Method for separating and analyzing circulating tumor cells

Abstract

The present disclosure provides a method of isolating and analyzing circulating tumor cells (CTCs) in a biological sample by using flow cytometry. By using this method, the responsiveness of a subject suffering from lung cancer or gastric cancer to a treatment process can be predicted in a rapid and simple manner.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 614,804, filed on December 26, 2023, which is incorporated herein by reference for all purposes. Technical Field

[0003] The present disclosure relates to the field of personalized medicine. More specifically, liquid biopsies are applied to the personalization and monitoring of treatment of cancer patients and for the diagnosis of cancer. The present disclosure relates to a method for identifying and characterizing CTC subpopulations (subgroups) within a population of circulating tumor cells (CTCs) in a biological sample, preferably in a blood sample of a patient. Background Art

[0004] 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), the major histological type of lung cancer, accounts for nearly 85% of lung cancer cases. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have shown significant efficacy in NSCLC patients with EGFR mutations. These inhibitors are associated with minimal side effects and have proven to improve quality of life, especially in patients carrying exon 19 deletions (E19del) or exon 21 point mutations (L858R).

[0005] Gastric cancer (GC) is one of the most common malignancies of the digestive tract. In 2022, global statistics reported approximately 968,784 new cases and approximately 660,175 deaths caused by GC. Although both the incidence and mortality have decreased recently, the detection rate and overall prognosis remain unsatisfactory. With the development of precision oncology in clinical practice, molecularly targeted therapy for GC has received a great deal of attention. Among them, therapies targeting human epidermal growth factor receptor 2 (Her2) have shown significant clinical benefits and are currently widely used in treatment regimens.

[0006] Obtaining cancer type-specific molecular information is crucial for guiding clinical practice of targeted therapy. Typically, such information is derived from solid tissue biopsies, which require invasive procedures to obtain. However, due to advanced stages of the disease or poor overall health, some patients may have contraindications or be intolerant to invasive biopsies, thus highlighting the need for safer and more convenient alternatives. In addition, for certain cancers, such as GC, biopsy results may lack sufficient accuracy.

[0007] Due to the extreme rarity of CTCs, detecting CTCs against the background of abundant white blood cells requires highly sophisticated methods. The challenges of CTC detection are further complicated by the cellular heterogeneity and plasticity, thus making the stable enrichment from biological samples particularly complex. Developing effective methods for analyzing these cells from minimal sample volumes is crucial for improving detection accuracy and enhancing its clinical applicability. Summary of the Invention

[0008] In response to the urgent needs in the field, the present disclosure provides a rapid and direct method for isolating and analyzing CTCs to facilitate the verification of EGFR mutations or Her2 status in these cells. Before initiating treatments such as EGFR TKIs, EGFR-targeted agents, or Her2-targeted therapies, the EGFR mutation or Her2 status is evaluated by separately isolating blood-derived epithelial cells from patients with lung cancer (such as NSCLC) or GC. Analyzing a panel of mixed CTCs significantly improves the detection rates of EGFR mutations and Her2 status compared to detection methods that rely on single CTC analysis or traditional immunohistochemistry (IHC).

[0009] In one embodiment, the present disclosure provides a method for analyzing circulating tumor cells (CTCs) in a biological sample of a cancer individual, comprising:

[0010] a) isolating white blood cells (WBCs) and CTCs from the biological sample;

[0011] b) gating the CTCs based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and vimentin (VIM); and

[0012] c) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2;

[0013] wherein steps b) and c) are performed by flow cytometry.

[0014] In a preferred embodiment, the biological sample is peripheral blood.

[0015] In a preferred embodiment, the biological sample is about 1 mL.

[0016] The method described herein enables the isolation and analysis of CTCs from the smallest possible amount of biological sample (such as 1 mL of peripheral blood).

[0017] In a preferred embodiment, the method may further comprise lysing red blood cells in the biological sample before step a).

[0018] In a preferred embodiment, steps b) and c) can be performed sequentially.

[0019] In a preferred embodiment, the cancer can be non-small cell lung cancer (NSCLC) or gastric cancer (GC).

[0020] In a preferred embodiment, the method may further comprise, if the CTC expression includes CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , EpCAM + , HLA-A.B.C + and VIM + markers, then identifying the cancer as non-small cell lung cancer (NSCLC).

[0021] In a preferred embodiment, the method may further comprise, if the CTC is mEGFR L858R + , then administering an EGFR tyrosine kinase inhibitor to the individual.

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

[0023] In a preferred embodiment, the method may further comprise, if the CTC expression includes CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + and HLA-A.B.C + markers, then identifying the cancer as gastric cancer (GC).

[0024] In a preferred embodiment, the method may further comprise administering to the individual at least one treatment selected from the following: Her2 tyrosine kinase inhibitor, anti-Her2 antibody, bispecific antibody, Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cells (if the CTC is Her2 + ).

[0025] In preferred embodiments, 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.

[0026] In another embodiment, the present disclosure provides a method of treating cancer in an individual in need thereof, comprising:

[0027] a) gating circulating tumor cells (CTCs) in biological samples derived from individuals based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-ABC and VIM;

[0028] b) testing CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; and

[0029] c) administering a cancer therapy to a subject, wherein

[0030] i) If the cancer is identified as non-small cell lung cancer (NSCLC) and the CTC is mEGFR L858R + , then the cancer therapy is an EGFR tyrosine kinase inhibitor, or

[0031] ii) If the cancer is identified as gastric cancer (GC), and the CTC is Her2 + , the cancer therapy includes at least one treatment selected from the following: Her2 tyrosine kinase inhibitors, anti-Her2 antibodies, bispecific antibodies, Her2-targeted antibody-drug conjugates and Her2-targeted CAR-T cells.

[0032] In preferred embodiments, the EGFR tyrosine kinase inhibitor may include erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, omotinib, lazertinib, and zolitinib.

[0033] In preferred embodiments, the Her2 tyrosine kinase inhibitor may include lapatinib; the anti-Her2 antibody may include trastuzumab, pertuzumab, tucatinib, and magituximab; the bispecific antibody may include zenidatuzumab; or the Her2-targeting antibody drug conjugate may include emtansine trastuzumab and destansine trastuzumab.

