Methods of using therapy-altered PD-l1 expression in treatment decisions for cancer therapy

Abstract

Methods of using levels PD-L1 expression by circulating cancer cells in the screening, monitoring, treatment and diagnosis of cancer in subjects are disclosed, along with methods of monitoring probability of progression free survival and / or overall survival of such subjects. The methods are based on assaying the level of PD-L1 expression in one or more of circulating tumor cells (CTCs), epithelial to mesenchymal transition CTCs (EMTCTCs), cancer associated macrophage-like cells (CAMLs), and cancer associated vascular endothelial cells (CAVEs) isolated from a subject having cancer, where the subject is undergoing immunotherapy treatment.

Description

METHODS OF USING THERAPY-ALTERED PD-L1 EXPRESSION IN TREATMENTDECISIONS FOR CANCER THERAPYBACKGROUND OF INVENTION

[0001] Cancer is the second leading cause of death in the United States, and 42% of men and 38% of women will develop cancer in their lifetimes[35'38]. Immunotherapy harnesses a patient’s own immune system to attack cancer, irrespective of the origin. The immune system is regulated by a network of checks and balances that evolved to attack foreign invaders like bacteria and viruses. However, cancer can evade the immune system by expressing proteins, such as PD-L1 and PD-L2, which inhibit the immune system from attacking cancer cells.

[0002] In particular, interactions between tumor cells and T cells involve contact between the major histocompatibility complexes (MHC) on tumor cells and the T cell receptor (TCR) on T cells

[0038] . Upon contact between the MHC and T cell receptor, the T cells are activated and the tumor cells are destroyed.

[0003] However, tumor cells can evade T cell immunosurveillance if they expresses the immune checkpoint protein PD-L1 on their surface. When present, PD-L1 binds to PD-1 expressed by T cells and activation of the T cell is blocked, thus suppressing T cell immunosurveillance.

[0004] Immune checkpoint inhibitors (ICI) have been developed that can block the PD- Ll / PD-1 interaction. Such inhibitors permit the T cell immunosurveillance mechanism to again function normally, and tumor cells can thus be destroyed through the normal immune response in the subject.

[0005] Blockage of CTLA-4 on T cells can have a similar effect. The first immunotherapeutic based on CTLA-4 was approved by the FDA for melanoma in 2011. The pace of FDA immunotherapy approvals increased in 2014 and by the end of 2016, there were 18 immunotherapy approvals for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), head and neck cancer, bladder cancer and Hodgkin’s lymphoma I39’43!. There are currently a number of open immunotherapy clinical trials indicating potential for broad efficacy across multiple tumors.

[0006] The key to the effective use of immune checkpoint inhibitors is determining whether a particular subject having cancer will respond to the drugs. If an antibody which binds to PD-L1or PD-1 and that serves as an immune checkpoint inhibitor is administered to a patient whose tumor cells do not express PD-L1, the treatment will be ineffective. As such antibody-based treatments are very expensive, it is important to have at least some indication that the patient will respond to the treatment.

[0007] Obtaining cancer cells via tumor biopsies for PD-L1 expression surveys has serious drawbacks that include pain and discomfort to patients, the inability to survey more than an isolated area of the tumor, and the potential for protein expression profiles to change over time in the tumor microenvironment.

[0008] Blood-based biopsies (BBB) are an improvement on tissue biopsies in that they can provide real time sequential tracking of cells shed by the tumor or that are otherwise tumor- associated cells. Blood samples can be more easily obtained, and obtained more often from a patient. Further, changes in protein expression profiles can be monitored over time. Circulating tumor cells (CTCs) are one type of cancer-associated cell that can easily be isolated from the peripheral blood and that can be used as a substitute for tumor cells obtained from tissue biopsies [i-4! CTCS aretumor cells broken off from the solid tumors into the blood stream. CTCs can be found in blood of carcinomas, sarcomas, neuroblastomas and melanomas patients.

[0009] Further knowledge regarding expression levels of PD-L1 under different circumstances, such as changes related to particular types of therapies, and the identification of additional cell types that can be assayed in blood-based biopsies will be critical in developing this technique for use in cancer patients that will benefit from treatment with immune checkpoint inhibitors.BRIEF SUMMARY OF INVENTION

[0010] The present invention generally relates to blood-based biomarkers and the cells that express them for use in screening, monitoring, and diagnosing cancer in a subject. The invention also relates to methods of making treatment decisions based on the presence or absence or change in expression of the biomarkers. The invention further relates to methods of predicting survival of a subject having cancer based on the presence or absence or change in expression of the biomarkers.

[0011] The methods defined herein will allow oncologists to select better combinations and sequences of conventional cytotoxic and immunotherapies, as well as identify patients likely to show durable responses to immunotherapy.

[0012] In more detail, the present invention is directed to leveraging new and existing knowledge regarding levels of PD-L1 expression in circulating cancer-associated cells from a subject having cancer in treatment decisions and in monitoring progression free survival (PFS) and / or overall survival (OS) in such subjects.

[0013] In a first embodiment, the invention is drawn to a method of monitoring probability of PFS and / or OS in a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, probability of PFS and / or OS in the subject is increased and when PD-L1 expression decreases between the first and second time point, probability of PFS and / or OS in the subject is decreased.

[0014] In a second embodiment, the invention is drawn to a method of determining probability of PFS and / or OS in a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, probability of PFS and / or OS in the subject is determined to be increasing and when PD-L1 expression decreases between the first and second time point, probability of PFS and / or OS in a subject is determined to be decreasing.

[0015] In a third embodiment, the invention is drawn to a method of making a treatment decision in a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein a cancer treatment is administered to the subject between the first and second time point, and wherein when PD-L1 expression decreases between the first and second time point, analternative treatment is administered to the subject. In certain aspects of this embodiment, the alternative treatment is not an ICI.

[0016] In a fourth embodiment, the invention is drawn a method of making a treatment decision for an immune checkpoint inhibitor in a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, an immune checkpoint inhibitor is indicated for administration to the subject.

[0017] In a fifth embodiment, the invention is drawn to a method of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein a cancer treatment is administered to the subject between the first and second time point, and wherein when PD-L1 expression increases between the first and second time point, administration of an immune checkpoint inhibitor is predicted to be beneficial for the subject.

[0018] In a sixth embodiment, the invention is drawn to a method of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression is high at the first and second time point, administration of an immune checkpoint inhibitor after the second time point is predicted to be beneficial for the subject.

[0019] In a seventh embodiment, the invention is drawn to a method of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer. The method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject havingcancer for PD-L1 expression, wherein when PD-L1 expression is low at the first time point and high at the second time point, administration of an immune checkpoint inhibitor after the second time point is predicted to be beneficial for the subject.

[0020] In certain of the relevant embodiments and aspects defined above, PD-L1 expression is assayed at one or more additional time points, such as 2, 3, 4, 5, 6, or more time points.Indeed, PD-L1 can be monitored in a continuous manner, such as once a day for one or more days, one or more weeks, or one or months.

[0021] In certain of the relevant embodiments and aspects defined above, a cancer treatment is administered to the subject between the first and second time point. The cancer treatment may be an immunotherapeutic agent. The cancer treatment may be an immune checkpoint inhibitor.

[0022] In certain of the relevant embodiments and aspects defined above, the immune checkpoint inhibitor is one or more of a PD-L1 antagonist, PD-1 antagonist, and a CTLA-4 antagonist.

[0023] In certain of the relevant embodiments and aspects defined above, the immune checkpoint inhibitor inhibits one or more of (i) binding between PD-L1 and PD-1, (ii) binding of PD-L1 to its binding partners, (iii) binding of PD-1 to its binding partners, and (iv) binding of CTLA-4 to its binding partners.

[0024] In certain of the relevant embodiments and aspects defined above, the immune checkpoint inhibitor is an antibody, such as a monoclonal antibody. In particular aspects, the immune checkpoint inhibitor is a human antibody, a humanized antibody, or a chimeric antibody.

[0025] Examples of specific immune checkpoint inhibitors include, but are not limited to, one or more of Nivolumab (Opdivo), Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Atezolizumab (Tecentriq), Tremelimumab, Durvalumab (MED14736) and Retifanlimab.

[0026] In certain of the relevant embodiments and aspects defined above, the cancer treatment includes, but is not limited to, immunotherapeutic agents, chemotherapeutic agents, radiotherapeutic agents, existing cancer drugs, CCR5 and CXCR4.

[0027] Examples of the cancer treatments include, but are not limited to, one or more of T- VEC, AM-0010, CXCR4 antagonist, TGF-beta kinase inhibitor galunisertib, anti-CSF-lR monoclonal antibody, Abemaciclib, Faslodex, necitumumab, AZD9291, Cyramza (ramucirumab), TPIV 200, Galunisertib, cancer vaccines, cytokines, cell-based therapies, bi- andmulti-specific antibodies, tumor-targeting mAbs, Rituximab, oncolytic viruses, reovirus, Blinatumomab, Sipuleucel-T, T-Vec, IL -2, IFN-a, Trastuzumab, Celuximab, bevacizumab, Tim- 3, BTLA, anti-IL-10, GM-CSF, anti-angiogenesis treatment, VEGF blockade, HMGB1, Nrpl, TAM receptor tyrosine kinases, Axl , MerTK, ALT-803, IL-15, Immunosuppressive Ligand Phosphatidylserine (PS), bavituximab, bevacizumab (anti-VEGF), coblmetinib (MEK inhibitor), vemurafenib (BRAF inhibitor), erlotinib (EGFR), alectinib (ALK inhibitor), bevacizumab (anti- VEGF), pazopanib (tyrosine kinase inhibitor), dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), sunitinib (RTK inhibitor), pazopanib (RTK inhibitor), sargramostim, VISTA, TIM-3, LAG-3, PRS-343, CD137 (4-lBB) / HER2 bispecific, USP7, anti-HER2, SEMA4D, CTLA-4, PD-1, PD-L1, and PD-L2.

