WO2025129332A1 - Method of prognosis and monitoring of cancer patient and of determining immunological status of a subject - Google Patents

Abstract

This invention relates to the prognosis, monitoring or diagnosis of cancer patients and of determining the immunological status of a subject. Particularly, the invention relates to a method of whole blood diagnostic test to monitor cancer patients' response to treatment with a medicament for modulating RAR related orphan receptors (RORs) and help predict patient outcomes. The prognosis, monitoring or diagnosis is achieved using a novel analysis method that uses the ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament for modulating RORs.

Description

METHOD OF PROGNOSIS AND MONITORING OF CANCER PATIENT AND OF DETERMINING IMMUNOLOGICAL STATUS OF A SUBJECT

FIELD OF THE INVENTION

[0001] This invention relates to the prognosis, monitoring or diagnosis of cancer patients and of determining the immunological status of a subject. Particularly, the invention relates to a method of whole blood diagnostic test to monitor cancer patients’ response to treatment with a medicament for modulating RAR related orphan receptors (RORs) and help predict patient outcomes. The prognosis, monitoring or diagnosis is achieved using a novel analysis method that uses the ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament for modulating RORs.

BACKGROUND

[0002] Many types of cancer are associated with an increased or decreased activity of retinoic-acid-receptor-related orphan nuclear receptors (RORs) alpha (RORa or RORA) and gamma (RORC, RORy or RORyt) or their controlled genes and gene products. Therefore, a novel cancer treatment revolves around the modulation of RORs.

[0003] The retinoic acid receptor-related (ROR) sub-family of orphan nuclear receptors was initially identified on the basis of sequence similarities to the retinoic acid and retinoid X receptor families. Through alternative promoter usage and exon splicing, the ROR genes encode different isoforms of RORa, [3 and y, which exhibit differential tissue expression and functions. RORyt is a differentially spliced isoform of RORy, that differs only in the N-terminus by the presence of 21 additional amino acids in RORy. The endogenous physiological ligands for RORyt have recently been identified as 7|3-27- dihydroxy cholesterol, and two other cholesterol biosynthetic intermediates.

[0004] RORyt is exclusively expressed in cells of the immune system including CD4⁺ CD8⁺ double positive thymocytes5, Th17, Tc17, and yb T cells, as well as a subset of innate lymphoid cells (ILCs) and regulatory T cells (Tregs). RORyt is a key transcription factor driving Th17 cell differentiation, and production of IL-17A, IL-17F and IL-22 in innate and adaptive immune cells, also termed “type 17” cells. Th17 cytokines, IL-17A, IL-17F, and IL-22, stimulate tissue cells to produce a panel of inflammatory chemokines, cytokines and metalloproteases, resulting in the recruitment of granulocytes to sites of inflammation. The Th17 cell subset has been shown to be the major pathogenic population in several models of autoimmune inflammation, including collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). RORyt deficient mice show impaired Th17 cell differentiation in vitro, significantly reduced Th17 cell populations in vivo, and decreased susceptibility to EAE and intestinal inflammation. RORyt-deficient T cells fail to induce colitis in the mouse T cell transfer model.

[0005] Human genetic studies have shown association of polymorphisms in the genes for Th17 cell-surface receptors, IL-23R and CCR6, with susceptibility to inflammatory bowel disease (IBD), multiple sclerosis (MS), rheumatoid arthritis (RA) ankylosing spondylitis (AS) and psoriasis. Clinical modulation of the IL-23/IL-17 pathway through biologies targeting IL-12/23, IL-23, IL-17A or IL-17RA has provided validation of its critical role in human autoimmune diseases. RORyt is a nuclear receptor target in the IL-23/IL-17 pathway and has been shown to be tractable to modulation by oral small molecules. Indeed, other nuclear receptors have been successfully targeted by orally available small molecules that are now marketed drugs.

[0006] A critical problem in the clinical management of many cancers is that it is highly heterogeneous.

[0007] Some cancer-specific prognostic biomarkers have been proposed in the prior art. However, a key question remains as to whether these biomarkers are adequate and accurate.

[0008] For example, a novel method for the prognosis, monitoring or diagnosis of cancer, including but not limited to prostate cancer, breast cancer, ovarian cancer, multiple myeloma, brain cancer, glioma, lung cancer, salivary cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, leukemia, melanoma, lymphoma, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, non-small cell lung carcinoma, gastroesophageal cancer among others, particularly for pancreatic ductal adenocarcinoma (PDAC), is the measure of IL-17A-producing CD8⁺ T-cell subpopulation cells (Tc17 cells) and IL-17A-producing CD4⁺ T-cell subpopulation cells (Th17 cells). However, while the prevalence of the second adaptive IL-17A-producing subpopulation, namely Th17 cells (CD4+RORyt+), associated with advanced tumor stage, it failed to correlate with shorter patient survival and to act as an independent prognostic marker for PDAC (Picard, Felix Simon Ruben et al. “IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts”, Gut vol. 72,8 (2023): 1510-1522. doi:10.1136/gutjnl-2022-327855). Further, the value of Tc17-cell and Th17-cell measurement as a prognostic and a monitoring tool has not been extensively studied and there is a lack of translational studies.

