WO2012007783A1 - Kits and methods for detecting the ability to induce an immunogenic cancer cell death in a subject - Google Patents

Abstract

The present invention relates to the fields of genetics, immunology and medicine. The present invention more specifically relates to in vitro or ex vivo methods for determining the susceptibility to a cancer treatment of a subject having a tumour. These methods comprise a step of determining the ability of the treatment, of the subject and/or of the tumour to induce an anticancer immune response, the inability of at least one of the treatment, the subject and the tumor to induce an anticancer immune response being indicative of a resistance of the subject to the therapeutic treatment of cancer. Inventors in particular identify genes specific of a human subject or of cancerous cells which can be used to predict or assess the sensitivity of a subject to a treatment of cancer. The invention also relates to particular compounds capable of activating or enhancing the immune system of a particular subject, when the subject is exposed to a therapeutic treatment of cancer or before such an exposition. It further relates to uses of such compounds, in particular to prepare a pharmaceutical composition to allow or improve the efficiency of a therapy of cancer in a subject in need thereof. The present invention in addition provides kits, methods for selecting a compound of interest, as well as pharmaceutical compositions and uses thereof.

Description

KITS AND METHODS FOR DETECTING THE ABILITY TO INDUCE AN IMMUNOGENIC CANCER CELL DEATH IN A SUBJECT

The present disclosure generally relates to the fields of genetics, immunology and medicine. The present invention more specifically relates to in vitro or ex vivo methods for determining the susceptibility to a cancer treatment of a subject having a tumour. These methods comprise a step of determining the ability of the treatment, of the subject and/or of the tumour to induce an anticancer immune response, the inability of at least one of the treatment, the subject and the tumor to induce an anticancer immune response being indicative of a resistance of the subject to the therapeutic treatment of cancer.

Inventors in particular identify genes specific of a human subject or of cancerous cells which can be used to predict or assess the sensitivity of a subject to a treatment of cancer.

The invention also relates to particular compounds capable of activating or enhancing the immune system of a particular subject, when the subject is exposed to a therapeutic treatment of cancer or before such an exposition. It further relates to uses of such compounds, in particular to prepare a pharmaceutical composition to allow or improve the efficiency of a therapy of cancer in a subject in need thereof.

The present invention in addition provides kits, methods for selecting a compound of interest, as well as pharmaceutical compositions and uses thereof.

BACKGROUND ART

Cancer is the major cause of mortality in most industrialized countries. Although several anti-cancer therapies are proposed, amongst which feature chemotherapy [anthracyclines such as daunorubicine, doxorubicin (DX), idarubicin and mitoxantrone (MTX), as well as oxali-platinum (oxaliplatin or OXP), cis-platinum (cisplatin or CDDP), and taxanes (paclitaxel or docetaxel) are considered as the most efficient cytotoxic agents of the oncologist armamentarium] and radiotherapy [XR], the benefits of said treatments still tends to be insufficient. Cytotoxic agents are supposed to directly destroy cancer cells by stimulating diverse cell death pathways. Nonetheless, several lines of evidence point to a critical contribution of the host immune system to the therapeutic activity mediated by tumoricidal agents (Zitvogel et al, 2008). Indeed, in some instances, the cell death modality triggered by chemotherapy or radiotherapy allows recognition of dying tumor cells by antigen presenting cells, thus eliciting a tumor-specific cognate immune response which is critical for tumor elimination.

However, most of standard chemotherapies induce a non-immunogenic apoptosis (Zitvogel et al, 2004; Steinman et al, 2004; Lake et al, 2006). Thus, even after an initially efficient chemotherapy, patients who do not develop an efficient antitumourous immune response are confronted to chemotherapy-resistant tumourous variants.

Inventors have shown for the first time that OXP and anthracyclines induce immunogenic cell death while other chemotherapeutic agents such as CDDP and alkylating agents tend to induce non-immunogenic cell death (Casares et al, 2005; Obeid et al, 2007). They have further observed that some patients were also resistant to treatments identified as inducing an immunogenic cell death.

Solutions to detect dysfunctions responsible for an absent or reduced response to existing treatments as well as compounds usable to overcome said dysfunctions therefore appear critical for the patient and are herein advantageously provided by inventors.

SUMMARY

The present invention is based on the observation by inventors that the cell death immunogenicity depends on the lethal stimulus, on the presence of specific signals produced by or exposed on tumor cells, as well as on the ability of the subject having the tumor, and in particular of the subject's immune system, to recognize said signals.

The present invention provides an in vitro or ex vivo method of assessing the sensitivity of a subject having a tumor to a treatment of cancer (in other words of determining susceptibility of a patient having a tumor to respond to a treatment of cancer), which method comprises a step of detecting the presence of an anticancer immune response of the subject undergoing the treatment of cancer, the absence of an anticancer immune response being indicative of a resistance of the subject to the treatment of cancer.

The method may be applied before and/or after exposition of the subject to the treatment of cancer.

In a particular embodiment, the therapeutic treatment of cancer is a conventional immunogenic treatment of cancer selected from a chemotherapy using a drug selected from an anthracyclin, a platin, a taxane and an antimitotic agent, preferably from an anthracyclin, a platin, and an antimitotic agent; and radiotherapy. The presence of cells selected from IL-17 producing γδ T lymphocytes, dendritic cells and cytotoxic T lymphocytes, in the tumor of the subject may in particular be indicative of an anticancer immune response and of a sensitivity of the subject to the treatment of cancer.

In vitro or ex vivo methods of assessing the sensitivity of a subject having a tumor to a treatment of cancer are further herein described. These methods comprise a step of determining the ability of the treatment, of the tumor and/or of the subject to induce an anticancer immune response, the inability of at least one of the treatment, the subject and the tumor to induce an anticancer immune response being indicative of a resistance of the subject to the treatment of cancer.

The presence, in the subject, of an alteration leading to the abnormal expression of an immune gene, as herein described, may in particular determine the inability of the subject to induce an anticancer immune response. The alteration may be a single nucleotide polymorphism (SNP).

The step of determining the ability of the tumor to induce an anticancer immune response may in particular consist in verifying the expression by tumor cells of an immunogenic cell death marker selected from a protein allowing or enhancing CRT exposure at the surface of tumor cells, and a protein expressed during the endoplasmic reticulum (ER) stress response and/or during the macroautophagic response of the subject's immune system. A method of selecting an optimal therapeutic treatment of cancer in a subject having a tumor is in addition herein described. This method comprises a step as previously described of assessing the sensitivity of the subject to a first treatment of cancer (herein also identified as "conventional treatment") and, if the subject is resistant to said first treatment of cancer, a step of selecting a "compensatory molecule", to be used, alone or in combination with the first treatment of cancer as the optimal therapeutic treatment of cancer for the subject.

A particular method of selecting an optimal therapeutic treatment of cancer in a subject having a tumor is a method comprising a step of assessing the sensitivity of the subject to a first treatment of cancer with a method as herein described, and, if the subject is resistant to said first treatment of cancer, selecting (i) a product allowing or enhancing the secretion of ATP, HMGBl, LysRS and/or IL-8, and/or the exposure of CRT, ERp57, LysRS and/or KDEL receptor at the surface of a tumour cell, (ii) a product stimulating the autophagy machinery and/or an ER stress response, (iii) a product recruiting and/or activating IL-17 producing γδ T lymphocytes, cytotoxic T cells and/or dendritic cells, (iv) a product promoting activation of the TLR4/myd88 pathway, or able to bypass said pathway, (v) a product triggering the P2RX7 (P2X purinoceptor 7) and/or the NALP3 inflammasome, (vi) a product allowing or enhancing the secretion of IL-lb, (vii) a product capable of stimulating intratumoral Vd2 T lymphocytes, and (viii) a product selected from an antiallergic drug, a neurotropic drug, an antihypertensive or cardiotropic drug, a spindle poison drug, an antimicrobial drug, an anti-osteoclastic drug, a diuretic drug, an oestrogen, and (ix) any combination thereof, to be used in combination with the first treatment of cancer as the optimal therapeutic treatment of cancer for the subject.

Also herein described are compensatory molecules for use in the treatment of cancer, preferably in combination with a conventional treatment of cancer, in particular a chemotherapeutic treatment of cancer, in a subject identified, by a method as previously described, as resistant to a conventional treatment of cancer.

The present invention further encompasses the use of such a compensatory molecule to prepare a pharmaceutical composition for treating a cancer in a subject identified, by a method as previously described, as resistant to a conventional treatment of cancer. Preferably, the pharmaceutical composition further comprises, as a combined preparation, a drug used in a conventional treatment of cancer, for simultaneous, separate or sequential use in the treatment of said cancer.

The present invention in particular encompasses a drug selected from (i) a product allowing or enhancing the secretion of ATP, HMGB1, LysRS and/or IL-8, and/or the exposure of CRT, ERp57, LysRS and/or KDEL receptor at the surface of a tumour cell, (ii) a product stimulating the autophagy machinery and/or an ER stress response, (iii) a product recruiting and/or activating IL-17 producing γδ T lymphocytes, cytotoxic T cells and/or dendritic cells, (iv) a product promoting activation of the TLR4/myd88 pathway, or able to bypass said pathway, (v) a product triggering the P2RX7 (P2X purinoceptor 7) and/or the NALP3 inflammasome, (vi) a product allowing or enhancing the secretion of IL-lb, (vii) a product capable of stimulating intratumoral Vd2 T lymphocytes, and (viii) a product selected from an anti-allergic drug, a neurotropic drug, an antihypertensive or cardiotropic drug, a spindle poison drug, an antimicrobial drug, an anti-osteoclastic drug, a diuretic drug, an oestrogen, and (ix) any combination thereof, for use in a treatment of cancer, preferably in combination with a conventional immunogenic treatment of cancer selected from a chemotherapy using a drug selected from an anthracyclin, a platin, a taxane and an antimitotic agent, preferably from an anthracyclin, a platin, and an antimitotic agent; and radiotherapy, in a subject identified as resistant to said conventional immunogenic treatment of cancer according to a method as herein described of assessing the sensitivity of a subject having a tumor to a therapeutic treatment of cancer. Induction of immunogenic cancer-cell death, using a compensatory molecule as herein described, allows the subject's immune system, thanks to the present invention, to contribute, through a "bystander effect", to the eradication of cancer cells and cancer stem cells which are resistant to conventional therapeutic treatments. Herein described is also a method of treating cancer comprising the administration to a subject in need thereof, as previously explained, of a compensatory molecule, preferably together with a drug used in a conventional treatment of cancer (as a combined preparation).

Further herein described are the following kits:

- A kit to detect the abnormal expression, in particular in a tumor biopsy, of a gene selected from CCR1, EIF2AK2, DNAJCIO, PDIA3, EIF2A, PPPICB, IKBKB, PPPICC, BAX and combinations thereof, in a tumor sample of the subject, the kit comprising (i) at least one pair of primers and (ii) at least one fluorescent probe, for example two different probes, allowing the quantitative detection of the expression of a gene selected from CCR1, EIF2AK2, DNAJCIO, PDIA3, EIF2A, PPPICB, IKBKB, PPPICC, BAX, and (iii) a leaflet providing the control quantitative expression values corresponding to at least one of said genes in a control population.

- A kit to detect the presence of a polymorphism associated with an abnormal expression of a gene selected from AHR and MTHFR (for example a kit to detect the presence of a polymorphism associated with an abnormal expression of such a gene), in a tumor or blood sample of the subject, the kit comprising (i) at least one pair of primers, and (ii) at least two differently labelled probes, the first probe recognizing the wild-type allele and the second probe recognizing the mutated allele of a gene selected from AHR and MTHFR.

- A kit comprising:

a. (i) at least one pair of primers, (ii) at least one fluorescent probe allowing the quantitative detection of the expression of a gene selected from CCR1, EIF2AK2, DNAJCIO, PDIA3, EIF2A, PPPICB, IKBKB, PPPICC and BAX and (iii) a leaflet providing the control quantitative expression values corresponding to at least one of said genes in a control population; and

b. (i) at least one pair of primers, and (ii) at least two differently labelled probes, the first probe recognizing the wild-type allele and the second probe recognizing the mutated allele of a gene selected from AHR and MTHFR.

FIGURES

Figure 1. Thl and Thl7 related genes expression in tumors post-chemotherapy. (A) MCA205 tumors were treated with Doxorubicin (DX) or PBS. Tumor growth was monitored before and 8 days post-chemotherapy.

(B) Gene expression in DX versus PBS group was tested by RT-PCR (Taqman) and shown as fold change 8 days after treatment (lower panel). A more than 2 fold change was used as threshold for significant differences.

(C) Measurements of protein levels of IFN-γ and IL-17 in tumor homogeneates by ELISA at different time points.

(D) AHR antagonist CH223191 was dissolved with DMSO and diluted in Olive Oil. Mice treated with either PBS or DX received a daily i.p. injection of CH223191 (2 mM, 100 μΐ) for 4 days starting from the day of DX (or PBS) treatment.

(E) Expression of IFN-γ and IL-17 in dissociated tumor beds was tested by intracellular staining gated on live, CD45.2⁺ and CD3⁺ cells at day 8 post-treatment. Each group contained at least 5 mice and each experiment was performed at least twice yielding identical results.

Each graph depicts means±SEM of tumor sizes (A, D) or protein expression (C) or percentages of positive cells (E). *p<0.05, **p<0.01, ***p<0.001.

Figure 2. CD8⁺ T cells and γδ T cells are the major sources of IFN-γ and IL-17 respectively post-chemotherapy.

(A) Single cell suspensions of MCA205 tumors (day 8 post-DX) were analyzed by flow cytometry. IFN-γ and IL-17 positive cells were gated within live CD45.2⁺ and CD3⁺ cells (TILs). Within this gate, the proportions of CD3⁺ CD8⁺ cells and CD3⁺ TCR δ⁺ cells were examined. A typical dot plot analysis is shown.

(B) A typical dot plot in one DX or PBS treated tumor showing IFN-γ production by CD8 and IL-17 production by γδ T is depicted (upper panel). The percentages of IFN-y⁺ and IL-

17⁺ T cells among CD4⁺, CD8 and TCR6⁺ TILs in PBS versus DX-treated tumors are indicated as means±SEM of 5 tumors per condition (lower panel).

(C) Absolute numbers of IFN-y⁺ CD8 T cells and IL-17⁺ γδ T cells per 1mm³ of tumor are indicated as means±SEM in 5 tumors treated with DX or PBS.

(D) Kinetic study of IL-17 and IFN-γ production by γδ T and CD8⁺T cells respectively analyzed by flow cytometry in tumors treated with PBS or DX. (E) Ki67 expression on γδ TILs 8 days after DX showed as means±SEM in 5 tumors treated with DX or PBS.

(F) Correlation between the percentages of γδ T17 and Tel TILs in all tumors (treated or not) was plotted for MCA205 sarcomas (each dot representing one tumor). *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Recruitment of both Tel and γδ T17 cells correlate with better outcome in radiotherapy of TS/A tumors..

(A) Established TS/A tumors were treated with local irradiation on day 7. Mice were segregated into responders and non responders according to their tumor regression (TR) or tumor progression (TP) after radiotherapy (n=5).

(B) Percentages of CD8 T cells and Tel among CD3⁺ TILs are indicated as means±SEM.

(C) Percentages of γδ T and γδ T17 cells among CD3⁺ TILs are indicated as means±SEM.

(D) Correlation between the percentages of γδ T17 and Tel TILs in all tumors (treated or not) was plotted for TS/A mammary cancers (each dot representing one tumor).

*p<0.05, **p<0.01, ***p<0.001.

Figure 4. IL-17 contributes to prophylactic and therapeutic responses to immunogenic chemotherapy.

(A) Role of IFN-γ and IL-17 in DX-mediated anti-tumor effects. Mice bearing established MCA205 sarcoma were treated with local DX and systemic neutralizing antibodies (against mouse IFN-γ (left panel) or IL-17. (right panel) or isotype control (Iso Ctrl) i.p. every 2 days (3 injections total) starting from the day of DX. Tumor sizes are plotted as means±SEM for 5 mice/group. The experiment was performed twice with identical results. (B-C) Role of IL-17/IL- 17RCC signaling pathway in the immunogenicity of cell death. Oxaliplatin (OX)-treated EG-7 cells were inoculated in the footpad of WT versus IL-17Rcc^{~ /_} mice (n=5) (B) or into WT mice along with anti-IL-17 neutralizing Ab (or isotype Ctrl Ab) (C) and OVA-specific IFN-γ secretion was measured in the draining lymph nodes. (D) Immunization with DX-treated MCA205 and rechallenge with a tumorigenic dose of live MCA205 were performed at day 0 and day 7 respectively in mouse with various genetic backgrounds (as indicated). The percentages of tumor free mice were scored at different time points. A representative experiment out of two is depicted including 6-10 mice /group.

*p<0.05, **p<0.01. Figure 5. The therapeutic activity of anthracyclines depended upon Vy4/6 γδ T cells.

Established MCA205 were treated locally with DX in various genetic backgrounds (A, C) or in WT mice in addition to systemic administration of neutralizing antibodies anti- CCL20 (or isotype Ctrl Ab) (B).

(D) A kinetic measurement of tumor sizes is plotted as means±SEM. A representative experiment out of two yielding identical results is shown.

*p<0.05, **p<0.01, ***p<0.001.

Figure 6. A DC/γδΤ cell cross-talk leading to IL-i -dependent IL-17 production. (A-B) Cocultures of naive LNs derived γδ T (A-B) or TCR δΤ (A) cells in the presence of recombinant mouse cytokines (1

IL-Ιβ or/and IL-23, 5 IL- 6) (A) with or without TCR cross-linking with anti-CD3e mAb pre-coated plates (5

Clone 145-2C11) (B).

(C) Triple or double mixed coculture of LNs derived γδ T cells and/or bone marrow- derived DC loaded or not with live or DX-treated MCA205 was monitored for IL-Ιβ and IL- 17A release with ELISA test at 48 hrs.

(D) DX-treated MCA205 loaded DC/ γδ T cell cross-talk was also performed in the presence of 20

IL-IRA (Amgen) or anti-IL-23 or IL-23R neutralizing antibodies or 10 μ^πιΐ IL-18BP or 20 μΜ CH-223191. IL-Ιβ and IL-17A release was measured at 48 hrs in ELISA.

(E) Na^'ive lymph node cells from C57bl/6 mice were seeded in 96 well plates, stimulated with cytokines indicated with or without anti-CD3 crosslinking in the presence of GolgiStop (BD Bioscience). IL-17 production and CD69 expression with or without CH- 223191 are depicted. Graphs depict means±SEM of triplicate wells of cytokine release assessed at 48 hrs in ELISA. A representative experiment out of 3-6 is depicted in each case.*p<0.05. Figure 7. Adoptive transfer of γδ T cells synergize with chemotherapy under condition that γδ T cells express IL-1R1.

(A) Tumor growth after an adoptive transfer of LN derived γδ T or TCR5^" T cells into tumor beds two days after local DX treatment in established MCA205 sarcoma.

(B) The synergistic effects between γδΤ cells and DX were analyzed comparing WT versus IL-1R1^"7" γδ T cells. Tumor sizes is plotted as means±SEM for 5 mice/group. A representative experiment out of two yielding identical results is shown. *p<0.05.

Figure 8. DX polarizes TILs towards a TH1 and TH17 pattern.

(A) The precise calculation of fold changes for individual cytokine or chemokine, which was significantly increased at day 8 post-DX, is depicted for Thl- and Thl7-like profiles as tested by low density array.

(B) The impact of AHR pharmacological inhibitor CH-223191 on pro-apoptotic effect of DX or MTX against MCA205 was measured. A reduction in mitochondrial membrane potential indicated by decreased DiOC6(3) fluorescence was used to show cell apoptosis. The experiment was performed twice with identical results.

Figure 9. Infiltration of CT26 tumors with Tel and γδ T17 cells after therapy with anthracyclines.

(A) Tumor growth kinetics after treatment of established CT26 colon cancers with PBS or DX. The graph depicts means±SEM of size in 5 tumors per condition.

(B) Single cell suspensions of CT26 tumors were analyzed by flow cytometry at day 8 post-DX. After gated on live cells, IL-17 production was checked in CD45.2⁺, CD3+, CD4⁺, TCR δ+ cells compared with their corresponding negative fractions. A typical dot plot is shown.

(C-D) The percentage of CD8 T cells among TILs and their IFN-γ production (C) and the percentage of γδ T cells among TILs and their IL-17 production (D) were examined in PBS versus DX-treated tumors by flow cytometry. Means±SEM of percentages in 5 tumors/group are indicated.

(E) Correlation between the percentages of γδ T17 and Tel TILs in all CT26 tumors (treated or not) was plotted (each dot representing one tumor).

*p<0.05, **p<0.01, ***p<0.001. Figure 10. Phenotype of tumor infiltrated γδ T cells after DX therapy.

Flow cytometry analyses of the γδ T17 cells in the gate of live CD45.2⁺, CD3⁺ T cells invading MCA205 tumors at day 8 post-DX after a staining using the antibodies indicated in the Y axis. A typical dot plot analysis is depicted. The experiment has been performed three times yielding identical results.

Figure 11. CCR6 does not contribute to the recruitment of γδ T17 in tumors.

Flow cytometry analyses of the γδ T17 cells in the gate of live, CD45.2⁺, CD3⁺ T cells invading MCA205 tumors at day 8 post-DX in WT (upper panel) versus CCR6 loss-of- function mice (lower panel).

A typical dot plot analysis is depicted with means±SEM for 5 mice.

Figure 12. IL-6 and TGF-β failed to play a role in the immunogenicity or therapeutic effects of anthracyclines.

(A) Established CT26 colon cancer was treated with doxorubicin (DX) in the presence of systemic administration of neutralizing anti-IL-6 Antibody (or isotype Control Antibody). Kinetic tumor growth with 5 animals/group was shown.

(B) . Mice were immunized with DX treated CT26 on the right flank and concomitantly challenged with live CT26 tumor cells on the opposite flank at day 0. In parallel, anti-TGF- β or a control peptide (100 μg/mouse) were administered systemically from day 0 to 10. Kinetic tumor growth with 5 mice/group is shown for one representative experiment. The experiment has been performed twice yielding identical results. *p<0.05, **p<0.01, ***p<0.001.

Figure 13. The single-nucleotide polymorphism (SNP) R554K or Arg554Lys (rs2066853) in AHR gene affects the efficacy of conventional anti-cancer therapy in a neoadjuvant setting (before surgery) in breast cancer patients treated with anthracyclines (n=239).

The proportion of pathological complete responses was compared in wild-type and mutated groups of patients. The Chi square test was used to test the genetic association between the primary endpoint and the AHR-R554Ks SNP. Figure 14. Oxaliplatin induced CRT exposure.

U20S cells stably expressing CRT-GFP treated with 1 μΜ mitoxantrone (MTX) 150 μΜ cisplatin (CDDP) or 300 μΜ oxaliplatin (OXP) for the indicated time have been analyzed by means of automated image acquisition and automated analysis. Data is depicted as (A) representative images (B) and normalized CRT-GFP granularity values as well as percent of cells exhibiting nuclear shrinkage. The data is depicted as mean ± s.e.m. of quadruplicates from a representative experiment. (C) Immunofluorescene was conducted on CRT-GFP expressing cells by means of staining with anti-CRT antibody and subsequent confocal image acquisition. (D) CRT has been cloned in frame n-terminal to a HaloTag^® sequence followed by a KDEL ER retention signal. (E) The impermeable HaloTag^® ligand forms covalent bonds exclusively with surface exposed HaloTag^®-CRT fusion protein, whereas intracellular HaloTag^®-CRT remains undetected. Figure 15. Mitochondrial cell death upon treatment with oxaliplatin and cisplatin.

U20S cells stably expressing CRT-GFP treated with 1 μΜ mitoxantrone (MTX) 150 μΜ cisplatin (CDDP) or 300 μΜ oxaliplatin (OXP) have been acquired by means of an automated microscope and subsequently subjected to automated analysis. The data is depicted as representative images (A) and (B) normalized Bax-GFP granularity values. The data represents mean ± s.e.m. of quadruplicates from a representative experiment. (C, D) U20S cells were treated with the indicated drugs at the indicated concentrations. 16 h after treatment, cell death was monitored by simultaneous staining with 3,3 dihexyloxacarbocyanine iodide (DiOC₆(3)) and propidium iodide (PI), and the percentage of dying (DiOC₆(3)^low PT, open bars) and dead (DiOC₆(3)^low PI⁺, closed bars) cells was determined by cytofluorometry.

The data represents means ± s.e.m. of triplicate determinations.

Figure 16. Oxaliplatin and cisplatin induced ATP release.