[0028] In certain of the relevant embodiments and aspects defined above, the assaying for PD-L1 expression may be by one or more of detecting PD-L1 protein expression or detecting PD-L1 mRNA production. PD-L1 protein expression may be detected, for example, via immunohistochemistry (IHC). IHC may be performed by membrane staining, cytoplasmic staining, or a combination thereof. IHC may be performed using an anti-PD-Ll antibody, i.e. an antibody having binding specificity for PD-L1. PD-L1 protein expression may be detected as a weak staining intensity by IHC, moderate staining intensity, or strong staining intensity. PD-L1 protein expression may also be detected as a low staining intensity by IHC, moderate staining intensity, or high staining intensity. PD-L1 protein expression may also be detected as inducible from low staining intensity to high staining intensity, or inducible from low staining intensity to moderate staining intensity, or inducible from moderate staining intensity to high staining intensity. PD-L1 protein expression may be detected as any staining of the isolated cells.

[0029] In certain aspects, IHC is performed using immunofluorescence (IF) staining where one or more antibodies with binding specificity for PD-L1 are utilized. Binding of the anti-PD- Ll antibody to PD-L1 may be detected via a fluorescent compound conjugated to the anti-PD-L l antibody or it may be detected via a detectable label-conjugated secondary antibody with binding specificity for the anti-PD-Ll antibody. Suitable detectable labels include fluorophores.

[0030] In certain of the relevant embodiments and aspects defined above, PD-L1 expression is detected when the level of PD-L1 expression is greater than PD-L1 expression is a population of stromal cells from a subject of the same species that does not have cancer.

[0031] In certain of the relevant embodiments and aspects defined above, CTCs, EMTCTCs, CAMLs, and CAVEs are isolated from blood obtained from the subject having cancer. In certain aspects, the blood is peripheral blood.

[0032] In certain of the relevant embodiments and aspects defined above, the subject having cancer may be undergoing treatment using one or more of a targeted agent, chemotherapy, or radiation therapy.

[0033] In certain of the relevant embodiments and aspects defined above, the cancer is a lung cancer, breast cancer, prostate cancer, pancreatic cancer, melanoma, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, neuroblastoma, sarcoma, osteosarcoma, esophageal, brain & ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, uterine cervix, or uterine corpus cancer. The cancer may be a solid tumor, such as solid tumor of a stage I, stage II, stage III, or stage IV cancer. The solid tumor may be a carcinoma, sarcoma, neuroblastoma or melanoma. Examples of lung cancer include, but are not limited to, non-small cell lung carcinoma (NSCLC).

[0034] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.BRIEF DESCRIPTION OF DRAWINGS

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0036] Figure 1. Blood based biopsy identified and subtyped circulating cells by DAPI, cytokeratin, EpCAM and CD45; then the QUAS-R fluorescence quenching technique was used to restain cells with RAD50 and PD-L1. (Fig. 1A) An example of a EMTCTC cluster of cells, weakly positive for cytokeratin, negative for EpCAM and negative for CD45. Box scale= 90pm. (Fig. IB) The samples were quenched by QUAS-R where the fluors were quenched without harming the protein epitopes

[0013] . The samples were then restained with PD-L1, RAD50 and PD- 1. Box scale= 90pm. (Fig. 1C) PD-L1 was measured by tracing the cell in Zen software which calculated the average intensity of each cell or cell cluster. Box scale= 35pm. (Fig. ID) RAD50 foci (red) were enumerated in each nucleus (Cyan). Box scale=35pm.

[0037] Figure 2. Individual and expanded images of DAPI, Cytokeratin and CD45 from Figure 3. (Fig. 2A) PDCTC with a filamentous cytokeratin signal, pathologically aberrant nuclei and no CD45. White arrow shows a typical white blood cells positive for DAPI and CD45. (Fig. 2B) EMTCTC with a diffuse cytokeratin signal, no CD45 and abnormal nuclei. (Fig. 2C) CAML with multiple aggregated nuclei and an enlarged cell that is both CD45+and cytokeratin# Boxes=65 pm.

[0038] Figure 3. Percentage of samples with cells that could be used to quantify PD-L1 at baseline (TO) and post induction of radiotherapy (Tl), blood based biopsies included two subtypes of circulating tumor cells and circulating stromal cells. Standard biopsies only had the initial baseline time points and only 22% of those samples had sufficient amount of tumor for analysis by PD-L1. Brown stain is PD-Ll / blue is hematoxylin. A blood based biopsy identified EMT tumor cells in 49% of baseline samples and in 66% of post therapy samples. Further, circulating stromal cells (CAMLs) were available in 81% of baseline samples and in 100% of follow up samples. Blue=DAPI, green=cytokeratin, purple=CD45, boxes=65 micron.

[0039] Figure 4. Testing and comparing the clinically approved IHC PD-L1 clones from DAKO and the BBB PD-L1 clone. (Fig. 4A) Clone 22c3 from patient ID# 8 sample which scored 1+ in 10% of the tumor. (Fig. 4B) Clone 28-8 from patient ID# 8 parallel sample which scored 2+ in 20% of the tumor. (Fig. 4C) A PD-L1 clone optimized for BBBs was used todetermine the number of cells positive for PD-L1 and the intensity of each cell found on the CellSieve™ microfilters. SI=pixel intensity quartile, %=percent of cells positive for the maximum pixel intensity quartile, N / A= no available sample to test.

[0040] Figure 5. Determining the thresholds for scoring PD-L1 expression in circulating cells. The PD-L1 signal of each BBB cell (n=374) was determined by Zen Blue and subtracted from it’s relative background for each image. The standard deviation of the background (n=373) was used as the threshold for a BBB IHC score of 0 (26% of all cells). Two times the standard deviation was used as the threshold for a BBB IHC score of 1 (42% of all cells). Twice the background was used as the threshold for a BBB IHC score of 2 (22% of all cells). All remaining intensities 2X-6X background were scored as >3 (10% of all cells).

[0041] Figure 6. Average PD-L1 changes in each individual patient before and after induction of radiotherapy, separated by cell type. (Fig. 6A and B) In CAML cells, an average PD-L1 increase was seen in 51% of patients, a decrease was seen in 22%, and 27% patients did not have CAMLs at one of the time points. In EMTCTCs, an average PD-L1 increase was seen in 29% of patients, a decrease was seen in 17% and 54% patients did not have EMTCTCs at one of the time points.

[0042] Figure 7. (Fig. 7A) Progression free survival (PFS) based on PD-L1 expression of immunotherapy in NSCLC at baseline before chemoradiation and immunotherapy. (Fig. 7B). PFS based on PD-L1 expression of immunotherapy of NSCLC patients right after chemoradiation. (Fig. 7C). PFS based on PD-L1 expression of immunotherapy of NSCLC patients approximately 30 days after chemoradiation.

[0043] Figure 8. (Fig. 8A). PFS based on change of PD-L1 expression in CTCs or CAMLs after SV-BR-l-TM therapy in combination with Retifanlimab. (Fig. 8B). Overall survival (OS) based on change of PD-L1 expression in CTCs or CAMLs after SV-BR-l-TM therapy in combination with Retifanlimab.

[0044] Figure 9. Leronlimab induction of PD-L1 expression in CTC / CAMLs.

[0045] Figure 10. Overall survival (OS) by leronlimab treatment + / - ICI therapy.

[0046] Figure 11. Comparing the PFS and OS of patients based on high / low PD-L1 expression at To and Tl. (Fig. 11A) PFS of patients with high and low PD-L1 expression at TO (HR=1.23, p=0.6286). (Fig. 1 IB) OS of patients with high and low PD-L1 expression at TO (HR=1.26, p= 0.7157). (Fig. 11C) PFS of patients with high and low PD-L1 expression at Tl(HR=2.60, p=0.0368). (Fig. 1 ID) OS of patients with high and low PD-L1 expression at Tl (HR=3.61, p= 0.0150).

[0047] Figure 12. Comparing the PFS and OS of patients based on high / low PD-L1 expression at To and Tl. (Fig. 12A) PFS of patients with high and low PD-L1 expression at TO (HR=0.61, p=0.4888). (Fig. 12B) OS of patients with high and low PD-L1 expression at TO (HR=0.91, p= 0.9154). (Fig. 12C) PFS of patients with high and low PD-L1 expression at Tl (HR=4.10, p=0.0009). (Fig. 12D) OS of patients with high and low PD-L1 expression at Tl (HR=4.63, p= 0.0010).

[0048] Figure 13. Monitoring PD-L1 and CAML size. CAML size initially dropped after the start of chemotherapy. The CAML size was steady for 4 cycles of chemotherapy, and at the same time the PD-L1 expression was increasing to high. Then the patient was given immunotherapy drug Pembrolizumab. However, CAML size increased, because the PD-L1 expression dropped to very low at the time of the immunotherapy. The CAML size increased further at the next time point because PD-L1 expression remained low.DETAILED DESCRIPTION OF THE INVENTIONI. Definitions

[0049] As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0050] As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., + / - 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.II. The Present Invention

[0051] Blood-based biopsies (BBB) provide real-time, sequential tracking of circulating tumor cells (CTCs) found in the peripheral blood and such assays can be used as a substitute totissue biopsies[1’4]. Assessing circulating tumor cells (CTCs) in the peripheral blood has the power to interrogate heterogeneous populations of CTCs, including CTC subtypes undergoing epithelial to mesenchymal transition (EMTCTCs)|2'3’5'9]and the prognostically relevant pathologically definable CTCs (PDCTCs)[6'101, both as a cancer diagnostic as well as a means for screening, monitoring treatment, and determining the susceptibility of a tumor in a particular subject to a particular treatment.