[0034] In another embodiment, the present disclosure provides a method for identifying an individual having cancer that is likely to respond to treatment with at least one 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 Her2-targeted CAR-T cells, comprising:

[0035] a) gating circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM;

[0036] b) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; and

[0037] c) identifying an individual having cancer that is likely to respond to treatment with:

[0038] i) an EGFR tyrosine kinase inhibitor, if the CTCs are from NSCLC and have CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-A.B.C + VIM + mEGFR L858R + ; or

[0039] ii) 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, if the CTCs are from GC and have CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 + EpCAM + HLA-A.B.C + Her2 + 。

[0040] 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.

[0041] By examining a mixed CTC population, flow-based methods enhance the sensitivity and efficiency of detecting these biomarkers, providing a more robust approach for identifying cancer-related mutations and markers in circulating tumor cells. This improvement highlights the potential of mixed CTC analysis as an alternative for cancer diagnosis and monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The patent application document contains at least one color-drawn figure. Upon request and payment of the necessary fees, the USPTO will provide a copy of the present patent application publication with color drawings.

[0043] Exemplary embodiments of the present application are described in detail below with reference to the following drawings:

[0044] Figure 1 Illustrates the phenotypic screening of H1975 (NSCLC, mutant EGFR L858R), A549 (NSCLC, wild-type EGFR), and MCF-7 (breast cancer, control) cancer cell lines, as well as healthy donor white blood cells.

[0045] Figure 2A -D illustrates the recovery validation test of H1975 cells in the form of a spike assay. Figure 2A Outlines the step-by-step process of sample preparation prior to validation. Figure 2B shows the selection process of CTC-related markers applied to each spiked sample. Figure 2C presents a dot plot for identifying recovered H1975 cells in the spiked group using specific target markers. Each panel shows representative data from six independent experiments. Figure 2D shows the recovered cell counts of spiked H1975 cells, which are summarized in a staggered bar graph and represented as mean ± SEM. Figure 2E illustrates the systematic recovery rate of all experiments calculated by linear regression analysis. Each data point represents an independent experiment.

[0046] Figure 3 Illustrates N87 (gastric cancer, Her2 + )、H520 (NSCLC, Her2 - control), and healthy donor white blood cells.

[0047] Figure 4A -D shows the recovery validation test of N87 cells in the form of a spike assay. Figure 4AThe step - by - step process of sample preparation before verification is outlined. Figure 4B shows the selection process of CTC - related markers applied to each spiked sample. Figure 4C presents a dot plot of using specific target markers to identify the recovered N87 cells in the spiked group. Each panel shows representative data from at least eight independent experiments. Figure 4D shows the recovered cell counts of spiked N87 cells, which are summarized in a staggered bar graph and represented as mean ± SEM. Figure 4E illustrates the systematic recovery rate of all experiments calculated by linear regression analysis. Each data point represents an independent experiment.

[0048] Figure 5 A - B illustrate the validation of a CTC - predictive biomarker detection platform for non - small cell lung cancer (NSCLC) using real samples. Figure 5 A shows that the same protocol (pedigree) applied to the H1975 recovery analysis is used to analyze peripheral blood samples from NSCLC and lung adenocarcinoma patients and healthy donors. Figure 5 B presents the validation results, which summarize (1) the percentage of CTCs in the CD45 - population and (2) the absolute CTC count (CTC#). The data are represented as mean ± SEM, where each data point represents an individual participant recruited in an IRB - approved study.

[0049] Figure 6 A - B illustrate the validation of a CTC - predictive biomarker detection platform for gastric cancer (GC) using real samples. Figure 6 A shows that the same protocol used in the GC recovery analysis is applied to analyze peripheral blood samples from GC patients and healthy donors. Figure 6 B presents the validation results, which summarize (1) the percentage of CTCs in the CD45 - population and (2) the absolute CTC count (CTC#). The data are represented as mean ± SEM, where each data point represents an individual participant recruited in an IRB - approved study. Detailed implementation

[0050] The foregoing and other aspects of the present disclosure will now be described in more detail with reference to other embodiments described herein. It should be understood that the present invention may be implemented in different forms and should not be construed as limited to the embodiments set forth herein. Instead, 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.

[0051] The terms used in the description of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention. As used in the description of the present invention and the appended claims, the singular forms "a", "an" and "the" are also intended to include the plural forms unless the context clearly indicates otherwise.

[0052] As used herein, the terms "comprising", "comprises", "including", "includes", "having", "has", "containing", "contains", "characterized by", or any other variation thereof are intended to cover non-exclusive inclusion, subject to any explicit limitations. For example, a composition, mixture, process, or method that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, or method.

[0053] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified. If in a claim, this would render the claim to exclude materials other than those recited, except for impurities ordinarily associated therewith. When the phrase "consisting of" appears in a clause of the claim body rather than immediately following the preamble, it only limits the elements recited in that clause; other elements are not excluded from the entire claim.

[0054] In cases where the applicant defines the invention or a part thereof using an open-ended term such as "comprising", it should be readily understood (unless otherwise stated) that the description should be interpreted as also using the term "consisting of" to describe the invention.

[0055] All numbers herein are understood to be modified by the term "about". As used herein, the term "about" is used to indicate that a value includes, for example, variations inherent in a measuring device, the method used to determine the value, or variations that exist among subjects being studied. Generally, the term is meant to encompass a variability of about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the circumstances.

[0056] The term "or" as used in a claim is intended to mean "and / or" unless explicitly indicated to refer to only alternative or alternative are mutually exclusive, although the present disclosure supports definitions that refer to only alternatives and "and / or".

[0057] As used herein, "subject" refers to an animal, including, for example, mammalian subjects diagnosed or suspected of having or developing cancer. In one embodiment, the term "subject" may refer to a vertebrate having cancer or a vertebrate believed to be in need of cancer treatment. Subjects include warm-blooded animals such as mammals, such as primates, more preferably humans. Non-human primates are also subjects. The term "subject" includes domestic 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.). Accordingly, veterinary uses and pharmaceutical formulations are contemplated herein.