[0009] The diversity of patients’ response involves both cell intrinsic factors and cell extrinsic factors, i.e., the microenvironment. Since tumor cells are constantly evolving, it is essential to develop new tools for risk stratification and accurate prediction of patient therapeutic response. Thus, new treatment strategies and molecular biomarkers for more successful patient stratification for effective clinical care are needed.

SUMMARY

[0010] In accordance with a first embodiment of the invention, there is provided a method of whole blood diagnostic test to diagnose or prognose cancer patients and help predict patient outcomes; which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample; wherein an increase in the ratio is indicative of cancer.

[0011] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes, which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment. [0012] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils, total lymphocytes and ratio of ROC+ neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament ; wherein an increase in the ratio is indicative of a poor response to the treatment.

[0013] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils and FOXP3+ Treg lymphocytes and ratio of RORC+ neutrophils versus FOXP3+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0014] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils and CD8+ Treg lymphocytes and ratio of RORC+ neutrophils versus CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0015] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring ratio of RORC+ neutrophils versus FOXP3+ Treg lymphocytes, and ratio of RORC+ neutrophils versus CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0016] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring ratio of RORC+ neutrophils, to ratio of F0XP3+ Treg lymphocytes to CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0017] In accordance with a further embodiment of the invention, the patient exhibits primary resistance to a treatment with a medicament.

[0018] In accordance with a further embodiment of the invention, the measurement of the ratio is done before and after treatment with a medicament for modulating RORs and one or more additional compounds selected from the group consisting of:

[0019] (a) a cytotoxic agent;

[0020] (b) an antimetabolite;

[0021] (c) an alkylating agent;

[0022] (d) an anthracycline;

[0023] (e) an antibiotic;

[0024] (f) an anti-mitotic agent;

[0025] (g) an hormone therapy;

[0026] (h) a signal transduction inhibitor;

[0027] (i) a gene expression modulator;

[0028] (j) an apoptosis inducer;

[0029] (k) an angiogenesis inhibitor;

[0030] (I) an immunotherapy agent;

[0031] (m) an Immune Checkpoint Inhibitor; and

[0032] (o) cancer vaccines.

[0033] In accordance with a further embodiment of the invention, the cytotoxic agent is selected from the group consisting of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, analogs or homologs thereof, and a combination thereof.

[0034] In accordance with a further embodiment of the invention, the antimetabolites is selected from the group consisting of methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, and a combination thereof.

[0035] In accordance with a further embodiment of the invention, the alkylating agent is selected from the group consisting of mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNll), lomustine (CCNll), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, and a combination thereof.

[0036] In accordance with a further embodiment of the invention, the anthracycline is selected from the group consisting of daunorubicin, doxorubicin, and a combination thereof.

[0037] In accordance with a further embodiment of the invention, the antibiotic is dactinomycin, bleomycin, mithramycin, anthramycin (AMC), and a combination thereof.

[0038] In accordance with a further embodiment of the invention, the anti-mitotic agent is selected from the group consisting of vincristine, vinblastine, and a combination thereof.

[0039] In accordance with a further embodiment of the invention, the signal transduction inhibitor is selected from the group consisting of imatinib, trastuzumab, and a combination thereof.

[0040] In accordance with a further embodiment of the invention, the gene expression modulator is selected from the group consisting of a siRNA, a shRNA, an antisense oligonucleotide, an HDAC inhibitor, and a combination thereof. [0041] In accordance with a further embodiment of the invention, the immunotherapy agent is selected from the group consisting of a monoclonal antibody, a chimeric antigen receptors (CARs) -T-Cell, and a combination thereof.

[0042] In accordance with a further embodiment of the invention, the hormone therapy is an luteinizing hormone-releasing hormone (LHRH) antagonist.

[0043] In accordance with a further embodiment of the invention, the apoptosis inducer is a recombinant human TNF-related apoptosis-inducing ligand (TRAIL).

[0044] According to some embodiments of the invention, the immune checkpoint inhibitor is a Programmed cell death 1 (PD-1 ) inhibitor, a Programmed Death-Ligand 1 (PDL-1 ) inhibitor, lymphocyte activation gene-3 (LAG-3) inhibitor, T cell immunoglobulin and mucin-domain containing-3 (TIM3) inhibitor, T cell immunoglobulin and ITIM domain (TIGIT) inhibitor, V-domain Ig suppressor of T cell activation (VISTA) inhibitor or a combination thereof.