[0052] Recently, another circulating cell associated with cancer has been identified in the peripheral blood of cancer patients that may be assayed in the methods defined herein. This cancer stromal cell subtype has been termed a cancer associated macrophage-like cell or CAML. CAMLs have been identified in the blood using a non-affinity microfiltration based method which captures both CTCs and CAMLs, and allows for singular or parallel analysis of these cancer specific circulating cell subtypes I1’6 6]. CAMLs are a recently defined circulating myeloid derived stromal cell, found in all the stages of invasive malignancy and in various solid malignancies (e.g. breast, prostate, non-small cell lung carcinoma (NSCLC), and pancreatic)[11,13, 14, 17] C AMLS are specialized myeloid polyploid cells in the blood in all stages of solid tumors and in blood cancers. They are easy to identify by their large size (greater than 25 pm), polyploid nucleus and morphologies: round, rod shaped, with one tail or two tails 180 degrees apart.CAMLs typically express CD31, CD14, CD45 and cytokeratin, and can also express EpCAM, CD146, CD11c and tie2[11, 13’14, 17]. While CAMLs appear to be cancer specific and disseminate from the organ sites of malignancy, it remains unknown if they actually reside at the primary tumor site or if they possess clinical utility.

[0053] Different subgroups of CTCs upregulate and / or down regulate phenotypes in relation to tumor progression, tumor spread, and in response to tumor treatments. The ability of individual cancer cells to transition states, such as the epithelial to mesenchymal transitions, leads to an additional circulating cancer cell subtype that may be assayed in the methods defined herein, namely epithelial to mesenchymal transition CTCs (EMTCTCs). EMT is a gradual morphogenetic process, and EMTCTCs encompass cells in various stages of transition[6]. EMTCTCs can be generally described by the down regulation of epithelial proteins, e.g. EpCAM and CK, and the upregulation of mesenchymal stem cell proteins, e.g. vimentin and CD34 l13l EMTCTC subtyping is typically performed using non-proteomic methods, i.e. mRNA expression or DNA analysis

[0013] .

[0054] A further circulating cell type associated with cancer that may be assayed in the methods defined herein is the cancer-associated vascular endothelial cell or CAVE. CAVEs are a subtype of circulating endothelial cells. Tumors require blood supply provided by tumor endothelial cells. CAVES are tumor endothelial cells that have broken off from the tumor site into the blood stream. CAVEs are often found in clusters. CAVEs express cytokeratin and various subtypes endothelial cell markers such as CD31, CD146, CD144, CD105, but do not express CD 14 or CD45 '34L

[0055] The utilization of such circulating cells has not been well studied in BBBs and the present invention is directed to methods of using CTCs, CAMLs, CAVEs and EMTCTCs in the screening, monitoring, diagnosis and treatment of different cancers, in particular those cancers in which associated CTCs, CAMLs, CAVEs and EMTCTCs express PD-L1 on their surface, .

[0056] The utilization of such circulating cells has not been well studied in BBBs and the present invention is directed to methods of using CTCs, CAMLs, CAVEs and EMTCTCs in screening, monitoring, and diagnosing cancer in a subject, based on the presence or absence or change in expression of biomarkers, such as PD-L1, on these cells. The invention also relates to methods of making treatment decisions based on the presence or absence or change in expression of biomarkers, such as PD-L1, on these cells. The invention further relates to methods of predicting survival of a subject having cancer based on the presence or absence or change in expression of biomarkers, such as PD-L1, on these cells.Methods of Monitoring Probability of PFS and / or OS

[0057] As indicated above, the invention is drawn to methods of monitoring probability of progression free survival (PFS) and / or overall survival (OS) in a subject having cancer. The methods comprise (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, probability of PFS and / or OS in the subject is increased and when PD-L1 expression decreases between the first and second time point, probability of PFS and / or OS in the subject is decreased.Methods of Determining Probability of PFS and / or OS

[0058] The invention is also drawn to methods of determining probability of PFS and / or OS in a subject having cancer. The methods comprise (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, probability of PFS and / or OS in the subject is determined to be increasing and when PD-L1 expression decreases between the first and second time point, probability of PFS and / or OS in a subject is determined to be decreasing.Methods for Making Treatment Decisions

[0059] In addition, the invention is drawn to methods of making treatment decisions in a subject having cancer. The methods comprise (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein a cancer treatment is administered to the subject between the first and second time point, and wherein when PD-L1 expression decreases between the first and second time point, an alternative treatment is administered to the subject. In certain aspects of this embodiment, the alternative treatment is not an ICI.

[0060] In a related embodiment, the invention is drawn methods of making treatments decision for an immune checkpoint inhibitor in a subject having cancer. The methods comprise (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, an immune checkpoint inhibitor is indicated for administration to the subject.Methods of Predicting Therapeutic Benefit of Immune Checkpoint Inhibitors

[0061] The invention is also drawn to methods of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer. The methods comprise (a) assaying oneor more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein a cancer treatment is administered to the subject between the first and second time point, and wherein when PD-L1 expression increases between the first and second time point, administration of an immune checkpoint inhibitor after the second time point is predicted to be beneficial for the subject.

[0062] In a related embodiment, the method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression is high at the first and second time point, administration of an immune checkpoint inhibitor after the second time point is predicted to be beneficial for the subject.

[0063] In a further related embodiment, the method comprises (a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and (b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression is low at the first time point and high at the second time point, administration of an immune checkpoint inhibitor is predicted to be beneficial for the subject.

[0064] In each of the embodiments and aspects of the invention related to methods of treatment, the methods can be practiced using immune checkpoint inhibitors alone or practiced in conjunction with additional means for treating and inhibiting cancer in a subject (e.g., the additional anti-cancer agents defined herein). Such additional means will be well known to the skilled artisan and include, but are not limited to means such as anti-cancer chemotherapeutics and radiotherapeutics and surgical removal of a tumor.

[0065] As used herein, the terms “treat”, “treating” and “treatment” have their ordinary and customary meanings, and include one or more of complete or partial clearance of a tumor or cancer from a subject, reducing the size of a tumor in a subject, killing cells of a tumor or cancer in a subject, and ameliorating a symptom of cancer or a tumor in a subject. Treatment means clearing, reducing, killing or ameliorating by about 1% to about 100% versus a subject to which an immune checkpoint inhibitor has not been administered. Preferably, the clearing, reducing,killing or ameliorating is about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5% or about 1%. The results of the treatment may be permanent or may continue for a period of days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1, 2, 3 or 4 weeks), months (such as 1, 2, 3, 4, 5, 6 or more months) or years (such as 1, 2, 3, 4, 5, 6 or more years).

[0066] As used herein, the terms “inhibit”, “inhibiting” and “inhibition” have their ordinary and customary meanings, and include one or more of, hindering, impeding, obstructing, deterring or restraining establishment of cancer or a tumor, development of cancer or a tumor, growth of cancer or a tumor and metastasis. Inhibition means hindering by about 1% to about 100% versus a subject to which an immune checkpoint inhibitor has not been administered. Preferably, the hindering is about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5% or about 1%. The methods of inhibition may be practiced in a subject prior to, concurrent with, or after the onset of clinical symptoms of cancer or a tumor. Thus, the subject may have cancer or a tumor, or merely be susceptible to developing cancer or a tumor. The results of the inhibition may be permanent or may continue for a period of days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1, 2, 3 or 4 weeks), months (such as 1, 2, 3, 4, 5, 6 or more months) or years (such as 1, 2, 3, 4, 5, 6 or more years).

[0067] The immune checkpoint inhibitors and pharmaceutical formulations comprising immune checkpoint inhibitors may be administered to a subject using different schedules, depending on the particular aim or goal of the method; the age and size of the subject; and the general health of the subject, to name only a few factors to be considered. In general, the immune checkpoint inhibitors and pharmaceutical formulations may be administered once, or twice, three times, four times, five times, six times or more, over a course of treatment or inhibition. The timing between each dose in a dosing schedule may range between days, weeks, months, or years, an includes administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more weeks. The same quantity of immune checkpoint inhibitor may be administered in each dose of the dosing schedule, or the amounts in each dose may vary. The identity of the immune checkpoint inhibitor may also vary or remain the same in each dose in a dosing schedule.

[0068] In each of the methods of the present invention, a “therapeutically effective amount” of an immune checkpoint inhibitor or pharmaceutical formulation comprising an immune checkpoint inhibitor is administered to a subject. The therapeutically effective amount will vary between subjects. However, the therapeutically effective amount is one that is sufficient to achieve the aim or goal of the method, whether inhibiting or treating. As an example, a therapeutically effective amount of an immune checkpoint inhibitor used in the methods of the invention is typically between about 0.1 pg to about 10,000 pg of immune checkpoint inhibitor per kg of body weight of the subject to which the peptide is administered. A therapeutically effective amount also includes between about 0.5 pg to about 5000 pg, between about 1 pg to about 500 pg, between about 10 pg to about 200 pg, between about 1 pg to about 800 pg, between about 10 pg to about 1000 pg, between about 50 pg to about 5000 pg, between about 50 pg to about 500 pg, between about 100 pg to about 1000 pg, between about 250 pg to about 2500 pg, between about 500 pg to about 2000 pg, between about 10 pg to about 800 pg, between about 10 pg to about 1000 pg, between about 1 pg to about 300 pg, and between about 10 pg to about 300 pg of immune checkpoint inhibitor per kg of body weight of the subject.