Cells were treated with mitoxantrone (MTX), oxaliplatin (OXP), or cisplatin (CDDP) at the indicated concentrations. 16 h post-treatment, the intracellular ATP was stained with quinacrine and the nuclei were counterstained with Hoechst 33342. The vital dye propidium iodide (PI) was used to visualize dead cells before acquisition by (A) automated fluorescence microscopy or (B) cytofluorometric analysis. In addition, the concentrations of intracellular (C) and extracellular (D) of ATP were monitored.

Results are means ± SEM of triplicate determinations. Figure 17. OXP-, but not CDDP- induced ER stress markers.

Cells stably expressing G3BP-GFP or GFP-LC3 were treated for 4 h with 1 mM sodium arsenate heptahydrate (NaHAsC^) or 10 μΜ rapamycin for 8 h as positive controls respectively. In addition the cells have been treated with 150 μΜ cisplatin (CDDP) or 300 μΜ oxaliplatin (OXP) for the indicated time to assess (A, B) the formation of stress granules and (C, D) the lipidation of LC3 as an indicator for autophagy. Representative images (A, C) and mean granularity values (B, D) of quadruplicates are shown.

(E) The phosphorylation status of eIF2cc has been assessed by immunobloting against the phosphoneoepitope Ser51 of eIF2cc by means of a monoclonal antibody. A polyclonal antibody has been used to visualize whole eIF2cc protein levels.

Figure 18. Thapsigargin restores CRT exposure in the presence of cisplatin.

Compounds from the ICCB known bioactive compounds library have been tested for their capacity to induce CRT-exposure. The library compounds were added at a concentration range from 90 nM to 48 μΜ in the presence (A) or absence (C) of 50 μΜ cisplatin (CDDP). The cells were incubated for 4 h and were acquired by means of automated microscopy. The data is depicted as dot plots and representative images (B).

To eliminate background produced by other library compounds the data from the library screen in the presence of cisplatin was plotted against data from a screen in the absence of CDDP. Mean as well as 95% percentile is depicted and the data represents doublets from two independent experiments.

(D) CRT exposure has been measured 4 h upon application of the indicated dose range of thapsigargin (THAPS) with and without 50 μΜ CDDP. Samples have been acquired in quadruplicates from 3 independent experiments and data is depicted as mean ± s.e.m.. Figure 19. Thapsigargin restores CRT-exposure of cisplatin treated cells in vitro and anti cancer immunogenicity in vivo. (A, B) U20S or HaloTag -CRT stably expressing U20S cells were assessed after a treatment with mitoxantrone (MTX), oxaliplatin (OXP) , cisplatin (CDDP), thapsigargin (THAPS) or cisplatin combined with thapsigargin by immunofluorescence staining or incubation with impermeable fluorescent HaloTag^® ligand respectively followed by flow cytometric analysis. CRT exposure upon combination of 150 μΜ CDDP with 1 μΜ THAPS was confirmed in (C) mouse lewis lung cell carcinoma, (D) CT26 and (E) MCA205 cells by means of immunostaining and following flow cytometric analysis.

(F) MCA205 cells have been used for tumor vaccination in vivo. Treated cells have been inoculated subcutaneously into the flank of C56BL/6 mice. The mice have been rechallenged after 6 days with living cells and the tumor growth is depicted in the survival plot (n=10).

Figure 20. THAPS exhibits no additional cytotoxicity.

(A) U20S cells were treated with the indicated drugs in the presence or absence of thapsigargin (THAPS) at the indicated concentrations. 16 h after treatment, cell death was monitored by simultaneous staining with 3,3 dihexyloxacarbocyanine iodide (DiOC₆(3)) and propidium iodide (PI), and the percentage of dying (DiOCe(3)^low PT, open bars) and dead (DiOC₆(3)^low PI⁺, closed bars) cells was determined by cytofluorometry.

The data represents means ± s.e.m. of triplicate determinations.

(B, C) U20S cells were treated with mitoxantrone (MTX), oxaliplatin (OXP), or cisplatin (CDDP) with and without THAPS at the indicated concentrations. 16 h post-treatment, the intracellular ATP was stained with quinacrine and the nuclei were counterstained with the Hoechst 33342. The vital dye propidium iodide (PI) was used to visualize dead cells before acquisition by (B) automated fluorescence microscopy or (C) cytofluorometric analysis. In addition, the concentrations of intracellular (D) and extracellular (E) of ATP were monitored.

Results are means ± SEM of triplicate determinations.

Figure 21. "CRT screen"

Compounds from the US drug compound library have been tested for their capacity to induce CRT-exposure. The library compounds were tested at a final concentration of 1 μΜ in CRT-GFP, H2B-RFP stably expressing U20S cells. The cells were incubated for 4 h and were acquired by means of automated microscopy. The data is depicted as dot plots representing normalized mean values (n=4).

Figure 22. Study profile

The Support Vector Machine (SVM) analyses were performed with the MEV software version 4.5. The LOOCV approach was used as SVM process to estimate the prediction accuracy of the molecular classifiers. The relevance of these classifiers was then tested with univariate (Fisher's exact test) and multivariate (logistic regression and ROC curves) methods.

Figure 23. « Calreticulin pathway» molecular classifiers based on 5 to 3 genes are detected in the two anthracycline treated cohorts (FEC) but not in the taxane treated cohort (TET).

The prediction accuracy of each molecular classifier was assessed by Fisher's exact test on the « pCR vs non pCR » contingency tables obtained from the LOOCV approach of SVM procedure. The SVM training parameters used were a polynomial kernal matrix with a diagonal factor of 1.3. The predictive value of the classifier was also evaluated by the sensibility (Se) and specificity (Spe) parameters. The "calreticulin pathway" was represented by an initial set of 43 genes in the cohort HOUSTON FEC and 53 genes in the cohort IGGO FEC/TET.

Figure 24. Three genes based - « Calreticulin pathway» molecular classifiers are independant predictive factors in the two anthracyclines treated cohorts (FEC) but not in the taxane treated cohort (TET).

A. Multivariate analyses using logistic regression were performed in each cohort to test the association of the molecular classifier with a pathological complete response (pCR) by taking into account the effects of classical clinical factors. The HOUSTON FEC cohort was matched on oestrogen receptor status. The IGGO FEC and TET cohorts were restricted to patients with oestrogen-receptor-negative tumours.

B. ROC curves were used to test the quality of the predictions.

C. The under-expression (down) or over-expression (up) of genes in the classifiers between pCR and non pCR groups are mentioned with the respective p- values of non parametric Mann- Whitney test. Adj P-value : adjusted P-value; 95% CI : 95% confidence interval ; AUC : area under ROC curve.

Figure 25. Search of a common « Calreticulin pathway» molecular classifier between the two anthracyclines treated cohorts (FEC).

A. The genes were classified by decreasing value according to the non parametric Mann- Whitney test used to compare the gene expressions between pCR and non-pCR groups. * These genes are represented by median values of replicates.

B. The p-values of genes from anthracyline-treated cohorts (FEC) were plotted. The common molecular classifier was constructed with the candidate genes located in the grey areas.

Figure 26. The common 3 genes based - « Calreticulin » molecular classifier is an independant predictive factor in the two anthracyclines treated cohorts (FEC) but not in the taxane treated cohort (TET).

A. The prediction accuracy of the common molecular classifier was assessed by Fisher's exact test on the « pCR vs non pCR » contingency tables obtained from the LOOCV approach of SVM procedure. The SVM training parameters used were a polynomial kernal matrix with a diagonal factor of 2. The predictive value of the classifier was also evaluated by the sensibility (Se), specificity (Spe), positive and negative predictive values (PPV and NPV) and accuracy parameters.

B. Multivariate analyses using logistic regression were performed in each cohort to test the association of the common molecular classifier with pathological complete response (pCR) by taking into account the effects of classical clinical factors.

C. The under-expression (down) or over-expression (up) of genes in the common classifier between pCR and non pCR groups are mentioned. * These gene expression differences were statistically significant between the pCR and non-pCR groups.

Figure 27. A molecular classifier (also herein identified as parameter signature or "algorithm") that integrates the "CALR pathway" signature and a MTHFR SNP is particularly efficient to predict the ability of a given subject to respond to anthracyclines in the HOUSTON FEC cohort. Multivariate analyses using logistic regression and ROC curves were performed to assess the prediction accuracy of four different models of classifiers.

DETAILED DESCRIPTION OF THE INVENTION Inventors have herein discovered an ordered sequence of molecular events in the pathway leading to the immunogenic cell death of tumour cells.

This pathway may be interrupted at several levels, by the loss of a positive mediator or by the presence of an inhibitor of such a positive mediator. The result of such an interruption will be the absence of reaction of the subject's immune system, in other words, the absence of "immunogenic cell death".

Inventors herein below identify particular products the detection of which can be used to determine if a subject will respond or not to a cancer treatment. Inventors further herein below provide methods which can be used (i) to determine the presence of an immunogenic response in a subject having a tumor, (ii) to determine the presence or level of exposure of particular proteins on the surface of tumour cells or of immune cells (as herein defined), (iii) to determine the presence or level of expression of particular proteins secreted by tumor cells or immune cells, (iv) to determine the susceptibility of a tumour cell to a cancer treatment, and/or (v) to determine if a subject will respond to a cancer treatment or will be resistant to said treatment.

"Immunogenic cell death" Cell death can be classified according to the morphological appearance of the lethal process (that may be apoptotic, necrotic, autophagic or associated with mitosis), enzymo logical criteria (with and without the involvement of nucleases or distinct classes of proteases, like caspases), functional aspects (programmed or accidental, physiological or pathological) or immunological characteristics (immunogenic or non-immunogenic) (Kroemer et al, 2009).

Thanks to the advancing comprehension of cellular demise, it has become clear that the textbook equation 'programmed cell death=apoptosis=caspase activation=non- immunogenic cell death', although applicable to some instances of cell death, constitutes an incorrect generalization, at several levels (Garg et ah, 2009). Thus, necrosis can be programmed both in its course and its occurrence (Vandenabeele et ah, 2008). Apoptosis can be lethal without caspase activation, and caspase activation does not necessarily cause cell death (Kroemer and Martin, 2005). Finally, cell death with an apoptotic appearance can be immunogenic (Casares et ah, 2005). These examples illustrate the urgent need to strive towards a more detailed comprehension of cell death subroutines.

Cell death is defined by Casares et al. (2005) as "immunogenic" if dying cells that express a specific antigen (such as the model antigen ovalbumin OVA or a tumor antigen), yet are uninfected (and hence lack pathogen-associated molecular patterns), and are injected subcutaneously into mice, in the absence of any adjuvant, cause a protective immune response against said specific antigen. Such a protective immune response precludes the growth of living transformed cells expressing the specific antigen injected into mice.

Inventors demonstrate that when cancer cells succumb to an immunogenic cell death (or immunogenic apoptosis) modality, they alert the immune system, which then mounts a therapeutic anti-cancer immune response and contributes to the eradication of residual tumor cells. Conversely, when cancer cells succumb to a non- immunogenic death modality, they fail to elicit such a protective immune response.

"Anti-cancer immune response"

The response from the immune system is herein called an "anti-cancer immune response" when it is directed against tumour cells, in particular cancerous cells. The anticancer immune response is allowed by a reaction from the immune system of the subject to the presence of cells, preferably of tumor cells, dying from an immunogenic cell death (as defined previously).

Preferably, the anti-cancer immune response allows, at least partly, the regression or destruction of the tumor. In the context of the present invention, the patient or subject is a mammal. In a particular embodiment, the mammal is a human being, whatever its age or sex. The patient may have a tumor. Unless otherwise specified in the present disclosure, the tumor is a malignant tumor.

An in vitro or ex vivo method of assessing the sensitivity of a subject having a tumor to a treatment of cancer is herein provided as a particular embodiment. This method comprises a step of detecting the presence of an anticancer immune response of the subject, the absence of an anticancer immune response being indicative of a resistance of the subject to the treatment of cancer.

Within the context of this invention, "non-responder" or "resistant" refers to the phenotype of a subject who does not respond to a treatment of cancer, in particular to a conventional treatment of cancer as previously defined, i.e. the volume of the tumor does not substantially decrease, or the symptoms of the cancer in the subject are not alleviated, or the cancer progresses, for example the volume of the tumor increases and/or the tumor generates local or distant metastasis. The terms "non-responder" or "resistant" also refer to the phenotype of a subject who will die from the cancer. Within the context of this invention, "responder" or "sensitive" refers to the phenotype of a patient who responds to a treatment of cancer, in particular to a conventional treatment of cancer as previously defined, i.e. the volume of the tumor is decreased, at least one of his symptoms is alleviated, or the development of the cancer is stopped, or slowed down.

A subject who responds to a cancer treatment is, in the sense of the present invention, a subject who typically has a much longer disease free survival chance than a patient who has been identified, with a method as herein described, as resistant to a treatment of cancer. Typically, a subject who responds to a cancer treatment is a subject who will be completely treated (cured). The sensitivity or susceptibility of a subject to a treatment of cancer indicates whether the subject is "responder" or "non-responder", in other words whether the subject will or will not, be at least partially treated (tumor growth retardation or regression), preferably be completely treated (cured), by said cancer treatment.

In a particular and preferred embodiment of the present invention, the subject is typically a subject undergoing a treatment of cancer, in particular a conventional treatment of cancer (preferably chemotherapy and/or radiotherapy). This means that, typically, before assessing the sensitivity of the subject to a particular treatment of cancer, this subject has been exposed to said particular treatment of cancer. The subject may have been exposed to part of a complete conventional treatment protocol, for example to at least one cycle of the all treatment protocol, for example two cycles of the all treatment protocol.

In another particular embodiment of the present invention, the method of assessing the sensitivity of a subject to a treatment of cancer is applied on a subject who has not been previously exposed to a treatment of cancer.

Methods herein described are predictive methods, i.e., methods capable of assessing the ability of a subject to mount an immune response in the context of an anthracycline, oxaliplatine or X Rays treatment as herein defined and not only prognostic methods, only capable of indicating whether the subject will survive to the cancer or die from the cancer.

In the context of the present invention, a "conventional treatment of cancer" may be selected from a chemotherapy, a radiotherapy, an hormonotherapy, an immunotherapy, a specific kinase inhibitor-based therapy, an antiangiogenic agent based-therapy, an antibody-based therapy, in particular a monoclonal antibody-based therapy, and surgery.

The term "conventionally" means that the therapy is applied or, if not routinely applied, is appropriate and at least recommended by health authorities. The "conventional" treatment is selected by the cancerologist depending on the specific cancer to be prevented or treated. In the present invention, the cancer is a cancer that is usually or conventionally treated with one of the following therapy: a chemotherapy, a radiotherapy, an hormonotherapy, an immunotherapy, a specific kinase inhibitor-based therapy, an antiangiogenic agent based- therapy, an antibody-based therapy and a surgery.

The cancer may be any kind of cancer or neoplasia. The cancer is preferably selected from a breast cancer, a prostate cancer, an oesophagus cancer, a colon cancer, a rectal cancer, a kidney cancer, a lung cancer, in particular a non-small cell lung cancer (NSCLC), a thyroid cancer, an osteosarcoma, a gastrointestinal sarcoma (GIST), a melanoma, a leukaemia, in particular an acute lymphoid leukemia, an Hodgkin lymphoma, and a neuroblastoma.

The tumour cell mentioned in the present invention is a cell obtained from a tumor of a subject suffering from a cancer, in particular from at least one of the previously identified cancers. The tumor cell is preferably selected from a carcinoma, a sarcoma, a lymphoma, a melanoma, a paediatric tumour and a leukaemia tumour.

It is to be understood that the expression "tumor cells" used to identify cells obtained from a tumor of a subject, is also used, in the present description, to identify circulating tumor cells (in the case of leukaemia for example), cells obtained from a tumor bed, or cells obtained from a metastase.

An hormonotherapy is a therapy leading to apoptosis or Fas ligands or soluble /membrane bound TRAIL (TNF-related-apoptosis-inducing-ligand) or soluble/membrane bound TNF (tumor necrosis factor) alpha (TNF a). Cancers sensitive to a hormonotherapy are conventionally treated using a compound such as an antiaromatase for example.

Cancers sensitive to an immunotherapy are conventionally treated using a compound selected for example from IL-2 (Inter leukine-2), IFN (Interferon) alpha (IFNa), and a vaccine.

Cancers sensitive to a specific kinase inhibitor-based therapy are conventionally treated using a compound selected for example from a tyrosine kinase inhibitor, a serine kinase inhibitor and a threonine kinase inhibitor.

Cancers sensitive to an antibody-based therapy, preferably to a monoclonal antibody-based therapy are conventionally treated using a specific antibody such as for example anti-CD20 (pan B-Cell antigen) or anti-Her2/Neu (Human Epidermal Growth Factor Receptor- 2/NEU). Preferably, the conventional treatment of cancer is a conventional chemotherapy or a conventional radiotherapy.

In the context of a conventional radiotherapy, the treatment may consist in exposing the subject to an irradiation selected for example from XR, gamma irradiations and/or UVC irradiations.

In the context of a conventional chemotherapy, the treatment may use a cytotoxic agent or cell death inducer (chemotherapeutic agent), in particular a genotoxic agent.

In a particular embodiment of the present invention, the chemotherapeutic agent is an agent selected for example from an anthracyclin, an antimitotic agent (spindle poison such as vincristine or vinblastine), a DNA intercalating agent, a taxane (such as docetaxel, larotaxel, cabazitaxel, paclitaxel (PG-paclitaxel and DHA-paclitaxel), ortataxel, tesetaxel, and taxoprexin), gemcitabine, etoposide, mitomycine C, an alkylating agent, a platin based component such as CDDP and OXP, and a TLR (Toll-like receptor)-3 ligand.

In a particular embodiment of the present invention, in particular when chemotherapy is administered to the subject before any surgical step, the chemotherapeutic agent is not a taxan, and preferably also not an antimitotic agent.

Particular anthracyclins may be selected, in the context of the present invention, from DX, daunorubicin, idarubicin and MTX. In a particular embodiment of the present invention, the antibody used in an antibody- based therapy is a cytotoxic antibody.

A particular breast cancer is a breast cancer conventionally treated with anthracyclins, taxanes, Herceptin, anti-PARP (Poly (ADP-ribose) polymerase), anti-PI3K (Phosphoinositide 3-kinase), mTOR (mammalian Target of Rapamycin) inhibitors, navelbine, gemcitabine, antioestrogens, antiaromatases, and/or a TLR-3 ligand, before or after a surgical step to remove breast tumor, preferably before such a surgical step.

A particular thyroid cancer is a thyroid cancer treated with radioactive iodine or tyrosine kinase inhibitors, preferably RET inhibitors.

A particular Hodgkin lymphoma is a Hodgkin lymphoma conventionally treated with

CHOP [Cyclophosphamide, Hydroxydaunorubicin, Oncovin (vincristine), and Prednisone and/or Prednisolone] or anthracyclines. A particular prostate cancer is a prostate cancer conventionally treated with taxanes and XR.

A particular colon cancer is a colon cancer conventionally treated with OXP and/or the combination of 5-fluorouracil (5 FU) and folinic acid.

A particular metastatic colon cancer is a metastatic colon cancer conventionally treated with 5 FU and OXP or irinothecan.

A particular rectal cancer is a rectal cancer conventionally treated with radiotherapy, preferably local radiotherapy, preferably together with CDDP and/or 5 FU.

A particular oesophagus cancer is an oesophagus cancer treated with CDDP, before or after a surgical step to remove the oesophagus tumor, preferably before such a surgical step, the administration of CDDP being preferably combined to the administration to the patient of a radiotherapy, preferably a local radiotherapy.

A particular kidney cancer is a kidney cancer conventionally treated with cytokines or anti- angiogenic drugs (sorafenib).

A particular lung cancer is a lung cancer conventionally treated with XR and platine or Permetrexed (Alimta®).

A particular early stage NSCLC is an NSCLC conventionally treated with CDDP and/or etoposide, or with taxanes and avastin [anti-VEGF (Vascular endothelial growth factor) antibody].

A particular osteosarcoma and a preferred GIST are respectively an osteosarcoma and a GIST conventionally treated with anthracyclins, imatinib (Gleevec®) and/or sunitinib. A particular melanoma is a melanoma conventionally treated with dacarbazine (DTIC); B - Raf inhibitors (PLX4032); sorafenib and/or temozolomide; electrochemotherapy; or isolated limb perfusion of TNFalpha, in particular of high doses of TNFalpha.

A particular neuroblastome is a neuroblastome conventionally treated with anthracyclines or alkylating agents, in particular in the context of an autologous bone marrow transplantation or of a stem cells transplantation.

A particular acute lymphoid leukemia is an acute lymphoid leukemia treated with anthracyclins, vinblastine and/or vincristine.

A particular multiple myeloma is a malignant hemopathy treated with anthracyclins, bortezomiv, revlimide, thalidomide and/or an alkylating agent, in particular in the context of an autologous bone marrow or stem cell transplantation. Conventional treatments of cancer, as described previously, in particular radio- and chemotherapy, are thought to mediate the direct elimination of tumour cells. Although different anti-tumour agents may kill tumor cells through an apparently homogenous apoptotic pathway, they differ in their ability to stimulate the subject's immune system. Indeed, there are circumstances in which anti-cancer therapy can induce a cellular death (immunogenic cell death) eliciting innate and cognate immune responses which in turn mediate part of the anti-tumour effect.

Inventors herein demonstrate that all cases of complete therapeutic success (cure) involve an immunological component.

As indicated previously, it is possible to distinguish between conventional treatments of cancer able to induce an immunogenic cell death, herein identified as "conventional immunogenic treatments", and conventional treatments of cancer which induce or tend to induce a non- immunogenic cell death, herein identified as "conventional non- immunogenic treatments".

As indicated previously, most of standard chemotherapies are known to induce a non- immunogenic apoptosis (Zitvogel et al, 2004 ; Steinman et al, 2004; Lake et al, 2006). OXP and anthracyclines in particular induce immunogenic cell death, as do radiotherapy (ionizing radiations), while other agents such as CDDP and alkylating agents tend to induce a non-immunogenic cell death (Casares et al, 2005; Obeid et al, 2007), as do etoposide, 5-FU and mitomycin C. A typical in vitro method used to assess the immunogenicity of a particular drug comprises the steps of:

(a) inducing the cell death or apoptosis of mammalian cells (for example cells from the CT26 or MCA205 mouse cell line), typically of mammalian cells capable of expressing calreticulin (CRT), by exposing said mammalian cells to a particular drug, for example 18 hours; (b) inoculating (for example intradermally) the dying mammalian cells from step (a) in a particular area (for example a flank) of the mammal, typically a mouse, to induce an immune response in this area of the mammal;

(c) inoculating (for example intradermally) the minimal tumorigenic dose of syngeneic live tumor cells in a distinct area (for example the opposite flank) from the same mammal, for example 7 days after step (b); and

(d) comparing the size of the tumor in the inoculated mammal with a control mammal also exposed to the minimal tumorigenic dose of syngeneic live tumor cells of step (c) [for example a mouse devoid of T lymphocyte], the stabilization or regression of the tumor in the inoculated mammal being indicative of the drug immunogenicity.

Inventors herein demonstrate that a subject having a tumor may however resist even to a conventional immunogenic treatment as previously identified and/or defined.

Herein provided is therefore an in vitro or ex vivo method of assessing the sensitivity of a subject having a tumor, as previously defined, to a treatment of cancer, in particular to a conventional immunogenic treatment, which method comprises a step of determining the ability of the subject and/or of the tumor to induce an anticancer immune response, the inability of at least one of the subject and of the tumor to induce an anticancer immune response being indicative of a resistance of the subject to the treatment of cancer.

Immune cells

Inventors demonstrate the critical role of subsets of cells from the immune system, herein identified as "immune cells", which reveal the presence of an anticancer immune response from a subject having a tumor. Preferably, this subject has been exposed to a treatment of cancer, in particular to at least one conventional treatment of cancer.

In a particular embodiment, a method of assessing the sensitivity of a subject having a tumor to a treatment of cancer is herein provided, wherein the method comprises a step of detecting the presence of immune cells selected in particular from γδ T lymphocytes, dendritic cells and cytotoxic T lymphocytes, in a tumor sample of the subject. In a preferred embodiment, the previously described method is applied on a subject who has not been exposed to a treatment of cancer. This method may further be applied to the same subject after said subject has been exposed to a treatment of cancer, in particular to a chemotherapeutic treatment of cancer, preferably to several cycles, for example two, three or four cycles of a complete chemotherapeutic treatment.

The method may further comprise a step of comparing the presence of immune cells in a tumor sample of the subject before and after exposition of the subject to a treatment of cancer as explained previously. This method may be applied in vitro or ex vivo on a biological sample or biopsy from the subject, in particular on a tumor sample or biopsy, on a biopsy of cells from the tumor bed, on cytospins, on cells from a metastase, or on circulating tumor cells.