[0045] In accordance with a further embodiment of the invention, the immune checkpoint inhibitor is Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or a combination thereof.

[0046] According to some embodiments of the invention, the method is for use as a population screening tool to determine immunological status of individuals and to prevent the development of cancer.

[0047] There is further provided a kit of parts for testing for prognosing and monitoring cancer therapy response comprising a means for detecting the ratio of neutrophils to lymphocytes in a blood sample.

[0048] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to diagnose or prognose patients suffering from emergency myelopoiesis and help predict patient outcomes; which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample; wherein an increase in the ratio is indicative of emergency myelopoiesis.

[0049] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor a patient’s response to a treatment and help predict a treatment outcomes, which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein said patient suffers from emergency myelopoiesis and wherein an increase in the ratio is indicative of a poor response to the treatment.

[0050] There is further provided a kit of parts for prognosing and monitoring a patient’s response to a treatment, comprising reagents used for detecting the level of total neutrophils and level of total lymphocytes in a blood sample, wherein the patient suffers from emergency myelopoiesis.

[0051] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:

[0053] Figs. 1 and 2 illustrate the Circulating RORy+ Neutrophils are increased in the patients with increased Neutrophil/Lymphocyte ratio. [0054] Fig. 3 illustrates that in the GE cancers there is increased RORy+ neutrophils particularly in the advancing cancer periphery.

[0055] Fig. 4 illustrates that there is a lower patient survival in those that have a higher RORy+ neutrophils in the cancer.

[0056] Fig. 5 illustrates that IL17+ Neutrophils also are present in the cancer (IL17+ is a surrogate marker of RORy+

[0057] Fig. 6 illustrates that RORy+ neutrophils express both IL17 and PD-L1 .

[0058] Fig. 7 illustrates that RORy+ neutrophils possess pro-tumour properties listed in the slide especially IL1A- tumour proliferation, HIF1A and PD-L1 (Cd274) Immunosuppression and PTEN - NETs formation.

[0059] Fig. 8 illustrates that gastroesophageal adenocarcinoma (GEA) patients with high neutrophil/Lymphocyte ratio have higher prevalence of circulating RORy+ neutrophils.

[0060] Fig. 9 illustrates that patients with a higher tissue NLR have a higher prevalence of intratumoural IL-17-expressing neutrophils. High circulating NLR associated with higher percentage of RORy+ neutrophils but high tissue NLR associated with high percentage of IL-17+ neutrophils.

[0061] Fig. 10 illustrates that all neutrophils are cytotoxic towards cancer cells.

[0062] Fig. 11 and Fig. 12 illustrate that there is no difference between cytotoxicity of HDNs and LDNs isolated from low NLR GEA patient.

[0063] Fig. 13 illustrates the circulating immune population of individuals with no tumor, with tumors of < 0.5 cm³ and with tumors of > 0.5 cm³

[0064] Fig. 14 illustrates the immune population in LNs and spleen of individuals with no tumor/controls, with tumors of < 0.5 cm³ and with tumors of > 0.5 cm³. [0065] Fig. 15 illustrates the microfluidic paper-based analytical chip for the detection and quantification of RORy+ neutrophils to be used in the kit of parts for testing, prognosing and monitoring cancer treatment response to a medicament.

DETAILED DESCRIPTION

[0066] The invention provides new methods to make a determination of risk of cancer progression and to identify the subject’s response to a treatment consisting of a medicament for modulating RAR related orphan receptors (RORs) or any cancer medicament or combination thereof. The methods of the invention may include identifying if a cancer patient will or will not respond to the treatment and identifying the cancer aggressiveness.

[0067] The present invention is useful to identify patients who are prone to developing cancer, identify a patient’s cancer prognosis and identify patients with good or poor prognoses or monitoring.

[0068] The RORs mediated cancer include prostate cancer, breast cancer, ovarian cancer, multiple myeloma, brain cancer, glioma, lung cancer, salivary cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, leukemia, melanoma, lymphoma, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, colon cancer and liver cancer.

[0069] For the first time, the present inventors have provided a method that allows a reliable prediction of cancer progression in response to a treatment, whereas methods of the prior art could not. The present inventors also provide, for the first time, a method of analysis of patient samples that is quick and easy to execute without requiring invasive procedures.

[0070] Unexpectedly, the measure of the ratio of neutrophils to lymphocytes in a patient’s blood sample has been proven to be a novel and accurate tool for the diagnosis, prognosis or monitoring of cancer development and response to treatment with a medicament modulating RORs. [0071] The present invention provides methods, biomarker panels and kits useful in predicting and monitoring cancer progression.