[0069] Appropriate doses and dosing schedules can readily be determined by techniques well known to those of ordinary skill in the art without undue experimentation. Such a determination will be based, in part, on the tolerability and efficacy of a particular dose.

[0070] Administration of the immune checkpoint inhibitor or pharmaceutical formulation may be via any of the means commonly known in the art of peptide delivery. Such routes include intravenous, intraperitoneal, intramuscular, subcutaneous and intradermal routes of administration, as well as nasal application, by inhalation, ophthalmically, orally, rectally, vaginally, or by any other mode that results in the immune checkpoint inhibitor or pharmaceutical formulation contacting mucosal tissues.

[0071] The pharmaceutical formulations of the invention comprise one or more immune checkpoint inhibitors and a pharmaceutically acceptable carrier. Suitable examples of carriers are well known to those skilled in the art and include water, water-for-inj ection, saline, buffered saline, dextrose, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), pol oxamer 407 and 188, hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids,polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terms specifically exclude cell culture medium. The formulations may further comprise stabilizing agents, buffers, antioxidants and preservatives, tonicity agents, bulking agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, and combinations thereof.Immune Checkpoint Inhibitors

[0072] As used herein, the term “immune checkpoint inhibitor” refers to a compound, such as a drug (including an antibody), that inhibits or blocks proteins expressed by cells of the immune system, such as T cells, and some types of cancer cells. These proteins inhibit immune responses and they can block T cells from killing cancer cells. When these proteins are blocked, inhibition of the immune system is overcome and T cells are able to kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7- 1 / B7-2. Immune checkpoint inhibitors thus seek to overcome one of cancer’s main defenses (i.e., T cells) against an immune system attack.

[0073] The immune checkpoint inhibitors of present invention include, but are not limited to, PD-L1 antagonists, PD-1 antagonists, and CTLA-4 antagonists.

[0074] The immune checkpoint inhibitors of present invention also include, but are not limited to, inhibitors one or more of (i) binding between PD-L1 and PD-1, (ii) binding of PD-L1 to its binding partner(s), (iii) binding of PD-1 to its binding partner(s), and (iv) binding of CTLA-4 to its binding partner(s).

[0075] The immune checkpoint inhibitors of present invention further include, but are not limited to, antibodies, such as monoclonal antibodies. In particular aspects, the immune checkpoint inhibitor is a human antibody, a humanized antibody, or a chimeric antibody. The immune checkpoint inhibitors also include fragments of antibodies that retain their inhibitory activity. Such antibody fragments include, but are not limited to, Fab fragments, F(ab')2 fragments, and single chain Fv (scFv). In one aspect, the immune checkpoint inhibitor is an antibody having binding specificity for PD-L1, PD-1 or CTLA-4, or antibody fragment thereof.

[0076] Examples of specific immune checkpoint inhibitors include, but are not limited to, one or more of Nivolumab (Opdivo), Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Atezolizumab (Tecentriq), Tremelimumab, Durvalumab (MED14736), and Retifanlimab.Cancer Treatments

[0077] In the embodiments and aspects of the invention directed to the treatment of cancer, the methods may include the administration of a therapeutically effective amount of one or more cancer treatments to the subject in addition to the immune checkpoint inhibitors.

[0078] The cancer treatments are only limited in that they be compatible with the immune checkpoint inhibitors that are also administered to the subject.

[0079] Additional cancer treatments include, but are not limited to, immunotherapeutic agents, chemotherapeutic agents, radiotherapeutic agents, existing cancer drugs, CCR5 and CXCR4. Examples of specific anti-cancer agents include, but are not limited to, one or more of T-VEC, AM-0010, CXCR4 antagonist, TGF -beta kinase inhibitor galunisertib, anti-CSF-lR monoclonal antibody, Abemaciclib, Faslodex, necitumumab, AZD9291, Cyramza (ramucirumab), TPIV 200, Galunisertib, cancer vaccines, cytokines, cell-based therapies, bi- and multi-specific antibodies, tumor-targeting mAbs, Rituximab, oncolytic viruses, reovirus, Blinatumomab, Sipuleucel-T, T-Vec, IL-2, IFN-a, Trastuzumab, Celuximab, bevacizumab, Tim- 3, BTLA, anti-IL-10, GM-CSF, anti-angiogenesis treatment, VEGF blockade, HMGB1, Nrpl, TAM receptor tyrosine kinases, Axl , MerTK, ALT-803, IL-15, Immunosuppressive Ligand Phosphatidyl serine (PS), bavituximab, bevacizumab (anti-VEGF), coblmetinib (MEK inhibitor), vemurafenib (BRAF inhibitor), erlotinib (EGFR), alectinib (ALK inhibitor), bevacizumab (anti- VEGF), pazopanib (tyrosine kinase inhibitor), dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), sunitinib (RTK inhibitor), pazopanib (RTK inhibitor), sargramostim, VISTA, TIM-3, LAG-3, PRS-343, CD137 (4-lBB) / HER2 bispecific, USP7, anti-HER2, SEMA4D, CTLA-4, PD-1, PD-L1, and PD-L2.Means for Assaying PD-L1 Expression

[0080] As will be apparent, the methods of the present invention are based on assaying, i.e. detecting and / or measuring PD-L1 expression in a cell. In one aspect of the invention, each of the methods defined herein can be used by simply determining whether a selected cell expresses PD-L1. Thus, these methods can be performed without the need to quantify the amount of PD-L1 expression in a cell. However, and in another aspect of the invention, each of the methods defined herein can be used by determining the relative or specific amount of PD-L1 expression by a cell. The relative amount may be determined, for example, by determining whether a cellexpresses more or less PD-L1 than another cell or standard. The specific amount may be determined, for example, by quantifying the level of PD-L1 expression in a cell.

[0081] The methods of the invention may be practiced by assaying PD-L1 expression at two separate time points. Thus, the method can be a simple assay of whether PD-L1 expression increases or decreases between two time points. The invention also encompasses methods based on assaying PD-L1 expression at one or more additional time points, such as 2, 3, 4, 5, 6, or more time points. Indeed, PD-L1 can be monitored in a continuous manner, such as once a day for one or more days, one or more weeks, or one or months.

[0082] PD-L1 expression may be assayed by one or more of detecting / measuring PD-L1 protein expression and detecting / measuring PD-L1 mRNA production. PD-L1 protein expression may be detected / measured / assayed, for example, via immunohistochemistry (IHC). For the sake of convenience, reference herein to immunohistochemistry (IHC) includes both immunochemistry performed on tissue, as well as immunochemistry performed on isolated cells (immunocytochemistry). IHC may be performed by membrane staining, cytoplasmic staining, or a combination thereof. IHC may be performed using an anti-PD-Ll antibody, such as, but not limited to, E1L3N, SP142.2, 28-8, 22C3, EPR19759, MIH2, MIH5, MIH6, ABM4E54, 130021, EPR20529, 10F.9G2, and CD274. PD-L1 protein expression may be detected / measured as a weak staining intensity, moderate staining intensity, or strong staining intensity. PD-L1 protein expression may also be detected as a low staining intensity, moderate staining intensity, or high staining intensity. PD-L1 protein expression may also be detected as inducible from low staining intensity to high staining intensity over time, or inducible from low staining intensity to moderate staining intensity over time, or inducible from moderate staining intensity to high staining intensity over time. PD-L1 protein expression may also be detected / measured simply as any staining whatsoever of the isolated cells, for example - any amount of staining above background.

[0083] In certain aspects, IHC is performed using immunofluorescence (IF) staining. One or more antibodies with binding specificity for PD-L1 may be utilized to detected PD-L1 protein expression. Binding of the anti-PD-Ll antibody to PD-L1 may be detected via a fluorescent compound or other detectable label conjugated to the anti-PD-Ll antibody or it may be detected via a fluorophore or other detectable label conjugated to a secondary antibody that, in turn, has binding specificity for the anti-PD-Ll antibody.

[0084] In certain of the relevant embodiments and aspects defined above, PD-L1 expression is determined to be detected when the level of PD-L1 expression is greater than PD-L1 expression is a population of stromal cells from a subject of the same species that does not have cancer.

[0085] In certain of the relevant embodiments and aspects defined above, PD-L1 expression is determined based on PD-L1 pixel intensity expressed by a cell as measured by the ZenBlue software using the area of the entire cell. The average pixel intensity of each cell is subtracted from the average pixel intensity of the local background for each image. The average pixel intensity of the cells may be quartiled into four different IHC groups and defined as 0-negative (pixel average 0-150), 1-low (pixel average 151-300), 2-medium (pixel average 301-750), and 3- high (pixel average 751+). Thus, for example, the amount of PD-L1 expressed by a CTC, CAML, or other cell expressing PD-L1 can be characterized as “low” when the pixel average is between 151-300, and the amount of PD-L1 expressed by a CTC, CAML, or other cell expressing PD-L1 can be characterized as “high” when the pixel average is greater than 751 as measured by the ZenBlue software.Source of Cells

[0086] The cells used in the methods of the present invention include one or more of CTCs, EMTCTCs, CAMLs, and CAVEs. Thus, the methods may be performed using one, two, three or all four of these types of circulating cells.

[0087] The cells may be obtained from any bodily fluid in which the cells can be found, including blood, such as peripheral blood. Blood samples may be collected in CellSave™ preservative tubes for example, and the blood may be processed with a Cell Sieve™ Microfdtration Assay using a low-pressure vacuum system, for example.Subjects

[0088] The subjects mentioned in the methods of the present invention will be a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.