The presence of immune cells in the tumor of a subject is indicative of the presence of an anticancer immune response in the subject who has been exposed to a treatment of cancer and reveals the sensitivity of the subject to the treatment of cancer (responder phenotype). The absence of immune cells in the tumor of a subject is indicative of the absence of an anticancer immune response in the subject who has been exposed to a treatment of cancer and reveals a resistance of the subject to the treatment of cancer (non responder phenotype).

The γδ T lymphocytes, the presence of which may be checked in the previously described method, are preferably selected from Vy4⁺ γδ T lymphocytes (mouse), in particular activated Vy4⁺ γδ T lymphocytes; νδ2 (or νδΐ in humans) T lymphocytes; Vy6⁺ γδ T lymphocytes, in particular activated Vy6⁺ γδ T lymphocytes; IL-17 producing γδ T lymphocytes (also herein called "γδ T17 cells"), in particular cells expressing RORyt (RAR-related orphan receptor), AHR (aryl hydrocarbone receptor), IL-23R, IL-17A and/or IL-22; γδ T lymphocytes expressing the IL-1 receptor (IL-IR or IL-lRl); and any combination of the previously mentioned γδ T lymphocytes such as, in particular IL-17 producing- Vy4⁺ and νγ6⁺γδ T lymphocytes, preferably expressing the IL-IR.

The previously mentioned γδ T lymphocytes populations identify populations of mammalian cells. Human γδ T lymphocytes have Υδ2 (circulating) T lymphocytes but no Vd2 (mucosal) T lymphocytes, contrary to mouse γδ T lymphocytes. Both populations of V52 and Vd2 T lymphocytes are however able to differentiate into VydT17 cells.

It is to note that the νγδ T lymphocytes, in particular those present in tumor beds, have the following phenotype: Ki67⁺, GzB⁺, CD69⁺ and IL-17⁺, when they are activated.

The dendritic cells, the presence of which may be checked in the previously described method, are preferably selected from myeloid cells (such as monocytic cells and macrophages) expressing langerin, MHC (major histocompatibility complex) class II, CCR2 (chemokine (C-C motif) receptor 2), CX3CR1 and/or Grl molecules in mice; myeloid cells expressing CD 14, CD 16, HLA dR (human leukocyte antigen disease resistance) molecule, langerin, CCR2 and/or CX3CR1 in humans; dendritic cells expressing CD 11c, MHC class II molecules, and/or CCR7 molecules; and IL-Ιβ producing dendritic cells. The cytotoxic T lymphocytes, the presence of which may be checked in the previously described method, are preferably selected from CD3+, CD4+ and/or CD8+ T lymphocytes, FOXP3 (forkhead box P3) T lymphocytes, Granzyme B/TIA (Tcell-restricted intracellular antigen) T lymphocytes, and Tel cells (IFN-γ producing CD8+ T lymphocytes). Other immune cells, the presence of which may be checked in the previously described method, are cells expressing a CRT receptor.

Such immune cells may be selected from cells expressing at least one of the following proteins: LRP1 (Low density lipoprotein receptor-related protein 1, CD91), Ca++-binding proteins such as SCARF 1 and SCARF2, MSRl (Macrophage scavenger receptor 1), SRA, CD59 (protectin), CD207 (langerin), and THSD1 (thrombospondin).

The detection step of the previously identified immune cells can be easily performed according to methods known by the man of the art such as immunochemistry, immunophenotyping, flow cytometry, Elispots assays (Panaretakis T. et al, 2009), classical tetramer stainings (Ghiringhelli F, et al, 2009), intracellular cytokine stainings, (Conforti R et al, 2010). In a particular embodiment of the present invention, the step of determining the presence of an anticancer immune response may consist in detecting and/or dosing, in a biological sample of the patient, for example in a blood or serum sample of the patient, the presence (or normal expression) of a particular cytokine, a particular chemokine, and/or of particular antibody, the absence or abnormal expression (in particular an insufficient amount), when compared to a standard expression (for example level of expression), of the particular cytokine, of the particular chemokine and/or of the particular antibody being indicative of an absent or insufficient anticancer immune response. The cytokine the presence of which is to be determined according to the previously described method may be selected from IL-lb, IL-7, IL-10, IL-12a, IL-12b, IL-15, IL-17, IL-21, IL-23, IL-27, IL-33, TNFa, LTbeta (lymphotoxin beta), IFNalpha, beta, lambda, gamma, and the following cytokine receptors [ST2/ILlrll, IL-1R1, IL-7r, IL-15Ra, IL- 21R, IL-23R, LtbR, AHR, Flt3 (fins-like tyrosine kinase receptor-3, CD135)] and the following transcription factors (RORc, RORgt, FOXP3, Ikaros, Id2, PU-1).

The chemokine the presence of which is to be determined according to the previously described method may be selected from CCL2 (Chemokine (C-C motif) ligand), CCL20/MIP3A, CCL5/RANTES, CCL7, CCL25, CXCL1, CXCL2, CXCL9/ITAC, CXCL10/IP10, CXCL12/SDF1, CXCL13, CXCL16/Bonzo, CX3CL1/Fractalkine, and their receptors (CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CCR2, CCR4, CCR5, CCR7, CX3CR1).

The antibody (Ab) the presence of which is to be determined according to the previously described method may be selected from anti-NY-ESOl Ab, anti-LAGEl Ab, anti-MICA/B Ab, anti-disulfide isomerase ERp5 Ab, anti-PARPl Ab, anti-ZNF707 (zinc finger protein) Ab in combination with PTMA (prothymosin, alpha), anti-CEP78 (centrosomal protein) Ab, anti-ODF2 (outer dense fiber of sperm tail 2) Ab, anti-SDCCAGl (serologically defined colon cancer antigen 1) Ab, anti-endothelin 1 (ET-1) ligand Ab, anti-endothelin B receptor (ET_BR) Ab and anti-Rgs5 (regulator of G protein signalling 5) Ab.

Immunogenic cell death-associated molecules found in the tumor The step of determining the ability of the tumor to induce an anticancer immune response consists in verifying, in the tumor cells (in particular in dying tumor cells, for example cells which have been exposed to a treatment of cancer), the presence of specific features herein disclosed and identified as "immunogenic cell death-associated molecules or signals" or "danger signals".

Inventors herein demonstrate that stressed and dying tumor cells emit a particular pattern of "danger signals". These immune cell death-associated molecules are either exposed on the surface of dying cells or secreted into the microenvironment. Thus, the combined action of 'find-me' signals (for the attraction of phagocytes) and 'eat-me' signals (for corps engulfment) together with the release of hidden molecules (which often signal danger and are usually secluded within live cells), influence the switch between silent corpse removal and inflammatory reactions that stimulate the cellular immune response. Inventors discovered that some of said immunogenic cell death-associated molecules are inherent to the tumor, i.e., independent from the subject having the tumor or from the treatment the subject may have been exposed to. Others only appear in or around the tumor (for example in a tumor bed) after an exposition of the subject having the tumor to a conventional treatment of cancer.

Inventors have discovered, in the present invention, that if the tumor cells of the subject do not correctly or normally express a functional immunogenic cell death-associated molecule, such as one of the particular proteins identified below, in particular do not secrete such a molecule or do not expose such a molecule at their surface (or otherwise secrete or expose such a molecule at an abnormal level compared to a standard level), an additional treatment, herein identified as "compensatory immunogenic treatment of cancer", should be administered to said patient, preferably in addition to a conventional treatment of cancer, to favour a reaction from the immune system against said tumour cells.

The exposure or secretion can be observed or determined before or after exposition of the subject to a conventional therapy as described previously, preferably after such an exposition, even more preferably before and after such an exposition. In a particular embodiment of the present invention, the method of determining the ability of a tumor to induce an anticancer immune response comprises a step of comparing the expression by tumor cells of functional immunogenic cell death-associated molecules before and after exposition of said tumor cells to a treatment of cancer.

An absent or abnormal (for example insufficient) level of expression of an immunogenic cell death-associated molecule by the tumor cell in response to a cancer treatment, in particular to a conventional one, indicates that the cell will not be, completely or partially, destroyed or eradicated by said cancer treatment.

In EP2084531, inventors have shown that the pre-apoptotic translocation of intracellular CRT (endo-CRT) to the plasma membrane surface (ecto-CRT) is a key feature of "immunogenic cell death". They demonstrated that when CRT is exposed on the surface of dying cells, it promotes their destruction by phagocytes such as dendritic cells. Phagocytes then interact with the immune system which is, in turn, responsible for the immune response. Inventors further demonstrated (i) that this effect is amplified when CRT is present in an increased amount on the surface of dying cells and (ii) that CRT is present in an increased amount on the surface of most tumour cells of a subject who has been exposed to a conventional treatment of cancer, in particular a cell death inducer (apoptosis inducer).

Inventors also showed that the proteins whose expression level and post-transcriptional modification regulate CRT exposure comprise in particular:

• at the level of ceramide metabolism: ceramide synthase, dihydroceramide desaturase, 3- ketosphingane reductase, serine palmitoyltransferase, sphingomyelin synthase, shingomyelinase, ceramidase, ceramide synthase, sphingosine kinase, sphingosine-1- phosphate phosphatase ;

• at the level of Bcl-2 proteins: Bax, Bak, Bok, Bcl-2, Bcl-XL, Mcl-1 as well as all the other multidomain or BH3-only proteins from the Bcl-2 family

• at the level of caspase-8 (CASP 8) activation and substrates: FADD (Fas-Associated protein with Death Domain), FLIP (FLICE-inhibitory protein), RIP (Receptor-interacting protein), TRADD (Tumor necrosis factor receptor type 1 -associated DEATH domain), BAP31 (B-cell receptor-associated protein 31); • at the level of the endoplasmic reticulum (ER) stress response: eIF2alpha (eIF2A), GCN2, HRI, PERK, PKR, PP1, GADD34, IREl, PERK and ATF6, BiP;

• at the level of the CRT translocation machinery: CRT, ERp57, KARS (lysyl-tRNA synthetase, LysRS), and KDEL receptor (Lys-Asp-Glu-Leu endoplasmic reticulum protein retention receptor).

In the context of the present invention, the step of determining the ability of the tumor to induce an anticancer immune response may for example consist in verifying the correct expression, by tumour cells, of a protein allowing or enhancing CRT exposure at the surface of the cells (herein considered as an immunogenic cell death-associated molecule or immunogenic cell death marker).

Such a protein may be anyone of the previously described proteins.

For example, such a protein may be selected in particular from CRT, CCL3 (MIP-1 -alpha) (SEQ ID NO: 456); CCR1 (MlPlalpha receptor, RANTES-R) (SEQ ID NO: 457); CCR2 (MCP-1 receptor) (SEQ ID NO: 458); IL-8 (C-X-C motif chemokine 8) (SEQ ID NO: 459); CXCR1 (IL-8 Receptor type 1) (SEQ ID NO: 460); CXCR2 (IL-8 Receptor type 2) (SEQ ID NO: 461); TNFRSF10A or TRAIL-receptor 1 (SEQ ID NO: 462), TNFRSF10B or TRAIL-receptor 2 (SEQ ID NO: 463), TNFRSF10C or TRAIL-receptor 3 (SEQ ID NO: 464), TNFRSFIOD or TRAIL-receptor 4 (SEQ ID NO: 465D), iNOS (Inducible NO synthase) (SEQ ID NO: 466); SOD2 (Superoxide dismutase 2) mitochondriale (SEQ ID NO: 467); E2AK3 (Eukaryotic translation initiation factor 2-alpha kinase 3, PERK) (SEQ ID NO: 468), in particular phosphorylated PERK; E2AK2 (EIF2AK2, Interferon-induced double-stranded RNA-activated protein kinase, PKR) (SEQ ID NO: 469); PP1 (Serine/threonine-protein phosphatases), in particular PP-1A (SEQ ID NO: 470), PP-1B (PPP1CB) (SEQ ID NO: 471) or PP-1G (PPP1CC) (SEQ ID NO: 472); PR15A (Protein phosphatase 1 regulatory subunit 15 A, GADD34) (SEQ ID NO: 473); eIF-2A, in particular phosphorylated eIF-2A (SEQ ID NO: 474); SERCA (Sarcoplasmic/endoplasmic reticulum calcium ATPases), in particular SERCA1 (SEQ ID NO: 475), SERCA2 (SEQ ID NO: 476), SERCA3 (SEQ ID NO: 477); MAP kinase 8 (MAPK8 or JNK1) (SEQ ID NO: 478); MAP kinase 9 (MAPK9 or JNK2) (SEQ ID NO: 479); IKBKA (IKK-alpha, IKKA, NFKBIKA, TCF-16) (SEQ ID NO: 480); IKBKB (IKK-beta, IKK2, NFKBIKB) (SEQ ID NO: 481); NEMO (IKBKG, IK -gamma, IKKAP1) (SEQ ID NO: 482); CASP-1 (IL-1BC, ICE), in particular activated CASP-1 (SEQ ID NO: 483); CASP-8 (MACH, FLICE) (SEQ ID NO: 484), in particular activated CASP-8; FADD (SEQ ID NO: 485); BAP31 (SEQ ID NO: 486) in particular cleaved BAP31; BAX (Bcl2-L-4) (SEQ ID NO: 487); BAK (Bcl2- L-7) (SEQ ID NO: 488); Bcl-2 (SEQ ID NO: 489); Bcl2-L-1 (Bcl-X) (SEQ ID NO: 490); ERp57 (protein disulfide-isomerase A3, PDIA3, ERp60) (SEQ ID NO: 491); and LysRS (SEQ ID NO: 492).

The step of determining the ability of the tumor to induce an anticancer immune response may also for example consist in verifying the correct expression (as defined previously), by tumour cells, of a protein expressed during the ER stress response and/or during the macroautophagic response of the subject's immune system (identified by inventors as involved in the immunogenic tumor cell death and herein considered as an immunogenic cell death-associated molecules).

Such a protein may be selected for example from AMBRAl (Activating Molecule in Beclin-1 -Regulated Autophagy), AMPK (5' adenosine monophosphate-activated protein kinase), ATG1, ATG5, ATG7, ATG10, ATG12, ATG14L (BARKOR), BCLN1 (Beclin 1), BIF1, CaMKK¾ (calcium/calmodulin-dependent protein kinase kinase), DAPK (death- associated protein kinase), DDIT3 (DNA damage inducible transcript 3) (CHOP, GADD153), DRAM (damage-regulated autophagy modulator), FIP200 (RB1CC1), Fox03 (forkhead box O transcription factor), GATE- 16 (Golgi-associated ATPase enhancer of 16 kDa), HDAC6 (histone deacetylase 6), HSPA5 (BiP (Binding immunoglobulin protein), GFP78, GP96), XBP1 (X-box binding protein 1), DNAJC3 (DnaJ homolog subfamily C member 3) (p58IPK), DNAJB9, DNAJB11, DNAJC10 (DNA sequence corresponding to SEQ ID NO: 527), EDEM1 (ER degradation-enhancing alpha-mannosidase-like 1), EDEM2, EDEM3, FIP200, GABARAP (gamma-amino butyric acid receptor-associated protein), LAMP-2 (lysosome-associated membrane protein type 2), LC3 (microtubule- associated protein 1 light chain 3) and its isoforms LC3A, LC3B and LC3C, the lipidated form of LC3 (LC3-II), mTOR, SERP1 (Stress-associated endoplasmic reticulum protein 1), SERP2, p62 (sequestosome 1/SQSTMl), PDIA6, PP1R15A (GADD34), Raptor, Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1 -interacting) TSC1 (tuberous sclerosis complex 1) and TSC2 (tuberous sclerosis complex 2). In a particular embodiment of the present invention, the presence, in a tumor sample of the subject, of an abnormal expression of a gene selected from CCR1, EIF2AK2, DNAJC10, PDIA3, EIF2A, PPPICB, IKBKB, PPPICC, and BAX, determines the inability of the subject to induce an anticancer immune response.

The expression is correct if the expressed protein is active or functional, i.e., in the context of the present invention, if the expressed protein is able to directly or indirectly induce a response from the immune system directed against the tumour cell.

In a particular method, the expression abnormality is a downregulation of the expression of CCR1, a downregulation of the expression of EIF2AK2, an upregulation of the expression of DNAJC10, and/or an upregulation of the expression of PDIA3. In another particular method, the expression abnormality is a downregulation of the expression of CCR1, a downregulation of the expression of EIF2AK2, and an upregulation of the expression of DNAJC10.

In a further particular method, the expression abnormality is a downregulation of the expression of CCR1, a downregulation of the expression of EIF2AK2, and an upregulation of the expression of PDIA3.

Methods usable by the man of the art to detect or quantify the previously mentioned proteins are well-known from the skilled man of the art and further identified below in the description.

When the tumour cells do not express or abnormally express the previously mentioned proteins, inventors herein indicates that a "compensatory immunogenic treatment of cancer" has to be applied to the subject having a tumor to induce a reaction of the immune system directed against said tumor. The present disclosure further relates to the abnormal expression of a gene which is specific to tumor cells.

The step of determining the ability of the tumor to induce an anticancer immune response may also consist in detecting the presence of an altered mutated nucleic acid, of an abnormal expression of the nucleic acid, or of an abnormal expression or activity of the protein encoded by the nucleic acid in a biological sample from the tumor's subject (as defined previously), the presence of said altered nucleic acid, abnormal expression of the nucleic acid, or abnormal expression or activity of the protein encoded by said nucleic acid being indicative of the inability for the tumor to induce an anticancer immune response, in particular when the subject having the tumor has been previously exposed to a treatment of cancer.

This detection step may indeed be performed before or after the administration to the subject having the tumor of at least part of a treatment of cancer, typically of at least part of a conventional treatment of cancer as previously explained. The detection step is preferably performed after such an administration, for example after one or two cycles of a complete treatment protocol.

The nucleic acid, mentioned in the previously described method, the alteration of which, abnormal expression of which, or the abnormal expression of the corresponding endogenous protein (protein encoded by said nucleic acid), should be detected, may be a gene encoding a protein selected from Eomes (SEQ ID NO: 493), IFNg (SEQ ID NO: 494), Tbx21 (Tbet) (SEQ ID NO: 495), IL-1R1 (SEQ ID NO: 496), FOXP3 (SEQ ID NO: 497), Ltb (SEQ ID NO: 498), LtbR (SEQ ID NO: 499), CXCL12 (SEQ ID NO: 500), CXCL13 (SEQ ID NO: 522), IL-33 (SEQ ID NO: 501), IL1RL1 (ST2) (SEQ ID NO: 502), IL-7r (SEQ ID NO: 503), IL-7 (SEQ ID NO: 504), Ccl5 (SEQ ID NO: 505), IL-21 (SEQ ID NO: 506), CXCL10 (IP-10) (SEQ ID NO: 507), CXCL2 (SEQ ID NO: 508), CXCL9 (Mig) (SEQ ID NO: 509), TNF-alpha (TNF-a) (SEQ ID NO: 510), IL-15 (SEQ ID NO: 511), AHR (SEQ ID NO: 1), IL-15ra (SEQ ID NO: 512), IL-lb (SEQ ID NO: 513), CXCL16 (SEQ ID NO: 514), CXCR6 (SEQ ID NO: 523), IL-10 (SEQ ID NO: 515), IL-27 (SEQ ID NO: 516), Ccl7 (SEQ ID NO: 517), IL-23r (SEQ ID NO: 518), CX3CL1 (SEQ ID NO: 519), CCL2 (SEQ ID NO: 520), IL-8 (SEQ ID NO: 521), CXCL11 (IT AC) (SEQ ID NO: 524), CXCR1 (SEQ ID NO: 525), CXCR2 (SEQ ID NO: 526), CCR1 (SEQ ID NO: 457), EIF2AK2 (SEQ ID NO: 469), DNAJC10 (SEQ ID NO: 527), PDIA3 (SEQ ID NO: 491), EIF2A (SEQ ID NO: 474), PPP1CB (SEQ ID NO: 471), 1KB KB (SEQ ID NO: 481), PPP1CC (SEQ ID NO: 472), and BAX (SEQ ID NO: 487).

In a particular embodiment of the present invention, the step of determining the ability of the tumor to induce an anticancer immune response may consist in determining alteration in a gene locus or in the expression of the protein encoded by said gene, in a biological sample of the patient, the presence of such an alteration being indicative of the inability of the tumor to induce an anticancer immune response.

In a particular embodiment, a method of determining the ability of a tumor to induce an anticancer immune response may comprise the following steps of (a) obtaining from the subject a test sample of tumoral DNA, cDNA or RNA, (b) contacting the test sample with at least one nucleic acid probe, wherein said nucleic acid is complementary to and specifically hybridises with a targeted altered nucleic acid sequence (one of the previously identified sequence) preferably comprising at least one point mutation, in particular a single nucleotide polymorphism (SNP), to form a hybridization sample, (c) maintaining the hybridization sample under conditions sufficient for the specific hybridization of the targeted nucleic acid sequence with the nucleic acid probe to occur, and (d) detecting whether there is specific hybridization of the altered targeted nucleic acid sequence with the nucleic acid probe.

Particular techniques the aim of which is to determine the abnormal (in particular low or absent) expression of a particular nucleic acid such as those described previously, or the abnormal expression of the corresponding endogenous protein (protein encoded by said nucleic acid) are detailed later in the description.

In a particular embodiment, if the tumor of a subject is not able to induce an anticancer immune response, the subject will be identified as resistant to conventional treatments of cancer. Inventors herein demonstrate that a "compensatory immunogenic treatment of cancer" as disclosed in the present description should be administered, preferably in addition to a conventional treatment of cancer, to such resistant subjects having a tumor which is not able to induce an efficient anticancer immune response, in order to allow such a response. immunogenic cell death-associated products found in the subject

Inventors herein demonstrate that the immunogenic cell death is also dependant from products, herein identified as "immunogenic cell death-associated products or signals", specific to the mammal, in particular to the human, i.e., independent from the presence of a tumor in the mammal subject, and independent from any treatment a mammal subject having a tumor may have been exposed to.

The step of determining the ability of the subject to induce an anticancer immune response may consist in detecting, using one of the previously identified methods (well known by the man skilled in the art), the presence of a mutated nucleic acid, of an abnormal expression of the nucleic acid, or of an abnormal expression or activity of the protein encoded by the nucleic acid in a biological sample (as defined previously) from the subject, the presence of said mutated nucleic acid, abnormal expression of the nucleic acid, or abnormal expression or activity of the protein encoded by said nucleic acid, being indicative of the inability for the subject to induce an anticancer immune response.

In a particular embodiment, if the subject is not able to induce an anticancer immune response, the subject will be identified as resistant or non-responder to conventional treatments of cancer as defined previously.

In a particular embodiment, a method of determining the ability of a subject to induce an anticancer immune response may comprise the following steps of (a) obtaining from the subject a test sample of DNA, preferably of genomic DNA, (b) contacting the test sample with at least one nucleic acid probe, wherein said nucleic acid is complementary to and specifically hybridises with a targeted mutated nucleic acid sequence (one of the below identified sequences) comprising a point mutation, preferably a single nucleotide polymorphism (SNP), to form a hybridization sample, (c) maintaining the hybridization sample under conditions sufficient for the specific hybridization of the targeted nucleic acid sequence with the nucleic acid probe to occur, and (d) detecting whether there is specific hybridization of the mutated targeted nucleic acid sequence with the nucleic acid probe.

If the subject has a tumor, the previously described detection step may be performed before and/or after any conventional treatment of cancer.

In a preferred embodiment, the step of determining the ability of a subject to induce an anticancer immune response may consist in detecting an abnormal nucleic acid sequence in a biological sample from the subject, the detection of such an abnormal nucleic acid sequence determining the inability of the subject to induce an anticancer immune response.

The method may in particular consist in verifying the presence, in the genome of the subject, of a mutated nucleic acid sequence leading to the abnormal expression of a gene involved in the "anti-cancer immune response", the presence of such a mutated nucleic acid sequence determining the inability of the subject to induce an anticancer immune response. These genes are herein identified under the term "immune genes". The nucleic acid, mentioned in the previously described methods, is typically located in an immune gene as defined previously and identified below.

In the context of the present invention, immune genes may be selected from anyone of the genes identified below in Table 1. Table 1 further identifies, for each immune gene, SNP(s) associated to a non-responder status of the subject (in other words, to the inability of the subject to induce an anticancer immune response).