[0058] "Administration" or "administering" is referred to herein as providing a modified T cell or pharmaceutical composition of the present application to a subject. By way of example and not limitation, administration can be by parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intra-abdominal, intra-capsular, intra-chondral, intracavitary, intra-coelomic, intracerebral, intraventricular, intra-colonic, intra-cervical, intra-gastric, intra-hepatic, intra-myocardial, intra-osseous, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal, intra-synovial, intra-thoracic, intra-uterine, intra-vesical, instillation, vaginal, rectal, oral, sublingual, intranasal, and transdermal. For example, injection can be 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 can be employed. Parenteral administration can be by, for example, instillation or by stepwise perfusion over time. Optionally or concurrently, administration can be by the oral route.

[0059] The use of the terms "treat" or "treatment" herein refers to the administration of a treatment or therapy to a subject for the purpose of curing, alleviating, mitigating, remedying, preventing, or improving a disorder, the symptoms of a disorder, a disease state secondary to a disorder, or a predisposition to a disorder. When used in the claims and / or the specification, the terms "inhibit", "reduce", or "prevent" or any variation of these terms includes any measurable decrease or complete inhibition to achieve the desired result.

[0060] 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 with the location and nature of the lesion or site to be targeted and includes, for example, regional, parenteral, intravenous, intramuscular, and / or systemic administration and formulations. For the target area, direct injection or injection into the vasculature or into and out of organs or tissues and into the blood vessels within organs or tissues is particularly contemplated. Local, regional, or systemic administration may also be appropriate.

[0061] "Circulating tumor cell", "CTC", and "CTCs", which are used interchangeably herein, refer to cells that have shed from a primary tumor into the vasculature and are circulating in the bloodstream. CTCs are considered to be the seeds for subsequent growth of additional tumors (metastases) in important distal organs, thereby triggering the mechanisms that cause the vast majority of cancer-related deaths.

[0062] As used herein, "CD45" refers to cluster of differentiation 45 (CD45), encoded by the PTPRC gene, also known as protein tyrosine phosphatase, receptor type C, and leukocyte common antigen. CD45 is used to identify white blood cells. Antibodies that bind CD45 can be used to detect CD45.

[0063] As used herein, "cytokeratin" refers to keratin-containing intermediate filaments found in the cytoskeleton of the cytoplasm of epithelial tissues. Cancer cells expressing cytokeratin lose their cytokeratin expression after undergoing epithelial-mesenchymal transition (EMT), where up to 20% of the cells have no detectable cytokeratin. "Cytokeratin-7", "keratin-7", "CK-7", and "sarcolectin", which are used interchangeably herein, refer to the protein encoded by the KRT7 gene in humans. "Cytokeratin-8", "keratin-8", and "CK-8", which are used interchangeably herein, refer to the protein encoded by the KRT8 gene in humans. Cytokeratins 7 and 8 are two closely related type II cytokeratins characteristic of simple epithelia. Cytokeratin 7 is less widespread than cytokeratin 8 and is expressed in sebaceous and sweat glands and some cells of the inner root sheath. Cytokeratin 8 is mainly found in non-squamous epithelial cells. Cytokeratin 7 is commonly present in lung adenocarcinoma, breast cancer, endometrioid tumors, and urothelial cell carcinoma of the bladder. The combination of cytokeratins 7 and 8 is a useful marker for differentiating adenocarcinoma and ductal carcinoma from squamous cell carcinoma. Antibodies that bind to cytokeratin 7 / 8 can be used to detect cytokeratin 7 / 8.

[0064] As 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 protein encoded by the KRT16 gene, and "cytokeratin-19", "keratin-19", and "CK-19" refer to the protein encoded by the KRT19 gene. Cytokeratins -14, 15, 16, and 19 are all type I cytokeratins and are collectively referred to as pancytokeratin (pan-CK). In cancer research, especially in the study of CTCs, pancytokeratin is commonly used as a biomarker for identifying and isolating tumor cells of epithelial origin, which typically overexpress cytokeratin relative to non-epithelial cells.

[0065] As used herein, "EpCAM" refers to epithelial cell adhesion molecule, a type I glycosylated membrane protein that is expressed at low levels in various human epithelial tissues but overexpressed in most solid cancers. To date, most studies have used EpCAM as a target marker for identifying potential CTCs. Its expression has been shown to be negatively correlated with cancer prognosis in patients.

[0066] As used interchangeably herein, "HLA-A.B.C", "major histocompatibility complex, class I, A, B, C", and "HLA class I A, B, and C" refer to human leukocyte antigens A, B, and C encoded by genes located on the short arm of chromosome 6 (6p21.3). HLA-A.B.C markers are commonly used to detect CTCs because they help distinguish CTCs from non-tumor cells in the bloodstream. Specifically, HLA-A.B.C supports the identification of epithelial cells, thereby differentiating CTCs from immune cells, detecting cells undergoing EMT, and providing insights into immune evasion and cancer progression.

[0067] As used interchangeably herein, "VIM" and "vimentin" refer to type III intermediate filament (IF) proteins expressed in mesenchymal cells, which are used herein as a major cytoskeletal component. Due to its role in the structure of mesenchymal cells, vimentin is often used as a marker for mesenchymal-derived cells and is used to identify cells undergoing EMT during normal development and metastasis, including the detection of CTCs.

[0068] As used interchangeably herein, the terms "sample", "test sample", "specimen", "biological sample", "sample from a subject", or "subject sample" refer to a sample or isolate of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes, which can be used directly as obtained from a subject or can be pre-treated, for example, by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to alter the properties of the sample in some manner as described herein or otherwise known in the art.

[0069] Methods well known in the art for collecting, manipulating, and processing urine, blood, serum, plasma, and other body fluids are used in the practice of the present disclosure. A test sample may contain additional components other than the analyte of interest, such as antibodies, antigens, haptens, hormones, drugs, enzymes, receptors, proteins, peptides, polypeptides, oligonucleotides, or polynucleotides. For example, a sample can be a whole blood sample obtained from a subject. It may be necessary or desirable to pre-treat the test sample (especially whole blood) prior to the immunoassay described herein, for example, using a pre-treatment reagent. Even in cases where pre-treatment is not required (e.g., most urine samples, pre-treated archived samples, etc.), pre-treatment of the sample is an option that may be performed only for convenience (e.g., as part of a protocol on a commercial platform). The sample can be used directly as obtained from a subject or after pre-treatment to alter the properties of the sample. Pre-treatment can include extraction, concentration, inactivation of interfering components, and / or addition of reagents.

