Method of isolating and analyzing circulating tumor cells

Abstract

To provide methods of isolating and analyzing circulating tumor cells.SOLUTION: Disclosed herein is a method of isolating and analyzing circulating tumor cells (CTCs) in a biological sample using flow cytometry. The method enables quick and simple prediction of responsiveness of a subject having lung cancer or gastric cancer to a course of treatment.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 614,804, filed on December 26, 2023, which is hereby incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates to the field of personalized medicine. More specifically, liquid biopsies are applied to individualize and monitor the treatment of patients with tumors and to diagnose tumors. The present disclosure relates to methods for identifying and characterizing sub - populations (sub - groups) of circulating tumor cells (CTCs) in a CTC population in a biological sample, preferably a blood sample of a patient.

Background Art

[0003] Lung cancer is the most common cause of cancer death worldwide, and the 5 - year survival rate of patients with this disease is less than 15%. Non - small cell lung cancer (NSCLC) is the major histological type of lung cancer, accounting for approximately 85% of lung cancer cases. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have demonstrated significant efficacy in NSCLC patients with EGFR mutations. These inhibitors are associated with minimal side effects and have been shown to improve quality of life, particularly in patients with exon 19 deletions (E19del) or exon 21 point mutations (L858R).

[0004] Gastric cancer (GC) is one of the most common malignant tumors in the digestive tract. In 2022, according to global statistics, approximately 968,784 new cases were reported, and the deaths caused by GC were approximately 660,175. Although both the incidence and mortality rates have recently decreased, the detection rate and overall prognosis are still not satisfactory. With the progress of precision oncology in clinical practice, molecularly targeted therapy for GC has received substantial attention. Among these, therapies targeting human epidermal growth factor receptor 2 (Her2) have shown significant clinical benefits and are now widely used in treatment regimens.

[0005] Obtaining molecular information specific to the type of cancer is essential in clinical practice to induce targeted therapy. Typically, such information is derived from invasive solid tissue biopsies that require invasive procedures to obtain. However, due to advanced disease stage or overall poor physical condition, some patients may be contraindicated or intolerant to those invasive biopsies, highlighting the need for safer and more convenient alternatives. In addition, in certain cancers, such as GC, biopsy results may lack sufficient accuracy.

[0006] Due to the extremely rare nature of CTCs, detecting CTCs within the background of a large number of white blood cells requires highly sophisticated methodologies. The challenge of CTC detection is further complicated by the heterogeneity and plasticity of these cells, particularly complicating consistent enrichment from biological samples. Developing an efficient approach to analyze these cells from a minimal sample volume is extremely important for enhancing detection accuracy and increasing its clinical applicability. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] In response to an urgent need in the art, the present disclosure provides a rapid and simple method for isolating and analyzing CTCs to facilitate confirmation of EGFR mutations or Her2 status in these cells. The EGFR mutation or Her2 status was evaluated by isolating blood-derived epithelial cells from patients with lung cancer, such as NSCLC or GC, prior to initiating treatment with therapies such as EGFR TKIs, EGFR-targeted agents, or Her2-targeted therapy, respectively. By analyzing a small group of mixed CTCs, the detection rates of EGFR mutations and Her2 status were significantly improved compared to single CTC analysis or detection methods that rely on conventional immunohistochemistry (IHC).

[0008] In one embodiment, the present disclosure is a method for analyzing circulating tumor cells (CTCs) in a biological sample of an individual having cancer, comprising a) Isolating white blood cells (WBC) and CTCs from a biological sample; b) Setting a gate for CTCs based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and vimentin (VIM); and c) Testing CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2; comprising providing a method in which steps b) and c) are performed by flow cytometry.

[0009] In a preferred embodiment, the biological sample is peripheral blood

[0010] In a preferred embodiment, the biological sample is approximately 1 mL.

[0011] The method described herein enables the isolation and analysis of CTCs from a minimal amount of biological sample, e.g., 1 mL of peripheral blood.

[0012] In a preferred embodiment, the method may further comprise lysing red blood cells in the biological sample prior to step a).

[0013] In a preferred embodiment, steps b) and c) may be performed continuously.

[0014] In a preferred embodiment, the cancer can be non-small cell lung cancer (NSCLC) or gastric cancer (GC).

[0015] In a preferred embodiment, the method may further comprise identifying the cancer as non-small cell lung cancer (NSCLC) if the CTCs express markers including CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , EpCAM + , HLA-A.B.C + , and VIM + .

[0016] In a preferred embodiment, the method further comprises administering an EGFR tyrosine kinase inhibitor to the individual if the CTC is mEGFR L858R. +

[0017] In a preferred embodiment, the EGFR tyrosine kinase inhibitor may include erlotinib, gefitinib, icotininib, afatinib, dacomitinib, osimertinib, rociletinib, ormutinib, lazertinib, and zolifertinib.

[0018] In a preferred embodiment, the method further comprises identifying the cancer as gastric cancer (GC) if the CTC expresses markers including CD45, - CK-7 / 8, + CK-14 / 15 / 16 / 19, + EpCAM, + and HLA-A.B.C. +

[0019] In a preferred embodiment, the method further comprises administering to the individual at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells if the CTC is Her2. +

[0020] In a preferred embodiment, the Her2 tyrosine kinase inhibitor may include lapatinib; the anti-Her2 antibody may include trastuzumab, pertuzumab, tucatinib, and margetuximab; the bispecific antibody may include zanidatamab; or the Her2-targeted antibody-drug conjugate may include trastuzumab emtansine and trastuzumab deruxtecan.

[0021] In another embodiment, the present disclosure provides a method of treating cancer in an individual in need thereof, a) Setting a gate for circulating tumor cells (CTCs) in a biological sample derived from an individual based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM; b) Testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2; and c) Administering cancer treatment to the individual, i) If the cancer is identified as non-small cell lung cancer (NSCLC) where the CTCs are mEGFR L858R + then the cancer treatment is an EGFR tyrosine kinase inhibitor, or ii) If the cancer is identified as gastric cancer (GC) where the CTCs are Her2 + then the cancer treatment comprises at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells. A method is provided that includes the above steps.

[0022] In a preferred embodiment, the EGFR tyrosine kinase inhibitor may include erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, ormutinib, lazertinib, and zolifertinib.