Table 1 :

Alteration/SNP Sequence

Polymorphism

Gene reference reference Coding status

SLC06A1 rsl0055840 C/G SEQ ID NO: 77 NONSYN

TNXB rsl009382 A/C/G/T SEQ ID NO: 78 NONSYN

P2RX7 rsl0160951 C/G SEQ ID NO: 79 NONSYN C5orf20 rs 1031844 G/T SEQ ID NO: 80 NONSYN

C6orfl0 rsl033500 C/T SEQ ID NO: 81 NONSYN

IL1RL1 rs 1041973 A/C SEQ ID NO: 82 NONSYN

HLA-DMB rsl042337 C/T SEQ ID NO: 83 SYNON

LENG9 rs 10423424 C/G SEQ ID NO: 84 NONSYN

TP53 rs 1042522 C/G SEQ ID NO: 85 NONSYN

UGT1A8 rsl042597 C/G/T SEQ ID NO: 86 NONSYN

NLRP2 rsl043673 A/C SEQ ID NO: 87 NONSYN

TAPBPL rsl045546 A/G SEQ ID NO: 88 NONSYN

BAT2 rsl046080 A/C SEQ ID NO: 89 NONSYN

MAGEA4 rs 1047246 C/G SEQ ID NO: 90 5UTR

CABYR rsl049683 A/C SEQ ID NO: 91 NONSYN

PHC1 rs 1049925 C/T SEQ ID NO: 92 NONSYN

ZNF615 rsl0500311 A/G SEQ ID NO: 93 NONSYN

KlkbW rsl052276 A/C/G/T SEQ ID NO: 94 NONSYN

SH3RF2 rsl056149 C/G SEQ ID NO: 95 NONSYN

ZNF83 rsl056185 A/C/G/T SEQ ID NO: 96 NONSYN

LRRC23 rsl057077 A/T SEQ ID NO: 97 NONSYN

ATF6 rsl058405 A/G/T SEQ ID NO: 98 NONSYN

IRF7 rsl061501 A/G SEQ ID NO: 99 SYNON

TNFRSF1B rs 1061622 G/T SEQ ID NO 100 NONSYN

MPHOSPH1 rs 1062465 A/T SEQ ID NO 101 NONSYN

ITGAL rs 1064524 C/T SEQ ID NO 102 NONSYN

BRDT rs 10747493 C/T SEQ ID NO 103 NONSYN

ECE1 rsl076669 C/T SEQ ID NO 104 NONSYN

DDX58 rsl0813831 A/G SEQ ID NO 105 NONSYN

ARMC3 rsl0828395 A/G SEQ ID NO 106 NONSYN

PZP rsl0842971 A/T SEQ ID NO 107 NONSYN

ZNF818 rsl0853858 A/G SEQ ID NO 108 NONSYN

TIGD6 rsl0875553 A/C/G/T SEQ ID NO 109 NONSYN

CTSS rs2230061 C/T SEQ ID NO 110 NONSYN

IFIH1 rsl0930046 C/T SEQ ID NO 111 NONSYN

LARS rsl0988 C/T SEQ ID NO 112 NONSYN

FAT2 rsl l05168 A/G SEQ ID NO 113 NONSYN

CEP290 rsl 1104738 C/T SEQ ID NO 114 NONSYN

FBX07 rsl l l07 C/T SEQ ID NO 115 NONSYN

GNLY rsl 1127 C/T SEQ ID NO 116 NONSYN

CCDC110 rsl 1132306 A/G SEQ ID NO 117 NONSYN

IL23R rsl 1209026 A/G SEQ ID NO 118 NONSYN

HSPB9 rsl 122326 A/C SEQ ID NO 119 NONSYN

CD86 rsl 129055 A/G SEQ ID NO 120 NONSYN

GAK rsl 134921 A/G SEQ ID NO 121 NONSYN

SP110 rsl 135791 C/T SEQ ID NO 122 NONSYN

LEPR rsl l37101 A/G SEQ ID NO 123 NONSYN

GSTP1 rsl 138272 C/T SEQ ID NO 124 NONSYN

IRAK3 rsl 152888 A/G SEQ ID NO 125 NONSYN BRDT rsl 156281 A/C SEQ ID NO 126 NONSYN

EGF rsl 1568943 A/G SEQ ID NO 127 NONSYN

EGF rsl 1569017 A/T SEQ ID NO 128 NONSYN

MAEL rsl 1578336 G/T SEQ ID NO 129 NONSYN

FAM196A rsl 1594560 A/C/G/T SEQ ID NO 130 NONSYN

ERCC1 rsl l615 C/T SEQ ID NO 131 SYNON

SIGLEC12 rsl 1668530 A/C/G/T SEQ ID NO 132 NONSYN

FCRLA rsl 1746 A/G SEQ ID NO 133 NONSYN

NUF2 rsl 1802875 C/T SEQ ID NO 134 NONSYN

CASC5 rsl l858113 C/T SEQ ID NO 135 NONSYN

DDX58 rsl2006123 A/G SEQ ID NO 136 3UTR

PLAC8L1 rsl2187913 A/T SEQ ID NO 137 NONSYN

ZNF816A rsl2459008 A/T SEQ ID NO 138 NONSYN

ZNF665 rsl 2460170 A/G SEQ ID NO 139 NONSYN

NLRP11 rsl2461110 A/C/G/T SEQ ID NO 140 NONSYN

NLRP4 rsl2462372 A/G SEQ ID NO 141 NONSYN

UGT1A5 rsl2475068 C/G SEQ ID NO 142 NONSYN

IFIH1 rsl 2479043 C/G SEQ ID NO 143 SYNON

C5orf20 rsl2520809 C/T SEQ ID NO 144 NONSYN

DDX58 rsl2555727 A/G SEQ ID NO 145 3UTR

MPHOSPH1 rsl2572012 A/T SEQ ID NO 146 NONSYN

C6orfl5 rsl265053 C/G SEQ ID NO 147 NONSYN

LY75 rsl2692566 A/C SEQ ID NO 148 NONSYN

IL1RL1 rsl2712142 A/C SEQ ID NO 149 3UTR

GDF3 rsl2819884 C/T SEQ ID NO 150 NONSYN

CCHCR1 rsl30076 C/T SEQ ID NO 151 NONSYN

CCDC36 rsl3068038 A/C SEQ ID NO 152 NONSYN

SLC06A1 rsl 3190449 A/G SEQ ID NO 153 NONSYN

RNF216 rsl3236790 C/T SEQ ID NO 154 NONSYN

TNFRSF10B rsl3265018 A/C/G SEQ ID NO 155 NONSYN

ZNF480 rsl3343641 C/T SEQ ID NO 156 NONSYN

HORMAD1 rsl336900 A/G SEQ ID NO 157 NONSYN

ILIRLI rsl3431828 C/T SEQ ID NO 158 5UTR

LY75 rsl397706 A/G SEQ ID NO 159 NONSYN

ILIRLI rsl420101 A/G SEQ ID NO 160 intron

RELL2 rsl 4251 A/C SEQ ID NO 161 NONSYN

FAT2 rsl432862 A/G SEQ ID NO 162 NONSYN

IL7R rsl494555 C/T SEQ ID NO 163 NONSYN

IL7R rsl494558 A/G SEQ ID NO 164 NONSYN

A2ML1 rsl558526 A/G SEQ ID NO 165 NONSYN

GP6 rsl613662 A/G SEQ ID NO 166 NONSYN

C6orf205 rsl634730 C/T SEQ ID NO 167 NONSYN

PCDH12 rsl 64075 A/C/G/T SEQ ID NO 168 NONSYN

PCDH12 rsl64515 A/C/G/T SEQ ID NO 169 NONSYN

GP6 rsl 654416 A/C/G/T SEQ ID NO 170 NONSYN

SP100 rsl 2724 A/G SEQ ID NO 171 NONSYN CD 180 rsl6875312 C/G SEQ ID NO 172 NONSYN

NME1-NME2 rs 16949649 C/T SEQ ID NO 173 flanking_5UTR

GSTP1 rsl695 A/G SEQ ID NO 174 NONSYN

CASC5 rsl6970911 A/G SEQ ID NO 175 NONSYN

ZNF615 rsl6983353 C/T SEQ ID NO 176 NONSYN

FCAR rsl6986050 A/G SEQ ID NO 177 NONSYN

PTPRH rsl6986309 A/C/G/T SEQ ID NO 178 NONSYN

TMC06 rsl7208187 C/G SEQ ID NO 179 NONSYN

SP100 rsl7275036 A/G SEQ ID NO 180 NONSYN

MPHOSPH1 rs 17484219 G/T SEQ ID NO 181 NONSYN

P2RX7 rsl7525809 C/T SEQ ID NO 182 NONSYN

MMP9 rsl7576 A/G SEQ ID NO 183 NONSYN

PSMB9 rsl7587 A/G SEQ ID NO 184 NONSYN

CASC5 rsl7747633 A/G SEQ ID NO 185 NONSYN

SPINK5 rsl7775319 A/G SEQ ID NO 186 NONSYN

LY75 rsl7827158 C/T SEQ ID NO 187 NONSYN

UGT1A8 rsl7863762 A/G SEQ ID NO 188 NONSYN

TLR7 rsl79008 A/C/T SEQ ID NO 189 NONSYN

ICAM1 rsl799969 A/G SEQ ID NO 190 NONSYN

IL6 rsl 800795 C/G SEQ ID NO 191 flanking_5UTR

MTHFR rsl801131 A/C SEQ ID NO 192 NONSYN

ESR1 rsl801132 C/G SEQ ID NO 193 SYNON

MTHFR rsl801133 C/T SEQ ID NO 194 NONSYN

ERBB2 rsl 136201 A/G SEQ ID NO 195 NONSYN

IL4R rsl801275 A/G SEQ ID NO 196 NONSYN

CD 180 rsl 803440 C/G SEQ ID NO 197 3UTR

IL4R rsl805011 A/C SEQ ID NO 198 NONSYN

IL4R rsl805015 C/T SEQ ID NO 199 NONSYN

IL4R rsl805016 G/T SEQ ID NO 200 NONSYN

TNXB rsl85819 A/C/G/T SEQ ID NO 201 NONSYN

A2ML1 rsl 860967 C/T SEQ ID NO 202 NONSYN

MFGE8 rsl878326 A/C/G/T SEQ ID NO 203 NONSYN

IL23R rsl 884444 G/T SEQ ID NO 204 NONSYN

MPHOSPH1 rsl886996 C/T SEQ ID NO 205 NONSYN

MPHOSPH1 rsl886997 A/G SEQ ID NO 206 NONSYN

ICOS rsl978595 C/T SEQ ID NO 207 flanking 5UTR

ZNF615 rsl978717 C/T SEQ ID NO 208 NONSYN

ZNF761 rsl984432 A/G SEQ ID NO 209 NONSYN

KLK2 rsl98977 C/T SEQ ID NO 210 NONSYN

IFIH1 rsl990760 C/T SEQ ID NO 211 NONSYN

MAGEA1 rs2008160 A/C/G/T SEQ ID NO 212 NONSYN

CLEC4A rs2024301 A/T SEQ ID NO 213 NONSYN

DPP A3 rs2024320 C/G SEQ ID NO 214 NONSYN

TAPBPL rs2041385 C/T SEQ ID NO 215 NONSYN

FAT2 rs2053028 A/C/G/T SEQ ID NO 216 NONSYN

IL13 rs20541 C/T SEQ ID NO 217 NONSYN IL4R rs2057768 A/G SEQ ID NO: 218 flanking 5UTR

FPR1 rs2070745 C/G SEQ ID NO: 219 NONSYN

IL4 rs2070874 C/T SEQ ID NO: 220 5UTR

BIRC5 rs2071214 A/G SEQ ID NO: 221 NONSYN

MAGEB3 rs2071309 C/T SEQ ID NO: 222 NONSYN

TAPBP rs2071888 C/G SEQ ID NO: 223 NONSYN

AKAP3 rs2072355 A/C/G/T SEQ ID NO: 224 NONSYN

ZBP1 rs2073145 A/G SEQ ID NO: 225 NONSYN

DHX58 rs2074158 A/G SEQ ID NO: 226 NONSYN

DHX58 rs2074160 A/G SEQ ID NO: 227 NONSYN

VARS2 rs2074506 A/C SEQ ID NO: 228 NONSYN

KLK9 rs2075802 A/C SEQ ID NO: 229 SYNON

SIGLEC9 rs2075803 A/G SEQ ID NO: 230 NONSYN

CEBPZ rs2098386 A/C/G/T SEQ ID NO: 231 NONSYN

ZNF347 rs2195310 A/G SEQ ID NO: 232 NONSYN

IL15RA rs2228059 A/C SEQ ID NO: 233 NONSYN

DCC rs2229080 C/G SEQ ID NO: 234 NONSYN

LTA rs2229094 C/T SEQ ID NO: 235 NONSYN

DOCK1 rs2229603 A/G SEQ ID NO: 236 NONSYN

ICAM3 rs2230399 C/G SEQ ID NO: 237 NONSYN

TNFAIP3 rs2230926 G/T SEQ ID NO: 238 NONSYN

GZMB rs2236338 A/G SEQ ID NO: 239 NONSYN

NCR2 rs2236369 C/T SEQ ID NO: 240 NONSYN

EGF rs2237051 A/G SEQ ID NO: 241 NONSYN

DPCR1 rs2240804 C/T SEQ ID NO: 242 NONSYN

LILRA4 rs2241384 C/T SEQ ID NO: 243 NONSYN

Klkbl4 rs2241414 A/C/G/T SEQ ID NO: 244 NONSYN

ATG16L1 rs2241880 C/T SEQ ID NO: 245 NONSYN

SLC44A4 rs2242665 A/G SEQ ID NO: 246 NONSYN

MGC23985 rs2250145 A/C/G/T SEQ ID NO: 247 NONSYN

MMP9 rs2250889 C/G SEQ ID NO: 248 NONSYN

IFNAR1 rs2257167 C/G SEQ ID NO: 249 NONSYN

CYP3A7 rs2257401 C/G SEQ ID NO: 250 NONSYN

SIGLEC9 rs2258983 A/C SEQ ID NO: 251 NONSYN

THG1L rs2270812 A/G SEQ ID NO: 252 NONSYN

NCR2 rs2273962 A/G SEQ ID NO: 253 NONSYN

LEMD1 rs2274702 G/T SEQ ID NO: 254 intron

MMP9 rsl7577 A/G SEQ ID NO: 255 NONSYN

FCRLA rs2275603 A/G SEQ ID NO: 256 NONSYN

PZP rs2277413 C/T SEQ ID NO: 257 NONSYN

CCDC33 rs2277603 A/G/T SEQ ID NO: 258 NONSYN

ZNF350 rs2278420 A/C/G/T SEQ ID NO: 259 NONSYN

WDR55 rs2286394 A/G SEQ ID NO: 260 NONSYN

PTPRH rs2288419 A/C/G/T SEQ ID NO: 261 NONSYN

PTPRH rs2288515 A/C/G/T SEQ ID NO: 262 NONSYN

PTPRH rs2288523 A/C/G/T SEQ ID NO: 263 NONSYN MORC1 rs2290057 C/T SEQ ID NO: 264 NONSYN

RBP5 rs2290237 A/T SEQ ID NO: 265 NONSYN

NOS2A rs2297518 A/G SEQ ID NO: 266 NONSYN

CDX1 rs2302275 C/G SEQ ID NO: 267 NONSYN

SPINK5 rs2303063 A/G SEQ ID NO: 268 NONSYN

THEG rs2303810 A/C SEQ ID NO: 269 NONSYN

FAT2 rs2304024 C/T SEQ ID NO: 270 NONSYN

FAT2 rs2304053 A/G SEQ ID NO: 271 NONSYN

GP6 rs2304167 A/C/G/T SEQ ID NO: 272 NONSYN

ORC4L rs2307394 A/G SEQ ID NO: 273 NONSYN

C19orf51 rs2365725 C/T SEQ ID NO: 274 NONSYN

CASC5 rs2412541 G/T SEQ ID NO: 275 NONSYN

CHRNA2 rs2472553 C/T SEQ ID NO: 276 NONSYN

PTPN22 rs2476601 C/T SEQ ID NO: 277 NONSYN

CCDC69 rs248427 A/C/G/T SEQ ID NO: 278 NONSYN

OLFML2B rs2499836 C/T SEQ ID NO: 279 NONSYN

PCDHA9 rs251354 C/G SEQ ID NO: 280 NONSYN

TRIM31 rs2523989 A/G SEQ ID NO: 281 NONSYN

LILRB3 rs255772 C/G SEQ ID NO: 282 intron

P2RX4 rs25644 A/G SEQ ID NO: 283 NONSYN

CD14 rs2569190 A/G SEQ ID NO: 284 5UTR

KLK14 rs2569491 A/C/G/T SEQ ID NO: 285 NONSYN

MORC1 rs2593943 A/G SEQ ID NO: 286 NONSYN

CXCL12 rs266088 C/T SEQ ID NO: 287 intron

NCR3 rs2736191 C/G SEQ ID NO: 288 flanking_5UTR

CAGE1 rs2876098 G/T SEQ ID NO: 289 NONSYN

PCDHB12 rs2910006 C/T SEQ ID NO: 290 NONSYN

PCDHB7 rs2910313 C/G SEQ ID NO: 291 NONSYN

KIR2DS4 rsl 130476 G/T SEQ ID NO: 292 NONSYN

SLU7 rs2961944 A/G SEQ ID NO: 293 NONSYN

CTLA4 rs3087243 A/G SEQ ID NO: 294 flanking 3UTR

C6orfl0 rs3129941 A/G SEQ ID NO: 295 NONSYN

C6orf47 rs3130617 C/T SEQ ID NO: 296 NONSYN

CDSN rs3130981 C/T SEQ ID NO: 297 NONSYN

CDSN rs3130984 C/T SEQ ID NO: 298 NONSYN

LECT2 rs31517 A/C/G/T SEQ ID NO: 299 NONSYN

ETFB si 130426 C/T SEQ ID NO: 300 NONSYN

SELL rsl l31498 C/T SEQ ID NO: 301 NONSYN

IL7R rs3194051 A/G SEQ ID NO: 302 NONSYN

SIGIRR rs3210908 A/G SEQ ID NO: 303 NONSYN

ERCC1 rs3212961 A/C SEQ ID NO: 304 intron

PZP rs3213831 C/T SEQ ID NO: 305 NONSYN

TEX 15 rs323344 A/C/G/T SEQ ID NO: 306 NONSYN

TEX 15 rs323345 A/C/G/T SEQ ID NO: 307 NONSYN

TEX 15 rs323346 A/C/G/T SEQ ID NO: 308 NONSYN

TEX 15 rs323347 A/C/G/T SEQ ID NO: 309 NONSYN ZNF528 rs324125 A/G SEQ ID NO: 310 flanking 5UTR

KIAA0141 rs351260 C/T SEQ ID NO: 311 NONSYN

ZNF701 rs366793 C/T SEQ ID NO: 312 NONSYN

CX3CR1 rs3732379 C/T SEQ ID NO: 313 NONSYN

CD 180 rs3733910 A/G SEQ ID NO: 314 SYNON

FAT2 rs3734055 A/C/G/T SEQ ID NO: 315 NONSYN

SYCE1 rs3737031 A/C/G/T SEQ ID NO: 316 NONSYN

C1RL rs3742089 A/G SEQ ID NO: 317 NONSYN

KIR3DL2 rs3745902 C/T SEQ ID NO: 318 NONSYN

IFIH1 rs3747517 A/G SEQ ID NO: 319 NONSYN

SYCE1 rs3747881 A/C/G/T SEQ ID NO: 320 NONSYN

SLC25A2 rs3749780 A/G/T SEQ ID NO: 321 NONSYN

C6orfl0 rs3749966 C/T SEQ ID NO: 322 NONSYN

P2RX7 rs3751142 A/C SEQ ID NO: 323 SYNON

P2RX7 rs3751143 G/T SEQ ID NO: 324 NONSYN

UGT1A5 rs3755321 A/G SEQ ID NO: 325 NONSYN

MPHOSPH1 rsl 129777 C/G SEQ ID NO: 326 NONSYN

MPHOSPH1 rs3758388 A/T SEQ ID NO: 327 NONSYN

MPHOSPH1 rs3758390 A/G SEQ ID NO: 328 NONSYN

CYP2C19 rs3758581 A/G SEQ ID NO: 329 NONSYN

NME1-NME2 rs3760468 A/T SEQ ID NO: 330 flanking_5UTR

NME1-NME2 rs3760469 G/T SEQ ID NO: 331 flanking 5UTR

MORC1 rs3762697 A/G SEQ ID NO: 332 NONSYN

IL1RL1 rs3771175 A/T SEQ ID NO: 333 3UTR

TLR3 rs3775291 A/G SEQ ID NO: 334 NONSYN

PCDHB6 rs3776096 C/T SEQ ID NO: 335 NONSYN

SPINK5 rs3777134 C/T SEQ ID NO: 336 NONSYN

PBK rs3779620 A/G SEQ ID NO: 337 NONSYN

TDRD6 rs3799277 C/T SEQ ID NO: 338 NONSYN

TIRAP rs3802813 A/G SEQ ID NO: 339 NONSYN

TIRAP rs3802814 A/G SEQ ID NO: 340 SYNON

FATE1 rs3810715 A/G SEQ ID NO: 341 NONSYN

Klkbl4 rs3815803 A/C/G/T SEQ ID NO: 342 NONSYN

ATP 1 OA rs3816800 C/G SEQ ID NO: 343 NONSYN

SIGLEC12 rs3829658 A/C/G/T SEQ ID NO: 344 NONSYN

IRAK2 rs3844283 C/G SEQ ID NO: 345 NONSYN

UGT1A5 rs3892170 C/G SEQ ID NO: 346 NONSYN

IL1R1 rs3917320 A/C SEQ ID NO: 347 SYNON

GALC rs398607 A/G SEQ ID NO: 348 NONSYN

FBXL21 rs40986 A/G SEQ ID NO: 349 NONSYN

NOTCH4 rs422951 A/C/G/T SEQ ID NO: 350 NONSYN

IRAK4 rs4251545 A/G SEQ ID NO: 351 NONSYN

CLEC4D rs4304840 A/G SEQ ID NO: 352 NONSYN

SKIV2L rs437179 G/T SEQ ID NO: 353 NONSYN

NLRP4 rs441827 C/T SEQ ID NO: 354 NONSYN

CTLA4 rs4553808 A/G SEQ ID NO: 355 flanking 5UTR SSX9 rs4598385 A/G SEQ ID NO: 356 NONSYN