[0089] The subject having cancer may be undergoing treatment for the cancer. Such treatments include, but are not limited to targeted agents, chemotherapy, and radiation therapy.The cancer may be one or more of lung cancer, breast cancer, prostate cancer, pancreatic cancer, melanoma, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, neuroblastoma, sarcoma, osteosarcoma, esophageal, brain & ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, uterine cervix, or uterine corpus cancer. The cancer may be a solid tumor, such as solid tumor of a stage I, stage II, stage III or stage IV cancer. The solid tumor may be, but is not limited to, carcinoma, sarcoma, neuroblastoma or melanoma. Example of lung cancers include, but are not limited to, non-small cell lung carcinoma (NSCLC).III. ExamplesCellSieve™ Low-Flow Microfdtration procedure

[0090] Blood samples (7.5 mL) collected in CellSave™ preservative tubes were processed with a CellSieve™ Microfiltration Assay using a low-pressure vacuum system[1, 12]. The CellSieve™ Microfiltration Assay isolates circulating cells based on size exclusion, >7 micron. A trained cytologist identified prognostically relevant pathologically definable CTCs (PDCTCs), epithelial to mesenchymal transition CTC cells (EMTCTCs), and CAMLs based on morphological features and the phenotypic expression of CD45, EpCAM, Cytokeratins 8, 18, 19 and DAPI (Figures 1 and 2)[1-6-12]using pre-established cytological features describedn> 14]. An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam and Zen2011 Blue (Carl Zeiss) was used for all imaging.Enumerating PDCTC / EMTCTC subtypes and CAMLs

[0091] The defining characteristics of the two most common CTC subtypes found in cancer patients (PDCTCS and EMTCTCs) and those for CAML identification were previously described[1, 6, 10'14]. Intact PDCTCs, EMTCTCs, and CAMLs were characterized (Figures 2 and 3)[1-6’10'14]. PDCTCs are CD45 negative, with filamentous cytokeratin positivity and DAPI positive nuclei with malignant pathological criteria, classified as the Cell Search® subtype of CTC[1’6’10'14]. EMTCTCs are CD45 negative with a diffuse cytokeratin signal and a DAPI positive nucleus with abnormal criteria, as previously defined[1, 6'9’12, 13]. CAMLs are described as enlarged (>30 pm), multinuclear cells with diffuse cytoplasmic cytokeratin staining, and / orCD45+ / CD14+|6‘n’14, 17, 18’311 All three cell types were identified and imaged by a trained CTC cytologist and confirmed by a pathologist. Apoptotic CTCs and CTCs that could not be cytologically classified as previously described were not included. After identification, cells were imaged and x-y axis of each cell was marked for future analysis. Samples were archived at 4°C for 1-3 years.QUAS-R quenching and restaining for PD-L1 and RAD50

[0092] After initial identification and quantification of PDCTCs, EMTCTCs and CAMLs, fluorescence was quenched and samples were restained with RAD50-DyLight 550 (Pierce Thermo), PD-L1 -Al exaFluor 488 (R&D systems) and DAPI nuclear stain (Figure 1). The QUAS-R (Quench, Underivatize, Amine-Strip and Restain) technique was used as previously described

[0013] . Briefly, after samples were imaged and marked filters were subjected to a sequential chemical treatment of quenching solution, Tris, and wash steps. After chemical quenching, filters were washed with PBS, incubated with 1XPBS / 2O%FBS and then incubated with antibodies against RAD50-AlexaFluor550 and PD-L1 -Al exaFluor 488 for 1 hour at room temp. After antibody incubation, filters are washed in IxPBST and slide mounted with Fluoromount-G / DAPI (Southern Biotech). Samples were oriented along the x / y axis and previously imaged cells were relocated using a Zen2011 Blue (Carl Zeiss) mark and find software. A Zen2011 Blue (Carl Zeiss) was used to process the images.Quantifying PD-L1 in primary tumor biopsies

[0093] PD-L1 expression from all available primary tumor biopsies were analyzed using both DAKO pharmdx clone 22c3 and DAKO pharmdx clone 28-8 according to manufacturer’s guidelines (Figure 4). Eight patients from the study had sufficient and available archived tumor samples to screen both clones and one sample had sufficient archived tumor for a single IHC test against clone 22c3. Both clones were stained according to standard operating procedures previously described I20-22-28].Quantifying RAD50 and PD-L1 in circulating cells

[0094] RAD50 loci formation was determined by enumerating the nuclear localized RAD50 loci in each cell (Figures 1 and 5)

[0019] . PD-L1 pixel intensity of each cell was measured by theZenBlue software by using the area of the entire cell. The average pixel intensity of each cell was subtracted from the average pixel intensity of the local background for each image (Figure 1C). The average pixel intensity of the cells was quartiled into 4 IHC groups: O-negative (pixel average 0-150), 1-low (pixel average 151-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+) (Figure 5). IHC range thresholds of PD-L1 intensity for IHC scoring were determined as: 150 pixel intensity was the standard deviation of the localized background signal, 300 pixel intensity was 2 times the standard deviation of the localized background, and 750 was two times the intensity of the localized background (Figure 5).Statistical Methods

[0095] Analyses were done in MATLAB R2013A using the counts from all subtypes and the known patient populations. For progression free survival analysis, the time to progression was defined as the interval between when TO blood sample was obtained to date of progression. Significance of the average changes in RAD50 foci formation and PD-L1 expression were determined by a Student’s T-test. A Pearson coefficient was used to determine the correlation between RAD50 foci and PD-L1 expression for individual measurements. Significance of Kaplan Meier plots were determined by log-rank analysis.Experiment 1

[0096] Forty-one patients with stage I-IV lung cancer were included in this prospective pilot study (Table 1). Anonymized peripheral blood samples were collected after written informed consent and according to the local IRB approval. Patients were recruited from July 2013 to May 2014 prior to starting radiotherapy for primary lung cancer. Four patients received Stereotactic Body Radiation Therapy (SBRT) for stage I disease and 37 patients received chemoradiation for stage II-IV disease with proton therapy (n=16) or Intensity-modulated radiation therapy (IMRT) (n=21). Anonymized blood samples (7.5 mb) were drawn and processed on site at the MD Anderson Cancer Center (MDA). Slides were anonymized then shipped and analyzed at Creatv MicroTech, Inc.’s clinical core laboratory. Anonymized biopsy samples from primary tumors were processed at MDA according to manufacturer’s protocols (DAKO). Results from institutions were not shared or communicated until completion of study.Table 1. Patient population overviewPDCTCs, EMTCTCs and CAMLs in LC patients

[0097] Prior reports indicated that the CTC subpopulation in NSCLC patients using the CellSearch® platform is typically found in only 0-5% of non-metastatic cases. In contrast, the EMTCTC population is typically found in -80% of non-metastatic patient populations, while CAMLs have not been extensively evaluated in NSCLC|3-6‘8’15‘17, 31’33]. In the first baseline blood sample taken prior to start of radiation therapy (TO) at least one cytokeratin positive cell (i.e. PDCTC, EMTCTC or CAML) (Figure 1 and 3) in 35 of the 41 samples (85%) was identified.

[0098] Patients then had a second follow up sample (Tl) taken 2-3 weeks after radiotherapy initiation or after the last fraction for SBRT patients. For Tl, there was at least one cytokeratin positive cell (i.e. PDCTC, EMTCTC or CAML) found in all 41 samples (100%). Specifically, EMTCTCs were found in 49% of TO samples and in 66% of T l samples. CAMLs were found in 81% of TO samples and in 100% of Tl samples (Figure 3). PDCTCs were found in only 1 sample at TO (2%) and in only 3 samples at Tl (7%) (Figure 3). Being that PDCTCs have been shown to be the same CTC population of cells isolated by the CellSearch® CTC System, these numbers are on par with previous reports[7'9, 15]. The CellSearch® system isolates CTCs in NSCLC patients ranging from -0-5% positivity in stage III NSCLC and 21-32% in stage IV[7'9,15]. As 35 of the patients were staged as I-III, 2-7% is within the range of the classical CTCpopulation|7‘9‘15]. The low incidence of the classical PDCTC population (Figure 3) is in contrast to EMTCTC and CAMLs which are present in 85% (TO) and 100% (Tl) of the samples. While it has been postulated that EMTCTCs alone may provide some increased sensitivity for liquid biopsies in NSCLC[7'9, 16], these results suggest that the combination of both EMTCTCs and CAMLs provides improved sensitivity analyzing tumor derived cells for blood based diagnosis.Comparison of PD-L1 levels in the primary tissue, CTCs and CAMLs

[0099] Available tissues from the original diagnostic biopsy were stained by IHC using 2 commercially available and CLIA-certified tests using clones 22c3 and 28-8 (DAKO). Useable tissue or cell blocks were only able to be retrieved from pathologic archives in 9 of 41 patients, and 1 of these 9 patients only had sufficient tissue for one IHC test (Figure 4). This was a result of tumor necrosis or small nodules resulting in insufficient mass to perform the PD-L1 IHC testing. Of the 9 archival samples, only 2 had positive PD-L1 staining with some variability in the expression scores and percentages between the 2 tests (Figure 4C). In comparison, PD-L1 expression was quantifiable in 85% of TO patient samples (n=35 / 41) and 100% (n=41 / 41) in Tl patient samples using the BBB. Specifically at TO, EMTCTCs and CAMLs showed low / negative (score 0 / 1) PD-L1 expression in 21 patients (60%), medium (score 2) expression in 9 patients (26%) and high (score 3) expression in 5 patients (14%) (Figure 4C).