[0072] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0073] Cancer is a devastating disease and one of the leading causes of death throughout the world. Many cancers are characterized by no early symptoms rendering early diagnosis difficult. Further, therapeutic treatments face the challenge of heterogeneity of response within a population of patients and within a given patient.

[0074] The modulation of ROR alpha and ROR gamma has the effect of blocking the production of IL17A, IL21 , IL17F, IL24, IL26 and other cytokines produced by either T helper 17, T cytotoxic 17, T folicular helper cells, and those produced by all variants or cytotoxic T cells, and Helper T cells as well as Variants of B cells that produce the aforementioned cytokines.

[0075] Th17 cells have a role in macrophage and leukocytes recruitment to cause inflammation. RORC and RORA are highly expressed in certain types of cancer.

[0076] Types of cancer that may be treated by compounds of the present invention include, but are not limited to, prostate cancer, breast cancer, brain cancer, glioma, lung cancer, salivary cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, ovarian cancer, multiple myeloma, leukemia, melanoma, lymphoma, gastric cancer, kidney cancer, pancreatic cancer, bladder cancer, colon cancer, metastatic cancer and liver cancer.

[0077] The invention provides new methods to make a determination of risk of cancer progression and to identify the subject’s response to a treatment consisting of a medicament for modulating RAR related orphan receptors (RORs) or any cancer medicament or combination thereof. The methods of the invention may include identifying if a cancer patient will or will not respond to the treatment and identifying the cancer aggressiveness.

[0078] The present invention is useful to identify patients who are prone to developing cancer, identify a patient’s cancer prognosis and identify patients with good or poor prognoses or monitoring.

[0079] The RORs mediated cancer include prostate cancer, breast cancer, ovarian cancer, multiple myeloma, brain cancer, glioma, lung cancer, salivary cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, leukemia, melanoma, lymphoma, gastric cancer, pancreatic cancer, kidney cancer, bladder cancer, colon cancer and liver cancer.

[0080] The immunological status of a subject can predict the subject’s likelihood of developing cancer. Individuals with inflamed immune systems are more prone to develop cancer. And may be used to monitor disease progression. The present approach is designed to render so-called ‘cold cancers’ that deflect all attempts from drug regimens and make the cancers ‘hot’, i.e. susceptible to allow chemo to enter the tumors.

[0081] In accordance with a first embodiment of the invention, there is provided a method of whole blood diagnostic test to diagnose or prognose cancer patients and help predict patient outcomes; which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample; wherein an increase in the ratio is indicative of cancer.

[0082] Once prognosis is determined, the results can be recorded in the subject’s medical file, which may assist in selecting a treatment regimen and/or determining prognosis of the subject.

[0083] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes, which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment. [0084] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils, total lymphocytes and ratio of ROC+ neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament ; wherein an increase in the ratio is indicative of a poor response to the treatment.

[0085] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils and FOXP3+ Treg lymphocytes and ratio of RORC+ neutrophils versus FOXP3+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0086] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils and CD8+ Treg lymphocytes and ratio of RORC+ neutrophils versus CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0087] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring ratio of RORC+ neutrophils versus FOXP3+ Treg lymphocytes, and ratio of RORC+ neutrophils versus CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0088] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring ratio of RORC+ neutrophils, to ratio of F0XP3+ Treg lymphocytes to CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein increased in the ratio is indicative of a poor response to the treatment.

[0089] In accordance with a further embodiment of the invention, the patient exhibits primary resistance to a treatment with a medicament.

[0090] In accordance with a further embodiment of the invention, the measurement of the ratio is done before and after treatment with a medicament for modulating RORs and one or more additional compounds selected from the group consisting of:

[0091] (a) a cytotoxic agent;

[0092] (b) an antimetabolite;

[0093] (c) an alkylating agent;

[0094] (d) an anthracycline;

[0095] (e) an antibiotic;

[0096] (f) an anti-mitotic agent;

[0097] (g) an hormone therapy;

[0098] (h) a signal transduction inhibitor;

[0099] (i) a gene expression modulator;

[00100] (j) an apoptosis inducer;

[00101] (k) an angiogenesis inhibitor;

[00102] (I) an immunotherapy agent;

[00103] (m) an Immune Checkpoint Inhibitor; and

[00104] (o) cancer vaccines.

[00105] In accordance with a further embodiment of the invention, the cytotoxic agent is selected from the group consisting of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, analogs or homologs thereof, and a combination thereof.

[00106] In accordance with a further embodiment of the invention, the antimetabolites is selected from the group consisting of methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, and a combination thereof.