FLJ41603 rs4629585 A/C SEQ ID NO: 357 NONSYN

VARS2 rs4678 C/T SEQ ID NO: 358 NONSYN

EGF rs4698803 A/T SEQ ID NO: 359 NONSYN

PCYOX1L rs4705336 C/G SEQ ID NO: 360 NONSYN

C19orf48 rs4801853 C/T SEQ ID NO: 361 NONSYN

ZNF578 rs4802965 A/G SEQ ID NO: 362 NONSYN

SAGE1 rs4829799 C/T SEQ ID NO: 363 NONSYN

TNFRSF10A rs20575 C/G SEQ ID NO: 364 NONSYN

CD14 rs4914 C/G SEQ ID NO: 365 SYNON

AHSG rs4918 C/G SEQ ID NO: 366 NONSYN

LOC283755 rs4931826 A/C SEQ ID NO: 367 NONSYN

SERPINA3 rs4934 A/G SEQ ID NO: 368 NONSYN

CCDC71 rs4955418 A/G SEQ ID NO: 369 NONSYN

CCDC71 rs4955419 A/T SEQ ID NO: 370 NONSYN

TLR4 rs4986790 A/G SEQ ID NO: 371 NONSYN

TLR4 rs4986791 C/T SEQ ID NO: 372 NONSYN

IFNAR2 rs2229207 C/T SEQ ID NO: 373 NONSYN

SELL rs2229969 C/T SEQ ID NO: 374 NONSYN

ILIRLI rs4988956 A/G SEQ ID NO: 375 NONSYN

ILIRLI rs4988957 C/T SEQ ID NO: 376 SYNON

ILIRLI rs4988958 C/T SEQ ID NO: 377 SYNON

TLR4 rs5030710 C/T SEQ ID NO: 378 SYNON

TLR4 rs5030719 G/T SEQ ID NO: 379 NONSYN

FPR1 rs5030878 C/T SEQ ID NO: 380 NONSYN

TLR4 rs5031050 A/T SEQ ID NO: 381 NONSYN

COP1 rs542571 A/T SEQ ID NO: 382 NONSYN

ICAM1 rs5498 A/G SEQ ID NO: 383 NONSYN

KLK1 rs5516 C/G SEQ ID NO: 384 NONSYN

KLK1 rs5517 A/C/G/T SEQ ID NO: 385 NONSYN

C6orfl0 rs560505 C/T SEQ ID NO: 386 NONSYN

TLR7 rs5741881 A/G SEQ ID NO: 387 SYNON

CTLA4 rs5742909 C/T SEQ ID NO: 388 flanking_5UTR

NOD2 rs5743291 A/G SEQ ID NO: 389 NONSYN

TLR9 rs5743846 A/G SEQ ID NO: 390 NONSYN

CASP1 rs580253 A/C/G/T SEQ ID NO: 391 SYNON

ITGB3 rs5918 C/T SEQ ID NO: 392 NONSYN

PASD1 rs5924658 C/G/T SEQ ID NO: 393 NONSYN

DDX53 rs5925720 G/T SEQ ID NO: 394 NONSYN

CTCFL rs6025606 A/C/G/T SEQ ID NO: 395 NONSYN

CCND1 rs9344 A/G SEQ ID NO: 396 SYNON

CTCFL rs6070122 C/G SEQ ID NO: 397 NONSYN

CTCFL rs6070128 C/G SEQ ID NO: 398 NONSYN

TMC4 rs641738 C/T SEQ ID NO: 399 NONSYN

SLC44A4 rs644827 C/T SEQ ID NO: 400 NONSYN

SIGLEC12 rs6509544 C/G SEQ ID NO: 401 NONSYN TNFRSF10A rs6557634 C/T SEQ ID NO: 402 NONSYN

FAT2 rs6650971 C/T SEQ ID NO: 403 NONSYN

SPANXN3 rs6654212 C/G SEQ ID NO: 404 NONSYN

UGT1A4 rs6755571 A/C SEQ ID NO: 405 NONSYN

UGT1A6 rs6759892 G/T SEQ ID NO: 406 NONSYN

CCDC110 rs6827370 C/T SEQ ID NO: 407 NONSYN

MYD88 rs6853 A/G SEQ ID NO: 408 3UTR

SH3TC2 rs6875902 A/C SEQ ID NO: 409 NONSYN

SPINK5 rs6892205 A/G SEQ ID NO: 410 NONSYN

IL7R rs6897932 C/T SEQ ID NO: 411 NONSYN

HCG9 rs6904029 A/G SEQ ID NO: 412 NONSYN

RPP21 rs6986 C/G SEQ ID NO: 413 NONSYN

IRAK2 rs708035 A/T SEQ ID NO: 414 NONSYN

NCAPD2 rs714774 C/G SEQ ID NO: 415 NONSYN

CASC5 rs7177192 C/G SEQ ID NO: 416 NONSYN

TULP2 rs7260579 C/T SEQ ID NO: 417 NONSYN

LIPI rs7278737 G/T SEQ ID NO: 418 NONSYN

LILRB4 rs731170 A/G SEQ ID NO: 419 NONSYN

CTLA4 rs733618 A/G SEQ ID NO: 420 flanking 5UTR

GSTM3 rs7483 A/G SEQ ID NO: 421 NONSYN

NLRP3 rs7525979 C/T SEQ ID NO: 422 SYNON

TRIM40 rs757259 C/T SEQ ID NO: 423 NONSYN

TRIM40 rs757262 A/G SEQ ID NO: 424 NONSYN

SH3RF2 rs758037 C/T SEQ ID NO: 425 NONSYN

FMR1NB rs764631 C/T SEQ ID NO: 426 NONSYN

CCDC110 rs7698680 A/T SEQ ID NO: 427 NONSYN

CCDC110 rs7699687 G/T SEQ ID NO: 428 NONSYN

TLR4 rs7869402 C/T SEQ ID NO: 429 3UTR

TLR4 rs7873784 C/G SEQ ID NO: 430 3UTR

P2RX7 rs7958311 A/G SEQ ID NO: 431 NONSYN

HSP90AA1 rs8005905 A/T SEQ ID NO: 432 NONSYN

ZNF614 rs8104890 C/T SEQ ID NO: 433 NONSYN

ZNF160 rs8105668 C/G SEQ ID NO: 434 NONSYN

BIRC8 rs8109165 A/G SEQ ID NO: 435 NONSYN

TULP2 rs8112811 C/T SEQ ID NO: 436 NONSYN

GZMB rs8192917 A/G SEQ ID NO: 437 NONSYN

SP100 rs836237 C/T SEQ ID NO: 438 flanking_3UTR

TLR7 rs864058 C/T SEQ ID NO: 439 SYNON

FPR1 rs867228 A/C SEQ ID NO: 440 NONSYN

LAG3 rs870849 C/T SEQ ID NO: 441 NONSYN

LOC284297 rs925878 A/G SEQ ID NO: 442 flanking 5UTR

C6orfl0 rs9268368 C/T SEQ ID NO: 443 NONSYN

C6orfl0 rs9268384 A/G SEQ ID NO: 444 NONSYN

PTPRH rs9304763 C/G SEQ ID NO: 445 SYNON

FLJ41603 rs9324624 C/T SEQ ID NO: 446 NONSYN

FAT2 rs9324700 A/C/G/T SEQ ID NO: 447 NONSYN ZNF614 rs9636139 A/G SEQ ID NO: 448 NONSYN

ZNF468 rs9749312 A/G SEQ ID NO: 449 3UTR

SLC36A3 rs978012 A/G SEQ ID NO: 450 NONSYN

NCR3 rs986475 C/T SEQ ID NO: 451 3UTR

CTAGE1 rs9946136 A/C/G/T SEQ ID NO: 452 NONSYN

Immune genes are preferably selected from^Hi? (BHLHE76) gene, MTHFR gene, DDX58 [DEAD (Asp-Glu-Ala-Asp) box polypeptide 58] or RIG1 gene, the tumor necrosis factor receptor superfamily member 10a (TNFRSF10A/TRAILR1/CD261) gene, the chemokine (C-X3-C motif) receptor 1 (CX3CRllFractalkine receptorlCMKBLRllGPR13IV28) gene, the sialic acid binding Ig-like lectin 5 (SIGLEC5/CD170/OBBP2/CD33L2) gene, the CPX chromosome region candidate 1 {CPXCRl I CT77) gene, the NLR family pyrin domain containing 4 (NLRP4I NALP4/PAN2/CT58) gene, the IFNG or IFNy receptor 1 (IFNGR1ICD119) gene, and the myotubularin related protein 15 (MTMR15IKIAA1018).

Table 2 identifies, for each identified immune gene, SNP(s) associated to a non-responder status of the subject (in other words, to the inability of the subject to induce an anticancer immune response).

Table 2:

Sequence

Gene Alteration/SNP Coding status

Polymorphism reference

reference

AHR rsl0250822 C/T SEQ ID NO 3 intron

rsl 1505406 C/T SEQ ID NO 4 intron rsl476080 A/C SEQ ID NO 5 intron rsl7779352 C/T SEQ ID NO 6 SYN rs2066853 A/G SEQ ID NO 7 NONSYN rs2074113 A/C SEQ ID NO 8 intron rs2158041 A/G SEQ ID NO 9 intron rs2282885 C/T SEQ ID NO 10 intron rs34938955 C/T SEQ ID NO 11 5UTR rs35225673 A/C/G/T SEQ ID NO 12 intron rs4986826 A/G SEQ ID NO 13 NONSYN rs713150 C/G SEQ ID NO 14 intron rs7796976 A/G SEQ ID NO 15 5UTR rs7811989 A/G SEQ ID NO 16 intron

MTHFR Rsl801133 C/T SEQ ID NO 194 NONSYN DDX58 rsl7217280 A/T SEQ ID NO: 17 NONSYN rs35253851 A/C SEQ ID NO: 18 NONSYN rs951618 A/G SEQ ID NO: 19 NONSYN rs35527044 G/T SEQ ID NO: 20 NONSYN rsl 1795404 A/C SEQ ID NO: 21 NONSYN rsl0813831 A/G SEQ ID NO: 22 NONSYN rsl 1899 C/T SEQ ID NO: 23 3UTR rsl0363 A/G SEQ ID NO: 24 3UTR rsl0970987 A/C/G/T SEQ ID NO: 25 SYN rs35050877 A/C/G/T SEQ ID NO: 26 3UTR rsl2236816 A/G SEQ ID NO: 27 3UTR rsl2235719 A/T SEQ ID NO: 28 5UTR

TNFRSF10A rs2230229 A/C/G/T SEQ ID NO: 29 NONSYN rsl7088980 G/T SEQ ID NO: 30 NONSYN rs20576 A/C SEQ ID NO: 31 NONSYN rs20575 C/G SEQ ID NO: 32 NONSYN rsl 1986840 C/G SEQ ID NO: 33 NONSYN rs20577 C/T SEQ ID NO: 34 NONSYN rs34737614 G/T SEQ ID NO: 35 NONSYN rs34127830 A/C/G/T SEQ ID NO: 36 3UTR rs2230230 C/T SEQ ID NO: 37 SYN rs3808537 C/T SEQ ID NO: 38 5UTR

CX3CR1 rs3732378 A/G SEQ ID NO: 39 NONSYN rs3732380 C/T SEQ ID NO: 40 NONSYN rs41535248 G/T SEQ ID NO: 41 NONSYN rsl 1715522 A/C SEQ ID NO: 42 flanking_5UTR rs7636125 C/G SEQ ID NO: 43 3UTR rsl 1710546 A/G SEQ ID NO: 44 3UTR rsl7038674 C/T SEQ ID NO: 45 3UTR rsl050592 C/T SEQ ID NO: 46 3UTR rs4986872 C/T SEQ ID NO: 47 SYN rsl7038679 A/G SEQ ID NO: 48 SYN

SIGLEC5 rs3829655 C/G SEQ ID NO: 49 NONSYN rs8108074 A/C/G/T SEQ ID NO: 50 NONSYN rs2278831 A/C/G/T SEQ ID NO: 51 NONSYN rs34553740 C/T SEQ ID NO: 52 NONSYN rsl973019 A/G SEQ ID NO: 53 NONSYN rsl7740650 A/C/G/T SEQ ID NO: 54 SYN rs8107754 A/C/G/T SEQ ID NO: 55 SYN

CPXCR1 rs5984611 A/G SEQ ID NO: 56 NONSYN rs5940915 A/C SEQ ID NO: 57 NONSYN rs41307393 C/G SEQ ID NO: 58 NONSYN rsl2556970 C/T SEQ ID NO: 59 3UTR

NLRP4 rs302453 A/T SEQ ID NO: 60 NONSYN rsl7857373 C/G SEQ ID NO: 61 NONSYN rsl7857374 A/C/T SEQ ID NO: 62 NONSYN rs34627915 A/G SEQ ID NO: 63 NONSYN rsl7854614 A/C SEQ ID NO: 64 NONSYN

IFNGR1 rsl327475 C/T SEQ ID NO: 65 intron

rsl887415 C/T SEQ ID NO: 66 NONSYN rsl7175350 A/C SEQ ID NO: 67 NONSYN rsl7175322 A/G SEQ ID NO: 68 NONSYN rsl 1575936 A/G SEQ ID NO: 69 NONSYN rs7769141 A/C SEQ ID NO: 70 3UTR rsl7181562 A/G SEQ ID NO: 71 3UTR rsl l914 G/T SEQ ID NO: 72 SYN

MTMR15 rs4779794 A G SEQ ID NO: 73 NONSYN

rsl7846417 A G SEQ ID NO: 74 NONSYN rs34722914 A G SEQ ID NO: 75 5UTR rs8023700 A/G SEQ ID NO: 76 3UTR

The method is typically performed on the nucleic acid obtained from cells of a biological sample (blood or serum for example) of the subject, for example on the genomic DNA obtained from blood or seric cells, in particular leukocytes, more preferably Peripheral Blood Mononuclear Cells (PBMC), which are non cancerous cells.

The method may also be performed on tumoral cells of the subject whose normal cells (non cancerous cells) have an altered genotype.

Inventors herein demonstrate that a "compensatory immunogenic treatment of cancer", as disclosed in the present description, should be administered, preferably in addition to a conventional treatment of cancer, to such resistant subjects which are not able to induce an anticancer immune response, in order to allow such a response.

Typically, the alteration in a nucleic acid sequence may be determined at the level of the selected gene (immune gene, specific to the subject, or tumor gene, specific to the tumor), for example AHR DNA, cDNA, RNA or polypeptide. Optionally, the detection is performed by sequencing all or part of the gene locus or by selective hybridization or amplification of all or part of the gene locus. More preferably a gene locus specific amplification is carried out before the alteration identification step. An alteration in the gene locus may be any form of mutation(s), deletion(s), rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations more specifically include point mutations. Deletions may encompass any region of two or more residues in a coding or non-coding portion of the gene locus, such as from two residues up to the entire gene or locus. Typical deletions affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions may occur as well. Insertions may encompass the addition of one or several residues in a coding or non-coding portion of the gene locus. Insertions may typically comprise an addition of between 1 and 50 base pairs in the gene locus. Rearrangement includes inversion of sequences. The gene locus alteration may result in the creation of stop codons, frameshift mutations, amino acid substitutions, particular RNA splicing or processing, product instability, truncated polypeptide production, etc. The alteration may result in the production of a polypeptide or protein with altered function, stability, targeting or structure. The alteration may also cause a reduction in protein expression or, alternatively, an increase in said production.

In a preferred embodiment, said alteration is a mutation, an insertion or a deletion of one or more bases. In a particular embodiment of the method according to the present invention, the alteration in the gene locus is selected from a point mutation, a deletion and an insertion in the gene or corresponding expression product, more preferably a point mutation and a deletion. The alteration may be determined at the level of the DNA, RNA or polypeptide. Within the context of this invention, the "gene locus", for example "the AHR gene locus", designates all sequences or products in a cell or organism including, regarding AHR for example, the AHR coding sequences, AHR non-coding sequences (e.g., introns), AHR regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer/silencer regions, terminator, 5'UTR, 3'UTR, etc.), all corresponding expression products, such as AHR RNAs (e.g., mRNAs) and AHR polypeptides (e.g., a pre-protein and a mature protein); as well as surrounding sequences of 20 kb region, preferably 15.3 kb region, upstream the starting codon (flanking the 5 'UTR region) of the AHR gene and 20 kb region, preferably 14.1 kb region, downstream the untranslated region (flanking the 3'UTR region). In a particular embodiment most alterations are not in the promoter sequence. In a particular embodiment of the present invention, the step of determining the ability of the subject to induce an anticancer immune response may consist in determining alteration in a gene locus (in particular an immune gene locus) or in the expression of the protein encoded by said gene, in a biological sample of the patient, the presence of such an alteration being indicative of the inability of the subject to induce an anticancer immune response.

Alteration of a nucleic acid sequence herein described (in relation with the tumor or with the subject) is preferably a mutation, an insertion or a deletion of one or more bases. More preferably said alteration is one or several single nucleotide polymorphism(s) (SNPs).

In a particular embodiment, the altered nucleic acid is a wild-type nucleic acid comprising at least one point mutation, preferably a single nucleotide polymorphism (SNP), for example a loss-of- function SNP, i.e., a SNP responsible for the absent or abnormal (non- functional) expression of the protein encoded by the nucleic acid. The wild-type nucleic acid may also comprise several single nucleotide polymorphism(s) (SNPs).

Once a first SNP has been identified in a genomic region of interest, more particularly in an immune gene locus, other additional SNPs in linkage disequilibrium with this first SNP can be identified. Indeed, any SNP in linkage disequilibrium with a first SNP associated with non-responder phenotype will be associated with this trait. Therefore, once the association has been demonstrated between a given SNP and non-responder phenotype, the discovery of additional SNPs associated with this trait can be of great interest in order to increase the density of SNPs in this particular region. Identification of additional SNPs in linkage disequilibrium with a given SNP involves: (a) amplifying a fragment from the genomic region comprising or surrounding a first SNP from a plurality of individuals; (b) identifying of second SNP in the genomic region harboring or surrounding said first SNP; (c) conducting a linkage disequilibrium analysis between said first SNP and second SNP; and (d) selecting said second SNP as being in linkage disequilibrium with said first marker. Sub-combinations comprising steps (b) and (c) are also contemplated. These SNPs in linkage disequilibrium can also be used in the methods according to the present invention, and more particularly in the methods to predict treatment response or ability to induce an anticancer immune response according to the present invention.

Mutations in a gene locus which are responsible for non-responder phenotype may be identified by comparing the sequences of the gene locus from patients presenting non- responder phenotype and responder phenotype. Based on the identified association of SNPs of the particular gene, the identified locus can be scanned for mutations. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the gene locus are scanned for mutations. Preferably, patients presenting non-responder phenotype carry the mutation shown to be associated with non- responder phenotype and responder phenotype do not carry the mutation or mutated allele associated with reduced cancer treatment response. The method used to detect such mutations generally comprises the following steps: amplification of a region of the gene locus of interest comprising a SNP or a group of SNPs associated with non responder phenotype from DNA samples of the gene locus from patients presenting non responder phenotype and responder phenotype; sequencing of the amplified region; comparison of DNA sequences of the corresponding genes from patients presenting non responder phenotype and responder phenotype; determination of mutations specific to patients presenting non responder phenotype.

In the AHR gene, the SNP may be more particularly selected from rsl0250822 (SEQ ID NO: 3), rsl 1505406 (SEQ ID NO: 4), rsl476080 (SEQ ID NO: 5), rsl7779352 (SEQ ID NO: 6), rs2066853 (SEQ ID NO: 7), rs2074113 (SEQ ID NO: 8), rs2158041 (SEQ ID NO: 9), rs2282885 (SEQ ID NO: 10), rs34938955 (SEQ ID NO: 11), rs35225673 (SEQ ID NO: 12), rs4986826 (SEQ ID NO: 13), rs713150 (SEQ ID NO: 14), rs7796976 (SEQ ID NO: 15), and rs7811989 (SEQ ID NO: 16).

A typical SNP in the AHR gene is rs2066853 (SEQ ID NO: 7). Such a SNP AHR A/G (R554K) is indicative of a subject being unable to induce an anticancer immune response. Such a subject is typically non-responder to conventional treatments of cancer. In the methylene tetrahydro folate reductase MTHFR gene, the SNP is preferably rsl801133 (SEQ ID NO: 194). Such a SNP MTHFR C/T (A222V) is indicative of a subject being able to induce a better anticancer immune response. Such a subject is typically responder to conventional treatments of cancer. In other words, the presence of a wild-type allele C in a subject is indicative of the inability of the subject to respond to conventional treatments of cancer.

A particular method herein described comprises, in addition to previously described steps, a step of controlling, in a tumor, blood or serum sample of the subject, the presence of a herein described single nucleotide polymorphism (SNP); the detection of at least one of: i. an abnormal expression of the proteins encoded by (i) a gene encoding CCR1, (ii) a gene encoding EIF2AK2, and (iii) a gene encoding DNAJC10 or PDIA3, and

ii. an alteration in the gene encoding MTHFR,

being indicative of a resistance of the subject to a therapeutic treatment of cancer.

Preferably, the alteration is a single nucleotide polymorphism (SNP) corresponding to rsl801133 (wild type allele C / mutated allele T) (SEQ ID NO: 194).

In the DDX58 gene, the SNP may be more particularly selected from rsl 7217280 (SEQ ID NO: 17), rs35253851 (SEQ ID NO: 18), rs951618 (SEQ ID NO: 19), rs35527044 (SEQ ID NO: 20), rsl 1795404 (SEQ ID NO: 21), rsl0813831 (SEQ ID NO: 22), rsl 1899 (SEQ ID NO: 23), rsl0363 (SEQ ID NO: 24), rsl0970987 (SEQ ID NO: 25), rs35050877 (SEQ ID NO: 26), rsl2236816 (SEQ ID NO: 27), and rsl2235719 (SEQ ID NO: 28).

In the TNFRSF10A gene, the SNP may be more particularly selected from rs2230229 (SEQ ID NO: 29), rsl7088980 (SEQ ID NO: 30), rs20576 (SEQ ID NO: 31), rs20575 (SEQ ID NO: 32), rsl 1986840 (SEQ ID NO: 33), rs20577 (SEQ ID NO: 34), rs34737614 (SEQ ID NO: 35), rs34127830 (SEQ ID NO: 36), rs2230230 (SEQ ID NO: 37) and rs3808537 (SEQ ID NO: 38).

In the CX3CR1 gene, the SNP may be more particularly selected from rs3732378 (SEQ ID NO: 39), rs3732380 (SEQ ID NO: 40), rs41535248 (SEQ ID NO: 41), rsl 1715522 (SEQ ID NO: 42), rs7636125 (SEQ ID NO: 43), rsl 1710546 (SEQ ID NO: 44), rsl7038674 (SEQ ID NO: 45), rsl050592 (SEQ ID NO: 46), rs4986872 (SEQ ID NO: 47), and rsl7038679 (SEQ ID NO: 48). In the SIGLEC5 gene, the SNP may be more particularly selected from rs3829655 (SEQ ID NO: 49), rs8108074 (SEQ ID NO: 50), rs2278831 (SEQ ID NO: 51), rs34553740 (SEQ ID NO: 52), rsl973019 (SEQ ID NO: 53), rsl7740650 (SEQ ID NO: 54), and rs8107754 (SEQ ID NO: 55).

In the CPXCR1 gene, the SNP may be more particularly selected from rs5984611 (SEQ ID NO: 56), rs5940915 (SEQ ID NO: 57), rs41307393 (SEQ ID NO: 58), and rsl2556970 (SEQ ID NO: 59).

In the NLRP4 gene, the SNP may be more particularly selected from rs302453 (SEQ ID NO: 60), rsl7857373 (SEQ ID NO: 61), rsl7857374 (SEQ ID NO: 62), rs34627915 (SEQ ID NO: 63) and rsl7854614 (SEQ ID NO: 64).

In the IFNGR1 gene, the SNP may be more particularly selected from rs 1327475 (SEQ ID NO: 65), rsl887415 (SEQ ID NO: 66), rsl7175350 (SEQ ID NO: 67), rsl7175322 (SEQ ID NO: 68), rsl 1575936 (SEQ ID NO: 69), rs7769141 (SEQ ID NO: 70), rsl7181562 (SEQ ID NO: 71), and rsl 1914 (SEQ ID NO: 72).

In the MTMR15 gene, the SNP may be more particularly selected from rs4779794 (SEQ ID NO: 73), rsl7846417 (SEQ ID NO: 74), rs34722914 (SEQ ID NO: 75), and rs8023700 (SEQ ID NO: 76).

The presence of an alteration in a nucleic acid may be easily detected by the man skilled in the art using methods of the art such as restriction digestion, sequencing, selective hybridisation (for example with a nucleic acid probe present on a nucleotide array), and/or selective amplification, as further explained below.

Alterations in a gene may also be detected by determining the presence of an altered RNA expression. Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, the presence of an altered quantity of RNA, etc. These may be detected by various techniques known in the art, including by sequencing all or part of the RNA or by selective hybridisation or selective amplification of all or part of said RNA, for instance.

The presence of an abnormal expression of a target nucleic acid (which may be a nucleic acid from the subject or from the tumor), such as one of those identified previously, may be detected in particular by real time quantitative reverse transcription PCR (qRT-PCR) using probes designed to hybridize within the target nucleic acid sequence (see O'Driscoll L. et al, 1993 and Yajima T. et al, 1998). In a further variant, the method comprises detecting the presence of an altered expression of the polypeptide or protein encoded by the gene of interest. Altered polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of polypeptide, the presence of an altered tissue distribution, etc. These may be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies), for instance.

In a particular embodiment, the detection of an abnormal protein expression may be easily performed, by the man skilled in the art, by measuring the cellular level of mRNA encoding a normal protein, a decreased level compared to a control or standard level being correlated to an abnormal protein expression.

Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing may be performed on the complete gene locus or, more preferably, on specific domains thereof, typically those known or suspected to carry deleterious mutations or other alterations.

Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction. Amplification may be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Preferred techniques use allele-specific PCR or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. Nucleic acid primers useful for amplifying sequences from the gene locus of interest are able to specifically hybridize with a portion of the gene locus that flank a target region of said locus, said target region being altered, for example in the case of the immune genes, in non responder patients. Another particular object of this invention resides in a nucleic acid primer useful for amplifying sequences from the gene or locus of interest including surrounding regions. Such primers are preferably complementary to, and hybridize specifically to nucleic acid sequences in the gene locus. Particular primers are able to specifically hybridize with a portion of the gene locus that flank a target region of said locus, said target region being altered, for example in the case of the immune genes, in non responders. Primers that can be used to amplify target region comprising SNPs may be designed based on their sequence or on the genomic sequence of a particular gene.

The invention also relates to a nucleic acid primer, said primer being complementary to and hybridizing specifically to a portion of a gene locus coding sequence (e.g., gene or RNA) altered in certain non responders subjects. In this regard, particular primers of this invention are specific for altered sequences in a gene locus or RNA. By using such primers, the detection of an amplification product indicates the presence of an alteration in the gene locus. In contrast, the absence of amplification product indicates that the specific alteration is not present in the considered sample.