[0023] In a preferred embodiment, the Her2 tyrosine kinase inhibitor may include lapatinib; the anti-Her2 antibodies may include trastuzumab, pertuzumab, tucatinib, and margetuximab; the bispecific antibody may include zanidatamab; or the Her2-targeted antibody-drug conjugate may include trastuzumab emtansine and trastuzumab deruxtecan.

[0024] In another embodiment, the present disclosure provides a method for identifying an individual having cancer that may respond to treatment with at least one treatment selected from the group consisting of an EGFR tyrosine kinase inhibitor, a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a Her2-targeted antibody-drug conjugate, and a Her2-targeted CAR-T cell, comprising: a) setting a gate for circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM; b) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2; and c) i) if the CTCs are derived from NSCLC and have - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-A.B.C + VIM + mEGFR L858R + identifying the individual as having cancer that may respond to treatment with an EGFR tyrosine kinase inhibitor or ii) if the CTCs are derived from GC and have - CK-7 / 8 + CK-14 / 15 / 16 / 19 + EpCAM + HLA-A.B.C + Her2 + identifying the individual as having cancer that is likely to respond to treatment with at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and a Her2-targeted CAR-T cell and providing a method comprising the step of for identifying an individual having cancer that may respond to treatment with at least one treatment selected from the group consisting of an EGFR tyrosine kinase inhibitor, a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a Her2-targeted antibody-drug conjugate, and a Her2-targeted CAR-T cell.

[0025] In another embodiment, the present disclosure provides a kit for use in the methods described herein, comprising one or more reagents for labeling CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, VIM, mEGFR L858R, and / or Her2.

[0026] By examining mixed populations of CTCs, flow cytometry-based methods enhance the sensitivity and efficiency of detecting these biomarkers and provide a more robust approach for identifying cancer-related mutations and markers in circulating tumor cells in the blood. This improvement highlights the potential of mixed CTC analysis as an alternative for cancer diagnosis and monitoring.

[0027] The file of this patent application contains at least one drawing executed in color. Copies of the patent application publication having color drawings are provided by the USPTO upon request and payment of the necessary fees.

[0028] Exemplary embodiments of the present application are described in detail below with reference to the following drawings.

Brief Description of the Drawings

[0029]

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DETAILED DESCRIPTION OF THE INVENTION

[0030] The foregoing and other aspects of the present disclosure will be described in more detail in relation to other aspects described herein. It should be recognized that the present invention can be embodied in different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0031] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0032] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," "characterized by," or any other variation thereof are intended to cover a non-exclusive inclusion that includes any limitations explicitly stated. For example, a composition, mixture, process, or method that includes a list of elements is not necessarily limited to those elements, and may include other elements not expressly described or inherent to such composition, mixture, process, or method.

[0033] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified. In a claim, such a transitional phrase closes the claim to the inclusion of materials other than those recited, except for impurities ordinarily associated therewith. When the phrase "consisting of" appears in the body of the claim, rather than immediately following the preamble, it limits only the elements recited in that clause; other elements are not excluded from the claim as a whole.

[0034] It should be readily understood that when the applicant defines the invention or a part thereof by terms without limitations such as "comprising" (unless otherwise specifically stated), the description should be construed as if the invention were so described using the term "consisting of."

[0035] All numbers recited herein are to be understood as being modified by the term "about." As used herein, the term "about" is used to indicate that a value includes, for example, variations due to the inherent error of the measuring device, the method used to determine the value, or variations that exist in the test subject tube. Typically, the term, depending on the situation, means a variation of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, or a variation of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0036] The use of the term "or" in the claims is used to mean "and / or" unless it is clear that only an alternative is meant or the alternatives are mutually exclusive, but the present disclosure supports definitions that refer only to alternatives and "and / or."

[0037] "Subject," as used herein, refers to an animal, including, for example, a mammalian subject diagnosed as having cancer or suspected of having or developing cancer. In one embodiment, the term "subject" can refer to a vertebrate having cancer or a vertebrate that is thought to be in need of treatment for cancer. Subjects include warm-blooded animals, such as mammals, such as primates, and more preferably humans. Non-human primates are likewise subjects. The term subject includes domesticated animals, such as cats, dogs, apes, etc., livestock (e.g., cows, horses, pigs, sheep, goats, etc.) and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary use and pharmaceutical formulations are contemplated herein.

[0038] "Administer" or "administration" as used herein refers to providing the modified T cells or pharmaceutical composition of the present application to a subject. By way of non-limiting example, administration can be effected parenterally, subcutaneously, intramuscularly, intravenously, intra-articularly, intratracheally, intra-abdominally, intra-synovially, intra-cartilaginously, intracavitarily, intracelially, intracerebellarly, intraventricularly, intra-colonically, intra-cervically, intra-gastrically, intra-hepatically, intra-myocardially, intra-osseously, intra-pelvicly, intra-pericardially, intra-peritoneally, intra-pleurally, intra-prostatically, intra-pulmonaryly, intra-rectally, intra-renal-ly, intra-retinally, intra-spinally, intra-synovially, intra-thoracically, intra-uterinely, intra-vesically, by bolus, vaginally, rectally, buccally, sublingually, intranasally, and transdermally. For example, an injection can be effected by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes may be used. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively or concurrently, administration can be by the oral route.

[0039] The use of the terms "treat" or "treatment" as used herein refers to administering treatment to a subject for the purpose of curing, alleviating, reducing, relieving, opening, preventing, or ameliorating a disorder, the symptoms of a disorder, a disease state secondary to a disorder, or a predisposition to a disorder. The terms "inhibit", "reduce", or "prevent", or any variation of these terms, when used in the claims and / or specification, include any measurable decrease or complete inhibition to achieve the desired result.

[0040] In certain embodiments, it is desirable to limit, reduce, or improve the size of a tumor or cancerous lesion. The route of administration will, of course, vary depending on the location and nature of the targeted lesion or site and can include, for example, local, parenteral, intravenous, intramuscular, and / or systemic administration and formulations. Direct injection into and from and within an organ or tissue and injection into the vasculature or a blood vessel are specifically contemplated with respect to the target area. Local, regional, or systemic administration may also be appropriate.

