drugs, CCR5 antagonists, CXCR4 antagonists, etc. Examples of cancer treatments include, but are not limited to, one or more of antibodies or antagonists that inhibit the activity of CCL3, CCL5 (RANTES), CCL7, or CCL8, leronlimab (PRO140), T-VEC, AM-0010, CXCR4 antagonists, the TGF-beta kinase inhibitor galunisertib, anti-CSF-1R monoclonal antibodies, abemaciclib, faslodex, and necrotizing agents. Situmumab, AZD9291, Cyramza (ramucirumab), TPIV200, Galunisertib, cancer vaccines, cytokines, cell-based therapies, bispecific and multispecific antibodies, tumor-targeted mAbs, rituximab, oncolytic viruses, reovirus, blinatumomab, sipuleucel-T, T-Vec, IL-2, IFN-α, trastuzumab, celuximab, bevacizumab, Tim-3, BTLA, anti-IL-10 , GM-CSF, antiangiogenic therapy, VEGF inhibition, HMGB1, Nrp1, TAM receptor tyrosine kinase, Axl, MerTK, ALT-803, IL-15, immunosuppressive ligand phosphatidylserine (PS), bavituximab, bevacizumab (anti-VEGF), cobrometinib (MEK inhibitor), vemurafenib (BRAF inhibitor), erlotinib (EGFR), alectinib (ALK inhibitor), bevacizumab (anti-VEGF), pazopanib (tyrosine kinase inhibitor), dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), durvalumab (anti-PD-L1), sunitinib (RTK inhibitor), pazopanib (RTK inhibitor), sargramostim, VISTA, TIM-3, LAG-3, PRS-343, CD137 (4-1BB) / HER2 bispecific antibody, USP7, anti-HER2, SEMA4D, CTLA-4, PD-1, PD-L1, and PD-L2. As a non-limiting example, the treatment may be a cancer vaccine, and the subject expresses at least one HLA allele. As a non-limiting example, the immunotherapy may be PD-L1 immunotherapy.

[0105] The subject referred to in the methods of the present invention may be a human, non-human primate, bird, horse, cow, goat, sheep, or companion animal such as a dog, cat, rodent, or other mammal. A subject with cancer may be undergoing treatment for the cancer. Such treatments include, but are not limited to, targeted agents, chemotherapy, radiation therapy, and the like.

[0106] Companion Diagnostics The information obtained by the method of the present invention can be used as a companion diagnostic or complementary diagnostic. A companion diagnostic or complementary diagnostic is a diagnostic test that can be used in conjunction with a therapeutic drug for a selected disease or condition. A companion diagnostic determines the suitability of a drug for treating a disease or condition in a particular patient; that is, a companion diagnostic helps predict whether a patient of interest will respond or not to the drug's effect on that disease or condition. Thus, a companion diagnostic is used in treatment decisions and provides information essential for the safe and effective use of the corresponding drug or biological product. [Example]

[0107] Sample collection and processing Whole blood samples (7.5 mL) collected in CELLSAVE (trademark) storage tubes were placed in a low-pressure vacuum system. [1,12] or syringe pump

[12] The cells were processed in a CELLSIEVE™ microfiltration assay using a cytometer. The CELLSIEVE™ microfiltration assay separates circulating cells based on size exclusion through pores with a diameter of 7-8 μm. A trained cytologist performed the filtration assay using a cytometer as described in the literature. [6,11,14] Using certain established cytological features, the morphological characteristics and phenotypic expression of CD45, EpCAM, cytokeratin 8, 18, 19, and DAPI were [1,6,12]Based on this, we identified CAMLs associated with prognosis. An Olympus BX54WI fluorescence microscope equipped with a Carl Zeiss AxioCam and Zen2011 Blue (Carl Zeiss) was used for all imaging.

