USE OF SK1 AS BIOMARKER FOR PREDICTING RESPONSE TO IMMUNECHECKPOINT INHIBITORS

Abstract

Immune checkpoint inhibitors (ICI) have revolutionized therapy for advanced cancer, however many patients still do not respond to treatment. However, the efficacy and effectiveness of these therapies varies greatly across individual patients and among different tumour types. A substantial unmet need is thus the development of biomarkers of response to ICI, in order to identify, before initiation of treatment, which patients are likely to experience a response to and clinical benefit from such treatments. Here, the inventors analyzed SPHK1 mRNA in tumor biopsies by in situ hybridization using the RNAscope technology in a cohort of 32 patients suffering from metastatic melanoma. They showed that elevated expression of SPHK1, encoding sphingosine kinase 1 (SK1), which produces the oncometabolite sphingosine-1-phosphate (S1P) is associated with a poor survival in metastatic melanoma patients treated with to the well-known immune-checkpoint inhibitor anti-PD-1 antibody. Accordingly, the present invention relates to the use of SK1 as biomarker for predicting response to immune-checkpoint inhibitors.

Claims

1. A method for determining whether a patient suffering from a cancer will achieve a response with an immune checkpoint inhibitor comprising i) determining the expression level of SK1 in a tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will not achieve a response when the level determined at step i) is higher than the predetermined reference value or concluding that the patient will achieve a response when the level determined at step i) is lower than the predetermined reference value.

2. The method of claim 1 wherein the patient suffer from a cancer selected from the group consisting of neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

3. The method of claim 1 wherein the patient suffers from melanoma.

4. The method of claim 1 wherein the patient suffers from a metastatic melanoma.

5. The method of claim 1 wherein the immune checkpoint inhibitor is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PD1 antibodies, anti-PDL1 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

6. The method of claim 1 wherein, the tumor sample is from a tumor resected from the patient.

7. The method of claim 1 wherein the tumor sample is from a biopsy performed in a primary tumor of the patient or in a metastatic sample distant from the primary tumor of the patient.

8. The method of claim 1 wherein the tumor sample is a sample of circulating tumor cells.

9. The method of claim 1 wherein the expression level of SK1 is determined by immunodetection.

10. The method of claim 1 wherein the expression level of SK1 is determined by detecting the quantity of mRNA encoding for SK1.

11. A method of treating a patient suffering from a cancer comprising i) determining the expression level of SK1 in a tumor sample obtained from the patient, ii) comparing the expression level determined at step i) with a predetermined reference value and (iii) administering to said patient a therapeutically effective amount of a SK1 inhibitor when the level determined at step i) is higher than the predetermined reference value.

12. The method of claim 11 wherein the SK1 inhibitor is administered with an immune checkpoint inhibitor as a combined preparation.

13. The method of claim 9, wherein the immunodetection is performed by immunohistochemistry (IHC) or immunofluorescence.

Description

FIGURES

[0075] FIG. 1. High SPHK1 expression correlates with poor survival of melanoma patients treated with anti-PD-1. (A) SPHK1 expression in human nevi (n=17) compared to primary melanoma (P, n=45) and in primary melanoma (P, n=25) compared to metastatic melanoma (M, n=44) was assessed using the Oncomine database. (B) Percentage of cancer cells positive for SPHK1 mRNA staining in metastatic melanoma tissues of 32 patients prior anti-PD-1 treatment. (C) Representative mRNA staining of low and high SPHK1 expression. Skin (P1-P3) or lymph node (P2-P4) biopsies from patients with metastatic melanoma. Percentages indicate the proportion of cancer cells positive for SPHK1 mRNA staining. Large and small blue lines represent 200 and 20 m, respectively. (D) Progression-Free Survival and (E) Overall Survival curves of patients with more than 50% of melanoma cells positive for SPHK1 (SPHK1 high) (n=11) or less than 50% (SPHK1 Low) (n=21). Survival times were calculated from the first day of the cycle of anti-PD-1 post biopsy. Statistical significance was determined by log-rank test.

EXAMPLE

[0076] Methods

[0077] Patient Cohorts

[0078] SPHK1 expression analysis in human nevi and melanomas was assessed in 2 different cohorts from Oncomine (Talantov Clin Cancer Res. 2005 Oct. 15; 11(20):7234-42; Xu Mol Cancer Res. 2008 May; 6(5):760-9).

[0079] Patient Survival Analyses

[0080] Protocol was approved by CPP du Sud-Ouest et Outre-Mer IV (Limoges, France). Informed, signed consents from metastatic melanoma patients were obtained. The major clinicopathologic characteristics and available treatment information of the cohorts are presented in Table B.

[0081] All survival times were calculated from the first day of the first cycle of anti-PD-1 therapy. Progression-free survival and overall survival were defined using the following first-event definitions: either relapse or death from any cause for PFS, and death from any cause for OS. Patients still alive were censored at their date of last follow-up. Comparison between groups (low expression vs high expression) was performed using log-rank test.