The invention also concerns the use of a nucleic acid primer or a pair of nucleic acid primers as mentioned above in a method of determining the treatment response of a subject having a tumor or in a method of assessing the response of a subject to a treatment of cancer.

Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s). A particular detection technique involves the use of a nucleic acid probe specific for wild-type or altered (immune or tumor) gene or corresponding RNA, followed by the detection of the presence of a hybrid. The probe may be in suspension or immobilized on a substrate or support (as in nucleic acid array or chips technologies). The probe is typically labeled to facilitate detection of hybrids.

In this regard, a particular embodiment of this invention comprises contacting the sample from the subject with a nucleic acid probe specific for an altered immune gene locus, and assessing the formation of an hybrid.

In a particularly preferred embodiment, the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for wild type gene locus and for various altered forms thereof. In this embodiment, it is possible to detect directly the presence of various forms of alterations in the gene locus in the sample. Also, various samples from various subjects may be treated in parallel. Within the context of this invention, a probe refers to a polynucleotide sequence which is complementary to and capable of specific hybridization with a (target portion of) gene or R A, and which is suitable for detecting polynucleotide polymorphisms associated with the gene alleles which predispose to or are associated with a reduced ability of the subject or of the tumor to induce an anticancer immune response ("mutated allele").

Probes are preferably perfectly complementary to the particular gene, RNA, or target portion thereof. Probes typically comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. It should be understood that longer probes may be used as well. A preferred probe of this invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a gene locus or RNA that carries an alteration.

The method of the invention employs a nucleic acid probe specific for an altered (e.g., a mutated) gene or RNA, i.e., a nucleic acid probe that specifically hybridizes to said altered gene or RNA and essentially does not hybridize to a gene or RNA lacking said alteration. Specificity indicates that hybridization to the target sequence generates a specific signal which can be distinguished from the signal generated through non-specific hybridization. Perfectly complementary sequences are preferred to design probes according to this invention. It should be understood, however, that certain mismatch may be tolerated, as long as the specific signal may be distinguished from non-specific hybridization.

The invention also concerns the use of a nucleic acid probe as described above in a method of determining cancer treatment response of a subject or in a method of assessing the response of a subject to a cancer treatment.

As indicated above, alteration in the (immune or tumor) gene locus may also be detected by screening for alteration(s) in polypeptide sequence or expression levels. In order to detect a protein on the cell surface, or, in order to detect the presence, in a cell, of a protein, immunohistochemistry (for example in a tumor bed), ELISA (for example in a blood or serum sample), immunoblotting (in particular Western blot), proteomics, or antibody-based biosensors directed against the protein of interest, as well as any other method known from the man of the art, can be applied to a tumour specimen as previously defined (see Obeid et al, 2007 which provide examples of such techniques).

Imunofluorescence staining or FACS (Fluorescent Activated Cell Sorting) analyses (flow cytometry analyses) is an example of an appropriate method to detect the translocation of a particular protein from the inside to the surface of a cell, in particular of a tumour cell that has been previously submitted to a treatment of cancer.

Contacting the sample with a ligand specific for a polypeptide encoded by a particular gene and determining the formation of a complex is also described.

Different types of ligands may be used, such as specific antibodies. In a specific embodiment, the sample is contacted with an antibody specific for a polypeptide encoded by a particular gene and the formation of a complex is determined. Various methods for detecting such a complex can be used, such as ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA).

Within the context of this invention, an antibody designates a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab '2, CDR regions, etc. Derivatives include single-chain antibodies, humanized antibodies, poly- functional antibodies, etc. An antibody specific for a polypeptide encoded by a particular gene designates an antibody that selectively binds said polypeptide, i.e., an antibody raised against said polypeptide or an epitope-containing fragment thereof. Although non-specific binding towards other antigens may occur, binding to the target polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding. Immunoblotting can in particular be used to measure the degradation of BAP31 , the phosphorylation of eIF2alpha, the presence of a protein selected for example from GCN2 and HRI or the activation of a protein selected for example from caspase 8, reticulon-3, PERK, PKR, Bax and Bak in a cell, in particular a tumor cell, more particularly in a tumor cell which has been previously exposed to a cancer treatment, in particular to a conventional cancer treatment. It is also disclosed kits to predict treatment response or to predict ability to induce an anticancer immune response comprising products and reagents for detecting in a sample from a subject the presence of an alteration in a gene locus or in the corresponding polypeptide or protein; in the gene or corresponding polypeptide or protein expression; and/or in the gene activity.

Such kits comprise any primer, any pair of primers, any nucleic acid probes (wild-type and mutant) and/or any ligand, preferably antibody, described in the present invention. Such kits can further comprise reagents and/or protocols for performing a hybridization, amplification or antigen-antibody immune reaction. Particular kits are the following kits:

- A kit to detect the abnormal expression of a gene selected from CCR1, EIF2Ak2, DNAJCIO, PDIA3, EIF2A, PPPICB, IKBKB, PPPICC, BAX and combinations thereof, in a tumor sample of the subject, the kit comprising (i) at least one pair of primers, in particular two, three, four, five, six, seven, eight, nine pairs of primers corresponding to the previously mentioned genes, and (ii) at least one probe, preferably a fluorescent probe, allowing the quantitative detection of the expression of a gene selected from CCR1, EIF2Ak2, DNAJCIO, PDIA3, EIF2A, PPPICB, IKBKB, PPPICC, BAX, preferably at least one fluorescent probe for each of the selected genes of the previously mentioned list of genes, and (iii) a leaflet providing the control quantitative expression values corresponding to at least one of said genes in a control population.

- A kit to detect an abnormal expression of a gene selected from AHR and MTHFR (in particular the presence of a polymorphism associated with an abnormal expression of such a gene) , in a tumor or blood sample of the subject, the kit comprising (i) at least one pair of primers, preferably two pairs of primers corresponding respectively to the AHR and to the MTHFR gene, and (ii) at least two differently labelled probes, preferably two differently labelled fluorescent probes, the first probe recognizing the wild-type allele and the second probe recognizing the mutated allele of a gene selected from AHR and MTHFR, preferably two differently labelled probes for each of said genes.

- A kit comprising:

1. (i) at least one pair of primers, (ii) at least two distinct probes, preferably different fluorescent probes, allowing the quantitative detection of the expression of a gene selected from CCR1, EIF2Ak2, DNAJC10, PDIA3, EIF2A, PPP1CB, IKBKB, PPP1CC and BAX and (iii) a leaflet providing the control quantitative expression values corresponding to at least one of said genes in a control population; and

2. (i) at least one pair of primers, and (ii) at least two differently labelled probes, the first probe recognizing the wild-type allele and the second probe recognizing the mutated allele of a gene selected from ^H ? and MTHFR.

The herein described kits may further comprise a micro-array or a 96-wells or 384-wells plate to be used for the herein described methods and read through quantitative PCR or multiplex technology.

Compensatory immunogenic treatment of cancer

Inventors advantageously herein provide a new strategy for treating cancer which consists in administering to the subject in need thereof an additional treatment herein identified as "compensatory immunogenic treatment of cancer". As explained previously, a typical subject is a subject resistant to a treatment of cancer, in particular to a conventional treatment of cancer. This compensatory immunogenic treatment of cancer will allow a reaction from the immune system of the subject having a tumor directed against the tumour cells, or will stimulate such a reaction.

Inventors have in particular discovered that such a compensatory immunogenic treatment of cancer is able to allow or improve the efficiency of a conventional therapy as described above, in a subject in need thereof. The compensatory immunogenic treatment of cancer according to the present invention typically involves the exogenous supply, administration for example, to the subject, of at least one compensatory product (molecule, compound, drug or therapeutic agent, cell), preferably together with a conventional therapeutic agent used in a treatment as described above (in order to obtain a therapeutic effect, preferably a synergistic effect), said conventional treatment being easily selected by the cancerologist, as exemplified previously, according to the nature of the cancer to be prevented or treated.

The function of a compensatory product, in the context of the present invention, is to allow the immune system to generate a cancer immune response in a subject identified, with a method herein described, as resistant to a treatment of cancer. As largely explained previously, such a resistance may be due to the inability of the treatment of cancer the subject has been exposed to, to the inability of the subject and/or to the inability of the tumor, to induce an anticancer immune response.

The compensatory product may be selected from a protein, as previously herein identified, i) allowing or enhancing CRT, ERp57, LysRS (KARS) and/or KDEL receptor exposure at the surface of tumor cells, ii) allowing or enhancing the secretion of ATP, HMGB1 (High- mobility group box 1), LysRS and/or IL-8, iii) stimulating the autophagy machinery, and/or an ER stress response, iv) recruiting and/or activating specific effectors in tumor beds, such as IL-17 producing γδ T lymphocytes, cytotoxic T cells and dendritic cells, v) promoting activation of the TLR4/myd88 pathway, vi) triggering the NALP3 (Nacht Domain-, Leucine-Rich Repeat-, and PYD-Containing Protein 3) inflammasome. The compensatory product may also be selected from (i) a product allowing or enhancing the secretion of ATP, HMGB1, LysRS and/or IL-8, and/or the exposure of CRT, ERp57, LysRS and/or KDEL receptor at the surface of a tumour cell, (ii) a product stimulating the autophagy machinery and/or an ER stress response, (iii) a product recruiting and/or activating IL-17 producing γδ T lymphocytes, cytotoxic T cells and/or dendritic cells, (iv) a product promoting activation of the TLR4/myd88 pathway, or able to bypass said pathway, (v) a product triggering the P2RX7 (P2X purinoceptor 7) and/or the NALP3 inflammasome, (vi) a product allowing or enhancing the secretion of IL-lb, (vii) a product capable of stimulating intratumoral Vd2 T lymphocytes, and (viii) a product selected from an anti-allergic drug, a neurotropic drug, an antihypertensive or cardiotropic drug, a spindle poison drug, an antimicrobial drug, an anti-osteoclastic drug, a diuretic drug, an oestrogen, and (ix) any combination thereof.

In the present invention, the term "endogenous" means that a particular protein (for example IL-lb) is produced by the cell as a wild-type protein. The wild-type protein has to be distinguished from the recombinant protein (for example rIL-lb), the recombinant protein whose activity, in particular regarding the immune system, is respectively substantially identical to that of the previously mentioned wild-type protein, but which need a human intervention to be produced by the cell.

In the present invention, the term "homologous variant" is used to designate any protein that comprises deleted or substituted amino acid(s), for example any wild-type or recombinant protein or protein fragment that exhibits the properties of the corresponding wild-type protein, in particular that is able to induce a response from the immune system, for example an immunogenic tumor cell death or apoptosis as previously defined.

A preferred "compensatory product" usable in the present invention that allows or enhances the secretion of ATP, HMGB1, LysRS and/or IL-8, and/or the exposure of CRT, ERp57, LysRS and/or KDEL receptor at the surface of a tumour cell, in particular of a dying tumor cell, can be selected from rCRT, rIL-8, inhibitors of PP1/GADD34, chloroquine, TLR7 agonists, antihistaminic drugs such as brompheniramine maleate, bumetanide, cyproheptadine, fenspiride, flunisolide, ketotifene, loratadine and/or cardiotrop drugs such as amlodipine besylate, atenolol, benazepril hydrochloride, nimodipine and/or antimicrobial such as cycloserine, diloxanide furoate, fluconazole, mebendazole, mefloquine and/or neurotrop drugs such as aripiprazole, bromocriptine mesylate, carbamazepine, clozapine, haloperidol, methysergide maleate, mianserin hydrochloride, mirtazapine, olanzapine, paroxetine hydrochloride, perphenazine, pizotyline malate, procyclidine hydrochloride, quetiapine fumarate, rapamycin, risperidone, sertraline hydrochloride, trazodone, ziprasidone and/or spindle poison drugs such as colchicine, doxorubicin, mitoxanthrone hydrochloride, vinblastine sulfate, vincristine sulphate and/or anti-osteoclast drugs such as etidronate disodium, estrogen such as estrone and homologous variant thereof.

A preferred "compensatory product" usable in the present invention for stimulating the autophagy machinery and/or an ER stress response may be selected from spermidin, resveratrol, and from an ER stress response inducer, such as thapsigargin (THAPS).

A preferred "compensatory product" usable in the present invention for recruiting and/or activating specific effectors in tumor beds, such as IL-17 producing γδ T lymphocytes, cytotoxic T cells and dendritic cells, may be selected from rIL-lb, rIL-17, rIL-22, a phosphoantigen, a V52 T lymphocytes activator, a leukotrien, a prostaglandin, and a chemokine.

A preferred "compensatory product" usable in the present invention for promoting activation of the TLR4/myd88 pathway, or able to bypass said pathway, may be selected from a TLR3 ligand such as such as Poly I:C, poly A:U; a TLR9 ligand such as CpG ODN (CpG oligodeoxynucleotides); and chloroquine.

A preferred "compensatory product" usable in the present invention for triggering the P2RX7 (P2X purinoceptor 7 ) and/or the NALP3 inflammasome, may be selected from a TLR7 agonist such as synthetic oligoribonucleotides containing arabinonucleotides, imiquimod and resiquimod; a TLR8 agonist such as polyGlO; a recombinant cytokine such as rIL-lb and IL-12; and an inhibitor of apyrases such as ecto-nucleoside-triphosphate- diphosphohydrolase (CD39) inhibitor (polyoxometalate 1), 6-N,N-Diethyl-D-beta-gamma- dibromomethylene adenosine triphosphate (ARL 67156), 2'(3')-0-(4-benzoylbenzoyl)- adenosine triphosphate, an antibody inhibiting the ecto-apyrase activity of CD39 and an antibody inhibiting the ecto-5'-nucleotidase activity.

A preferred "compensatory product" usable in the present invention that allows or enhances the secretion of IL-lb, in particular by an immune cell (as herein defined), can be selected from recombinant IL-12 (rIL-12) and/or recombinant IL-lb (rIL-lb). Such a recombinant cytokine may advantageously be used in combination with a molecule selected from an anti-PDl (Programmed Death 1) molecule, a B7-DCFc molecule, an antibody directed against CTLA4 (anti-Cytotoxic T-Lymphocyte Antigen 4 Ab) or against 4-1BBL (anti-4-lBBL Ab), a metronomic cyclophosphamide and any combination thereof.

The compensatory molecule may also be a molecule capable of stimulating intratumoral Vd2 T lymphocytes such as a molecule selected from a phosphoantigen (such as bromohydrinpyrophosphate or BrHPP, phosphostim®) and a lipid. Such a compensatory molecule is preferably used in combination with a conventional chemo therapeutic agent in particular in patients who do not correctly express IL-17.

The compensatory molecule may more particularly be selected from an anti-allergic drug, in particular an anti-histaminic drug or an anti-inflammatory drug; a neurotropic drug, in particular an antidepressant drug, an antipsychotic drug, an antiparkinsonian drug, an anti- headache drug, an analgesic drug, an anticonvulsant drug and an immunosuppressive drug; an antihypertensive or cardiotropic drug; a spindle poison drug such as an antineoplastic drug, an antimitotic drug and an antigout drug; an antimicrobial drug, in particular an anthelmintic drug, an amebicide drug, an antibacterial drug, an antifungal drug and an antimalarial drug; an anti-osteoclastic drug; a diuretic drug; an oestrogen; and any combination thereof.

In particular embodiments of the present invention:

- the anti-histaminic drug may be selected from antazoline phosphate, azelastine hydrochloride, brompheniramine maleate, cyclizine, cyproheptadine, ketotifene, fenspiride, loratadine and terfenadine.

- the anti-inflammatory drug may be flunisolide.

- the antidepressant drug may be selected from sertraline hydrochloride, paroxetine hydrochloride, mianserin hydrochloride, trazodone and mirtazapine.

- the antipsychotic drug may be selected from ketanserin tartrate, risperidone, olanzapine, quetiapine fumarate, ziprasidone, clozapine, aripiprazole, haloperidol and perphenazine.

- the antigout drug may be colchicine. - the antiparkinsonian drug may be selected from procyclidine hydrochloride and bromocriptine mesylate.

- the anti-headache drug may be selected from methylsergide maleate and pizotyline malate.

- the analgesic drug may be carbamazepine.

- the anticonvulsant drug may be carbamazepine.

- the immunosuppressive drug may be rapamycin.

- the antihypertensive or cardiotropic drug may be selected from atenolol, benazepril hydrochloride, amlodipine besylate and nimodipine.

- the antineoplastic drug may be selected from MTX, DX, vinblastine sulphate and vincristine sulphate.

- the antimitotic drug may be colchicine.

- the amebicide drug may be diloxanide furoate.

- the anthelmintic drug may be mebendazole.

- the antibacterial drug may be selected from cycloserine

- the antifungal drug may be fluconazole.

- the antimalarial drug may be mefloquine.

- the anti-osteoclastic drug may be etidronate disodium.

- the diuretic drug may be bumetanide.

- the oestrogen may be estrone.

The compensatory molecule may further be selected from an histamine HI antagonist such as antazoline phosphate, azelastine hydrochloride, brompheniramine maleate, cyclizine, cyproheptadine, ketotifene, fenspiride, loratadine or terfenadine; a 5HT uptake inhibitor such as sertraline hydrochloride or paroxetine hydrochloride; a Ca channel blocker such as amlodipine besylate or nimodipine; a spindle poison such as colchicine, vinblastine sulphate or vincristine sulfate; a topoisomerase II inhibitor such as MTX or DX; a dopamine antagonist such as haloperidol; a dopamine and serotonin antagonist such as risperidone, olanzapine, or clozapine; a glucose uptake inhibitor such as mebendazole; an inhibitor of alanine racemase such as cycloserine; a norepinephrine reuptake inhibitor such as mianserin hydrochloride; an alpha2-adrenergic receptor antagonist such as mirtazapine; a ergosterol synthesis inhibitor such as fluconazole; a 5HT antagonist such as ketanserin tartrate, pizotyline lalate; a beta adrenergic blocker such as atenolol; a ACE inhibitor such as benazepril hydrochloride; a bone resorption inhibitor such as etidronate disodium; an anticholinergic such as benserazide hydrochloride, biperiden, carbidopa, cyclopentolate hydrochloride, dibucaine hydrochloride, dicyclomine hydrochloride, doxepin hydrochloride, ethopropazine hydrochloride, maprotiline hydrochloride, mepenzolate bromide, nortriptyline, protryptiline hydrochloride, oxybutynin chloride, procyclidine hydrochloride, pyrimethamine, quinidine gluconate, solifenacin, trimipramine maleate; a prolactine inhibitor such as bromocriptine mesylate; a FRAP inhibitor such as rapamycin, a steroid such as flunisolide; an adrenergic agonist such as adrenaline bitartrate, xylometazoline hydrochloride, naphazoline hydrochloride; and any combination thereof.

The compensatory molecule may further be selected from an acetamide, an alkaloid derived from periwinkle, an alkaloid derived from ergot, an anthracycline, a benzimidazole, a benzodiazepine, a butyrophenone, a dibenzoazepine, a dibenzocycloheptene, a dibenzodiazepine, a dihydropyridine, a diphosphonate, a phenylpiperidine, a propanol and a thiazole derivative.

A particular acetamide may be selected from for example acetaminosalol, acetanilide, aminitrozol, bufexamac, citiolone, clofexamide chlorhydrate, clofezone, fenoxedil chlorhydrate, guanfacine chlorhydrate, lidocaine, lidocaine chlorhydrate, mefexamide chlorhydrate, oxetacaine, salicylate de picolamine, thiamphenicol, thiamphenicol aminoacetate acetylcysteinate, thiamphenicol aminoacetate chlorhydrate and valpromide.

A particular alkaloid derived from periwinkle may be selected from for example vindesine sulphate and vinorelbine ditartrate.

A particular alkaloid derived from ergot may be selected from for example lisuride maleate acide, methylergometrine maleate, methysergide maleate acide and nicergoline. A particular anthracycline may be selected from for example aclarubicine chlorhydrate, daunorubicine chlorhydrate, epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine and zorubicine chlorhydrate. A particular benzimidazole may be selected from for example albendazole, astemizole, bendazol, benperidol, candesartan cilexetil, chlormidazole chlorhydrate, clemizole hexachlorophenate, clemizole penicilline, clemizole undecylenate, domperidone, flubendazole, lansoprazole, mibefradil dichlorhydrate, mizolastine, omeprazole, oxatomide, pantoprazole sodique, pimozide, rabeprazole sodique, telmisartan and tiabendazol.

A particular benzodiazepine may be selected from for example alprazolam, bromazepam, brotizolam, chlordiazepoxide, clobazam, clonazepam, clorazepate dipotassique, clotiazepam, cloxazolam, delorazepam, diazepam, estazolam, flunitrazepam, ketazolam, loflazepate d'ethyle, loprazolam mesilate, lorazepam, lormetazepam, medazepam, midazolam chlorhydrate, nitrazepam, nordazepam, oxazepam, pirenzepine dichlorhydrate, prazepam, temazepam, tetrazepam, tofisopam and triazolam.

A particular butyrophenone may be selected from for example benperidol, buflomedil chlorhydrate, droperidol, fluanisone, haloperidol decanoate, moperone chlorhydrate, pipamperone dichlorhydrate, primaperone chlorhydrate and trifluperidol chlorhydrate A particular dibenzoazepine may be selected from for example carpipramine dichlorhydrate, clomipramine chlorhydrate, desipramine chlorhydrate, imipramine chlorhydrate, metapramine fumarate, opipramol dichlorhydrate, prozapine chlorhydrate, quinupramine, trimipramine maleate and trimipramine mesilate. A particular dibenzocycloheptene may be selected from for example amineptine chlorhydrate, amitriptyline, amitriptyline chlorhydrate, cyproheptadine chlorhydrate, demexiptiline chlorhydrate, nortriptyline chlorhydrate, noxiptiline chlorhydrate and protriptyline chlorhydrate. A particular dibenzodiazepine may be selected from for example clozapine and dibenzepine chlorhydrate. A particular dihydropyridine may be selected from for example felodipine, isradipine, lacidipine, nicardipine chlorhydrate, nifedipine and nitrendipine.

A particular diphosphonate may be selected from for example alendronate monosodique, clodronate disodique, ibandronate sodique, pamidronate disodique and tiludronate disodique.

A particular phenylpiperidine may be selected from for example remifentanil chlorhydrate and sufentanil.

A particular propanol may be selected from for example acranil, alprenolol chlorhydrate, bufeniode, buphenine chlorhydrate, bupranolol chlorhydrate, chlorobutanol, cimepanol, clofedanol, corbadrine chlorhydrate, cyclopentobarbital ephedrine, dimercaprol, dioxethedrine chlorhydrate, dioxyphedrine chlorhydrate, diphepanol, ephedrine, ephedrine chlorhydrate, ephedrine levulinate, ephedrine sulfate, fenalcomine chlorhydrate, ifenprodil tartrate, inosiplex, isoxsuprine chlorhydrate, metaraminol bitartrate, methoxamine chlorhydrate, metoprolol succinate, metoprolol tartrate, nadolol, ornidazole, oxprenolol chlorhydrate, penbutolol sulfate, phenylpropanolamine chlorhydrate, pindolol, pridinol chlorhydrate, pseudoephedrine chlorhydrate, pseudoephedrine sulfate, racephedrine chlorhydrate, ritodrine chlorhydrate, secnidazole, suloctidil, tertatolol chlorhydrate, trihexyphenidyle chlorhydrate, zipeprol and zipeprol dichlorhydrate

A particular thiazole derivative may be selected from for example azathioprine, bifonazole, butoconazole nitrate, carbimazole, clotrimazole, dacarbazine, econazole nitrate, eprosartan mesilate, etomidate chlorhydrate, fenticonazole nitrate, histamine dichlorhydrate, imiquimod, isoconazole nitrate, ketoconazole, metronidazole, metronidazole benzoate, miconazole, miconazole nitrate, nimorazole, ondansetron chlorhydrate, ornidazole, oxiconazole nitrate, secnidazole, sertaconazole nitrate, sulconazole nitrate, thiamazole, imidazole and tioconazole.

A further object of the present invention relates to the use of at least one compensatory molecule, from the molecules identified previously, to prepare a pharmaceutical composition that is preferably intended to be administered in combination with a distinct product, typically an agent used in a treatment of cancer, in particular in a conventional treatment of cancer as mentioned previously (for example a non immunogenic treatment), to prevent or treat a cancer as defined above, in a mammal, preferably a human.

In this context, the compensatory molecule can be considered as an adjuvant to the conventional therapeutic drug.