[0108] micronucleus <Experimental Example 1> Micronuclei (MN) are the result of the body's DNA repair mechanisms and are formed due to intrachromosomal abnormalities, which represent subclonal cancer populations with high cell viability and resistance to drug therapy. MN are often observed as small fragments of nucleic acid detached from the primary nucleus in circulating stromal cells (CStCs) as a result of DNA damage. 1,2 (Figures 2 and 3). Expression of programmed cell death ligand (PD-L1) appears to be upregulated in CStCs where DNA has been damaged and undergoing repair mechanisms, e.g., cells forming MNs. To determine prognostic significance for clinical outcome, CAMLs were evaluated for the presence of MNs and cellular PD-L1 expression in patients with metastatic breast cancer (mBC).

[0109] In a prospective pilot study of n = 76 patients with metastatic breast cancer (mBC) who were starting a new line of treatment, MN formation in CAML was enumerated. Whole blood (7.5 mL) was collected, filtered for CAML, and then PD-L1 14 Staining for PD-L1 was performed. DAPI was used to identify MNs, which are small (<3 μm), DAPI+ round structures present within the cytoplasm, separate from the primary nuclei. The number of MNs was compared with PD-L1 expression in all CAMLs, and the presence of MNs was compared with all available clinical variables. Patient hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) were analyzed using 2-year censored univariate analysis according to RECIST v1.1.

[0110] MN was identified in 59% (n=45 / 76) of CAML patients. The presence of MN within CAML significantly predicted worse PFS and OS over 24 months (Figures 5 and 6). MN-positive CAML had significantly higher PD-L1 expression than MN-negative CAML (p=0.0082) (Figure 7). Regression analysis revealed a significant linear relationship between the number of MN within CAML and PD-L1 expression (R 2 =0.9821, p=0.0089).

[0111] <Experimental Example 2> Although MN formation in CAML was rare in colorectal cancer (n = 25), breast cancer (n = 97), and various other cancers, MN formation was observed in 44% of all evaluated colorectal cancer patients and 60% of all evaluated breast cancer patients. Furthermore, the presence of CAML MN was an independent prognostic factor for progression-free survival (PFS) (HR = 17.2, 95% CI 3.6-80.9, p = 0.001) and overall survival (OS) (HR = 70.3, 95% CI 6.6-752.8, p = 0.002) in colorectal cancer patients (Figure 8), and for PFS (HR = 2.3, 95% CI 1.4-3.9, p = 0.0019) and OS (HR = 2.0, 95% CI 1.1-3.5, p = 0.0362) in breast cancer patients. Thus, the presence of MN was associated with poor prognosis for PFS and OS in these subjects.

[0112] Furthermore, in an analysis of n = 130 cancer patients with various cancer types, the presence of MN in CAML was found to predict worse PFS (HR = 2.4, 95% CI 1.4-1.2, p = 0.0030) and OS (HR = 2.6, 95% CI 1.3-5.3, p = 0.0097) (Figure 9).

[0113] <Experimental Example 3> Further assays were performed to assess the co-expression of MN and PD-L1 in CAML from breast cancer patients. PD-L1 expression was found to be statistically higher in patients with a higher number of MN in CAML (Figures 10 and 11), and the presence of MN correlated with a better response to PD-L1 / PD-1 immunotherapy (data not shown).

[0114] <Experimental Example 4> In patients with serial samples from baseline before and after cancer treatment, changes in the number of MNs in CAML provide useful information for the patient. Patients with adenocarcinoma (n = 25) were treated with FOLFOX or FOLFIRI (Table 1). Figure 12 shows the MN information in CAML analyzed for various parameters. Figure 12a is a linear regression plot showing the relationship between the number of CAMLs and the number of MNs for each patient in the baseline sample. Figure 12b plots the number of MNs in patients currently receiving chemotherapy (i.e., genotoxic agents) or newly diagnosed, untreated patients. Figure 12c shows data on the average number of MNs associated with nonmetastatic (stage III) or metastatic (stage IV) spread. Figure 12d shows the frequency of MNs in all CAMLs (n = 776). For micronuclei, 91% (blue) of CAMLs were negative, and only 9% were positive for at least one micronucleus. [Table 1]