[0041] "Circulating tumor cells", "CTC", and "CTCs" are used interchangeably herein and refer to cells that have detached from a primary tumor into the vasculature and are circulating in the bloodstream. CTCs are thought to be seeds for subsequent growth (metastasis) of further tumors in important distal organs and induce mechanisms that are the cause of the majority of cancer-related deaths.

[0042] "CD45", as used herein, refers to cluster of differentiation 45 (CD45), also known as protein tyrosine phosphatase, receptor type C, and leukocyte common antigen, and is encoded by the PTPRC gene. CD45 is used to identify leukocytes. Antibodies that bind to CD45 may be used to detect CD45.

[0043] "Cytokeratin", as used herein, refers to keratin-containing intermediate filaments found in the cytoplasmic cytoskeleton of epithelial tissues. Cytokeratin-expressing cancer cells lose their cytokeratin expression after undergoing epithelial-mesenchymal transition (EMT), and up to 20% of the cells do not have detectable cytokeratin. "Cytokeratin-7", "Keratin-7", "CK-7", and "Sarcoleptin" are used interchangeably herein and refer to the protein encoded by the KRT7 gene in humans. "Cytokeratin-8", "Keratin-8", and "CK-8" are used interchangeably herein and refer to the protein encoded by the KRT8 gene in humans. Cytokeratins 7 and 8 are two closely related type II cytokeratins that are characteristic of simple epithelia. Cytokeratin 7 is not as widespread as cytokeratin 8 and is expressed in cells of the sebaceous and sweat glands, and part of the inner root sheath. Cytokeratin 8 is mainly found in non-squamous epithelial cells. Cytokeratin 7 is usually present in the lung, breast adenocarcinoma, endometrial tumors, and transitional cell carcinoma of the bladder. The combination of cytokeratins 7 and 8 is a useful marker for distinguishing adenocarcinoma and ductal carcinoma of the breast from squamous cell carcinoma. Antibodies that bind to cytokeratin 7 / 8 may be used to detect cytokeratin 7 / 8.

[0044] When used interchangeably herein, "cytokeratin-14", "keratin-14", and "CK-14" refer to the protein encoded by the KRT14 gene in humans. Similarly, "cytokeratin-15", "keratin-15", and "CK-15" refer to the protein encoded by the KRT15 gene, "cytokeratin-16", "keratin-16", and "CK-16" refer to the gene encoded by the KRT16 gene, and "cytokeratin-19", "keratin-19", and "CK-19" refer to the gene encoded by the KRT19 gene. Cytokeratin-14, 15, 16, and 19 are all type I cytokeratins and are collectively known as pan-cytokeratin (pan-CK). In cancer research, especially in CTC research, pan-cytokeratin is often used as a biomarker to identify and isolate tumor cells of epithelial origin that typically overexpress cytokeratin compared to non-epithelial cells.

[0045] "EpCAM", as used herein, refers to epithelial cell adhesion molecule, a type I glycosylated membrane protein that is expressed at low levels in various human epithelial tissues but is overexpressed in most solid cancers. To date, most studies have used EpCAM as a target marker to identify potential CTCs. Its expression has been shown to be inversely proportional to the prognosis of cancer in patients.

[0046] "HLA-A.B.C", "major histocompatibility complex, class I, A, B, C", and "HLA class I A, B, C" are used interchangeably herein and refer to human leukocyte antigens A, B, and C, which are encoded by genes located on the short arm of chromosome 6 (6p21.3). The HLA-A.B.C marker is often used in the detection of CTCs because it helps to distinguish CTCs from non-tumor cells in the bloodstream. Specifically, HLA-A.B.C supports the identification of epithelial cells, discriminates CTCs from immune cells, detects cells undergoing EMT, and provides insights into immune evasion and cancer progression.

[0047] "VIM" and "vimentin" are used interchangeably herein and refer to type III intermediate filament (IF) proteins expressed in mesenchymal cells that serve as a major cytoskeletal component. Because of its role in mesenchymal cell structure, vimentin is often used as a marker for mesenchymal-derived cells to identify cells undergoing EMT in both normal development and the progression of metastasis, including the detection of CTCs.

[0048] The terms "sample", "test sample", "specimen", "biological sample", "sample from a subject", or "subject sample" are used interchangeably herein and mean a sample or isolate of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, white blood cells or monocytes, which can be obtained from a subject and used directly, or can be pre-treated, for example, by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the characteristics of the sample in some of the methods considered herein or in other methods known in the art.

[0049] Methods well known in the art for collecting, handling, and processing urine, blood, serum, and plasma, and other body fluids are used in the practice of the present disclosure. A test sample can include additional moieties in addition to the analyte of interest, such as an antibody, antigen, hapten, hormone, drug, enzyme, receptor, protein, peptide, polypeptide, oligonucleotide, or polynucleotide. For example, the sample can be a whole blood sample obtained from a subject. A test sample, particularly whole blood, may need to be treated, for example, with a pretreatment reagent, prior to the immunoassay described herein or may be desirable. Even when pretreatment is not necessary (e.g., most urine samples, pre-treated stored samples, etc.), pretreatment of the sample can be an option carried out for mere convenience (e.g., as part of the protocol of a commercial platform). The sample can be used directly as obtained from the subject or after pretreatment to modify the characteristics of the sample. Pretreatment can include extraction, concentration, inactivation of interfering components, and / or addition of reagents.

[0050] Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety for the teachings relevant to the passages and / or paragraphs introducing the references.

[0051] Method for isolating and analyzing circulating tumor cells in blood In one embodiment, a method for isolating and analyzing a population of circulating tumor cells (CTCs) in a biological sample from an individual having cancer comprises: a) isolating white blood cells (WBCs) and CTCs from the biological sample; b) setting a gate for CTCs based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and vimentin (VIM); and c) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2; comprising: Steps b) and c) are performed by flow cytometry.

[0052] The biological sample may contain a population of CTCs. The population of CTCs may contain one or more subpopulations of CTCs. The composition of the subpopulation of circulating tumor cells (CTCs) is characteristic for a particular tumor, e.g., a particular solid tumor. Thus, the CTC subpopulation and each subgroup of CTCs are surrogate markers for cancer, tumor, and / or metastasis. The subgroup of CTCs is further characteristic for a particular individual, e.g., a patient. The CTC composition of the individual subgroups in the biological sample is specific for a particular patient. This can be used to predict and monitor treatment and disease progression or regression, respectively. Thus, the population of CTCs or the CTC subgroups and their respective amounts (ratios) in a sample identified by the methods of the present disclosure can be used as specific biomarkers for each tumor, therapeutic treatment, and individual patient, and these biomarkers, e.g., mEGFR L858R or Her2, are suitable, for example, for personalized medicine.