[0070] Unless otherwise defined, all 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 by reference in their entirety for the teachings relevant to the sentences and / or paragraphs in which they are cited.

[0071] Method for isolating and analyzing circulating tumor cells

[0072] In one embodiment, a method for isolating and analyzing a population of circulating tumor cells (CTCs) in a biological sample from an individual with cancer comprises the following steps:

[0073] a) isolating white blood cells (WBCs) and CTCs from the biological sample;

[0074] b) gating the CTCs based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and vimentin (VIM); and

[0075] c) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2;

[0076] wherein steps b) and c) are performed by flow cytometry.

[0077] The biological sample may contain a population of CTCs. The population of CTCs may contain one or more CTC subgroups. The composition of the circulating tumor cell (CTC) subgroup is characteristic of a particular tumor (e.g., a particular solid tumor). Thus, CTC subpopulations and their corresponding CTC subgroups are surrogate markers for cancer, tumors, and / or metastases. CTC subgroups are additional characteristics of a particular individual (e.g., a patient). The composition of a single subgroup of CTCs in a biological sample is specific to a particular patient. It can be used separately to predict and monitor treatment and disease progression or regression. Thus, the CTC population or the CTC subgroup and the corresponding amount (ratio) in a sample identified by the methods of the present disclosure can be used as specific biomarkers for the corresponding tumor, treatment, and individual patient. For example, these biomarkers (e.g., mEGFR L858R or Her2) are suitable for personalized medicine.

[0078] The biological sample described herein is obtained from an individual. The individual can be human, preferably a patient with 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.

[0079] In one embodiment, any method effective for removing cells that may interfere with CTC analysis can be used to isolate WBCs and CTCs. For example, WBCs and CTCs can be separated by 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 differential centrifugation. In one embodiment, WBCs and CTCs are separated by lysis of RBCs. In another embodiment, WBCs and CTCs are separated by RBC lysis followed by centrifugation.

[0080] In one embodiment, the method includes the step of gating WBCs and CTCs based on forward scatter (FSC) and CD45 to exclude cell debris and cell clumps and deplete blood cells to obtain CTC-like cells. In one embodiment, blood cells are depleted using anti-CD45 antibodies. If CD45 expression is negative, circulating tumor cells can be confirmed. Given that CTCs are extremely rare compared to other circulating cells, the isolation of CTCs involves identifying and excluding cells that express the pan-leukocyte marker CD45.

[0081] Additionally, the method includes 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 a biological sample, and simultaneously testing for mutant epidermal growth factor receptor (mEGFR) L858R or Her2 in the CTCs to confirm a subset of CTCs, thereby determining the appropriate drug for treatment.

[0082] In one embodiment, both steps b) and c) in the method of the present disclosure are performed by flow cytometry in order to isolate and analyze CTCs in one experiment. In another embodiment, steps b) and c) can be performed sequentially.

[0083] In one embodiment, depending on the requirements of the analysis method, the biological sample can be peripheral blood, preferably about 1 mL.

[0084] In the present invention, the methods described herein allow for the isolation and analysis of CTCs from a small amount of biological sample (e.g., 1 mL of peripheral blood).

[0085] In another embodiment, the method can further include lysing the red blood cells in the biological sample before step a).

[0086] The research of the present invention discloses a method configured to detect a CTC concentration equal to or higher than 2 CTCs per 1 mL of whole blood. In another embodiment, the method is configured to detect a CTC concentration equal to or higher than 5 CTCs per 1 mL of whole blood. In other words, if there are two or more CTCs in whole blood, the CTCs can be isolated for further phenotypic analysis, such as mEGFR L858R or Her2. The results of the examples show that even when there are as few as two CTCs in 1 mL of biological sample, the R 2 value of the test results is still good, indicating strong sensitivity for low-concentration detection.

[0087] In one embodiment, the cancer can be lung cancer, including small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC).

[0088] In a preferred embodiment, the lung cancer is NSCLC, and its phenotype is CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-A.B.C + VIM + 。

[0089] In one embodiment, the method may further include, if the CTCs express the markers CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , EpCAM + , HLA-A.B.C + and VIM + , then identifying the cancer as NSCLC.

[0090] The ability to detect and characterize CTCs has the potential to contribute to the treatment of cancer patients. The method may further include, if the CTCs are identified as mEGFR L858R + , then administering an EGFR tyrosine kinase inhibitor to the individual.

[0091] Examples of EGFR tyrosine kinase inhibitors may include erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, omitinib, lazertinib, and zolitinib.

[0092] In one embodiment, the cancer can be gastric cancer (GC), such as Her2-positive GC.

[0093] In a preferred embodiment, the GC has the phenotype CD45 - CK-7 / 8 +CK-14 / 15 / 16 / 19 + EpCAM + HLA-A.B.C + 。

[0094] In one embodiment, the method may further comprise, if the CTCs express the marker CD45 - 、CK-7 / 8 + 、CK-14 / 15 / 16 / 19 + 、EpCAM + and HLA-A.B.C + ,then identifying the cancer as GC.

[0095] If the CTCs are identified as Her2 + ,then the individual may be administered at least one treatment selected from Her2 tyrosine kinase inhibitors, anti-Her2 antibodies, bispecific antibodies, Her2-targeted antibody-drug conjugates, and Her2-targeted CAR-T cells.

[0096] Examples of Her2 tyrosine kinase inhibitors may include lapatinib; examples of anti-Her2 antibodies may include trastuzumab, pertuzumab, tucatinib, and margetuximab; an example of a bispecific antibody may be zandertuzumab; and examples of Her2-targeted antibody-drug conjugates may include trastuzumab emtansine and deruxtecan.