[0115] Figure 13 shows two case studies tracking CAML MN by blood sampling. Figure 13a corresponds to Patient A. After progression on maintenance therapy (Xeloda, red box), FOLFOX (orange box) was initiated. After four cycles of treatment, the number of CAML MN increased, which correlated with tumor growth (+17%). Treatment was discontinued due to infection. The number of CAML MN further increased, and new lesions were discovered at cycle 6. FOLFOX was then restarted, and the number of CAML MN continued to increase, leading to the patient's withdrawal from the study. Figure 13b corresponds to Patient B. After progressive disease on FOLFOX, a single-agent CCR5 inhibitor (leronlimab, blue box) was initiated, which resulted in a corresponding decrease in the number of CAML MN, which correlated with a decrease in tumor size (-39%). Thereafter, the number of CAML MNs increased slightly, which correlated with a slight increase in tumor size (+11%), at which point FOLFIRI (purple box) was added to leronlimab. After the introduction of FOLFIRI, a decrease in the number of CAML MNs was observed, which correlated with stable disease.

[0116] extracellular vesicles <Experimental Example 1> The budding of extracellular structures in CAML has been observed in patients with metastatic non-small cell lung cancer (mNSCLC). In a prospective analysis of n=40 mNSCLC samples, we enumerated extracellular vesicles (EVs) in CAML to determine whether their formation impacts clinical outcome. These preliminary results suggest that EV budding from CAML predicts poor clinical outcome and may serve as a mechanism for the formation and systemic dissemination of cancer EVs.

[0117] We initiated a single-blind pilot study to assess extracellular vesicle formation in CAMLs from mNSCLC patients using pretreatment blood samples to determine their frequency and clinical utility. De-identified blood was obtained, filtered to isolate CAMLs, and stained for cytokeratin, CD45, CD31, and PD-L1. Extracellular vesicle budding was observed as small (<1 μm) spherical protrusions protruding from the cell periphery. EVs were quantified, and hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) were compared using univariate analysis censored at 24 months. Imaged EVs were also characterized by PD-L1 biomarker expression. CAML was identified in 88% (n=35 / 40) of all samples. EV budding was identified in 60% (n=21 / 35) of CAML cases The presence of EV budding in CAML was associated with significantly worse PFS (HR=3.39, 95% CI=1.4-8.2, p=0.0137) (Figure 14, top panel). The presence of EV budding in CAML was associated with significantly worse OS (HR=3.57, 95% CI=1.4-9.4, p=0.0201) (Figure 14, bottom panel). EV demonstrated PD-L1 positive expression in 57% (n=12 / 21) of CAMLs (Figure 15).

[0118] EV budding found in phagocytic stromal cells in the blood, along with positive tumor biomarkers, predicts faster disease progression and worse survival. These findings suggest that CAML may be the cell of origin for some cancer EVs and can be assessed by liquid biopsy analysis.

[0119] <Experimental Example 2> In an analysis of n=130 patients with various cancer types (breast cancer n=29, colorectal cancer n=25, esophageal cancer n=1, lung cancer n=43, ovarian cancer n=1, pancreatic cancer n=19, prostate cancer n=2, sarcoma n=10), the presence of CAML EVs was found to predict worse PFS (HR=4.0, 95% CI 2.4-6.8, p<0.0001) and OS (HR=5.9, 95% CI 3.1-11.5, p<0.0001) (Figure 16).

[0120] Figure 17 shows the proportion of CAML with EVs over a 15-month follow-up period in one patient undergoing chemoradiotherapy (CRT). The patient received CRT at baseline (BL) and had a 33% decrease in the presence of CAML EVs at T1 blood draw after CRT completion. The presence of CAML EVs increased from T2 to T5, at which point the patient had disease progression confirmed by radiological imaging (PET / CT). The patient was initiated on ipilimumab, but subsequently developed further metastases at T7, indicating disease progression.