[0053] The biological sample described in this specification is obtained from one individual. The individual can be a human, preferably a patient having cancer. In another embodiment, the individual can be any mammal. In a preferred embodiment, the biological sample to be isolated and analyzed is a human sample, such as whole blood.

[0054] In one embodiment, the WBCs and CTCs can be isolated using any method that effectively removes cells that can interfere with the analysis of CTCs. For example, the WBCs and CTCs can be isolated through techniques such as centrifugation, red blood cell (RBC) lysis, or Ficoll-Paque. Centrifugation can include density gradient centrifugation, low-speed centrifugation, high-speed centrifugation, or fractionation centrifugation. In one embodiment, the WBCs and CTCs are isolated by RBC lysis. In another embodiment, the WBCs and CTCs are isolated by centrifugation after RBC lysis.

[0055] In one embodiment, the method includes the step of setting gates for WBCs and CTCs based on forward scatter light (FSC) and CD45 to exclude cell debris and aggregates and to deplete hematopoietic cells to obtain CTC-like cells. In one embodiment, the hematopoietic cells are depleted using an anti-CD45 antibody. If the CD45 expression is negative, circulating tumor cells in the blood can be confirmed. Considering that CTCs are extremely rare compared to other circulating blood cells, the isolation of CTCs involves the identification and exclusion of cells expressing the pan-leukocyte marker CD45.

[0056] In addition, the method includes the step of testing the cells for cytokeratin-7 / 8 (CK-7 / 8), cytokeratin-14 / 15 / 16 / 19 (CK-14 / 15 / 16 / 19), epithelial cell adhesion molecule (EpCAM), human leukocyte antigen-A.B.C (HLA-A.B.C), and vimentin (VIM) to identify CTCs in one biological sample, and testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2 to identify a subset of CTCs for appropriate drugs for treatment.

[0057] In one embodiment, steps b) and c) in the method of the present disclosure are all carried out by flow cytometry to isolate and analyze CTCs in one experiment. In another embodiment, steps b) and c) can be carried out continuously.

[0058] In one embodiment, the biological sample can be peripheral blood, preferably about 1 mL, depending on the requirements of the analysis method.

[0059] In the present invention, the method described herein enables the isolation and analysis of CTCs from a small amount of biological sample, for example, 1 mL of peripheral blood.

[0060] In another embodiment, the method can further include a step of lysing red blood cells in the biological sample before step a).

[0061] The tests of the present invention show a method configured to detect a CTC concentration of 2 or more CTCs per mL of whole blood. In another embodiment, the method is configured to detect a CTC concentration of 5 or more CTCs per mL of whole blood. In other words, when there are 2 or more CTCs in the whole blood, the CTCs can be isolated for further analysis based on a phenotype, such as mEGFR L858R or Her2. The results of this example show that even when there are as few as 2 CTCs in 1 mL of the biological sample, the R 2 value of the test result is still excellent, demonstrating strong sensitivity for low-concentration detection.

[0062] In one embodiment, the cancer can be lung cancer, including small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0063] In a preferred embodiment, the lung cancer is CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-A.B.C + VIM +It is NSCLC having the phenotype.

[0064] In one embodiment, the method may further include the step of identifying that the cancer is NSCLC if the CTC expresses the markers CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , EpCAM + , HLA-A.B.C + , and VIM + .

[0065] The ability to detect and characterize CTCs has potential to assist in the treatment of cancer patients. The method may further include the step of administering an EGFR tyrosine kinase inhibitor to an individual if the CTC is identified as being mEGFR L858R + .

[0066] Examples of EGFR tyrosine kinase inhibitors may include erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, ormutinib, lazertinib, and zolifertinib.

[0067] In one embodiment, the cancer may be gastric cancer (GC), such as Her2-positive GC.

[0068] In a preferred embodiment, GC has the phenotype of CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , HLA-A.B.C + .

[0069] In one embodiment, the method may further include the step of identifying that the cancer is GC if the CTC expresses the markers CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , and HLA-A.B.C + .

[0070] If an individual is identified as having CTCs that are Her2 + then at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells can be administered.

[0071] Examples of Her2 tyrosine kinase inhibitors can include lapatinib; examples of anti-Her2 antibodies can include trastuzumab, pertuzumab, tucatinib, and margetuximab; examples of bispecific antibodies can include zanidatamab; and examples of Her2-targeted antibody-drug conjugates can include trastuzumab emtansine and trastuzumab deruxtecan.

[0072] In another aspect, the present disclosure is a method of treating cancer in an individual in need thereof, a) setting a gate for circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM, b) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2, and c) administering cancer treatment to the individual, i) if the cancer is identified as non-small cell lung cancer (NSCLC) in which the CTCs are mEGFR L858R + then the cancer treatment is an EGFR tyrosine kinase inhibitor, or ii) if the cancer is identified as gastric cancer (GC) in which the CTCs are Her2 + then the cancer treatment includes at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells A method comprising the steps is provided.

[0073] In a preferred embodiment, the EGFR tyrosine kinase inhibitor may include erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, ormutinib, lazertinib, and zolifertinib.

[0074] In a preferred embodiment, the Her2 tyrosine kinase inhibitor may include lapatinib; the anti-Her2 antibody may include trastuzumab, pertuzumab, tucatinib, and margetuximab; the bispecific antibody may include zanidatamab; or the Her2-targeted antibody-drug conjugate may include trastuzumab emtansine and trastuzumab deruxtecan.

[0075] In another embodiment, the present disclosure provides a method for identifying an individual having cancer who may respond to treatment by at least one treatment selected from the group consisting of an EGFR tyrosine kinase inhibitor, a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a Her2-targeted antibody-drug conjugate, and a Her2-targeted CAR-T cell, comprising: a) setting a gate for circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM; b) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2; and c) i) if the CTCs are derived from NSCLC and have CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-A.B.C + VIM + mEGFR L858R + then an EGFR tyrosine kinase inhibitor, or ii) if the CTCs are derived from GC and have CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 + EpCAM + HLA-A.B.C+ Her2 + If it has, at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells A step of identifying an individual having cancer that may respond to treatment with A method is provided that includes

[0076] In one aspect, the present disclosure provides a kit for use in the methods described herein, the kit comprising one or more reagents for labeling CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, VIM, mEGFR L858R, and / or Her2.