In a particular embodiment, if the patient having a tumor is to be exposed to an immunogenic conventional cancer treatment as previously defined, and if the tumor is identified, using a method as herein described, as not able to induce an anticancer immune response, then a compensatory product should be administered to the subject, preferably together with the first exposition, for example administration, of the immunogenic conventional cancer treatment (for example chemotherapeutic drug, ionizing radiation, etc.).

Such a compensatory product may be selected in particular from a recombinant CRT (rCRT) and a recombinant IL-8 (rIL-8).

In such a situation where the tumor is not able to induce an anticancer immune response, the compensatory product is preferably to be administered in the tumor or in the tumor bed. In another particular embodiment, (i) if the patient having a tumor is to be exposed to an immunogenic conventional cancer treatment as previously defined, for example a chemotherapy using anthracyclines, (ii) if the tumor, for example a breast tumor, is identified, using a method as herein described, as able to induce an anticancer immune response, and (iii) if the subject is identified, using a method as herein described, as not able to induce an anticancer immune response, because, for example, of the presence of a SNP [as identified previously, in particular rs2066853 (SEQ ID NO: 7)] in her AHR gene, then a compensatory product should be administered to the subject, preferably with a conventional treatment of cancer.

In a preferred embodiment, the conventional treatment of cancer is a chemotherapy and the compensatory product is administered after each cycle of the all chemotherapeutic treatment, preferably two, three, four or five days after the exposition of the subject to a cycle of the chemotherapeutic treatment. Such a compensatory product may be selected for example from IL-17 producing γδΤ cells, phosphantigens such as biphosphonates (zoledronate) and clodronate.

In a particular embodiment of the present invention, the absence of IL-17 producing γδ T lymphocytes in the tumor of a subject or the presence of SNP in the genomic DNA of the subject (such as in the AHR gene) for example, is indicative of the absence of an anticancer immune response in the subject who has been exposed to a conventional treatment of cancer, in particular to a chemotherapeutic treatment of cancer, and reveals a resistance of the subject to the treatment of cancer.

A compensatory product should thus be administered to this subject, preferably locally in the tumor, preferably together with the conventional treatment of cancer. Such a compensatory product may be selected from IL-17 producing γδ T lymphocytes; recombinant IL-22 (rIL-22) and/or IL-17 (rIL-17); phosphoantigenic synthetic ligands of γδ T lymphocytes [preferably together with recombinant IL-lb (rIL-lb) and/or IL-23 (rlL- 23)], such as bromohydrin pyrophosphate (BrHPP, active pharmaceutical ingredient in Phosphostim) and νδ2 T lymphocytes activators such as biphosphonates (zoledronate) and/or clodronate.

The previously mentioned compensatory products are preferably injected locally into the tumor or in a tumor bed.

Also herein provided, is a pharmaceutical composition comprising such a compensatory molecule or a combination of identical or different compensatory molecules, in association with a pharmaceutically acceptable excipient or diluent.

Appropriate excipient, diluant or carrier usable in the all present invention may be selected for example from saline, isotonic, sterile or buffered solutions, etc. They can further comprise stabilizing, sweetening and/or surface-active agents, etc. They can be formulated in the form of ampoules, flasks, tablets, or capsules, by using techniques of galenic known per se. The pharmaceutical composition mentioned previously may be administered to the subject in need thereof, before, during and/or after any treatment of cancer described previously. It is preferably administered during or after said treatment, for example 24 hours, two days, three days or four days after the treatment.

For example, in a particular embodiment of the present invention, the compensatory products are IL-17 producing γδ T lymphocytes which may be advantageously administered to a subject in need thereof, two days after exposition of said subject to a treatment of cancer using radiotherapy or a chemotherapy wherein, for example, DX is administered to the subject.

Also herein described are compensatory molecules for use in the treatment of cancer, preferably in combination with a conventional treatment of cancer, in particular a chemotherapeutic treatment of cancer, in a subject identified, by a method as herein described, as resistant to a conventional treatment of cancer.

Method to prevent or treat a disease

The present invention also relates to a method for preventing or treating a cancer, as herein defined, comprising the administration to a mammal, in particular a human, in need thereof, of at least one compound selected from the previously described compensatory molecules, preferably together with (in combination with) a distinct therapeutic agent, typically an agent used in a conventional treatment of cancer as defined previously. A subject in need of a compensatory molecule is subject that has been tested and identified as resistant to a treatment of cancer according to the method described above.

In a particular embodiment of the present invention, the previously described method for treating cancer is performed on a subject having a tumor before surgical resection thereof. In another particular embodiment of the present invention, the previously described method for treating cancer is performed on a subject having a tumor after surgical resection thereof. The above method to treat a disease may comprise a step of directly injecting at least one selected compensatory molecule in the tumour, or in the tumor bed, of the subject in need thereof. Screening methods

The present invention also provides a method for screening or selecting a compound that is able to modify the activity of the immune system towards a tumor cell, in particular to trigger an immunogenic tumor cell death, the method comprising a step of detecting and/or measuring the level of expression, by a particular tumor cell, of a functional immunogenic cell death-associated molecule as herein described, in the presence of a test compound, wherein a modified expression in comparison with a control cell that has not been exposed to or contacted with the test compound, is indicative of the capacity of said compound to modify the activity of the immune system towards said cell.

The present invention further provides a method for screening a compound usable for treating a cancer, as a compensatory product according to the present invention, in a subject having an altered nucleic acid, an altered nucleic acid expression, or an abnormal expression or activity of the protein corresponding to said nucleic acid, said method comprising determining in vitro, in vivo or ex vivo the ability of a test compound to (i) restore a functional expression of said altered or abnormal protein (ii) modulate (i.e., induce, increase, or decrease) the expression or activity of said protein, or (iii) modulate the expression or activity of a ligand of said protein. The compounds identified with one of the herein described screening methods may be used, in the context of the present invention, as compensatory molecules.

Other characteristics and advantages of the invention are given in the following experimental section (with reference to figures 1 to 27), which should be regarded as illustrative and not limiting the scope of the present application. EXPERIMENTAL PART

Example 1: IL-13-dependent contribution of IL-17 producing γδΤ cells in the efficacy of cytotoxic anticancer therapies

By triggering an immunogenic cell death modality, some anticancer compounds including anthracyclines elicit tumor-specific IFN-γ producing CD8⁺ T cells that are mandatory for therapeutic success. This adaptive immune response depends on IL-Ιβ produced by DC confronted with or exposed to anthracycline treated tumor cells. Inventors analyzed the influence of immunogenic chemotherapy on the tumor microenvironment to identify inflammatory components which link innate and cognate immune responses. Inventors herein demonstrate that distinct subsets of γδ T lymphocytes (Vy4⁺ and Vy6 ) colonized tumors, where they proliferate and become potent IL-17 producers upon chemotherapy. In the present experiment, IL-17A production by γδ T cells fully depended on the DC mediated IL-Ιβ production and aryl hydrocarbon receptors also contributed to this process. In νγ4/6^_/" mice or in the absence of a functional IL-17/IL-17 receptor (IL-17R) pathway or upon blockade of AHR, the response to immunogenic cell death or the efficacy of chemotherapy was compromised. Conversely, adoptive transfer of γδ T cells increased the efficacy of anthracycline-based chemotherapy, under the condition that these cells express the IL-1R1. Therefore, IL-17 producing γδΤ cells or lymphocyte^ T17 cells) represent a novel link between cell death and cognate immunity during anticancer chemotherapy.

While the contribution of IFN-γ to tumor surveillance and anticancer immune responses is clearly established, that of the IL-17A/IL-17R signaling pathway remains controversial (Kryczek et al, 2009; Martin-Orozco et al, 2009; Wang et al, 2009). In tumor models where CD4⁺ T cells are the source of IL-17, this cytokine promotes IL-6-mediated Stat3 activation, acting as a pro-tumorigenic trigger (Kortylewski et al, 2009; Wang et al, 2009). Thus, inventors supposed that IL-17 could be one of the factors that link chronic inflammation to cancer development. However, in adoptive transfer experiments, IL-17- producing CD8⁺ T cells could reduce the volume of large established tumor, presumably by differentiation into long-lasting IFN-γ producers (Hinrichs et al., 2009). Therefore, the source and/or the targets of IL-17 must determine whether this cytokine enhances or reduces tumorigenesis. Interestingly, it appears that the production of IL-17 is strongly dependent on signaling via aromatic AHR, a ligand-activated transcription factor widely expressed in many tissues including lymphoid organs. In particular, Thl7 cells and dendritic cells express high levels of AHR. Activation of AhR by yet elusive endogenous ligands markedly increased the proportions of Thl7 cells and their production of IL-17 (Veldhoen et al., 2008). However, before the present invention it was unknown whether and how AHR impacts on anticancer immune responses at the level of IL-17 production.

Similarly, the contribution of γδ T cells in tumor immunosurveillance is still elusive (Hayday, 2009). In humans, γδ 1 T cells have been shown to either mediate immunosuppressive activities (Peng et al, 2007) or to be associated with a reduced occurrence of cancers in transplanted patients bearing a CMV infection (Dechanet et al., 1999). In contrast, γδ 2 T cells can be activated by various synthetic ligands to produce Thl-like cytokines and exhibit cytotoxic functions against tumors (Kabelitz et al., 2007). Although various γδ T cell subsets have been reported to be able to produce IL-17 during microbial infection or autoimmune disorders of mice (O'Brien et al., 2009; Shibata et al., 2007), no data are available on the incidence and functional relevance of IL-17-producing γδ T cells in cancer. γδΤ17 cells have been reported to share most phenotypic markers with Thl7 (expression of CCR6, RORyt, AHR, IL-23R, IL-17A, IL-22) (Martin et al, 2009). They depend upon TGF-β but not IL-23 or IL-6 for their generation and maintenance (Do et al.) and they were unrestricted by Vy usage (although they were mostly Vy2Vy3 in the context of mycobacteria (Martin et al, 2009) and Vy4 in experimental autoimmune encephalitis (Sutton et al, 2009)). Recent work suggests that thymic selection does little to constrain γδΤ cell antigen specificities, but instead determines their effector fate. When triggered through the T cell receptor, ligand-experienced cells make IFN-γ, whereas ligand-na^'ive γδΤ cells produce IL-17 (Jensen et al, 2008).

It is herein demonstrated that a therapy-induced immunogenic cancer cell death which stimulates a therapeutic anti-cancer immune response influences the composition and the architecture of the immune infiltrate present in tumors, which in turn contributes to the control of residual tumor cells.

Inventors herein show that, in response to an immunogenic chemotherapy with anthracyclines and OXP, an early infiltration by γδ T17 cells is a prerequisite for optimal colonization of tumor beds by CD8 T lymphocytes, eventually leading to tumor growth retardation or regression. Inventors demonstrate that DC producing IL-Ιβ in response to dying tumor cells and AHR signaling determine and optimize IL-17 release by γδ T cells. Finally, they show that both γδ T cells and the IL-17/IL17 receptor signaling are required for inducing an optimal anticancer response of a subject undergoing a chemotherapy and that the adoptive transfer of γδ T17 cells increases the therapeutic efficacy of an anticancer chemotherapy.

Material and Methods

Mice. Wild type C57bl/6 (H-2^b) and BALB/c (H-2^d) mice aged between 7-12 weeks were purchased from Harlan (Gannat, France). Nude mice were bred in the animal facility of IGR. TCR δ^_/" (H-2^b), IL-1R1^"7" (H-2^b) and IL-17Ra ^/_(H-2^b) mice were bred at CDTA, Orleans, France through BR and PP (as for TCR δ^{~ ~}). νγ4γ6^_/~ mice (H-2^b) were kindly provided by GM and KI. IL-23pl9^_/" (H-2^b) were kindly provided by FP. CDld^_/" and CCR6 ^{~ ~} (H-2^b) were bred at St Vincent de Paul Hospital AP-HP, Paris, France and provided by KB. The experimental protocols were approved by the Animal Care and Use Committee in the animal facility of Institut Gustave Roussy. Cell lines and reagents. CT26 (H-2^d) colon cancer, MCA205 sarcoma (H-2^b), TS/A mammalian cancer (H-2^d) and EL-4 thymoma (H-2^b) were cultured in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM non-essential amino acids, and 10 mM HEPES at 37° C, 5% C0₂. All media were purchased from GIBCO, France. AHR antagonist CH223191 was from Calbiochem. Recombinant mouse interleukin-ΐβ (IL-Ιβ), IL-23, IL-6, TGF-β and IL- 18 BPd/Fc were from R&D system. DX hydrochloride (D1515) and MTX dihydrochloride (M6545) were from Sigma Aldrich. Mouse IL-17, IL-Ιβ ELISA kits were purchased from eBioscience. Mouse IL-22, IL-23 ELISA kits were purchased from R&D system. Antibodies for CD45.2 (clone 104), CD3e (clone 145-2C11), CD4 (clone GK1.5), CD8cc (clone 53-6.7), TCR §clone GL-3, CD69 (clone H1.2F3), IL-17A (clone TC11-18H10) or IFN-γ (clone XMG1.2) for surface or intracellular staining were from BD bioscience or eBioscience. Neutralizing antibodies for IL-17 (MAB421), IFN-γ (XMG1.2), CCL20 (MAB760), IL-23 (AF1619), IL-23R (MAB1686) and IL-6 (MAB406) were from R&D system. LIVE/DEAD Fixable Dead Cell Stain Kit, DiOC6(3) and DAPI were purchased from Molecular Probes, Invitrogen. CpG oligodeoxynucleotide (ODN) 1668 was from MWG Biotech AG.

Tumor models and chemo/radiotherapy. 0.8 million MCA205 or CT26 or TS/A tumor cells were inoculated subcutaneously near the thigh into C57B1/6 (H-2^b) or BALB/c (H-2^d) mice. Anthracyclines-based chemotherapy was performed in MCA205 and CT26 models by intratumoral injecting DX (2 mM, 50 μΐ) when tumors reached the size 25-40mm². Radiotherapy was performed by local X-ray irradiation (10 Gy, RT250, Phillips) at the unshielded tumor area when TS/A tumor reached the size 40-60 mm².

Gene Expression Assays. Tumors from mice either treated with DX or PBS were removed 8 days after treatment. Whole RNA was extracted using RNeasy Mini Kit, QIAGEN from pieces of tumor homogenates. 5 μg of RNA from each sample were reverse-transcribed using Quantitect Reverse Transcription Kit (QIAGEN). Gene expression assays were performed with TaqMan® 96 well Plates customized to test cytokines, chemokines as well as transcription factors using StepOnePlus™ Real-Time PCR System. PPIA was chosen as the endogenous control to perform normalization between different samples.

Tumor dissection and flow cytometry. Tumor burdens were carefully removed, cut into small pieces with scissors within digesting buffer (400 U/ml Collagenase IV and 150 U/ml DNase I in RPMI1640) and incubated for 30 min at 37° C. Single cell suspension was obtained by grinding the digested tissue and filtering through 70 μΜ cell strainer. After washing with PBS, cells were resuspended at 2>< 10⁷/ml, blocked with 10 μg/ml anti- CD 16/CD32 (eBioscience) in PBS containing 2% mouse serum for 5min at 4° C. 2.5 μg/ml of antibodies were used for surface staining at 4°C, 30 min. LIVE/DEAD Fixable Dead Cell Stain Kit was used to distinguish live and dead cells. For intracellular staining, freshly isolated cells were treated with, 50 ng/ml PMA, ^g/ml ionomycin and Golgi-stop (BD Pharmingen), 4hrs, 37°C in RPMI containing 2% mouse serum (Janvier, France). Cells were then washed with PBS and stainied with anti-IFN-y(PE-cy7) and anti-IL-17 (PE) using BD Cytofix/Cytoperm™ Kit following the instructions. Protein extraction. Tumors were mechanically dissociated with lysis buffer (T-PER Tissue Protein Extraction Reagent, PIERCE) containing protease inhibitor (complete Mini EDTA- free, Roche). Tumor lysate was then centrifuged at lOOOOxg, 5min, 4°C to obtain supernatant.

Purification and adoptive transfer of γδΓ cells. Naive C57B1/6 mice aged between 8-10 weeks were sacrificed and the skin-draining lymph nodes (LNs) including inguinal, popliteal, superficial cervical, axillary and brachial LNs were collected. LNs were squeezed with tweezers gently in digesting buffer, kept at 37° C for 20 min and then pass through 40 μΜ cell strainer to get single cell suspension. Dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotec) before purifying γδΤ cells with TCRy/5⁺ T Cell Isolation Kit (Miltenyi Biotec). An autoMACS™ Separator was used with the recommended programs. Purity of this isolation normally reached 95%. The TCR δ CD3⁺ cells fraction was also collected from the final separation step and was called 'non γδΤ' cells for some experiments. Day 2 after DX or PBS treatment, 2.5^X 10⁵ cells were injected directly into the tumor with insulin syringes in adoptive transfer setting.

T cell priming and tumor vaccination. EG7 cells were pretreated either with 5 μg/ml OXP or left untreated for 24hrs, washed thoroughly and injected at 1 million/50 μΐ into the syngeneic mice foodpad. CpG/OVA (CpG (5 μg/mouse), OVA (1 mg/mouse)) and PBS injection were used as positive and negative controls. In some setting, neutralizing antibody (200 μg/mouse) for IL-17A or isotype control antibody was injected i.p. 5 days later, the popliteal lymph node cells were harvested, seeded in 96 well plate at 3>< 10⁵/well and restimulated with 1 mg/ml OVA protein. IFN-γ secretion was measured by OptEIA™ Mouse IFN-γ ELISA kit (BD Bioscience). MCA205 cells were treated with 2 μΜ MTX for 18hrs, washed thoroughly and injected into left flank subcutaneously at 0.3 million/mouse. PBS was used as control. Mice were rechallenged with 5xl0⁴ live MCA205 cells in the right flank 7 days later. Tumor growth was monitored every 2-3 days.

DC-tumor mixed lymphocyte cultures. DC were propagated in Iscoves's medium (Sigma Aldrich) supplemented with J558 supernatant, 100 Ul/ml Penicillin, 100 μg/ml Streptomycin, 2 mM L-glutamine, 50 μΜ 2-mercaptoethanol (Sigma), 10% heat- inactivated and filtered, 10% FCS and 40ng/ml GM-CSF. DC were used between day 8 and 12 when the proportion of CDl lc/MHC class 11+ cells was > 80%. In mixed cocultures, DC were seeded at 10⁵/100 μΐ/well in U bottom 96 well plates. Tumor cells were treated with 25μΜ DX or 2 μΜ MTX for 16hrs, washed in PBS and added into these wells at 7.5x l0⁵/100 μΐ/well. 2^χ 10⁴/50 μΙγδΤ cells were added into the wells 12 hrs later. Supernatant was collected 48 hrs later.

Statistical analyses of experimental data. All results are expressed as means±standard error of the mean (SEM) or as ranges when appropriate. For two groups, normal distributions were compared by Student's t test. Non-normal samplings were compared using the Mann- Whitney's test or Wilcoxon matched paired test when appropriate. The log-rank test was used for analysis of Kaplan-Meier survival curve. Statistical analyses were performed using Prism 5 software (GraphPad, San Diego, CA). P values of < 0.05 were considered significant.

Results

Patterns of cytokine/chemokine production post-chemotherapy

Anthracyclines induce immune responses that culminate in CD8⁺ T cell- and IFN-y/IFN-γ R dependent antitumor effects (Ghiringhelli et al., 2009). To further study chemotherapy- induced immune effectors at the site of tumor retardation, inventors performed quantitative RT-PCR to compare the transcription profile of 40 immune gene products expressed in MCA205 tumors which were regressing in response to chemotherapy with the anthracycline DX 8 days post-treatment, with that of progressing tumors due to the absence of treatment (PBS control) (Fig. 1A). Several Thl -related gene products were specifically induced in regressing tumors (Fig. IB). Thus, the Thl transcription factors Eomes and Tbx21 (also called T-bet), as well as the end product IFN-γ, were increased 4-5 fold in doxorubicine (DX) versus PBS -treated tumors (Fig. 8A). Unsupervised hierarchical clustering indicates that IFN-γ production correlates with that of the transcription factor Tbx21, which is the quintessential Thl transcription factor. By day 3-7, the protein levels of IFN-γ also increased in regressing MCA205 sarcoma (Fig. 1C). Other surrogate markers of Thl responses (lymphotoxin-β, Ccl5, CxcllO, Cxcl9, TNF-a) were also significantly induced at the mR A level following anthracycline treatment (Fig. IB). Unexpectedly, another set of gene products were also overexpressed in the context of anthracyc line-induced tumor regression. These genes encoded IL-7R, IL-21, AHR, Cxcl2 and Foxp3, suggesting that inflammation and/or tissue repair took place in the tumor bed (Fig. IB, Fig. 8A). Indeed, on days 3 to day 8 post-chemotherapy, the protein levels of the inflammatory cytokine IL-17 were significantly increased within tumor homogenates (Fig. 1C, right panel). Reenforcing this finding, we show that AHR, a sensor of small chemical compounds, is involved in the success of anthracyclines based therapy in this model. CH- 223191 is a pure antagonist of AHR since it does not have any agonist actions up to 100 μΜ (Kim et al, 2006). Blocking AHR with CH-223191 markedly reduced the efficacy of DX on established cancers in vivo (Fig. ID) although CH-223191 had no cell-autonomous effects on the tumor cells, alone or in combination with anthracyclines (Fig. 8B). Moreover, DX (compared with PBS) induced a 3-fold increase in the proportions of both IFN-γ and IL-17 producing tumor infiltrating lymphocytes (TILs) as tested by flow cytometry (Fig. IE). All together, these data show that chemotherapy modify the chemokine/cytokine tumor microenvironment, leading to early Thl7-geered inflammation together with a marked Thl polarization. γδ T lymphocytes are the major source of IL-17 in several models of anticancer chemotherapy.

To identify the cellular source of IFN-γ and IL-17, TILs were immunophenotyped by a combination of cell surface staining and intracellular detection of the cytokines with flow cytometry. Careful analyses revealed that 8 days post-chemotherapy in MCA205 sarcomas, the major source of IFN-γ were CD8⁺ T cells, while that of IL-17 were mostly TCR δ⁺ T cells rather than CD4⁺ Thl 7 cells (Fig. 2A). Inventors further analyzed the IFN-γ and IL- 17 production by each subset of TILs. It turned out that CD4⁺ T cells could produce IFN-γ and a small amount of IL-17 while CD8 T and γδ T cells were polarized to become potent producers of IFN-γ and IL-17 respectively. Doxorubicin-based chemotherapy substantially enhanced IFN-γ production by CD8⁺ and CD4⁺ TILs as well as IL-17 production by γδ TILs (Fig. 2B) and induced a more intense infiltration of these cytokine producers (Fig. 2C). A kinetic study indicated that γδ TILs invade MCA205 tumor beds at early time points (Fig. 2D), rapidly divide (as indicated by the expression of Ki67) (Fig. 2E) and produce IL-17 shortly after chemotherapy, with significant increases over the background 4 days after anthracyline injection (Fig. 2D, left panel). This early induction of IL-17 contrasts with the comparatively late induction of IFN-γ production by CD8⁺ T cells, which emerged 8 days after chemotherapy (Fig. 2D, right panel).

To generalize these findings, inventors systematically immunophenotyped TILs in CT26 colon cancer treated by a single intratumoral injection of DX which significantly retarded tumor growth (Fig. 9A). Indeed, the majority of IL-17+ TILs were CD45⁺CD3^bright cells and they failed to express CD4 but were positively stained with anti-TCR δ specific antibodies (Fig. 9B). Consistently, chemotherapy increased the frequency of IFN-γ producing CD8⁺ T lymphocytes (Tel) (Fig. 9C) and IL-17-producing γδ T cells (γδ T17) (Fig. 9D) among TILs. Next, inventors monitored transplantable TS/A mammary carcinomas treated with local radiotherapy which operates in a T cell-dependent manner (Apetoh et al., 2007). Irradiation of TS/A tumors led either to tumor regression (TR) or to no response and hence tumor progression (TP) (Fig. 3A). An accumulation of both Tel (Fig. 3B) and γδ T17 (Fig. 3C) lymphocytes was found in those tumors that responded to radiotherapy, but not in those that continued to proliferate or in untreated controls. Importantly, in all three tumor models that were tested, a clear correlation was observed between invading γδ T17 and Tel cells in tumor beds (Fig. 2F, Fig. 3D, Fig. 9E). Thus, chemotherapy triggers the accumulation of cytokine producing TILs in the tumor bed. This applies to IFN-y-producing CD8⁺ T cells, which have previously been shown to contribute to the chemotherapy- induced anticancer immune response (Ghiringhelli et al, 2009), as well as to IL-17-producing γδ T cells, which inventors decided to characterize at the functional level.