[0121] <Experimental Example 3> In a prospective analysis of patients with mNSCLC (n=104), EV budding was enumerated in CAML to determine its clinical significance in progression-free survival (PFS) and overall survival (OS), and further subtype classification based on treatment with or without standard-of-care PD-L1 immunotherapy (IMT). These preliminary data suggest that EV-positive (EV+) CAML predicts a poor prognosis in mNSCLC.

[0122] We initiated a single-blind, multi-prospective study to investigate the relationship between EV budding in CAML and progression-free survival (PFS) and overall survival (OS) before initiating a new line of treatment for mNSCLC. De-identified blood (7.5 mL) was collected from n=104 patients with pathologically confirmed mNSCLC and filtered to isolate CAML. EV budding was measured using tumor / EV markers (i.e., cytokeratin, CD163, or CD31) and an immune-specific marker (PD-L1). Blood was filtered using CELLSIEVE™ microfiltration, and CAML EV budding was characterized as small (≤5 μm) globular protrusions arising from the cell cytoplasm. CAML EVs were quantified by their presence (EV+) or absence (EV-), and hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) at 60 months were compared using censored univariate and multivariate analyses.

[0123] CAML was identified in 93% (n=97 / 104) of all samples. EV budding was identified in 62% (n=60 / 97) of samples with CAML. EV(+) CAML was associated with significantly worse PFS (HR=1.67, p=0.0410) and OS (HR=1.88, p=0.0180) (panels a and b of Figure 18, respectively). EV(+) patients who did not receive immunotherapy (IMT) had significantly worse PFS (HR=2.51, p=0.0251) and OS (HR=2.32, p=0.0407) (Figure 19). EV(+) patients benefited from additional IMT, demonstrating prolonged mPFS and mOS (Figure 19).

[0124] These results indicate that EV budding in phagocytic stromal cells present in the blood of mNSCLC patients predicts worse PFS and OS (Figure 18 and Table 2). The worsening PFS and OS due to the presence of EVs in CAML is improved by the addition of PD-L1 immunotherapy (Figure 19). [Table 2]

[0125] Multinuclear expansion Although multinucleated nuclear proliferation (EPN) is a common criterion for identifying CAML, the degree of cellular nuclear proliferation and multinucleated proliferation may correlate with both cell size and cancer-related cellular abnormalities. In an evaluation of patients with various cancers (n=130), patients with larger nuclear mass, as measured by nuclear area, were found to have more aggressive disease, as measured by progression-free survival (PFS) and overall survival (OS). Among CAML patients, patients with a nuclear area of ​​465 μm or greater were significantly more aggressive than those with a nuclear mass of 465 μm or greater. 2 Patients with >1% CI had worse PFS (HR=2.8, 95% CI 1.7-4.8, p=0.0002) and OS (HR=2.9, 95% CI 1.5-5.5, p=0.0025) (Figure 20).

[0126] Internalization in intact cells Previous studies have shown that patients with internalized cells (e.g., CTCs) within CAML have worse clinical outcomes than those without. However, aberrant internalization of non-CTC cells, such as leukocytes, and other internal masses of unknown origin can also be observed in CAML. However, this internalization of CAML has been shown to be more prevalent in patients with advanced disease stages. We evaluated CAML in various cancer types and found that CAML encapsulating whole or partial WBCs or whole or partial CTCs, or CAML associated with WBCs, tended to exhibit worse clinical outcomes, including worse PFS (HR = 1.6, 95% CI 0.9-2.6, p = 0.1094) and OS (HR = 1.6, 95% CI 0.8-3.0, p = 0.2173) (Figure 21). We also evaluated CAML across various cancer types and found that CAML with internalized non-disintegrated cellular debris or other internal structures was associated with worse clinical outcomes, including worse PFS (HR=2.9, 95% CI 1.5-5.8, p=0.0047) and OS (HR=2.1, 95% CI 0.9-5.0, p=0.1356) (Figure 22).