[0077] The following examples regarding specific aspects for carrying out the present invention are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example

[0078] (Example 1) Phenotypic screening of cell lines H1975 (NSCLC, mutant EGFR L858R), A549 (NSCLC, wild-type EGFR), MCF-7 (breast cancer), and in addition healthy donor leukocytes To establish a CTC detection platform based on flow cytometry, a series of gating strategies must first be developed and validated. In the case of the mutant EGFR (mEGFR) L858R NSCLC detection platform, cell lines from various cancers were utilized, including H1975 (NSCLC, with mutant EGFR L858R), A549 (NSCLC, wild-type EGFR), and MCF-7 (breast cancer, serving as an indicator control). These cell lines were used to screen for CTC markers and the mutated NSCLC marker, mEGFR L858R. In addition, white blood cells from healthy donors with lysed red blood cells (RBCs) were used as healthy controls (CTC marker negative controls) and subjected to phenotypic screening by flow cytometry.

[0079] CTCs are typically identified using biomarkers that are specifically expressed in cancer cells but not in normal blood cells. Many cancers, particularly those with an epithelial cell phenotype, express high levels of epithelial cell surface antigens and cytokeratins, which are common markers of epithelial differentiation. Therefore, epithelial cell adhesion molecule (EpCAM) and cytokeratins are frequently used to identify cancer cells in a mixed population and distinguish them from other cells such as white blood cells (WBCs).

[0080] In Example 1, three cell surface markers, EpCAM, CD45, and HLA-A.B.C, were used together with two intracellular markers, CK-7 / 8 and CK-14 / 15 / 16 / 19, to distinguish cancer cells from WBCs and other blood cells. In addition, vimentin (VIM) was used to distinguish NSCLC from other cancers (in this example, breast cancer), while a mutant EGFR L858R marker was used to distinguish between wild-type and L858R EGFR in NSCLC.

[0081] To verify the effectiveness of these markers, human tumor cell lines (H1975, A549 for NSCLC, and MCF-7 for breast cancer) were stained with various antibodies: ECD-anti-human CD45, BV510-anti-human EpCAM, FITC-anti-CK-7 / 8, Alexa Fluor 647-anti-CK-14 / 15 / 16 / 19, Alexa Fluor 700-anti-HLA-A.B.C, Alexa Fluor 405-anti-vimentin, and PE-anti-mEGFR antibody (EGF receptor (L858R variant-specific) (43B2) rabbit mAb (PE conjugate), Cell Signaling Technology, catalog number 64716) together with leukocytes from healthy donors.

[0082] As shown in Figure 1, using this approach, H1975, A549, and MCF-7 cells were successfully identified as CTCs. These three cell lines exclusively expressed EpCAM, CK-7 / 8, and HLA-A.B.C, while most cells were negative for CD45 and CK-14 / 15 / 16 / 19. In contrast, no CTCs were detected in healthy donor samples serving as negative controls. This demonstrates that the method can effectively discriminate CTCs from blood cells using flow cytometry. Furthermore, NSCLC cells can be distinguished from breast cancer cells based on vimentin expression, and the presence of the L858R mutation in EGFR could be specifically identified using the L858R antibody.

[0083] In summary, the phenotype of mEGFR L858R NSCLC (H1975 cells) was characterized based on the expression of circulating tumor cell (CTC)-related markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , EpCAM + , HLA-A.B.C + , and VIM + . Similarly, the targeting marker was the mutant EGFR (L858R) +It included. The A549 cell line is also a certain type of NSCLC, but has wild-type EGFR and was used as a negative control for mutant EGFR (L858R specific); similarly, the MCF-7 cell line originating from breast cancer was also utilized as a negative control for mutant EGFR (L858R specific). The A549 and MCF-7 cell lines are cancer-like and show CTC markers, but they do not express mEGFR L858R, providing a clear contrast for evaluating L858R positivity in H1975 cells. This comparison further emphasizes the potential of the flow cytometry-based method for identifying and characterizing L858R-positive CTCs in NSCLC.

[0084] The flow cytometry screening results of each group are presented in row units consisting of a set of biaxial panels. The dashed square indicates the gating region of interest for a specific cell population, and the dashed arrow represents the gating strategy and logic. The solid arrow indicates the intensity or expression level of the specific marker.

[0085] (Example 2) Spike experiment using H1975 cells To evaluate the ability of flow cytometry to detect rare mEGFR L858R NSCLC cells at low concentrations, a spike assay of H1975 cells was performed. A small number of H1975 cells were added to healthy whole blood to stimulate low concentrations of NSCLC CTCs. The spike samples were analyzed by applying the gating strategy from Example 1 for detecting mEGFR L858R NSCLC, and the sensitivity and recovery rate of the method in identifying rare CTCs in normal blood cells were evaluated.

[0086] 2.1. Sample processing As shown in Figure 2A, whole blood samples alone and mixtures of whole blood and H1975 aliquots were first prepared.

[0087] H1975 cells were harvested by trypsinization at 90% confluence. After counting the cells, aliquots of cell suspensions containing 2, 5, 10, or 25 cells (spike-in groups) were prepared and added to 1 mL of peripheral blood from healthy donors. Samples of peripheral blood only were used as negative controls (sham-treated groups), and H1975 cells (cells alone group) were used as positive controls (index positive controls) for CTC gate setting analysis.

[0088] The sham-treated groups and spike-in groups were processed for red blood cell (RBC) lysis. Cells from all groups were stained with ECD-anti-human CD45, BV510-anti-human EpCAM, FITC-anti-CK-7 / 8, Alexa Fluor 647-anti-CK-14 / 15 / 16 / 19, Alexa Fluor 700-anti-HLA-A.B.C, Alexa Fluor 405-anti-vimentin, and PE-anti-mEGFR antibody (EGF receptor (L858R variant-specific) (43B2) rabbit mAb (PE conjugate), Cell Signaling Technology, catalog number 64716), or isotype controls, and analyzed using FACS / flow cytometry to identify spiked H1975 cells.