[00107] In accordance with a further embodiment of the invention, the alkylating agent is selected from the group consisting of mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNll), lomustine (CCNll), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, and a combination thereof.

[00108] In accordance with a further embodiment of the invention, the anthracycline is selected from the group consisting of daunorubicin, doxorubicin, and a combination thereof.

[00109] In accordance with a further embodiment of the invention, the antibiotic is dactinomycin, bleomycin, mithramycin, anthramycin (AMC), and a combination thereof.

[00110] In accordance with a further embodiment of the invention, the anti-mitotic agent is selected from the group consisting of vincristine, vinblastine, and a combination thereof.

[00111] In accordance with a further embodiment of the invention, the signal transduction inhibitor is selected from the group consisting of imatinib, trastuzumab, and a combination thereof.

[00112] In accordance with a further embodiment of the invention, the gene expression modulator is selected from the group consisting of a siRNA, a shRNA, an antisense oligonucleotide, an HDAC inhibitor, and a combination thereof. [00113] In accordance with a further embodiment of the invention, the immunotherapy agent is selected from the group consisting of a monoclonal antibody, a chimeric antigen receptors (CARs) -T-Cell, and a combination thereof.

[00114] In accordance with a further embodiment of the invention, the hormone therapy is an luteinizing hormone-releasing hormone (LHRH) antagonist.

[00115] In accordance with a further embodiment of the invention, the apoptosis inducer is a recombinant human TNF-related apoptosis-inducing ligand (TRAIL).

[00116] According to some embodiments of the invention, the immune checkpoint inhibitor is a Programmed cell death 1 (PD-1 ) inhibitor, a Programmed Death-Ligand 1 (PDL-1 ) inhibitor, lymphocyte activation gene-3 (LAG-3) inhibitor, T cell immunoglobulin and mucin-domain containing-3 (TIM3) inhibitor, T cell immunoglobulin and ITIM domain (TIGIT) inhibitor, V-domain Ig suppressor of T cell activation (VISTA) inhibitor or a combination thereof.

[00117] In accordance with a further embodiment of the invention, the immune checkpoint inhibitor is Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or a combination thereof.

[00118] According to some embodiments of the invention, the method is for use as a population screening tool to determine immunological status of individuals and to prevent the development of cancer.

[00119] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to diagnose or prognose patients suffering from emergency myelopoiesis and help predict patient outcomes; which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample; wherein an increase in the ratio is indicative of emergency myelopoiesis.

[00120] In accordance with a further embodiment of the invention, there is provided a method of whole blood diagnostic test to monitor a patient’s response to a treatment and help predict a treatment outcomes, which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein said patient suffers from emergency myelopoiesis and wherein an increase in the ratio is indicative of a poor response to the treatment.

[00121] As mentioned, the prediction of the prognosis of a subject can be used to select the treatment regimen of a subject and thereby treat the subject in need thereof.

[00122] Gastroesophageal adenocarcinoma (GEA) patients with a high circulating neutrophil-to-lymphocyte ratio (NLR>4) have worse oncological outcomes. One phenomenon that explains this phenotype is the induction of emergency myelopoiesis, a process by which the cancer cells secrete various factors that skew the normal hematopoiesis towards increased production of RORy+ neutrophils. Currently, there are no therapies targeting a high NLR, but RORy+ neutrophils may potentially be an adequate target.

[00123] High NLR GEA patients have a higher prevalence of RORy+ neutrophils in their blood. This subset of neutrophils, both within the circulation and the tumour, has tumour-promoting properties. Additionally, RORy inhibition in the murine gastric model resulted in reduced tumour burden and in the establishment of a less tumour-permissive microenvironment.

[00124] However, running a flow cytometry experiment for every GEA patient at various timepoints is time-consuming, expensive and would require specialized equipment and personnel.

[00125] Kits may allow a lab technician, a patient at home, a primary care physician, a nurse, a pharmacist, a specialist, etc. to carry out the predicting and monitoring cancer progression process, ie blood collection. Said kits may contain reagents to isolate, stain and quantify neutrophils and lymphocytes. Said quantification may be done by fluorescence. A microfluidic paper-based analytical chip may be used for the detection and quantification of RORy+ neutrophils. [00126] There is further provided a kit of parts for testing for prognosing and monitoring cancer therapy response, and patients suffering from emergency myelopoiesis, comprising a means for detecting the ratio of neutrophils to lymphocytes in a blood sample.

[00127] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.

[00128] The following Examples are provided to illustrate the invention and are not to be construed as limiting the invention in any manner. The scope of the invention is defined by the appended claims.

EXAMPLE 1

Quantify prevalence of RORy+ neutrophils in blood and in tumor of GEA patients

[00129] Results are shown in Figs. 1 to 7.