[0097] In another aspect, the present disclosure provides a method of treating cancer in an individual in need thereof, comprising:

[0098] a) gating circulating tumor cells in a biological sample derived from the individual based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM;

[0099] b) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; and

[0100] c) administering a cancer therapy to the individual, wherein

[0101] i) if the cancer is identified as non-small cell lung cancer (NSCLC), and the CTCs are mEGFR L858R + ,then the cancer therapy is an EGFR tyrosine kinase inhibitor, or

[0102] ii) if the cancer is identified as gastric cancer (GC), and the CTCs are Her2 +, the cancer therapy includes at least one treatment selected from the following: Her2 tyrosine kinase inhibitor, anti-Her2 antibody, bispecific antibody, Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cell.

[0103] In a preferred aspect, the EGFR tyrosine kinase inhibitor may include erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, omitinib, lazertinib, and zolitinib.

[0104] In a preferred aspect, the Her2 tyrosine kinase inhibitor may include lapatinib; the anti-Her2 antibody may include trastuzumab, pertuzumab, tucatinib, and margetuximab; the bispecific antibody may include zenidatamab; or the Her2-targeted antibody-drug conjugate may include trastuzumab emtansine and deruxtecan.

[0105] In another aspect, the present disclosure provides a method for identifying an individual with cancer who may respond to at least one treatment selected from the following: EGFR tyrosine kinase inhibitor, Her2 tyrosine kinase inhibitor, anti-Her2 antibody, Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cell, the method comprising:

[0106] a) Gating circulating tumor cells in a biological sample derived from the individual based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-A.B.C, and VIM;

[0107] b) Testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; and

[0108] c) Identifying an individual with cancer who may respond to the following treatment:

[0109] i) EGFR tyrosine kinase inhibitor, 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 + ; or

[0110] ii) at least one treatment selected from the following: Her2 tyrosine kinase inhibitor, anti-Her2 antibody, bispecific antibody, Her2-targeted antibody-drug conjugate, and Her2-targeted CAR-T cell, if the CTCs are derived from GC and have CD45- CK-7 / 8 + CK-14 / 15 / 16 / 19 + EpCAM + HLA-A.B.C + Her2 + 。

[0111] In one aspect, the present disclosure provides a kit for the methods described herein, which comprises 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.

[0112] The following examples for implementing specific aspects of the present invention are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0113] Examples

[0114] Example 1: Phenotypic screening of cell lines H1975 (NSCLC, mutant EGFR L858R), A549 (NSCLC, wild-type EGFR), MCF-7 (breast cancer) in addition to healthy donor leukocytes

[0115] To establish a flow-based CTC detection platform, a series of gating strategies must first be developed and validated. For the mutant EGFR (mEGFR) L858R NSCLC detection platform, cell lines from various cancers were used, including H1975 (NSCLC, with mutant EGFR L858R), A549 (NSCLC, wild-type EGFR), and MCF-7 (breast cancer, used as an indicator control). These cell lines were used to screen for CTC markers and the mutant NSCLC marker, mEGFR L858R. In addition, leukocytes from healthy donors (where red blood cells (RBC) were lysed) were used as healthy controls (negative controls for CTC markers) and were phenotypically screened by flow cytometry.

[0116] CTCs are typically identified using biomarkers that are specifically expressed on cancer cells but not on 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. Thus, epithelial cell adhesion molecule (EpCAM) and cytokeratins are often used to identify cancer cells in a mixed population and thereby distinguish them from other cells such as white blood cells (WBC).

[0117] In Example 1, three cell surface markers, EpCAM, CD45, and HLA-A.B.C, and two intracellular markers, CK-7 / 8 and CK-14 / 15 / 16 / 19, were used to distinguish cancer cells from WBCs and other blood cells. Additionally, vimentin (VIM) was used to distinguish NSCLC from other cancers (breast cancer in this example), and the mutant EGFR L858R marker was used to distinguish the wild type and L858R EGFR of NSCLC.

[0118] 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 together with white blood cells from healthy donors: ECD-anti-human CD45, BV510-anti-human EpCAM, FITC-anti-CK-7 / 8, Alexa Fluor647-anti-CK-14 / 15 / 16 / 19, Alexa Fluor 700-anti-HLA-A.B.C, Alexa Fluor405-anti-vimentin, and PE-anti-mEGFR antibody (EGF receptor (L858R mutant specific) (43B2) rabbit mAb (PE conjugate), Cell Signaling Technology Cat#64716).

[0119] As Figure 1 shown, using this method, H1975, A549, and MCF-7 cells were successfully identified as CTCs. These three cell lines specifically 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 the healthy donor samples used as negative controls. This indicates that this method effectively distinguishes CTCs from blood cells using flow cytometry. Additionally, based on vimentin expression, NSCLC cells can be distinguished from breast cancer cells, and the presence of the L858R mutation in EGFR can be specifically identified using the L858R antibody.

[0120] 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 + , as well as target markers (including mutant EGFR (L858R) +)。The A549 cell line (also a type of NSCLC but with wild-type EGFR) was used as a negative control for mutant EGFR (L858R specific); similarly, the MCF-7 cell line derived from breast cancer was also used as a negative control for mutant EGFR (L858R specific). Although the A549 and MCF-7 cell lines are cancerous and exhibit CTC markers, they do not express mEGFR L858R, thus providing a clear contrast for evaluating L858R positivity in H1975 cells. This comparison further highlights the potential of the flow cytometry-based method in discriminating and characterizing L858R-positive CTCs in NSCLC.

[0121] The flow cytometry screening results for each group are presented in a row-by-row manner and consist of biaxial panel sets. The dashed boxes indicate the gated regions of interest for specific cell populations, while the dashed arrows represent the gating strategy and logic. The solid arrows indicate the intensity or expression level of specific markers.

[0122] Example 2: Spiking experiment using H1975 cells

[0123] To evaluate the ability of flow cytometry to detect rare mEGFR L858R NSCLC cells at low concentrations, a spiking experiment with H1975 cells was conducted. A small number of H1975 cells were added to healthy whole blood to simulate low concentrations of NSCLC CTCs. The gating strategy used in Example 1 for detecting mEGFR L858R NSCLC was applied to analyze the spiked samples, thereby evaluating the sensitivity and recovery rate of the method in discriminating rare CTCs among normal blood cells.

[0124] 2.1. Sample processing

[0125] As Figure 2A shown, separate whole blood samples and mixtures of whole blood and H1975 aliquots were first prepared.