[0089] 2.2. Identification of spiked H1975 cells by FACS The gating strategy was the same as that used for H1975 phenotype screening established in Example 1.

[0090] First, cell debris, clumps, and white blood cells were excluded using forward scatter light (FSC) and CD45 - (left panel of Figure 2B). Next, consensus CTC-related markers were identified: CK-7 / 8 in the middle panel of Figure 2B + and CK-14 / 15 / 16 / 19 - . Next, additional consensus CTC-related markers were identified: EpCAM in the right panel of Figure 2B + and HLA-A.B.C + . Finally, VIM +was used as an additional NSCLC CTC marker, and the target marker mEGFR (L858R specific) that is specifically expressed on H1975 + was identified as shown in Figure 2C. H1975 cells were successfully detected in spiked samples with 2 - 25 H1975 cells added to 1 mL of whole blood. The mEGFR (L858R) mutation was observed in 100% of the identified CTCs.

[0091] 2.3. Linear analysis and recovery rate evaluation of spiked H1975 cells The number of detected H1975 cells was compared with the expected number of cells to evaluate the recovery of the method. Identification of the recovered H1975 cells was performed by screening for specific targeting markers: CD45 - , CK - 7 / 8 + , CK - 14 / 15 / 16 / 19 - , EpCAM + , HLA - A.B.C + , VIM and mEGFR (L858R) + . The number of spiked cells and the number of detected cells were plotted on the x - axis and y - axis respectively (Figure 2D). A strong linear relationship was observed between the expected tumor cells and the detected tumor cells, and the R 2 value was 0.86 (Figure 2E). These results demonstrate that the disclosed method can detect one cell in a mixed population of one million cells, which is essential for identifying rare cell types such as circulating tumor cells (CTCs) in blood.

[0092] The average recovery rate was calculated to be 51.3% (Figure 2E), which demonstrates that most of the spiked cells were recovered through the concentration process and that it is possible to detect as few as 2 tumor cells in 1 mL of blood. Overall, the method of the present disclosure is an effective method for increasing the ability to detect CTCs by flow cytometry.

[0093] CTCs are present at extremely low concentrations within large cell populations, making their detection difficult. However, by utilizing a detection platform based on flow cytometry, the efficiency of identifying target cells is significantly enhanced compared to polymerase chain reaction (PCR), which is the current clinical practice. The method not only reduces the total number of cells required for analysis but also minimizes the sample volume needed to detect the cells of interest.

[0094] (Example 3) Clinical validation of a CTC prediction biomarker detection platform for NSCLC using actual samples The above results suggest that the CTC detection platform for NSCLC can identify a small number of tumor cell lines spiked in whole blood with a high recovery rate. To further validate the clinical effectiveness of the same flow cytometry protocol used in the H1975 spike assay, whole blood samples were collected from 26 cancer patients and 10 healthy subjects for CTC detection (Table 1). For cancer patients, all blood samples were collected before the start of any anti-cancer treatment. The performance of the CTC detection platform in discriminating between L858R NSCLC patients and healthy donors was evaluated. In addition, the feasibility of detecting the EGFR L858R mutation in CTCs using a flow cytometry-based method compared to wild type or other mutations was also investigated. Next, the results were compared with standard diagnostic methods to evaluate the accuracy and reliability of the flow cytometry-based method for detecting this specific EGFR mutation in CTCs.

[0095] Peripheral blood samples from NSCLC patients and healthy donors were analyzed using a method consistent with that used in the H1975 spike assay. Circulating tumor cells (CTCs) in the blood were identified using the following markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , HLA-A.B.C, EpCAM + , VIM + , and mEGFR (L858R) +Characterized using and applied to clinical samples as shown in Figure 5A. L858R + Summarize the CTC detection results as the percentage of CTCs within the population and the absolute CTC count (CTC#), and as shown in Figure 5B for CD45 - The data is from L858R + CTCs can be detected in samples from NSCLC patients, indicating that the same markers were not detected in samples from healthy donors. The results are statistically significant and highlight the potential of this method for identifying L858R CTCs in NSCLC patients + Highlight the potential of this method for identifying CTCs

[0096] Summarize the clinical validation results in Table 2, which compares the standard diagnostic procedure (PCR on solid tissue biopsy) with the CTC detection platform (flow cytometry for liquid biopsy, the method introduced in the present invention). As shown in Table 2, the consistency between the PCR and flow cytometry-based methods is high, with an 87.5% (7 / 8) agreement in the identification of positive cases and an 82.4% (14 / 17) agreement in the identification of negative cases. These findings demonstrate that the reliability of the flow cytometry-based liquid biopsy method closely matches that of the established PCR solid tissue biopsy approach, while offering potential advantages in terms of sensitivity and non-invasiveness

[0097]

Table 1

[0098]

Table 2

[0099] (Example 4) Phenotypic screening of healthy donor leukocytes along with cell lines N87 (gastric cancer, Her2 + ) and H520 (NSCLC, Her2 - control) Her2 +To further establish a flow cytometry-based CTC detection platform for gastric cancer (GC), a series of gating strategies were first developed and verified. N87 (GC, Her2 + ) and H520 (NSCLC, Her2 - serving as controls) were utilized from various cancer cell lines. These cell lines were used to screen for CTC markers and Her2 markers. In addition, white blood cells from healthy donors with lysed red blood cells (RBC) were used as healthy controls (CTC marker negative controls) and subjected to phenotypic screening by flow cytometry.

[0100] CTCs are typically identified using biomarkers that are specifically expressed on cancer cells but not on normal blood cells. Many cancers, especially those with an epithelial cell phenotype, express high levels of epithelial cell surface antigens and cytokeratins, which are common markers of epithelial differentiation. As a result, epithelial cell adhesion molecule (EpCAM) and cytokeratin are often used to identify cancer cells in a mixed population and distinguish them from other cells such as white blood cells (WBC).

[0101] In Example 4, three cell surface markers, EpCAM, CD45, and HLA-A.B.C, were used together with two intracellular markers, CK-7 / 8 and CK-14 / 15 / 16 / 19, to distinguish cancer cells from WBC and other blood cells. In addition, the Her2 marker was used to detect Her2-positive gastric cancer.