EXAMPLE 2

Identification of neutrophils that inhibit response to cancer therapy

[00130] Julia Kargl et al. (JCI Insight. 2019;4(24):e130850) showed that in patients treated with Immune checkpoint inhibitors PD1 inhibitors, the presence of high intracancer neutrophils and a high ratio of neutrophils to CD8 + lymphocytes in the cancer is predictive of either no response or progression of cancer under PD1 immunotherapy. In a mouse model when they combined an anti-neutrophil and inhibitors, the cancer responded to treatment with immunotherapy anti PD1 inhibitor. [00131] In accordance with the present invention, we further define these neutrophils that inhibit response to cancer therapy and behave in suppressing delivery to cancers of CD8+ lymphocytes as being these RORC+ Neutrophils.

EXAMPLE 3

Kit: use of a microfluidic paper-based analytical chip for the detection and quantification of RORy+ neutrophils

[00132] The proposed device may be a microfluidic paper-based analytical device (uPADs), the membranes of which will be made with nitrocellulose.

[00133] Preparation of Tetrazine-modified Nanoparticles (Tz-FNP): Fluorescent nanoparticles with amine-terminated ends are modified with 2,5-dioxopyrrolidin-1 -yl 5-(4- (1 ,2,4,5-tetrazin-3-yl) benzylamino)-5-oxopentanoate (Tz-NHS) to generate tetrazinemodified nanoparticles (Tz-NP). This reaction is performed in PBS containing 5% dimethylformamide (DMF) for 3 hours at room temperature. The optimal size of Tz-FNP for optimal uptake by neutrophils needs to be troubleshooted and confirmed.

[00134] Preparation of TCP-Modified Antibodies (TCO-Ab): Monoclonal antibodies (CD66b and RORy) are modified with (E)-cyclooct-4-enyl-2,5-dioxopyrrolidin-1 -yl carbonate (TCO-NHS). TCO-NHS was left to react with 5mg of antibody in 10% DMF for 3 hours at room temperature. The optimal concentration of each antibody needs to be troubleshooted and confirmed for optimal visualization and binding.

[00135] Preparation of nanoparticles-antibodies immunoconjugate: TCO-Ab are to be reacted with 0.25mg of Tz-FNP for 3 hours at room temperature and purified using Sephadex. The stability and longevity of the immunoconjugate at room temperature need to be tested.

[00136] Preparation of blood sample: Since RORy is a nuclear receptor, the cells/neutrophils need to be semi-permeabilize to allow the migration of the immunoconjugate into the neutrophils. To do so, various different permeabilization/fixation agent will be tested: Tween20, Triton X-100, methanol and commercially available permeabilization/fixation buffer. This step requires an incubation of 20 to 30m in depending on the agent used. The signal to background ratio will be assessed for each technique and the agent with the lower ratio will be chosen for subsequent trials. This is to ensure the accuracy and reliability of the fluorescence reading.

[00137] Nanoparticle targeting and detection: The immunoconjugate is then transferred to the conjugation channel/pad (binding affinity to the material of the pad, binding period and stability need to be confirmed), where they will bind to the targets of interest present in the analyte (blood in our case). The optimal volume of blood as well as the corresponding volume of immunoconjugate will need to be troubleshooted. The binding period will also need to be assessed. Ideally, the aim is to have a binding period of 15 minutes or less.

[00138] Data read-out and analysis: Since we want to have both quantitative and qualitative data, the proposed device needs to include a transducer that will convert the fluorescence intensity to a quantifiable measurement. A mini fluorescence reader can be attached to the uPADs for easy reading and analysis. The detection of the presence/absence of RORy+ neutrophils will be determined by the presence/absence of fluorescence at the two test lines.

[00139] The immunoconjugate will already be bound to the conjugation pad in the kit and the user will only have to add their sample, rendering user-friendly.

[00140] The microfluidic paper-based analytical chip for the detection and quantification of RORy+ neutrophils is shown in Fig. 15.

[00141] While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure. References

1 . Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of COVID- 19 associated with acute respiratory distress syndrome. Lancet Respir Med. 2020 Feb 18.

2. Ardain A, Porterfield JZ, KI verpris HN, Leslie A. Type 3 ILCs in Lung Disease. Front Immunol. Frontiers; 2019; 10:92.

3. Mikacenic C, Hansen EE, Radella F, Gharib SA, Stapleton RD, Wurfel MM. Interleukin- 17A Is Associated With Alveolar Inflammation and Poor Outcomes in Acute Respiratory Distress Syndrome. Crit Care Med. 2016 Mar;44(3):496-502.