[0100] At TO, expression of PD-L1 in the circulating cells closely paralleled the IHC biopsy results for 2 IHC positive stained samples using the 28-8 IHC clone results (Figure 4C). Three patients had concordant negative PD-L1 tissue by IHC and low (0 / 1) expression on circulating cells, but 3 patients had discordant results with negative tissue IHC PD-L1 but 2 / 3 scores on the circulating cells, and 1 patient lacked circulating cells in the TO sample (Figure 4). Given the limited number of samples, a proper statistical analysis was not possible. However, these results suggest primary biopsies inconsistently provide sufficient tissue for assaying PD-L1 expression while a blood based biopsy (BBB) approach could measure intrinsic levels and monitor changes of PD-L1 expression in circulating cells originating from cell populations found at the primary lung tumor.Dynamic expression of PD-L1 in circulating cells

[0101] There have been suggestions that PD-L1 may be induced in tumors by various cytotoxic therapies, including radiation|20'25'26'30’441. To determine if this could be seen using a blood based biopsy (BBB), PD-L1 staining was evaluated at the TO and T1 time points. A normalized comparative scoring system was developed in a similar manner to the classical 0-3 PD-L1 expression IHC tissue biopsy scoring (Figures 5 and 6). After staining and imaging, PD- L1 expression and the local background for all 373 cells found in LC patients were measured. The local background of each image averaged 375±150 pixel intensity (Figure 5). To account for the localized background effect, the background of each image was subtracted from each measured cell, yielding a corrected PD-L1 pixel intensity range of 17-3090 (Figure 5). The cells were then grouped with the corrected pixel intensities using the standard deviation of background of 0-150 pixels as a score of 0 (26% of cells) and 2 times the standard deviation (151-300 pixels) as a score of 1 (low expression, 42% of cells). Medium expression, a score of 2 (22% of cells) was determined as being 2 times the mean background signal (301-750 pixel) and high expression or score of 3 (10% of cells) was set at >2 times the mean background signal (>750 pixels) (Figure 5).

[0102] Pixel intensity of PD-L1 in EMTCTCs averaged 384±484 at TO and 672±669 at T1 (p=0.021), while CAMLs had an average of 182±89 at TO and 282±169 at T1 (p=0.004) (data not shown). Regression analysis found a weak, but significant, positive correlation between RAD50 and PD-L1 from TO to T1 (Pearson R2=0.079, p<0.0001, n=373). While RAD50 was reliably induced from TO to T1 among patients, changes in PD-L1 expression in individual patients was far more variable (data not shown). Three distinct patterns of PD-L1 expression were found between TO and T1 in the 35 patients who were assessable for both time points. Eighteen patients (51%) had no / low PD-L1 expression at both time points, 6 patients (17%) had persistently medium / high PD-L1 at both time points and 11 patients (32%) saw an increase from a low 0 / 1 score to a 2 / 3 score (data not shown).Experiment 2A

[0103] As indicated above, PD-L1 can be expressed on circulating tumor cells (CTCs), cancer associated macrophage-like cells (CAMLs), and epithelial to mesenchymal transitionCTCs (EMTCTCs) isolated from subjects having cancer. Tt may also be expressed on cancer associated vascular endothelial cells (CAVEs) (data not shown).

[0104] Moreover, other experiments suggested that PD-L1 expression on CTCs, CAMLs, and EMTCTCs changed over time from a baseline at the start of treatment in patients that received immunotherapy simultaneously with chemoradiation therapy.

[0105] In present experiment, the concept that other cancer therapies might also alter PD-L1 expression was investigated. As discussed below, these experiment shows that PD-L1 expression changed for a percentage of breast cancer patients treated by a combination of a cancer vaccine with a check point inhibitor. Similarly, PD-L1 expression changed for a variety of cancer patients who received different therapies in combination with immunotherapy. Furthermore, those patients whose PD-L1 expression was determined to be high or increased had a higher degree of progression free survival (PFS) and overall survival (OS) compared to patients whose PD-L1 expression was determined to be low. This data can be used in methods of predicting PFS and OS in subjects having cancer.

[0106] As reported above in Experiment 1, PD-L1 expression of NSCLC patients changed after treatment by chemoradiation. In the present experiment, local / locally advanced NSCLC patients (n=96) were treated with one of the immunotherapy drugs (Atezolizumab, Durvalumab or Pembrolizumab) in combination with chemoradiation. Blood samples were taken at three different time points: Baseline (before therapy), just after chemoradiation, and approximately 30 days after chemoradiation. Figure 7A is a plot of progression free survival (PFS) at baseline. Low expression is the solid line, and high expression is the dashed line. At baseline, PFS of high or low PD-L1 expression did not stratify the responders. Figure 7B is a plot of PFS just after chemoradiation. PFS of high PD-L1 is improving. Figure 7C is the plot of PFS approximately 30 days after chemoradiation. There is a significant difference in PFS between the high and low PD- L1 expressing patients at this time point. The explanation is that chemoradiation changed PD-L1 expression of many patients. A few high PD-L1 expression patients at baseline become low PD- L1 after chemoradiation. In contrast, many more low PD-L1 expression patients at baseline become high PD-L1 after chemoradiation. Thus, monitoring PD-L1 over time is important. A change in PD-L1 expression due to chemoradiation was found for other cancers (data not shown).Experiment 2B

[0107] Other therapeutic agents were tested in further experiments. Breast cancer vaccine SV-BR-l-GM, in combination with check point inhibitor Retifanlimab, an immunotherapy drug, were used to treat breast cancer. In more detail, SV-BR-l-GM treatment includes low pre-dose cyclophosphamide, intradermal inoculation of ~20 million irradiated SV-BR-l-GM cells, postdose local interferon-a and an anti-PD-1 inhibitor (Retifanlimib), with cycles every 3 weeks. Blinded anonymized blood samples were taken at baseline (BL), prior to starting SV-BR-l-GM therapy (n=44), a 2nd sample (Tl) taken after therapy initiation (~23 days) and if possible, a third sample (T2) taken at the standard tumor assessment (~75 days).

[0108] To evaluate the predictive value of CTCs / CAMLs and their PD-L1 expressions, cells were isolated and quantified using the LifeTracDx® liquid biopsy test. The quantities of CTCs & CAMLs and their respective PD-L1 expression were analyzed based on PFS using RECIST vl.l and OS hazard ratios (HRs) by censored univariate analysis at 24 months.

[0109] Blood samples were available from 93% (n=41 / 44) of all patients. At BL, CTCs were found in 42% (n=17 / 41) and CAMLs in 90% (n=37 / 41). BL CTCs predicted significantly worse OS, but not PFS (Table 2). Tl samples were available from 88% (n=36 / 41) of patients.Decreases in number of CTCs or CAMLs after SV-BR-l-GM therapy were seen in 40% of patients, correlating with better PFS. PD-L1 in CTCs or CAMLs at BL was not associated with improved clinical responses (Table 2).Table 2. Hazard ratio comparisons of CTCs and CAMLs after SV-BR-l-GM therapy

[0110] An increase in PD-L1 in CTCs or CAMLs after SV-BR-l-GM therapy was seen in 42% (n=15 / 36) of patients, correlating with better PFS (Figure 8A) and OS (Figures 8B).

[0111] SV-BR-l-GM therapy thus appeared to upregulate PD-L1 in n=15 patients which correlated with better responses to combination treatment with the anti-PD-1 check point inhibitor Retifanlimab.Experiment 2C

[0112] Additional data on change of PD-L1 expressions for a variety of cancers treated by a variety of therapies in combination with immunotherapies was obtained. A pilot study to monitor the peripheral blood of metastatic cancer patients undergoing systemic treatment with immunotherapy in combination with other therapies, was evaluated for PD-L1 expression on CAMLs prior to and post immunotherapy induction with clinical correlation at 24 months.

[0113] In particular, metastatic cancer (n=46), breast cancer (n=14), non-small cell lung cancer (n=24), and renal cell cancer (n=8) metastatic cancer, all starting new lines of systemic chemotherapy in combination with immunotherapy (pembrolizumab [n=30], nivolumab [n=13], Durva [n=l ], or atezolizumab [n=3]) for newly diagnosed recurrent metastasis (n=24) or with previously treated progressive metastatic disease (n=22) was assayed. CAMLs were isolated from 7.5 ml of blood at baseline (TO) using the LifeTracDx® PD-L1 test and scored PD-L1 as high or low in the manner described above. If possible, a follow-up sample (Tl) was taken (~48 days) after the induction of immunotherapy. Patient’s PFS and OS were analyzed by censored univariate analysis based on RECIST vl.l over two years.

[0114] TO PD-L1 CAML data was available for 89% (n=41 / 46) of patients, with 39% (n=l 6 / 41) having high CAML PD-L1 which was not correlated with improved PFS (HR=0.9, p=0.985, CI=0.5-1.9) or OS (HR=1.0, p=0.899, CI=0.4-2.2).

[0115] Tl PD-L1 CAML data was available for 74% (n=34 / 46) of patients, with 53% (n=18 / 34) having high CAML PD-L1, which significantly correlated with improved PFS (HR=2.9, p=0.012, CI=1.3-6.4) and OS (HR=5.5, p=0.0005, CI=2.2-13.6).

[0116] In comparing CAML PD-L1 change post immunotherapy, it was determined that patients with consistently low PD-L1 at TO & Tl had the poorest responses, median PFS (mPFS)=2.3 months and median OS (mOS)=5.5 months. In contrast, patients with consistently high PD-L1 at TO & Tl had better responses, mPFS=6.1 months and mOS=16.7 months. Further, patients who increased in CAML PD-L1 after immunotherapy also had good response rates, mPFS=10.3 months and mOS=14.8 months.