[0126] H1975 cells were harvested at 90% confluence by trypsin treatment. After cell counting, cell suspensions containing 2, 5, 10, or 25 cells (spiked groups) were prepared and added to 1 mL of peripheral blood from healthy donors. Only the peripheral blood samples were used as negative controls (mock-treated groups), while samples containing only H1975 cells (single-cell groups) were used as positive controls for CTC gating analysis (indicating positive controls).

[0127] The mock treatment group and the spiked group were subjected to red blood cell 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 mutant - specific) (43B2) rabbit mAb (PE conjugate), Cell Signaling Technology Cat#64716) or isotype.

[0128] 2.2. Identification of spiked H1975 cells by FACS

[0129] The gating strategy was the same as that determined for H1975 phenotypic screening in Example 1.

[0130] Initially, cell debris, cell clumps, and white blood cells were excluded using forward scatter (FSC) and CD45 (left panel of Figure 2B). Next, common CTC - related markers were identified: CK - 7 / 8 in the middle panel of Figure 2B + and CK - 14 / 15 / 16 / 19 - . Subsequently, additional common 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) + specifically expressed on H1975 was identified, as shown in Figure 2C. H1975 cells were successfully detected in spiked samples, with 2 to 25 H1975 cells added to 1 mL of whole blood. The mEGFR (L858R) mutation was observed in 100% of the identified CTCs.

[0131] 2.3. Linear analysis and recovery rate assessment of spiked H1975 cells

[0132] The number of detected H1975 cells was compared with the expected cell count to evaluate the recovery rate of the method. The identification of recovered H1975 cells was performed by screening for specific target 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 count of detected cells were plotted on the x-axis and y-axis, respectively (Figure 2D). A strong linear relationship was observed between the predicted and detected tumor cells, with an R 2 value of 0.86 (Figure 2E). These results indicate that the disclosed method can detect single cells in a mixed population of millions of cells, which is necessary for identifying rare cell types such as circulating tumor cells (CTCs).

[0133] The average recovery rate was calculated to be 51.3% (Figure 2E), indicating that most of the spiked cells were recovered through the enrichment process, and as few as 2 tumor cells in 1 mL of whole blood could be detected. Overall, the method disclosed herein is an effective method for detecting CTCs by flow cytometry.

[0134] CTCs exist at extremely low concentrations within a larger cell population, which makes their detection challenging. However, compared to current clinical practice, polymerase chain reaction (PCR), the efficiency of identifying target cells is significantly improved by utilizing a flow-based detection platform. This method not only reduces the total number of cells that need to be analyzed but also minimizes the sample volume required to detect cells of interest.

[0135] Example 3: Clinical Validation of a CTC Predictive Biomarker Detection Platform for NSCLC Using Real Samples

[0136] The above results indicate that the CTC detection platform for NSCLC can identify a small number of tumor cell lines spiked into whole blood by the recovery rate. To further validate the clinical utility of the same flow cytometry protocol used in the H1975 spiking experiment, 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 any anti-cancer treatment was initiated. The performance of the CTC detection platform in differentiating L858R NSCLC patients from healthy donors was evaluated. In addition, the feasibility of using a flow-based method to detect the EGFR L858R mutation (as opposed to wild-type or other mutations) in CTCs was investigated. The results were then compared with standard diagnostic methods to evaluate the accuracy and reliability of the flow-based method for detecting this specific EGFR mutation in CTCs.

[0137] Peripheral blood samples from NSCLC patients and healthy donors were analyzed using a method consistent with that used in the H1975 spiking experiment. Circulating tumor cells (CTCs) were characterized using the following markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 - , HLA-A.B.C, EpCAM + , VIM +and mEGFR (L858R) + , and applied to clinical samples, such as Figure 5 shown in A. The L858R + CTC detection results were summarized as the percentage of CTCs and the absolute CTC count (CTC#) in the CD45 - population, as shown in Figure 5 B. The data showed that L858R + CTCs could be detected in clinical samples from NSCLC patients, while the same marker was not detected in samples from healthy donors. The results were statistically significant, highlighting the potential of this method to identify L858R + CTCs in NSCLC patients.

[0138] The clinical validation results are summarized in Table 2, which compares the standard diagnostic procedure (PCR of solid biopsy) with the CTC detection platform (flow cytometry of liquid biopsy, the method proposed in the present invention). As shown in Table 2, there was a high degree of agreement between PCR and the flow-based method, with 87.5% (7 / 8) agreement in identifying positive cases and 82.4% (14 / 17) agreement in identifying negative cases. These findings demonstrated the reliability of the flow cytometry-based liquid biopsy method, which was in close agreement with the established PCR solid biopsy method, while having potential advantages in terms of sensitivity and non-invasiveness.

[0139] Table 1. Patient demographics

[0140]

[0141]

[0142] Table 2. Clinical validation correlation results of the CTC predictive biomarker detection platform (flow cytometry of liquid biopsy) and the standard diagnostic procedure (PCR of solid biopsy)

[0143]

[0144] Example 4: Phenotypic screening of N87 (gastric cancer, Her2+) and H520 (non-small cell lung cancer, Her2- control) cell lines and healthy donor leukocytes

[0145] To further establish a flow-based CTC detection platform for Her2 + gastric cancer (GC), a series of gating strategies must first be developed and validated. Cell lines from various cancers were used, including N87 (GC, Her2 +) and H520 (NSCLC, used as Her2 control). These cell lines were used to screen for CTC markers and Her2 markers. In addition, white blood cells from healthy donors (red blood cells (RBC) lysed) were used as healthy controls (negative controls for CTC markers) and phenotypically screened by flow cytometry.

[0146] 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. Thus, epithelial cell adhesion molecule (EpCAM) and cytokeratins are often used to identify cancer cells in a mixed population, thereby distinguishing them from other cells (such as white blood cells (WBCs)).

[0147] In Example 4, three cell surface markers, EpCAM, CD45, and HLA-A.B.C, and two intracellular markers, CK-7 / 8 and CK-14 / 15 / 16 / 19, were used to distinguish cancer cells from WBCs and other blood cells. In addition, Her2 markers were used to detect Her2-positive gastric cancer.