4. Li JT, Melton AC, Su G, Hamm DE, LaFemina M, Howard J, et al. Unexpected Role for Adaptive a[3Th17 Cells in Acute Respiratory Distress Syndrome. The Journal of Immunology. American Association of Immunologists; 2015 Jul 1 ; 195(1 ):87-95.

5. Buonocore S, Ahem PP, Uhlig HH, Ivanov II, Littman DR, Maloy KJ, et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature. 2010 Apr 29;464(7293): 1371-5.

6. Xu S, Cao X. Interleukin-17 and its expanding biological functions. Cell Mol Immunol. Nature Publishing Group; 2010 May;7(3): 164-74.

7. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille J J, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL- 174- T helper cells. Cell. 2006 Sep 22; 126(6): 1121-33.

8. Ding Q, Liu G-Q, Zeng Y-Y, Zhu J-J, Liu Z-Y, Zhang X, et al. Role of IL-17 in LPSinduced acute lung injury: an in vivo study. Oncotarget. 2017 Nov 7;8(55):93704-11 .

9. Crowe CR, Chen K, Pociask DA, Alcorn JF, Krivich C, Enelow Rl, et al. Critical role of IL-17RA in immunopathology of influenza infection. The Journal of Immunology. American Association of Immunologists; 2009 Oct 15;183(8):5301-10.

10. Li C, Yang P, Sun Y, Li T, Wang Z, Zou Z, et al. IL-17 response mediates acute lung injury induced by the 2009 pandemic influenza A (H1 N1 ) virus. Cell Res. Nature Publishing Group; 2012 Mar;22(3):528-38.

11. Du J, Han J-C, Zhang Y-J, Qi G-B, Li H-B, Zhang Y-J, et al. Single-Nucleotide Polymorphisms of IL-17 Gene Are Associated with Asthma Susceptibility in an Asian Population. Med Sci Monit. 2016 Mar 8;22:780-7.

12. Xie M, Cheng B, Ding Y, Wang C, Chen J. Correlations of IL-17 and NF-KB gene polymorphisms with susceptibility and prognosis in acute respiratory distress syndrome in a Chinese population. Biosci Rep. 2019 Feb 28;39(2):860.

13. Nicholls JM, Poon LLM, Lee KC, Ng WF, Lai ST, Leung CY, et al. Lung pathology of fatal severe acute respiratory syndrome. The Lancet. 2003 May 24;361 (9371 ): 1773-8. 14. Wong CK, Lam CWK, Wu AKL, Ip WK, Lee NLS, Chan IHS, et al. Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol. John Wiley & Sons, Ltd; 2004 Apr;136(1 ):95-103.

15. Faure E, Poissy J, Goffard A, Fournier C, Kipnis E, Titecat M, et al. Distinct immune response in two MERS-CoV-infected patients: can we go from bench to bedside? Baldanti F, editor. PLoS ONE. 2014;9(2):e88716.

16. Mahallawi WH, Khabour OF, Zhang Q, Makhdoum HM, Suliman BA. MERS-CoV infection in humans is associated with a pro-inflammatory Th1 and Th17 cytokine profile. Cytokine. 2018 Apr; 104:8-13.

17. Bermejo-Martin JF, Ortiz de Lejarazu R, Pumarola T, Rello J, Almansa R, Ram rez P, et al. Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza. Crit Care. 2009; 13(6): R201 .

18. Haun, J. B., Devaraj, N. K., Marinelli, B. S., Lee, H., & Weissleder, R. (2011 ). Probing intracellular biomarkers and mediators of cell activation using nanosensors and bioorthogonal chemistry. ACS nano, 5(4), 3204-3213. https://doi.Org/10.1021/nn200333m

19. Picard FSR, Lutz V,Brichkina A,et al.GutEpubahead of print: [21 Dec 2023], doi: 10.1136/gutjnl-2022-327855

20. Harrisson S, et al, Clinical Landscape in NASH, VOLUME 21 , ISSUE 8, P2001 -2014, JULY 2023, DOI:https://doi.org/10.1016/j.cgh.2023.03.041

21. Dufour J-F,Anstee QM,Bugianesi E,et al.Gut2022;71 :2123-2134

Claims

CLAIMS:

1 . A method of whole blood diagnostic test to diagnose or prognose cancer patients and help predict patient outcomes; which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample; wherein an increase in the ratio is indicative of cancer.

2. A method of whole blood diagnostic test to monitor cancer patients’ response to treatment and help predict patient outcomes, which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment.

3. A method of whole blood diagnostic test to monitor cancer patients’ response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils, total lymphocytes and ratio of ROC+ neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment.

4. A method of whole blood diagnostic test to monitor cancer patients’ response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils and FOXP3+ Treg lymphocytes and ratio of RORC+ neutrophils versus FOXP3+ Treg lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment.