[0127] Combination of characteristics Based on the experimental results described above, combinations of the above observable biological cell characteristics were evaluated, and it was found that various combinations could stratify patients with poor clinical outcomes. The optimal combination was CAML MN, CAML EV, and nuclei 465 μm in a multi-tumor set of n = 130. 2 These are combinations of CAMLs that predict worse PFS (HR=4.2, 95% CI 2.5-7.0, p<0.0001) and OS (HR=5.0, 95% CI 2.7-9.3, p<0.0001) (Figure 23).

[0128] Metastatic disease Further analysis revealed that of n = 40 patients initially treated for non-metastatic disease, n = 18 patients were re-diagnosed with metastatic disease within 2 years, and 72% were found to have metastatic disease within 10 months. Among n = 22 patients without metastases at re-diagnosis, 1 had CAML MN, 1 had CAML cellular debris, 5 had CAML WBCs, and 1 had CAML EVs. Among n = 18 patients whose metastatic disease progressed within 2 years, 5 had CAML MN, 4 had CAML cellular debris, 7 had CAML WBCs, 1 had CAML-binding CTCs, and 13 had CAML EVs. This indicates that the presence of CAML EVs predicted patients who would rapidly relapse with metastatic disease with 85% accuracy. Furthermore, the presence of CAML EVs or CAML MNs in a patient's blood rapidly re-diagnosed patients as having metastatic disease with 90% accuracy. Because samples were collected before treatment initiation, this suggests that CAML with MN or EV may be initially underdiagnosed and may identify patients who likely had metastatic disease at the time of blood collection.

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Claims

1. 1. A method for predicting overall survival (OS) and / or progression-free survival (PFS) of a subject having cancer, comprising measuring the presence of one or more of the following in circulating cells or produced by circulating cells from a biological sample from the subject having cancer, wherein the presence of one or more of (a), (b), (c), (d), and (e) predicts shorter OS and / or PFS than a subject having the same cancer in the absence of the presence of one or more of (a), (b), (c), (d), or (e).

2. 1. A method for predicting the presence of metastatic spread and / or metastatic progression in a subject having cancer, comprising measuring the presence of one or more of: (a) MNs, (b) EVs, (c) EPNs, (d) internalized intact cells, and (e) internal cell debris in circulating cells from a biological sample from the subject having cancer, wherein the presence of one or more of (a), (b), (c), (d), and (e) predicts the presence of metastatic spread and / or metastatic progression in the subject.

3. 1. A method of predicting cancer progression in a subject having cancer, comprising measuring the presence of one or more of: (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject having cancer, wherein the method predicts that the cancer will progress in the subject if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and / or additional biological samples but not present in or produced by circulating cells of the first biological sample, and predicts that the cancer will not progress in the subject if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the first biological sample but not present in or produced by circulating cells of the second and / or additional biological samples.

4. 1. A method of predicting cancer progression in a subject having cancer, comprising measuring the presence of one or more of: (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject having cancer, wherein 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, and wherein one or more of (a), (b), (c), (d), or (e) are present in the first biological sample and a second biological sample at a later date, and any additional biological samples obtained from the subject after at least one cancer treatment. is present in or produced by circulating cells of the first biological sample but not present in or produced by circulating cells of the second and / or additional biological samples, and predicts that cancer will not progress in the subject if one or more of (a), (b), (c), (d), or (e) is present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample.

5. 1. A method of predicting therapeutic response in a subject having cancer, comprising measuring the presence of one or more of: (a) MN, (b) EV, (c) EPN, (d) internalized intact cells, and (e) internal cell debris in circulating cells or produced by circulating cells in a first biological sample, a second biological sample at a later date, and any additional biological samples obtained from the subject having cancer, wherein 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, and wherein (a), (b), (c), (d), or (e) cysts are present. the subject is predicted to respond to treatment if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample; and the subject is predicted to not respond to treatment if one or more of (a), (b), (c), (d), or (e) are present in or produced by circulating cells of the second and any / or additional biological samples but not present in or produced by circulating cells of the first biological sample.