[0117] This data suggests that any therapy for cancer may have the potential of changing the PD-L1 expression on the tumor cells. The LifeTracDx® PD-L1 test can monitor dynamic changes in PD-L1 in circulating tumor immune cells and predicts clinical benefit to PD-L1 immunotherapies in an array of cancer types, in combination with other therapies.Experiment 3

[0118] Metastatic triple negative breast cancer (mTNBC) is an aggressive subtype of breast cancer that has poor clinical outcomes. In previously treated recurrent mTNBC, median overall survival (mOS) and percent survival at 3 years was determined to be 6.6 months and 1.9% for 3rdline chemotherapy, -11.8 months and 10.5% for >2 line sacituzumab govitecan, and -23 months and 22% for 1stline pembrolizumab + chemotherapy.

[0119] CCR5 is a G protein coupled receptor that is overexpressed in -95% of mTNBC and has been identified as a potential drug target, blocking tumor motility and spread. Leronlimab is a humanized monoclonal antibody that binds to and inhibits CCR5, blocking CCR5-mediated function independently of hormone status.

[0120] A retrospective follow-up was conducted on FDA Phase I clinical trials where 28 patients with mTNBC were treated with leronlimab across three clinical trials ((NCT03838367, N=10), (NCT04313075, N=16), (NCT04504942, N=2)). Treatment on NCT03838367 included leronlimab with carboplatin. NCT04313075 and NCT04504942_allowed physician’s choice treatment. Leronlimab was administered weekly (350 mg (n=10), 525 mg (n=l 5) or 700 mg (n=3)). 7 patients were treated with leronlimab in combination with atezolizumab (n=4), or subsequently with pembrolizumab (n=2) or nivolumab (n=l).

[0121] In the retrospective follow-up, LifeTracDx® was used to analyze PD-L1 expression on CTCs and CAMLs as described above. The cells analyzed had been collected at baseline and at 30-90 days after the start of therapy. The microscope signal for PD-L1 fluorescent channel is in the AF555 channel. For the microscope and filter cubes utilized, the PD-L1 fluorescent channel with signal was: 1) 0-399 is PD-L1 low, 2) 400-799 is PD-L1 medium, and 3) > 800 is PD-L1 high. The signal ranges will change base on the microscope, fluorescent cube and the level of the filter background.

[0122] Upregulation of PD-L1 in circulating cells (i.e. CTCs or CAMLs) was identified in 81% (n=17 / 21) of patients after leronlimab, and in 88% (n=15 / 17) of patients who received a 525 mg or 700 mg dose (Figure 9).

[0123] 7 patients were treated with an immune checkpoint inhibitor (ICI) concurrently or subsequently with leronlimab. Of these patients, 2 were found to express low levels of PD-L1 in their CTCs / CAMLs, while 5 patients were found to express either medium or high levels. N=5 / 5 patients who induced medium or high level PD-L1 expression in their CTCs / CAMLs and were treated with an immune checkpoint inhibitor (ICI) concurrently or subsequently with leronlimab were alive after 48 months (these n=5 patients had 4 median lines of any prior therapy). (Figures 9 and 10). The data in Figures 9 and 10 indicates that blood test for PD-L1 expression is predictive and accurate for survival.Experiment 3

[0124] A pilot study to monitor the peripheral blood of n=46 metastatic cancer patients undergoing systemic treatment with immunotherapies (IMT) in combination with other therapies was undertaken to evaluate CAML PD-L1 expression prior to and post IMT induction with clinical correlation at 2 years.

[0125] In the study, breast (n=14), non-small cell lung (n=24), and renal cell (n=8) cancer patients were starting new lines of systemic chemotherapy in combination with IMT (pembrolizumab [n=30], nivolumab [n=13], Durva [n=l], or atezolizumab [n=3]) for newly diagnosed recurrent metastasis (n=24) or for previously treated progressive metastatic disease (n=22). See Table 3.Table 3. Clinical Parameters

[0126] CAMLs were isolated from 7.5 ml baseline (TO) blood using the LifeTracDx® PD-L1 test and scored PD-L1 expression as high or low (0=Neg, l=Low, 2=High, 3=Very High). If possible, a follow-up sample (Tl) was taken (~48 days) after IMT induction. Patients’ progressive free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate analysis based on RECIST vl.l over 2 years.[00127J TO samples were available for 98% (n=45 / 46) of patients and Tl samples were available for 76% (n=35 / 46) of patients. At TO, high CAML PD-L1 expression was not correlated with improved PFS (HR=1.23, p=0.6286) (Figure 11A) or OS (HR=1.26, p= 0.7157) (Figure 1 IB). At Tl , high CAML PD-L1 expression was significantly correlated with improved PFS (HR-2.60, p-0.0368) (Figure 11C) and OS (HR=3.61, p= 0.0150) (Figure 11D). Patients with consistently low PD-L1 at TO & Tl had the poorest responses (mPFS = 2.0 months, mOS = 6.8 months). Patients with consistently high PD-L1 had the best responses (mPFS = 10.1 months and mOS = 20.9 months). See Table 4.Table 4. CAML PD-L1 Changes Between TO and T1

[0128] The results demonstrate a cancer agnostic blood-based biopsy that may be used to monitor dynamic PD-L1 expression changes in CAMLs. Monitoring PD-L1 expression in CAMLs appears to offer a dynamic perspective on immune response modulation over time. In the three cancers assayed, high PD-L1 expression in CAMLs after starting IMT appeared prognostic for increased IMT benefit.Experiment 4

[0129] A prospective pilot study was undertaken to monitor the peripheral blood of n=67 metastatic breast cancer (mBC) patients undergoing systemic treatment with immunotherapies (IMT) in combination with other therapies, and to evaluate CAML PD-L1 score prior to and post IMT induction with clinical outcome analysis at 2 years.

[0130] In the study, new lines of systemic therapy were started in combination with IMT for active progressive metastatic disease in mBC patients (n=67). Demographics can be found in Table 5. CAMLs were isolated from 7.5 ml baseline (TO) blood using the LifeTracDx® PD-L1 test and scored PD-L1 as high or low. If possible, a follow-up sample (Tl) was taken (~47 days) after IMT induction. Patient’s progressive free survival (PFS) and overall survival (OS) hazard ratios (HRs) were analyzed by censored univariate analysis based on RECIST vl .1 over 2 years.Table 5. Demographic table.

[0131] Baseline TO CAML PD-L1 expression data was available for 94% (n=63 / 67) of patients. 44% (n=28 / 63) of patients had high CAML PD-L1 expression which was not correlated with PFS or OS (Figure 12A and B). Post IMT, T1 CAML PD-L1 expression data was available for 78% (n=52 / 67) of patients. 58% (n=30 / 52) of patients had high CAML PD-L1 expression which significantly correlated with improved PFS and improved OS (Figure 12C and D). Patients with consistently low CAML PD-L1 expression at TO & T1 had the poorest responses (Table 6). Patients with increased CAML PD-L1 expression after IMT induction had better responses (Figure 12C and D; Table 6). Patients with consistently high CAML PD-L1 expression at TO & T1 had the best response rates (Table 6; Figure 13).Table 6. CAML PD-L1 Changes Between TO and T1 .[00132J The results demonstrate a blood-based biopsy which can monitor dynamic PD-L1 changes in circulating tumor immune cells and may predict enhanced clinical benefit to PD-L1 IMTs in mBC.Experiment 5

[0133] In metastatic breast cancer (mBC), anti-programmed cell death ligand (PD-L1) / PD-1 immune checkpoint inhibitors (ICIs), e.g. Pembrolizumab, are FDA approved for use in a subpopulation of mBC patients with a PD-L1 combined positive score (CPS) of >10, showing median progression-free survival (mPFS) of 9.7 months and median overall survival (mOS) of >24 months. However, 62% of patients have <10 CPS, which may also benefit from ICIs (i.e. >1 CPS have mPFS = 7.6 months vs 5.6 for chemotherapy). One hypothesis to why low PD-L1 patients respond to ICI is a dynamic PD-L1 upregulation after new therapy induction, requiring a biomarker that can monitor PD-L1 and subsequent benefit to ICIs.

[0134] Interestingly, recent studies have identified PD-L1 expressing myeloid derived cells that disseminate into the blood from primary tumors, tumor-macrophage fusion cells (TMFCs), which may predict ICI response. However, dynamic changes in TMFC PD-L1 during ICI treatment and its relationship to tumor CPS is unknown.

[0135] In this study, PD-L1 expression was monitored in TMFCs during ICI treatment and compared to tumor CPS in mBC patients to evaluate their associations with PFS & OS at 24 months.

[0136] A prospective pilot study of n=43 patients was conducted with pathologically confirmed mBC prior to receiving anti-PD-l / PD-Ll ICIs. CellSieve microfilters isolated TMFCs from 7.5 ml peripheral blood samples at 4 time points, prior to induction of PD-L1 ICI (TO) andat monthly timepoints (T1-T3) for up to 4 months after TCI induction. TMFCs were identified by their enlarged cell size (>30 pm) and textured polyploid nucleus. Average PD-L1 expressions in TMFC were measured and categorized as negative / low or high. Pearson’s correlation compared average TMFC PD-L1 to CPS PD-L1 from tissue IHC. TMFC PD-L1 expression and CPS score were both compared to patients’ PFS and OS by Cox proportional univariate and multivariate analysis over 24 months.