[0102] To verify the effectiveness of these markers, human tumor cell lines (Her2 + N87 for GC and Her2 - H520 for NSCLC) were stained with various antibodies: ECD-anti-human CD45, BV510-anti-human EpCAM, FITC-anti-CK-7 / 8, Alexa Fluor 647-anti-CK-14 / 15 / 16 / 19, Alexa Fluor 700-anti-HLA-A.B.C, and PE / Cy7-anti-Her2 antibody together with white blood cells from healthy donors.

[0103] As shown in Figure 3, this approach successfully identified N87 and H520 cells as CTCs. Both cell lines exclusively expressed EpCAM, CK-7 / 8, CK-14 / 15 / 16 / 19, and HLA-A.B.C, and most of the cells were negative for CD45. In contrast, CTCs were not detected in healthy donor samples that served as negative controls. This finding demonstrates that the method can effectively distinguish CTCs from other blood cells using flow cytometry. Furthermore, N87 and H520 cells can be distinguished from each other based on the expression of Her2, highlighting that the method can discriminate between CTC populations.

[0104] In summary, the phenotype of Her2-positive GC cells (N87 cells) was characterized based on the expression of blood circulating tumor cell (CTC)-related markers including CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , and HLA-A.B.C + , as well as the targeting marker Her2 + . H520 cells are cancer-like and show CTC markers, but they do not express Her2, providing a clear contrast for evaluating Her2 positivity in N87 cells. This comparison further emphasizes the potential of the flow cytometry-based method for identifying and characterizing Her2-positive CTCs in GC.

[0105] The flow cytometry screening results for each group are presented row by row in a set of biaxial panels. The dashed rectangles indicate the target gate setting regions for specific cell populations, and the dashed arrows represent the gating strategies and logic. The solid arrows indicate the intensity or expression level of specific markers.

[0106] (Example 5) Spike experiment using N87 cells An N87 cell spike assay was performed to evaluate the ability of flow cytometry to detect low concentrations of Her2-positive gastric cancer cells. A small number of N87 cells were added to healthy whole blood to stimulate low concentrations of GC CTCs. The spike samples were analyzed by applying the gating strategy from Example 4 for detecting Her2-positive GCs, and the sensitivity and recovery rate of the method in the identification of rare CTCs in normal blood cells were evaluated.

[0107] 5.1. Sample processing As shown in Figure 4A, whole blood samples alone and mixtures of whole blood and N87 aliquots were first prepared.

[0108] N87 cells were harvested by trypsinization at 90% confluence. After counting the cells, aliquots of cell suspensions containing 2, 5, 10, or 25 cells (spike-in groups) were prepared and added to 1 mL of peripheral blood from healthy donors. Samples of peripheral blood only were used as negative controls (mock-treatment groups), and samples containing N87 cells only (cell-alone groups) were used as positive controls (index positive controls) for CTC gating analysis.

[0109] The mock-treatment groups and spike-in groups were processed for red blood cell (RBC) lysis. Cells from all groups were stained with ECD-anti-human CD45, BV510-anti-human EpCAM, FITC-anti-CK-7 / 8, Alexa Fluor 647-anti-CK-14 / 15 / 16 / 19, Alexa Fluor 700-anti-HLA-A.B.C, and PE / Cy7-anti-Her2 antibodies, analyzed using FACS / flow cytometry, and spike-in N87 cells were identified.

[0110] 5.2. Isolation and analysis of spike-in N87 cells by FACS The gating strategy was the same as that used for N87 phenotype screening established in Example 4.

[0111] First, cell debris, clumps, and white blood cells were removed by forward scatter light (FSC) and CD45 -Excluded using (left panel of Figure 4B). Next, consensus CTC-related markers were identified: CK-7 / 8 in the middle panel of Figure 4B + and CK-14 / 15 / 16 / 19 + . Next, additional consensus CTC-related markers were identified: EpCAM in the right panel of Figure 4B + and HLA-A.B.C + . Finally, as shown in Figure 4C, the target marker Her2 + specifically expressed on N87 was identified. N87 cells were successfully detected in spiked samples with 2 - 25 N87 cells added to 1 mL of whole blood. Her2 + cells were observed in 100% of the identified CTCs.

[0112] 5.3. Linear analysis and recovery rate evaluation of spiked N87 cells The number of detected N87 cells was compared with the expected number of cells to evaluate the recovery of the method. Identification of the recovered N87 cells was performed by screening for specific targeting markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , HLA-A.B.C + , and Her2 + . The number of spiked cells and the number of detected cells were plotted on the x-axis and y-axis respectively (Figure 4D). A strong linear relationship was observed between the expected tumor cells and the detected tumor cells, and the R 2 value was 0.6103 (Figure 4E). These results demonstrate that the disclosed method can detect one cell in a mixed population of one million cells, which is essential for identifying rare cell types such as circulating tumor cells (CTCs) in blood.

[0113] When the average recovery rate was calculated, it was 61% (Figure 4E), demonstrating that most of the spiked cells were recovered through the enrichment process and that as few as two tumor cells per 1 mL of whole blood could be detected. Overall, the method of the present disclosure is an effective way to increase the ability to detect CTCs by flow cytometry.

[0114] CTCs are present at extremely low concentrations within a large cell population, making their detection difficult. However, by utilizing a detection platform based on flow cytometry, the efficiency of identifying Her2-positive GCs is significantly enhanced compared to immunohistochemistry (IHC), which is the current clinical practice. This approach not only reduces the total number of cells required for analysis but also minimizes the sample volume needed to detect the cells of interest.

[0115] (Example 6) Verification of a CTC prediction biomarker detection platform for GC using actual samples The above results suggest that the CTC detection platform for GC is capable of identifying a small number of tumor cell lines spiked in whole blood with a high recovery rate. To further verify the clinical effectiveness of the same flow cytometry protocol used in the N87 spike assay, whole blood samples were collected from 14 cancer patients and 10 healthy subjects for CTC detection (Table 3). All blood samples from cancer patients were collected before the start of anti-cancer treatment. Her2 + The performance of the CTC detection platform in discriminating between GC patients and healthy donors was evaluated. In addition, the correlation between standard diagnostic methods and the CTC detection method was also evaluated.