6. 10. The method of claim 1, wherein the OS and / or PFS is at least 12 months.

7. 10. The method of claim 1, wherein the OS and / or PFS is at least 24 months.

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

9. The circulating cells 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) a cell size of about 20-300 μm in size, and (c) a morphological shape selected from the group consisting of spindle-shaped, tadpole-shaped, round, oval, two-legged, two or more-legged, thin-legged, and irregular; 6. The method according to claim 1, wherein the

10. The circulating cells have the following additional characteristics: (d) CD14 expression; (e) CD45 expression; (f) EpCAM expression; (g) vimentin expression; (h) PD-L1 expression, (i) CD11c marker expression on monocytes and macrophages; (j) endothelial CD146 marker expression; (k) endothelial and macrophage CD202b marker expression; (l) endothelial, macrophage and leukocyte CD31 marker expression; and (m) epithelial cancer cell CK8, 18, and / or 19 marker expression; 10. The method of claim 9, comprising one or more of:

11. 6. The method of claim 1, wherein the source of the biological sample is one or more of peripheral blood, blood, lymph nodes, bone marrow, cerebrospinal fluid, and urine.

12. The method of claim 11 , wherein the biological sample is antecubital vein blood, inferior vena cava blood, femoral vein blood, portal vein blood, or jugular vein blood.

13. 6. The method of any one of claims 1 to 5, wherein the cancer is stage I cancer, stage II cancer, stage III cancer, stage IV cancer, carcinoma, sarcoma, neuroblastoma, melanoma, epithelial cell carcinoma, lung cancer, breast cancer, prostate cancer, pancreatic cancer, bladder cancer, kidney cancer, head and neck cancer, colon cancer, liver cancer, ovarian cancer, osteosarcoma, esophageal cancer, brain and ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, cervix, uterine corpus cancer, or other solid tumor.

14. 6. The method of any one of claims 1 to 5, wherein circulating cells are separated from the biological sample for said measurement using one or more techniques selected from the group consisting of size exclusion, immunocapture, red blood cell lysis, leukocyte depletion, polymeric polysaccharides such as FICOLL®, electrophoresis, dielectrophoresis, flow cytometry, magnetic levitation, and various microfluidic chips, slits, channels, hydrodynamic size-based sorting, grouping, trapping, enrichment of large cells, removal of small cells, or combinations thereof.

15. 15. The method of claim 14, wherein circulating cells are isolated from the biological sample using a size exclusion method using a microfilter.

16. 16. The method of claim 15, wherein the microfilter has a pore size ranging from about 5 μm to about 20 μm.

17. 17. The method of claim 16, wherein the microfilter pores have circular, racetrack, oval, square, and rectangular pore shapes.

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

19. 15. The method of claim 14, wherein circulating cells are separated by any of physical size-based sorting using a microfluidic chip, hydrodynamic size-based sorting, grouping, trapping, immunocapture, enrichment of large cells, or size-based removal of small cells.

20. 6. The method of claim 1, wherein a microfiltration assay is used to separate circulating cells from the biological sample for said measurement.

21. 6. The method of claim 4 or claim 5, wherein the treatment is one or more of chemotherapy, monotherapy, combination drug therapy, immunotherapy, radiation therapy, chemoradiotherapy, radiation combined with one or more drugs, chemoradiotherapy combined with one or more drugs, cancer vaccine, and cell therapy.

22. 22. The method of claim 21, wherein the treatment is a cancer vaccine and the subject expresses at least one HLA allele.

23. 4. The method of any one of claims 1 to 3, wherein the subject is being treated with one or more of chemotherapy, monotherapy, combination drug therapy, immunotherapy, radiation therapy, chemoradiotherapy, radiation combined with one or more drugs, chemoradiotherapy combined with one or more drugs, a cancer vaccine, and cell therapy.

24. 24. The method of claim 23, wherein the immunotherapy is PD-L1 immunotherapy.

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