[0082] In Situ mRNA Hybridization

[0083] In situ detection of SPHK1 transcripts in Formalin-Fixed, Paraffin-Embedded Tissues was performed using the RNAscope assay with RNAScope 2.5 VS ProbeHs-SPHK1 and the ACD RNAscope 2.0 Red kit (Advanced Cell Diagnostics). Assay specificity was assessed measuring the signal in positive and negative control samples. Positivity of endothelial cells was used as an intrinsic positive control. Cases with positive intrinsic control and no signal in tumor cells were considered as negative. Quantification was assessed by evaluating the percentage of positive tumor cells blinded to clinical response to treatment.

[0084] Results

[0085] Analysis of two different cohorts from the Oncomine database indicated that SPHK1 (encoding SK1) transcript levels were higher in human primary melanomas as compared to nevi (FIG. 1A, left panel); SPHK1 expression was further increased in metastatic melanomas (FIG. 1A, right panel), suggesting that SPHK1 expression might be associated with melanoma progression.

[0086] In order to evaluate whether SPHK1 expression is related to the clinical outcome of advanced melanoma patients receiving anti-PD-1 therapy (Table B), we analyzed SPHK1 mRNA in tumor biopsies by in situ hybridization using the RNAscope technology. According to the distribution of the percentage of tumor cells positive for SPHK1, two groups of patients named SPHK1 Low (<50%) and SPHK1 High (>50%) were defined (FIG. 1B). FIG. 2C shows representative SPHK1 staining for these two groups. Kaplan-Meier analysis revealed that patients with low SPHK1 expression had longer PFS and OS than those with high SPHK1 expression (p=0.0112 and p=0.0445, respectively) (FIGS. 1D and E). These findings support the hypothesis that SPHK1 expression represents a potential biomarker to predict tumor progression and resistance to anti-PD1 in metastatic melanoma patients.

TABLE-US-00003 TABLE B Clinical characteristics of the anti-PD-1 cohort. Continuous variables were presented as median with range (min-max) and categorical variables were summarized by frequencies and percentages. Total SK1low (<=50%) SK1high (>50%) N = 32 N = 21 N = 11 Gender(n = 32) Male 21 (65.6%) 15 (71.4%) 6 (54.5%) Female 11 (34.4%) 6 (28.6%) 5 (45.5%) Age at treatment initiation (n = 32) <=65 years 13 (40.6%) 8 (38.1%) 5 (45.5%) >65 years 19 (59.4%) 13 (61.9%) 6 (54.5%) Who Performance Status (n = 31) 0 20 (64.5%) 13 (65.0%) 7 (63.6%) 1 11 (35.5%) 7 (35.0%) 4 (36.4%) Missing 1 1 0 Stage (n = 32) IIIc 5 (15.6%) 5 (23.8%) 0 (0.0%) IV 5 (15.6%) 4 (19.0%) 1 (9.1%) IVa 5 (15.6%) 4 (19.0%) 1 (9.1%) IVb 5 (15.6%) 1 (4.8%) 4 (36.4%) IVc 12 (37.5%) 7 (33.3%) 5 (45.5%) Histological subtype (n = 32) Mucosal 1 (3.1%) 1 (4.8%) 0 (0.0%) Cutaneous 30 (93.8%) 20 (95.2%) 10 (90.9%) Other 1 (3.1%) 0 (0.0%) 1 (9.1%) BRAF(n = 31) No 22 (71.0%) 12 (60.0%) 10 (90.9%) Yes 9 (29.0%) 8 (40.0%) 1 (9.1%) Missing 1 1 0 NRAS(n = 27) No 17 (63.0%) 9 (56.3%) 8 (72.7%) Yes 10 (37.0%) 7 (43.8%) 3 (27.3%) Missing 5 5 0 Treatment line (n=32) 1 23 (71.9%) 14 (66.7%) 9 (81.8%) 2 6 (18.8%) 5 (23.8%) 1 (9.1%) 3 3 (9.4%) 2 (9.5%) 1 (9.1%) Treatment line (n = 32) <2 23 (71.9%) 14 (66.7%) 9 (81.8%) >=2 9 (28.1%) 7 (33.3%) 2 (18.2%) DCI(n = 32) Pembrolizumab 22 (68.8%) 13 (61.9%) 9 (81.8%) Nivolumab 10 (31.3%) 8 (38.1%) 2 (18.2%)

REFERENCES

[0087] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0088] 8. V. Albinet et al., Dual role of sphingosine kinase-1 in promoting the differentiation of dermal fibroblasts and the dissemination of melanoma cells. Oncogene 33, 3364-3373 (2014). [0089] 9. M. Mrad et al., Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting Ml macrophage response in melanoma. Oncotarget 7, 71873-71886 (2016). [0090] 10. N. J. Pyne, J. Ohotski, R. Bittman, S. Pyne, The role of sphingosine 1-phosphate in inflammation and cancer. Adv Biol Regul 54, 121-129 (2014). [0091] 11. N. J. Pyne, S. Pyne, Sphingosine 1-phosphate and cancer. Nat Rev Cancer 10, 489-503 (2010). [0092] 12. C. S. Garris, V. A. Blaho, T. Hla, M. H. Han, Sphingosine-1-phosphate receptor 1 signalling in T cells: trafficking and beyond. Immunology 142, 347-353 (2014). [0093] 13. S. Spiegel, S. Milstien, The outs and the ins of sphingosine-1-phosphate in immunity. Nat Rev Immunol 11, 403-415 (2011).