[0116] Peripheral blood samples from gastric cancer (GC) patients and healthy donors were analyzed using a method consistent with the method used in the GC recovery assay. Circulating tumor cells (CTCs) in the blood were identified using the following markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM +, HLA-A.B.C + , and Her2 + were used for characterization. These markers were applied to clinical samples as shown in Figure 6A. Her2 + CTC detection results were summarized as the percentage of CTCs and the absolute CTC count (CTC#) within the CD45 - population as shown in Figure 6B. The data indicate that CTCs can be detected in clinical samples from GC patients, but the same markers were not detected in samples from healthy donors. The results are statistically significant, highlighting the potential of this method for the identification of Her2 + CTCs in GC patients.

[0117] Clinical validation results are summarized in Table 4, which compares the standard diagnostic procedure (IHC of solid tissue biopsy) with the CTC detection platform (flow cytometry of liquid biopsy, the method introduced in the present invention). As shown in Table 4, the agreement between the IHC-based and flow cytometry-based methods is complete in the identification of positive cases, with all 7 positive cases detected by both methods (100% agreement). Furthermore, both methods also agreed in all 7 negative cases, and no false positives or false negatives were observed.

[0118] These results demonstrate a perfect correlation between the IHC-based solid tissue biopsy and the flow cytometry-based liquid biopsy method, indicating the high accuracy and reliability of the flow cytometry method. The flow cytometry-based detection method not only agrees with IHC in the identification of both positive and negative cases but also offers the advantage of being non-invasive, making it a promising alternative for the clinical diagnosis of Her2 + CTCs in GC patients.

[0119]

Table 3

[0120]

Table 4

[0121] Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision many other means and / or structures for performing the functions described herein and / or for obtaining one or more of the results and / or advantages thereof, and such variations and / or modifications are considered to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will recognize that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend upon the particular application or applications for which the teachings of the invention are used. Those skilled in the art will be able to recognize or confirm, using nothing more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it is understood that within the scope of the appended claims and their equivalents, embodiments of the invention may be practiced otherwise than as specifically described and claimed. Embodiments of the invention of the present disclosure are directed to each and every distinct feature, system, product, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, products, materials, kits, and / or methods is included within the scope of the invention of the present disclosure if such features, systems, products, materials, kits, and / or methods are not mutually inconsistent.

[0122] Similarly, it should be understood that in any method of the claims that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited, unless expressly indicated to the contrary.

Claims

1. A method for analyzing circulating tumor cells (CTCs) in a biological sample of an individual having cancer, comprising: a) isolating white blood cells (WBCs) and CTCs from the biological sample; b) setting a gate for CTCs based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and vimentin (VIM); and c) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2. The method according to claim 1, wherein steps b) and c) are performed by flow cytometry.

2. The method according to claim 1, wherein the biological sample is peripheral blood.

3. The method according to claim 2, wherein the biological sample is about 1 mL.

4. The method according to claim 1, wherein the biological sample contains more than 5 CTCs per mL of the biological sample.

5. The method according to claim 1, further comprising lysing red blood cells in the biological sample prior to step a).

6. The method according to claim 1, wherein steps b) and c) are performed continuously.

7.

8. If CTC expresses markers including CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , EpCAM + , HLA-A.B.C + , and VIM + , the method according to claim 1, further comprising the step of identifying that the cancer is non-small cell lung cancer (NSCLC).

9. If CTC is mEGFR L858R + The method according to claim 7, further comprising the step of administering an EGFR tyrosine kinase inhibitor to the individual if so. The method according to claim 8, wherein the EGFR tyrosine kinase inhibitor comprises erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, olmutinib, lazertinib, and zolifertinib.

10.

11. If CTC expresses markers including CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , and HLA-A.B.C + , the method according to claim 1, further comprising the step of identifying CTCs derived from gastric cancer (GC).

12. When CTC is Her2 + The method according to claim 10, further comprising the step of administering to the individual at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells. The method according to claim 11, wherein the Her2 tyrosine kinase inhibitor comprises lapatinib; the anti-Her2 antibody comprises trastuzumab, pertuzumab, tucatinib, and margetuximab; the bispecific antibody comprises zanidatamab; or the Her2-targeted antibody-drug conjugate comprises trastuzumab emtansine and trastuzumab deruxtecan.

13. A method for treating cancer in an individual in need thereof, comprising: a) setting a gate for circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM; b) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2; and c) administering cancer treatment to the individual.

14. i) If the cancer is identified as non-small cell lung cancer (NSCLC) with CTC being mEGFR L858R + then the cancer treatment is an EGFR tyrosine kinase inhibitor, or ii) If the cancer is identified as gastric cancer (GC) where the CTC is Her2 + then the cancer treatment comprises the step of including at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells

15.

16. The method according to claim 13, wherein the EGFR tyrosine kinase inhibitor comprises erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, olmutinib, lazertinib and zolifertinib.

15. The method according to claim 13, wherein the Her2 tyrosine kinase inhibitor comprises lapatinib; the anti-Her2 antibody comprises trastuzumab, pertuzumab, tucatinib and margetuximab; the bispecific antibody comprises zanidatamab; or the Her2-targeted antibody-drug conjugate comprises trastuzumab emtansine and trastuzumab deruxtecan.

16. A method for identifying an individual having cancer that may respond to treatment by at least one treatment selected from the group consisting of an EGFR tyrosine kinase inhibitor, a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a Her2-targeted antibody-drug conjugate, and a Her2-targeted CAR-T cell, comprising: a) setting a gate for circulating tumor cells (CTCs) in the blood based on forward scatter light (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM in a biological sample derived from the individual; b) testing the CTCs for mutated epidermal growth factor receptor (mEGFR) L858R or Her2, and c) i) If CTC is derived from NSCLC and CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-A.B.C + VIM + mEGFR L858R + is present, an EGFR tyrosine kinase inhibitor; or ii) The CTC is derived from GC and CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 + EpCAM + HLA-A.B.C + Her2 + If it has, at least one treatment selected from the group consisting of a Her2 tyrosine kinase inhibitor, an anti-Her2 antibody, a bispecific antibody, a Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells identifying an individual having cancer that may respond to treatment by A method comprising the steps of.

17. A kit for use in the method according to claim 1, comprising one or more reagents for labeling CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, VIM, mEGFR L858R, and / or Her2.

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