5. A method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring RORC+ neutrophils and CD8+ Treg lymphocytes and ratio of RORC+ neutrophils versus CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment.

6. A method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring ratio of RORC+ neutrophils versus FOXP3+ Treg lymphocytes, and ratio of RORC+ neutrophils versus CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament; wherein an increase in the ratio is indicative of a poor response to the treatment.

7. A method of whole blood diagnostic test to monitor cancer patients response to treatment and help predict patient outcomes; which comprises measuring ratio of RORC+ neutrophils, to ratio of FOXP3+ Treg lymphocytes to CD8+ Treg lymphocytes in a blood sample before and after treatment with a medicament ; wherein an increase in the ratio is indicative of a poor response to the treatment.

8. The method of any one of claims 2 to 9, wherein said patient exhibits primary resistance to a treatment with a medicament.

9. The method of any one of claims 2 to 8, wherein the measurement of the ratio is done before and after treatment with at least one medicament for modulating RORs and one or more additional compounds selected from the group consisting of :

(a) a cytotoxic agent;

(b) an antimetabolite; (c) an alkylating agent;

(d) an anthracycline;

(e) an antibiotic;

(f) an anti-mitotic agent;

(g) an hormone therapy;

(h) a signal transduction inhibitor;

(i) a gene expression modulator;

(j) an apoptosis inducer;

(k) an angiogenesis inhibitor;

(l) an immunotherapy agent;

(m) an Immune Checkpoint Inhibitor;

(n) cancer vaccines.

10. The method of claim 9, wherein

• the cytotoxic agent is selected from the group consisting of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, analogs or homologs thereof, and a combination thereof;

• the antimetabolites is selected from the group consisting of methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, and a combination thereof;

• the alkylating agent is selected from the group consisting of mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNll), lomustine (CCNll), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, and a combination thereof;

• the anthracycline is selected from the group consisting of daunorubicin, doxorubicin, and a combination thereof;

• the antibiotic is dactinomycin, bleomycin, mithramycin, anthramycin (AMC), and a combination thereof;

• the anti-mitotic agent is selected from the group consisting of vincristine, vinblastine, and a combination thereof;

• the signal transduction inhibitor is selected from the group consisting of imatinib, trastuzumab, and a combination thereof;

• the gene expression modulator is selected from the group consisting of a siRNA, a shRNA, an antisense oligonucleotide, an HDAC inhibitor, and a combination thereof;

• the immunotherapy agent is selected from the group consisting of a monoclonal antibody, a chimeric antigen receptors (CARs) -T-Cell, and a combination thereof;

• the hormone therapy is an luteinizing hormone-releasing hormone (LHRH) antagonist; and

• the apoptosis inducer is a recombinant human TNF-related apoptosisinducing ligand (TRAIL).

11. The method of claim 9, wherein said immune checkpoint inhibitor is a Programmed cell death 1 (PD-1 ) inhibitor, a Programmed Death-Ligand 1 (PDL-1 ) inhibitor, lymphocyte activation gene-3 (LAG-3) inhibitor, T cell immunoglobulin and mucin-domain containing-3 (TIM3) inhibitor, T cell immunoglobulin and ITIM domain (TIGIT) inhibitor, V-domain Ig suppressor of T cell activation (VISTA) inhibitor or a combination thereof.

12. The method of claim 9, wherein said immune checkpoint inhibitor is Pembrolizumab, Nivolumab, Atezolizumab, Durvalumab, or a combination thereof.

13. The method of any one of claims 1 to 10, for use as a population screening tool to determine immunological status of individuals and to prevent the development of cancer.

14. A kit of parts for testing, prognosing and monitoring cancer treatment response to a medicament according to the method of any one of claims 1 to 13, comprising reagents used for detecting the level of total neutrophils and level of total lymphocytes in a blood sample.

15. A method of whole blood diagnostic test to diagnose or prognose patients suffering from emergency myelopoiesis and help predict patient outcomes; which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample; wherein an increase in the ratio is indicative of emergency myelopoiesis.

16. A method of whole blood diagnostic test to monitor a patient’s response to a treatment and help predict a treatment outcomes, which comprises measuring total neutrophils, total lymphocytes and ratio of total neutrophils versus total lymphocytes in a blood sample before and after treatment with a medicament; wherein said patient suffers from emergency myelopoiesis and wherein an increase in the ratio is indicative of a poor response to the treatment.

17. A kit of parts for prognosing and monitoring a patient’s response to a treatment according to the method of claim 15 or 16, comprising reagents used for detecting the level of total neutrophils and level of total lymphocytes in a blood sample, wherein the patient suffers from emergency myelopoiesis.