[0137] 95.3% (n=41 / 43) of patients provided a TO sample. 90.7% (n=39 / 43) of patients provided a T1 sample (-28 days after ICI). 67.4% (n=29 / 43) of patients provided a T2 sample (-71 days after ICI), and 41.9% (n=18 / 43) of patients provided a T3 sample (-117 days after ICI).

[0138] mPFS of patients with CPS >10, 1-10, <1 was 8.5, 7, 2 months, respectively (>10 CPS vs <10 CPS HR=1.5, p=0.9180), and mOS was 12.1, 9.0, 18.9 months, respectively (>10 CPS vs <10 CPS HR=0.6, p=0.9981).

[0139] No significant correlations were identified between CPS and TO TMFC PD-L1 (p=0.6109). Further, patients with high TMFC PD-L1 at T2 (HR=3.1, p=0.0475) had significantly better PFS, while T1 (HR=1.8, p=0.1843) and T3 (HR=4.0, p=0.0686) trended toward better PFS, but not for OS. Combining patients with high TMFC PD-L1 at any time point had significantly improved PFS (HR=2.8, 95%CI=1.4-5.5, p=0.0052), but not OS, compared to patients with consistently low TMFC PD-L1.

[0140] In view of these results, tumor PD-L1 CPS was not found to be correlated with clinical outcomes. However, high TMFC PD-L1 expression at any time point correlated with improved PFS, suggesting that monitoring PD-L1 in TMFCs may serve as a real-time biomarker to more accurately indicate responses to ICI therapies.

[0141] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.REFERENCES

[0142] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. Each cited patent and publication is incorporated herein by reference in its entirety. All of the following references have been cited in this application:1. Adams DL, Zhu P, Makarova OV, Martin SS, Charpentier M, Chumsri S, et al. The systematic study of circulating tumor cell isolation using lithographic microfilters. RSC Advances. 2014;4:4334-42.2. Lianidou ES, Markou A. Circulating tumor cells in breast cancer: detection systems, molecular characterization, and future challenges. Clinical chemistry. 2011;57: 1242-55.3. Pantel K, Brakenhoff RH, Brandt B. Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nature reviews Cancer. 2008;8:329-40.4. Paterlini-Brechot P, Benali NL. Circulating tumor cells (CTC) detection: clinical impact and future directions. 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Claims

WHAT IS CLAIMED IS:

1. A method of monitoring probability of progression free survival and / or overall survival in a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, probability of progression free survival and / or overall survival in the subject is increased and when PD-L1 expression decreases between the first and second time point, probability of progression free survival and / or overall survival in the subject is decreased.

2. A method of determining probability of progression free survival and / or overall survival in a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, probability of progression free survival and / or overall survival in the subject is determined to be increasing and when PD-L1 expression decreases between the first and second time point, probability of progression free survival and / or overall survival in a subject is determined to be decreasing.

3. A method of making a treatment decision in a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein a cancer treatment is administered to the subject between the first and second time point, and wherein when PD-L1 expression decreases between the first and second time point, an alternative treatment is administered to the subject.

4. A method of making a treatment decision for an immune checkpoint inhibitor in a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression increases between the first and second time point, an immune checkpoint inhibitor is indicated for administration to the subject.

5. A method of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein a cancer treatment is administered to the subject between the first and second time point, and wherein when PD-L1 expression increases between the first and second time point, administration of an immune checkpoint inhibitor is predicted to be beneficial for the subject.

6. A method of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression is high at the first and second time point, administration of an immune checkpoint inhibitor after the second time point is predicted to be beneficial for the subject.

7. A method of predicting therapeutic benefit of an immune checkpoint inhibitor for a subject having cancer, comprising:(a) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a first time point from a subject having cancer for PD-L1 expression, and(b) assaying one or more of CTCs, EMTCTCs, CAMLs, and CAVEs isolated at a second time point from a subject having cancer for PD-L1 expression, wherein when PD-L1 expression is low at the first time point and high at the second time point, administration of an immune checkpoint inhibitor after the second time point is predicted to be beneficial for the subject.

8. The method of any one of claims 1, 2, 4, 6 and 7, wherein a cancer treatment is administered to the subject between the first and second time point.

9. The method of claim 8, wherein the cancer treatment is an immunotherapeutic agent.

10. The method of claim 8, wherein the cancer treatment is an immune checkpoint inhibitor.

11. The method of any one of claims 1-7, wherein PD-L1 expression is assayed at one or more additional time points.

12. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor is one or more of a PD-L1 antagonist, PD-1 antagonist, and a CTLA-4 antagonist.

13. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor inhibits binding between PD-L1 and PD-1.

14. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor inhibits binding of PD-L1 to its binding partners.

15. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor inhibits binding of PD-1 to its binding partners.

16. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor inhibits binding of CTLA-4 to its binding partners.

17. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor is an antibody.

18. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor is a monoclonal antibody.

19. The method of any one of claims 4-7, wherein the immune checkpoint inhibitor is a human antibody, a humanized antibody, or a chimeric antibody.

20. The method of any one of claim 4-7, wherein the immune checkpoint inhibitor is one or more of Nivolumab (Opdivo), Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Atezolizumab (Tecentriq), Tremelimumab, Durvalumab (MED14736), and Retifanlimab.

21. The method of claim 3 or 5, wherein the cancer treatment is selected from the group consisting of immunotherapeutic agents, chemotherapeutic agents, radiotherapeutic agents, cancer drugs, CCR5 and CXCR4.

22. The method of claim 18, wherein the cancer treatment is selected from the group consisting of T-VEC, AM-0010, CXCR4 antagonist, TGF-beta kinase inhibitor galunisertib, anti-CSF-lR monoclonal antibody, Abemaciclib, Faslodex, necitumumab, AZD9291, Cyramza (ramucirumab), TPIV 200, Galunisertib, cancer vaccines, cytokines, cell-based therapies, bi- and multi-specific antibodies, tumor-targeting mAbs, Rituximab, oncolytic viruses, reovirus, Blinatumomab, Sipuleucel-T, T-Vec, IL-2, IFN-a, Trastuzumab, Celuximab, bevacizumab, Tim- 3, BTLA, anti-IL-10, GM-CSF, anti-angiogenesis treatment, VEGF blockade, HMGB1, Nrpl, TAM receptor tyrosine kinases, Axl , MerTK, ALT-803, IL-15, Immunosuppressive Ligand Phosphatidyl serine (PS), bavituximab, bevacizumab (anti-VEGF), coblmetinib (MEK inhibitor), vemurafenib (BRAF inhibitor), erlotinib (EGFR), alectinib (ALK inhibitor), bevacizumab (anti- VEGF), pazopanib (tyrosine kinase inhibitor), dabrafenib (BRAF inhibitor), trametinib (MEK inhibitor), sunitinib (RTK inhibitor), pazopanib (RTK inhibitor), sargramostim, VISTA, TIM-3, LAG-3, PRS-343, CD137 (4-lBB) / HER2 bispecific, USP7, anti-HER2, SEMA4D, CTLA-4, PD-1, PD-L1, and PD-L2.

23. The method of any one of claims 1-7, wherein the assaying for PD-L1 expression is one or more of detecting PD-L1 protein expression and detecting PD-L1 mRNA production.

24. The method of claim 23, wherein the PD-L1 protein expression is detected via immunohistochemistry (IHC).

25. The method of claim 24, wherein IHC is performed by membrane staining, cytoplasmic staining, or a combination thereof.

26. The method of claim 24 or 25, wherein IHC is performed using an anti-PD-Ll antibody.

27. The method of any one of claims 24-26, wherein PD-L1 protein expression is detected as a low staining intensity by IHC.

28. The method of any one of claims 24-26, wherein PD-L1 protein expression is detected as a high staining intensity by IHC.

29. The method of any one of claims 24-26, wherein PD-L1 protein expression is detected as inducible by IHC.

30. The method of any one of claims 24-26, wherein PD-L1 protein expression is detected as any staining of the isolated cells.

31. The method of claim 24, wherein IHC is performed using immunofluorescence (IF) staining, wherein one or more antibodies with binding specificity for PD-L1 are utilized.

32. The method of claim 31, wherein binding of the anti-PD-Ll antibody to PD-L1 is detected via a fluorescent compound conjugated to the anti-PD-Ll antibody.

33. The method of claim 31, wherein binding of the anti-PD-Ll antibody to PD-L1 is detected via a fluorophore-conjugated secondary antibody with binding specificity for the anti- PD-Ll antibody.

34. The method of any one of claims 1-7, wherein PD-L1 expression is detected when the level of PD-L1 expression is greater than PD-L1 expression is a population of stromal cells from a subject of the same species that does not have cancer.

35. The method of any one of claims 1-7, wherein CTCs, EMTCTCs, CAMLs, and CAVEs are isolated from blood obtained from the subject having cancer.

36. The method of claim 35, wherein the blood is peripheral blood.

37. The method of any one of claims 1-7, wherein the subject having cancer is undergoing treatment using one or more of a targeted agent, chemotherapy, or radiation therapy.

38. The method of any one of claims 1-7, wherein the cancer is a solid tumor.

39. The method of claim 38, wherein the solid tumor is of a stage I, stage II, stage III or stage IV cancer.

40. The method of claim 39, wherein the solid tumor is a carcinoma, sarcoma, neuroblastoma or melanoma.

41. The method of any one of claims 1-7, wherein the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, melanoma, bladder cancer, kidney cancer, head and neck cancer, colorectal cancer, liver cancer, ovarian cancer, neuroblastoma, sarcoma, osteosarcoma, esophageal, brain & ONS, larynx, bronchus, oral cavity and pharynx, stomach, testis, thyroid, uterine cervix, or uterine corpus cancer.

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