[0148] To verify the effectiveness of these markers, human tumor cell lines (for Her2 + N87 of GC, for Her2 - H520 of NSCLC) were stained with various antibodies together with white blood cells from healthy donors: 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-human HLA-A.B.C, and PE / Cy7-anti-Her2 7 antibody.

[0149] As Figure 3 shown, the method successfully identified N87 and H520 cells as CTCs. The two cell lines specifically expressed EpCAM, CK-7 / 8, CK-14 / 15 / 16 / 19, and HLA-A.B.C, and most of the cells were CD45 negative. In contrast, no CTCs were detected in the healthy donor samples used as negative controls. This finding indicates that the method effectively distinguishes CTCs from other blood cells using flow cytometry. In addition, N87 and H520 cells can be distinguished from each other based on Her2 expression, highlighting the method's ability to distinguish CTC populations.

[0150] In summary, the phenotype of Her2-positive GC cells (N87 cells) is based on circulating tumor cell (CTC)-related markers, including CD45 -, CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + and HLA-A.B.C + ) and the expression of the target biomarker Her2 + was characterized. Although H520 cells are cancerous and exhibit CTC biomarkers, they do not express Her2, thus providing a clear comparison for evaluating Her2 positivity in N87 cells. This comparison further highlights the potential of flow cytometry-based methods to identify and characterize Her2-positive CTCs in GC.

[0151] The flow cytometry screening results for each group are presented row by row and consist of a set of biaxial plots. The dashed boxes indicate the gated regions of interest for specific cell populations, while the dashed arrows represent the gating strategy and logic. The solid arrows represent the intensity or expression level of specific biomarkers.

[0152] Example 5: Spike-in experiment using N87 cells

[0153] To evaluate the ability of flow cytometry to detect low concentrations of Her2-positive gastric cancer cells, a spike-in experiment with N87 cells was conducted. A small number of N87 cells were added to healthy whole blood to simulate low concentrations of GC CTCs. The gating strategy used in Example 4 for detecting Her2-positive GC was applied to analyze the spiked samples, thereby evaluating the sensitivity and recovery rate of the method for discriminating rare CTCs among normal blood cells.

[0154] 5.1. Sample preparation

[0155] As Figure 4A shown, individual whole blood samples and mixtures of whole blood and N87 aliquots were prepared first.

[0156] N87 cells were harvested at 90% confluence by trypsin treatment. After cell counting, cell suspensions containing 2, 5, 10, or 25 cells (spiked groups) were prepared and added to 1 mL of peripheral blood from healthy donors. Only the peripheral blood sample was used as a negative control (mock-treated group), while the sample containing only N87 cells (single-cell group) was used as a positive control for CTC gating analysis (indicator positive control).

[0157] The false treatment group and the spiked group were subjected to red blood cell lysis (RBC). 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, and analyzed using FACS / flow cytometry to identify the spiked N87 cells.

[0158] 5.2. Isolation and analysis of spiked N87 cells by FACS

[0159] The gating strategy was the same as the gating strategy determined in Example 4 for N87 phenotype screening.

[0160] Initially, cell debris, cell clumps, and white blood cells were excluded using forward scatter (FSC) and CD45 (left panel of Figure 4B). Next, common CTC-related markers were identified: CK-7 / 8 in the middle panel of Figure 4B + and CK-14 / 15 / 16 / 19 + . Subsequently, additional common CTC-related markers were identified: EpCAM in the right panel of Figure 4B + and HLA-A.B.C + . Finally, the target marker Her2 + was identified, which was specifically expressed on N87, as shown in Figure 4C. N87 cells were successfully detected in the spiked samples, with 2 - 25 N87 cells added to 1 mL of whole blood. Her2 + cells were observed in 100% of the identified CTCs.

[0161] 5.3. Linear analysis and recovery rate assessment of spiked N87 cells

[0162] The number of detected N87 cells was compared with the expected cell count to evaluate the recovery rate of the method. Identification of the recovered N87 cells was performed by screening for specific target markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , HLA-A.B.C + and Her2 + . The number of spiked cells and the detected cell count were plotted on the x-axis and y-axis, respectively (Figure 4D). A strong linear relationship was observed between the predicted and detected tumor cells, R 2The value was 0.6103 (Figure 4E). These results indicate that the disclosed method can detect single cells in a mixed population of millions of cells, which is necessary for identifying rare cell types such as circulating tumor cells (CTCs).

[0163] The average recovery rate was calculated to be 61% (Figure 4E), indicating that most of the spiked cells were recovered through the enrichment process and as few as 2 tumor cells in 1 mL of whole blood could be detected. Overall, the method disclosed herein is an effective method for enhancing the ability to detect CTCs by flow cytometry.

[0164] In a larger cell population, CTCs exist at extremely low concentrations, making their detection challenging. However, compared with current clinical practice (immunohistochemistry (IHC)), the efficiency of differentiating Her2-positive GCs was significantly improved by using a flow-based detection platform. This method not only reduces the total number of cells that need to be analyzed but also minimizes the sample volume required to detect the cells of interest.

[0165] Example 6: Validation of the CTC Predictive Biomarker Detection Platform for GC Using Real Samples

[0166] The above results indicate that the CTC detection platform for GC is capable of differentiating a small number of tumor cell lines spiked into whole blood with a high recovery rate. To validate the clinical utility of the same flow cytometry protocol used in the N87 spiking experiment, whole blood samples were collected from 14 cancer patients and 10 healthy subjects for CTC detection (Table 3). Blood samples were collected from all cancer patients before the start of anti-cancer treatment. The performance of the CTC detection platform in differentiating Her2 + GC patients from healthy donors was evaluated. In addition, the correlation between the standard diagnostic method and the CTC detection method was also evaluated.

[0167] Peripheral blood samples from gastric cancer (GC) patients and healthy donors were analyzed using a method consistent with that used in the GC recovery experiment. Circulating tumor cells (CTCs) were characterized using the following markers: CD45 - , CK-7 / 8 + , CK-14 / 15 / 16 / 19 + , EpCAM + , HLA-A.B.C + and Her2 + . These markers were applied to clinical samples as Figure 6 shown in A. The Her2 + CTC detection results were summarized as the percentage of CTCs and the absolute CTC count (CTC#) in the CD45 - population, as Figure 6As shown in B. The data indicate that CTCs can be detected in clinical samples from GC patients, while the same markers are not detected in samples from healthy donors. The results are statistically significant, highlighting the potential of this method in identifying Her2 + CTCs in GC patients.

[0168] The clinical validation results are summarized in Table 4, which compares the standard diagnostic procedure (IHC of solid biopsy) with the CTC detection platform (flow cytometry of liquid biopsy, the method proposed in the present invention). As shown in Table 4, the agreement between IHC and the flow-based method in differentiating positive cases is very good, with 7 / 7 (100% agreement) of positive cases detected by both methods. In addition, both methods are consistent for all 7 negative cases (100% agreement), with no false positives or false negatives.

[0169] These results demonstrate the perfect correlation between the IHC-based solid biopsy and the flow cytometry-based liquid biopsy method, showing the high accuracy and reliability of the flow cytometry method. The flow-based detection method not only matches IHC in differentiating positive and negative cases, but also has the advantage of being non-invasive, making it a promising alternative for the clinical diagnosis of Her2 + CTCs in GC patients.

[0170] Table 3. Patient demographics

[0171]

[0172] Table 4. Clinical validation correlation results of the CTC predictive biomarker detection platform (flow cytometry of liquid biopsy) and the standard diagnostic procedure (IHC of solid biopsy)

[0173]

[0174] Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of these variations and / or modifications is considered to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend upon one or more specific applications of the teachings of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced otherwise than as specifically described and claimed. Embodiments of the invention disclosed herein relate to each and every separate feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more of such features, systems, articles, materials, kits, and / or methods, if not mutually inconsistent, is included within the scope of the invention disclosed herein.

[0175] It should also be understood that, unless expressly stated to the contrary, in any method that includes more than one step or act as claimed in this application, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

1. A method for analyzing circulating tumor cells (CTCs) in a biological sample of an individual suffering from cancer, comprising: a) separating white blood cells (WBC) and CTCs from the biological sample; b) gating the CTCs based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-ABC, and vimentin (VIM); and c) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; Wherein steps b) and c) are performed by flow cytometry. The method according to claim 1 , wherein the biological sample is peripheral blood. The method of claim 2 , wherein the biological sample is about 1 mL.

4. The method of claim 1, wherein the biological sample comprises more than 5 CTCs per milliliter of the biological sample. The method according to claim 1 , further comprising lysing red blood cells in the biological sample before step a). The method according to claim 1 , wherein steps b) and c) are performed sequentially.

7. The method according to claim 1, further comprising, if the CTCs express CD45 - ,CK-7 / 8 + ,CK-14 / 15 / 16 / 19 - EpCAM + , HLA-ABC + and VIM + If the cancer is detected by Western blot, the cancer is identified as non-small cell lung cancer (NSCLC).

8. The method according to claim 7, further comprising, if the CTC is mEGFR L858R + , an EGFR tyrosine kinase inhibitor is administered to the individual.

9. The method of claim 8, wherein the EGFR tyrosine kinase inhibitor comprises erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, omotinib, lazertinib and zolitinib.

10. The method according to claim 1, further comprising, if the CTC expresses CD45 - ,CK-7 / 8 + ,CK-14 / 15 / 16 / 19 + EpCAM + and HLA-ABC + If the CTC is detected by the expression of a marker, it is identified that the CTC is derived from gastric cancer (GC).

11. The method according to claim 10, further comprising, if the CTC is Her2 + , the individual is administered at least one treatment selected from 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.

12. The method of claim 11, wherein the Her2 tyrosine kinase inhibitor comprises lapatinib; the anti-Her2 antibodies comprise trastuzumab, pertuzumab, tucatinib, and magituximab; the bispecific antibody comprises zenidatuzumab; or the Her2-targeting antibody-drug conjugate comprises emtansinetrastuzumab and dexamethasonetrastuzumab.

13. A method for treating cancer in an individual in need thereof, comprising: a) gating circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-ABC and VIM; b) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; and c) administering a cancer therapy to the individual, wherein i) If the cancer is identified as non-small cell lung cancer (NSCLC) and the CTC is mEGFR L858R + , then the cancer therapy is an EGFR tyrosine kinase inhibitor, or ii) If the cancer is identified as gastric cancer (GC) and the CTC is Her2 + , the cancer therapy includes at least one selected from the group consisting of Her2 tyrosine kinase inhibitors, anti-Her2 antibodies, bispecific antibodies, Her2-targeted antibody-drug conjugates and Her2-targeted CAR-T cells.

14. The method of claim 13, wherein the EGFR tyrosine kinase inhibitor comprises erlotinib, gefitinib, icotinib, afatinib, dacomitinib, osimertinib, rociletinib, omotinib, lazertinib and zolitinib.

15. The method of claim 13, wherein the Her2 tyrosine kinase inhibitor comprises lapatinib; the anti-Her2 antibodies comprise trastuzumab, pertuzumab, tucatinib, and magituximab; the bispecific antibody comprises zenidatuzumab; or the Her2-targeting antibody-drug conjugate comprises emtansinetrastuzumab and dexamethasone.

16. A method for identifying an individual with cancer that is likely to respond to 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: a) gating circulating tumor cells (CTCs) in a biological sample derived from the individual based on forward scatter (FSC), CD45, CK-7 / 8, CK-14 / 15 / 16 / 19, EpCAM, HLA-ABC and VIM; b) testing the CTCs for mutant epidermal growth factor receptor (mEGFR) L858R or Her2; and c) Identify individuals with cancer that may respond to treatment with: i) EGFR tyrosine kinase inhibitors, if the CTCs are derived from NSCLC and have CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 - EpCAM + HLA-ABC + VIM + mEGFR L858R + ;or ii) at least one treatment selected from the group consisting of: Her2 tyrosine kinase inhibitors, anti-Her2 antibodies, bispecific antibodies, Her2-targeted antibody drug conjugates, and Her2-targeted CAR-T cells, if the CTC is derived from GC and has CD45 - CK-7 / 8 + CK-14 / 15 / 16 / 19 + EpCAM + HLA-ABC + Her2 + .

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

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