Most γδ T17 TILs had an effector memory phenotype which was preponderantly CD44⁺ CD62L^" CD69⁺ granzyme B⁺. γδ T17 TILs did not express CD27, CD 122, Scart 2 (a marker of γδ T17 cells residing in skin draining lymph nodes), CD24, c-kit or NKG2D (Fig. 10). Flow cytometry indicated that around 60% of tumor filtrating γδ T17 utilized Vy4 chain (Fig. 10) but expression of Vyl and Vy7 chain was rarely found (data not shown). We then sorted γδ T17 TILs which do not express Vyl, Vy4 or Vy7 and performed single-cell PCRs (Boucontet et al, 2005) to examine their Vy chain usage. These experiments revealed that 21 out of 23 clones contained a functional Vy6 rearrangement identical to the one found in fetal γδ T cells, indicating that most γδ T17 TILs express either Vy4 or Vy6.

Inventors conclude that, during chemotherapy or radiotherapy-induced tumor regression, distinct subsets of γδ T cells accumulate in tumor beds and become γδ T17 cells, correlating with (and presumably preceding) the accumulation of Tel cells.

The IL-17/IL-17R pathway is involved in the immunogenicity of cell death

Since both γδ T17 and Tel cells accumulated within tumors after chemotherapy or radiotherapy in a coordinated fashion, inventors determined whether neutralizing antibodies directed against their signature cytokines IL-17 and IFN-γ could mitigate the efficacy of anticancer therapies. The neutralization of either IFN-γ or IL-17 negatively affected the growth-retarding effect of DX against MCA205 tumors (Fig. 4A). Inventors have reported that specific anti-tumor immune response relies on CD8⁺ T cells which could be primed by tumor cells undergoing immunogenic cell death and developed a system in which IFN-γ production by OVA-specific T cells could be triggered by OXP -treated EG7 cells (Ghiringhelli et al, 2009). Inventors utilize this system to check whether IL-17 is involved in initiating the specific anti-tumor response, comparing normal wild type (WT) with IL-17Rcc ^/_ mice. In this assay, the absence of IL-17RCC fully abolished antigen- specific T cell priming in response to dying cells, yet had no negative effect to T cell priming by OVA holoprotein admixed with CpG oligodeoxynucleotides (Fig. 4B). Consistently, a neutralizing anti-IL-17A antibody (but not the isotype control antibody) markedly impaired the OVA-specific T cell response to OXP-treated EG7 cells (Fig. 4C). Since Thl/Tcl immune responses against dying tumor cells mediate a prophylactic protection against a rechallenge with live tumor cells (Apetoh et al., 2007; Ghiringhelli et al, 2009), inventors addressed the functional relevance of IL-17/IL-17RCC pathway in such a protective immunity. The subcutaneous injection of anthracycline mitoxanthrone (MTX) treated MCA205 sarcoma cells could protect WT mice (but not athymic nude mice) against rechallenge with live MCA205 tumor cells (Fig. 4D). The efficacy of this vaccination was attenuated in IL-17RCC ^{~ ~} mice. Since IL-17 was not significantly produced by CD4⁺ T cells, neither in the draining LN (not shown) nor in tumor beds during chemotherapy (Fig. 2A, 2B, Fig. 9B), they refrained from investigating Thl7 cells and rather focused on γδΤ and NKT cells as potential IL-17 producers (Mills, 2008; Pichavant et al, 2008) that might contribute to the anticancer vaccination by dying tumor cells. While CDld^{~ ~} mice, which lack NKT population (Godfrey et al, 2009), were undistinguishable from WT controls in their ability to protect themselves against the live tumor cells rechallenge after dying tumor cell vaccine, νγ4/6^_/" mice (Sunaga et al, 1997) exhibited a reduced capacity to mount an anticancer immune response (Fig. 4D). These results suggest that IL-17, IL-17R, as well as γδ T17 cells, all play an important role in the afferent phase of the immune response against dying tumor cells that includes T cell priming for IFN-γ production. γδ T lymphocytes are indispensable for the efficacy of chemotherapy

To further evaluate the contribution of γδ T cells to the therapeutic action of mitoxanthrone on established MCA205 sarcomas, such tumors were implanted in age and sex matched WT, TCR δ ^{~ ~}, νγ4/6^_/" and then subjected to systemic chemotherapy. As compared to wild type controls, the absence of the TCR δ chain, as well as Vy4 and Vy6 γδ T cells greatly reduced the efficacy of chemotherapy (Fig. 5A).

Expression of CCR6 is a hallmark of Thl7 cells at the phenotypic and functional (Reboldi et al, 2009) levels during some inflammatory processes. Inventors therefore analyzed the role of CCR6 in the efficacy of chemotherapy. Since CCL20 was abundant in tumor tissues post-chemotherapy (data not shown), they assessed whether γδ T17 cells could be recruited in a CCL20/CCR6-dependent manner. The tumoricidal activity of DX against CT26 was not affected by repetitive systemic injections of neutralizing anti-CCL20 mAb before and during anthracyclines treatment (Fig. 5B). Consistently, anthracyclines treatment against established MCA205 sarcoma remained efficient in CCR6 loss-of-function mice (Fig. 5C). Moreover, CCR6 deficiency did not influence tumor infiltration by γδ T17 (Fig. 11). Therefore, both νγ4/γ6 γδ Τ cells and their effector molecular pathway IL-17/IL-17R are involved in the prophylactic and therapeutic efficacy of anticancer agents while CCR6 signaling seems to be indispensable for their colonization of tumor bed. IL-1 -dependent activation of γδ T lymphocytes To explore the molecular requirements for γδ T17 cell activation in situ, inventors sorted γδ T cells from the skin-draining lymph nodes (LNs) of naive mice (representing about 1% of the T cell pool contained in LN). Among these γδ T cells, around 70% harbored the Vy4 TCR and they vigorously produced IL-17 (but not IFN-γ) upon stimulation with PMA/ionomycine (data not shown) (Do et al). In contrast to Thl7 cells (Ivanov et al, 2006), LN resident γδ T cells failed to produce IL-17 in response to TGF-β or IL-6, alone or in combination with IL-Ιβ (Fig. 6A). However, LN-resident γδ T cells potently secreted IL-17A (and IL-22, not shown) in response to the combined stimulation with IL-Ιβ plus IL-23 (Fig. 6A). TCR engagement also synergized with IL-Ιβ (and to a lesser extent with IL-23) to trigger IL-17 secretion by LN-resident γδ T cells (Fig. 6B). It is noteworthy that these stimuli specifically activated IL-17 (Fig. 6 A, 6B) but not IFN-γ production (data not shown) by γδ T cells. Since Vy4⁺ and Vy6⁺ γδΤ cells were activated (as indicated by their Ki67⁺, GzB⁺, CD69⁺, IL-17⁺ phenotype) within tumor beds after chemotherapy, inventors addressed the question as to whether dying tumor cells could directly or indirectly (through myeloid antigen presenting cells) promote the activation of Vy4⁺ and Vy6⁺ T cells. Although doxorubicin-treated MCA205 cells failed to directly induce IL-17 (or IL-22, not shown) secretion by γδ T cells, they did so indirectly. Thus, bone marrow-derived DC (DC) that had been loaded with doxorubicin-treated MCA205 cells (Fig. 6C) or CT26 cells (not shown) but not with live tumor cells markedly stimulated the release of IL-17 (and IL- 22, not shown) by γδ T cells (Fig. 6C). As a quality control for in vitro generated DC, the expression of CDl lc, MHC class II, CDl lb and F4/80 was assessed. Only 'qualified' DC preparations that contain functional DC (>80% CD1 lc MHCII ) rather than macrophages (>70% CDl lb⁺F4/80⁺CDl lc) can activate γδ T cells for IL-17 production when they encountered DX treated tumor cells (data not shown).

Dying tumor cells (exposed to doxorubicin) but not live tumor cells could trigger IL-Ιβ production by DC (Fig. 6C and (Ghiringhelli et al, 2009)). The IL-17 production by γδ T cells was dependent on IL-Ιβ since the IL-lRl/IL-Ιβ antagonist IL-IRA entirely abrogated the DC/γδ T cell cross-talk in the presence of dying cells while IL-Ιβ production was not modified (Fig.6D). Blocking AHR could also hamper IL-17 production in this co- culture system. In contrast, neutralizing IL-23 or blocking IL-23 R or blocking IL-18/IL- 18R interaction failed to damp down the production of IL-17 (and IL-22, not shown) by the combination of DC, dying tumor cells and γδ T cells (Fig. 6D). It is conceivable that the y5TCR might be engaged by an MHC class I-like molecule presented by DC because the IL-17 production by γδ T cells was significantly improved by cell contact or a TCR cross- linking. Indeed, the supernatants (containing at least IL-Ιβ) of BMDCs loaded with dying cells could not entirely substitute for dying cell loaded DC in these in vitro assays (not shown). Blocking AHR markedly attenuated IL-17 production by γδ T cells, both at the level of cytokine release (not shown) and on a per cell basis (Fig. 6E, left panel) in response to CD3 cross-linking and IL-Ιβ and/or IL-23, yet did not affect γδ T cell viability (not shown) or their activation pattern (Fig. 6E, right panel).

Since inventors found that IL-Ιβ was required for the production of IL-17 by γδ T cell in vitro, they assumed that γδ T cells might be activated locally by this cytokine. Indeed, the adoptive transfer of γδ T cells (instead of the non γδ T cells purified from na^'ive skin LNs) into tumor beds two days post-DX ameliorated the efficacy of chemotherapy (Fig. 7A) while infusion of γδ T cells into non-treated tumors (failing to release IL-Ιβ) could not control tumor outgrowth (Fig. 7A). However, when γδ T cells were derived from IL-1R1 loss-of-function mice, the synergistic antitumor effects of doxorubicine and adoptively transferred γδ T cells were lost (Fig. 7B), demonstrating the key role of endogenous IL-Ιβ in driving the γδ T cell response.

Altogether, the present data indicate that chemotherapy-induced cell death stimulates DC to release IL-Ιβ, which in turn is required for IL-17 production by γδ T cells, γδ T cells can act as enhancers of the immunological component of anticancer immune therapies, provided that they express the IL-1R.

Conclusions

Example 1 demonstrate a critical role for a subset of γδ T cells, particularly the Vy4 and Vy6-expressing subsets, which produce the effector cytokine IL-17, in the adaptive immune response against dying tumor cells which contributes to the efficacy of anthracycline-based conventional anticancer chemotherapy. Inventor demonstrate that the IL-17/IL-17RCC signalling pathway is required for the priming of IFN-y-secreting antigen- specific T cells by tumor cells exposed to chemotherapy (Fig. 4B, 4C). This tumor-specific Tel -mediated immune response is essential for the protective anticancer immunity that is triggered by immunization with dying tumor cells (Fig. 4D) because this protective immune response is lost in athymic nude mice (Fig. 4D) or when CD8+ T cells are depleted (Casares et al, 2005) or when the IFN-y/IFN-yR system is blocked either by injection of neutralizing antibodies or knockout of IFN-γ (Ghiringhelli et al., 2009). Accordingly, inventors found that the absence of IL-17RCC reduced the capacity of mice to mount a protective immune response against dying tumor cells (Fig. 4D).

When exploring the source of IL-17 production elicited by dying tumor cells, inventors found that γδ Τ cells were the quantitatively and functionally most important IL-17 producers, based on several observations. First, in the context of anticancer chemotherapy, γδ Τ17 cells accumulated within tumors (Fig. 2B, 2C, 3C, Fig. 9D). Indeed, most IL-17 producing cells were positive for γδ Τ markers (Fig. 2A, Fig. 9B). Secondly, antigen- specific CD4⁺ T cells in lymph nodes (LNs) draining the dying tumor cells are polarized to a Thl cytokine (IL-2 and IFN-γ) secretion pattern (Ghiringhelli et al, 2009) instead of a Thl7 pattern (data not shown). Also, IL-6 and TGF-β, two key regulatory cytokines essential for the differentiation of Thl7 cells (Ivanov et al, 2006) were dispensable for the efficacy of chemotherapy (Fig. 12A, 12B), suggesting that Thl7 cells are not required for the anticancer immune response that amplify the effect of chemotherapy. Thirdly, when popliteal lymph nodes were recovered from mice that had been injected with dying (but not live) tumor cells through footpad, the re-stimulation of LN-resident cells using anti-CD3e Ab+IL-23 readily enhanced IL-17 production (not shown), a feature common to memory T cells, especially innate NKT (Rachitskaya et al, 2008) and γδ T cells (Sutton et al, 2009) (Fig. 6B). Fourthly, the subset of NKT cell capable of producing IL-17 in LN (CD103⁺CD4^"NK1.1^"CCR6⁺ CDld tetramer⁺) (Doisne et al, 2009) did not appear to be specifically triggered by dying cells in vivo (not shown). Moreover, CDld^"7" mice, which lack NKT cells, were indistinguishable from WT mice when the efficacy of chemotherapy was assessed in prophylactic vaccination settings (Fig. 4D). Finally, knockout Vy4/6 or TCR δ attenuated the protective antitumor vaccination with dying tumor cells (Fig. 4D) and reduced the efficacy of the anthracyc line-based chemotherapy on established tumors (Fig. 5A). In the context of immune responses stimulated by dying cancer cells, it clearly appears that IL-Ι , an inflammatory cytokine that is produced by dendritic cells (DC), plays a major role in stimulating IL-17 production and the anticancer function of γδ T cells. The key role of IL-Ιβ in regulating γδ T cells function was shown by using IL-IRA in cocultures of DC/ γδ T cells in the presence of dying tumor cells (Fig. 6D). Also, γδ T cells that lack IL-1R cannot amplify the tumoricidal action of anthracyclines as IL-1R expressing γδ T cells do (Fig. 7B). Interestingly, DC-mediated IL-Ιβ secretion was also found mandatory for the polarization of CD8⁺ T cells towards a Tel pattern (Ghiringhelli et al, 2009). The herein provided results demonstrate the importance of DC, γδ T17 cells and Tel cells (IFN-γ producing CD8⁺ T lymphocytes) to favor optimal anticancer immune responses. Inventors noticed a strong correlation between γδ T17 and Tel cells post- chemotherapy in three different tumor models and the fact that the emergence of IL-17 production precedes that of IFN-γ production by Tumor Infiltrating Lymphocytes (TILs). It is well possible that besides helping developing Tel response, γδ T17 cells might enhance the chemoattraction of Tel effector cells into the tumor beds. These results are compatible with observations obtained in a cancer-unrelated context, microbial infection, in which γδ T17 associated with Thl responses to exert protective immune response (Umemura et al, 2007). As IL-17 could not directly induce IFN-γ production or enhance proliferation of CD8⁺T cells (data not shown), the present results imply a causal relationship between the presence of γδ T17 cells and the recruitment of antitumor effector Tel cells into tumor beds.

Example 2: the single-nucleotide polymorphism R554K (rs2066853) in AHR gene (affects the efficacy of conventional anti-cancer therapy in a neoadjuvant setting (before surgery) breast cancer patients

The inventors observed that the single-nucleotide polymorphism (SNP) R554K (rs2066853 - SEQ ID NO: 7) in AHR gene (NCBI Reference Sequences: AHR genomic DNA : NC 000007.13 (SEQ ID NO: 1); AHR niRNA : NM 001621.3 (SEQ ID NO: 2)) affects the efficacy of conventional anti-cancer therapy in a neoadjuvant setting in breast cancer patients (n=239). Indeed, the proportion of pathological complete responses was higher in AHR wild-type group than in mutated group of patients treated with anthracyclines (24.3% in patients carrying the AHR normal allele versus 11.4% in patient with the R554K mutated allele; p=0.02 by Chi² analysis). In other words, the AHR R554K mutated allele enhanced the probability of relapse in patients treated with anthracyclines. Other AHR polymorphisms have the same predictive value, in particular the following AHR SNPs: rsl0250822 (SEQ ID NO: 3), rsl 1505406 (SEQ ID NO: 4), rsl476080 (SEQ ID NO: 5), rsl7779352 (SEQ ID NO: 6), rs2074113 (SEQ ID NO: 8), rs2158041 (SEQ ID NO: 9), rs2282885 (SEQ ID NO: 10), rs34938955 (SEQ ID NO: 11), rs35225673 (SEQ ID NO: 12), rs4986826 (SEQ ID NO: 13), rs713150 (SEQ ID NO: 14), rs7796976 (SEQ ID NO: 15), and rs7811989 (SEQ ID NO: 16).

The numbers and percentages of patients enrolled in the case-control study are displayed in a contingency table (Table 3) based on the primary endpoint (pathological complete response) and the genotype of AHR-R554K (rs2066853) SNP.

Table 3:

Materials and methods Clinical study design

The inventors retrospectively constructed patient database using data obtained from Institut Gustave Roussy (France). All patients provided written informed consent for enrollment in the study. Eligible patients had histologically confirmed sporadic breast cancer. All patients received an anthracycline-based chemotherapy before surgery (FEC protocol in neoadjuvant setting). This study was based on a retrospective cohort (n=197 - patients not treated with Herceptin) and a case-control cohort (n=42) matched for age, tumor grade and hormone receptors. The primary endpoint of the study was the pathogical complete response. After generation of the patient database and collection of genomic DNA samples, genotyping and statistical analyses were performed in a blinded fashion. A total of 239 patients fulfilled the inclusion criteria. Chi square test was used to compare the distribution of clinical characteristics across the two genotype groups. All analyses were carried out using SPSS software, version 16 (IBM SPSS Statistics, France).

Genotyping

DNA was isolated from frozen blood leukocytes from subjects. The TAQMAN Drug

Metabolism Genotyping assay ID: C 11 170747 20 was used to genotype the AHR G/A polymorphism (rs2066853). Briefly, 10 ng of genomic DNA was mixed with 5 of 2X TaqMan Genotyping Master Mix (Applied Biosystems) and 0,25 μΐ, of 40X genotyping assay in a final volume of 10 μί. Temperature cycling and real time fluorescence measurement were done using an StepOnePlus System (Applied Biosystems). The genotypes were assigned to each subject, by comparing the signals from the two fluorescent probes, FAM and VIC, and calculating the -log(F AM/VIC) ratio for each data point with the StepOne software v2.0(Applied Biosystems). The other AHR SNPs have been tested with the same procedure. Examples of the following TAQMAN Genotyping assays have been used : rsl0250822 (SEQ ID NO: 3) (TAQMAN Genotyping assay ID :

C 2541466 10), rsl476080 (SEQ ID NO: 5) (TAQMAN Genotyping assay ID :

C 8302430 10), rs2282885 (SEQ ID NO: 10) (TAQMAN Genotyping assay ID :

C 2541460_1_), rs2158041(SEQ ID NO: 9) (TAQMAN Genotyping assay ID :

C 2541454_30), rs713150 (SEQ ID NO: 14) (TAQMAN Genotyping assay ID : C 2541463J0), rs7796976 (SEQ ID NO: 15) (TAQMAN Genotyping assay ID :

C_30633941_10), rs2074113 (SEQ ID NO: 8) (TAQMAN Genotyping assay ID : C_16163703_10), rs7811989 (SEQ ID NO: 16) (TAQMAN Genotyping assay ID : C_29150577_20) rs4986826 (SEQ ID NO: 13) (TAQMAN Genotyping assay ID : C_25650166_20), rsl7779352 (SEQ ID NO: 6) (TAQMAN Genotyping assay ID : C_25650165_20). Example 3: Restoration of the immunogenicity of cisplatin-induced cancer cell death

In this example, inventors specifically addressed the question why CDDP - in contrast to the related compound OXP - fails to induce immunogenic cell death. To address this question, they monitored several cell lines that express a series of cell death-relevant biosensors or biomarkers, allowing them to map the defect in the CRT exposure pathway elicited by CDDP. Furthermore, they designed a screening system allowing them to identify compounds that are inert with regard to apoptotic signalling, yet can restore CDDP's capacity to induce CRT exposure and to stimulate immunogenic cell death.

Materials and Methods Reagents and materials

Cell death was induced with MTX, CDDP (Sigma, Saint Louis, USA) or OXP (Sanofi- Aventis, Paris, France). Quinacrine and THAPS were purchased from Sigma. PeIF2a and eIF2a antibody has been purchased from cell signaling. Cell culture media and selection antibiotics were from Gibco.

Cell culture

U20S, 293FT and HeLa were cultured in DMEM medium supplemented with 10% (v/v) fetal calf serum, 1 mM sodium pyruvate and 10 mM Hepes buffer. CT26, Lewis lung cell carcinoma and MC205 cells were grown in RPMI supplemented with identical components. U20S clones were selected with 1 mg/ml G418 (Gibco) or Zeocin or 5 μg/ml Blasticidine and stable clones were kept under 200 μg/ml or 1 μg/ml selection respectively.

Viral transduction

Lentiviral particles for the transduction of cells with H2B-RFP have been produced in 293FT cells by means of the ViraPower lentiviral expression system (Invitrogen) following the manufacturer's instructions. For this purpose an H2B-RFP cDNA sequence has been cloned into the pLenti6 vector by means of the gateway system. HT-CRT stably expressing U20S cells

The HaloTag® sequence was amplified from a pHT2 plasmid (Promega) as Notl restriction fragment with the STOP codon removed from the Halotag sequence. For the PCR amplification the following primers were used: Forward :5'- AAGCGGCCGCAATGGGATCCGAAATCGGTAC-3 ' (SEQ ID NO: 453); Reverse : 5'- AAGCGGCCGCGCCGGCCAGCCCGGGGAGCC-3' (SEQ ID NO: 454). PCR products were isolated on agarose gel, purified using the QIAquick Gel Extraction kit (Qiagen), and digested with the restriction enzyme Notl (Bio labs). The digested PCR product was ligated into the CRT-GFP plasmids at the Notl restriction site after removing the GFP sequence. Transfection of U20S cells with the HaloTag®-CRT was carried out with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Stable clones stably were selected by means of Zeocin selection.

CRT-GFP, Bax-GFP; G3BP-GFP; GFP-LC3 stably expressing U20S cells

U20S cells have been transfected by means of Lipofectamin 2000 following the manufacturers instruction with either CRT-GFP, Bax-GFP, G3BP-GFP or LC3-GFP cDNA. Subsequently the cells have been stably selected using G418 selection antibiotic (Gibco). Resistant cells have been single cell sorted with a FACSvantage cell sorter and GFP expressing clones have been selected. Some of the clones have further been stably transduced with lentiviral particles expressing H2B-RFP. These cells have again been single cell sorted to identify double fluorescent clones.

Compound screen for CRT-exposing drugs death assays

One day prior to the experiment, 5 x 10³ U20S cells stably expressing CRT-GFP and H2B-RFP were seeded into 96-well Black/Clear Imaging Plates pre-treated with poly-L- lysine (BD Biosciences, San Jose, CA, USA). The ICCB known bioactive compounds library (Enzo life science) (BML2840), comprising 480 distincts compounds, was added at a concentration range from 90 nM to 48 μΜ in the presence or absence of 50 μΜ CDDP. The cells were incubated for 4 h at 37°C and subsequently fixed with 4 % paraformaldehyde (PFA) for 20 min. After washing with PBS 4 viewfields per well were acquired by means of a BD pathway 855 automated microscope. The images were segmented and analyzed for GFP-granularity and nuclear shape area using the BD Atto Vision software version 1.6 before data mining. The data was statistically evaluated using graph pad. To avoid inter plate variations the data has been intra plate normalized by calculating the ratio to untreated controls for each datapoint. Cell death assays

6 10⁵ U20S cells were treated with the indicated cell death inducers for 16 h at the indicated concentration. Cell death was quantified by cytofluorometric analysis using a FACS Vantage (Becton Dickinson, Mountain View, USA) as described previously (REF). Thus, cells were stained with 40 nM 3,3 dihexyloxacarbocyanine iodide (DiOC₆(3); Molecular Probes, Eugene, OR, USA) for 30 min at 37°C and concomitantly with 1 μg/ml propidium iodide (PI; Sigma Aldrich) for 30 min at 37°C to determine the mitochondrial transmembrane potential. Data were statistically evaluated using CellQuest Pro software (Becton Dickinson, Mountain View, USA).

Quinacrine Immunofluorescence

U20S cells were treated with the indicated cell death inducers for 16 h at the indicated concentrations. Subsequently, cells were labeled with quinacrine as described previously (Martins et ah, 2009). In short, cells were labeled with 1 μΜ quinacrine in Krebs-Ringer solution (125 mM NaCl, 5 mM KC1, 1 mM MgS0₄, 0,7 mM KH₂P0₄, 2 mM CaCl₂, 6 mM glucose and 25 mM Hepes, pH 7.4) for 30 min at 37°C. Thereafter, cells were stained with 1 μg/ml PI (Invitrogen) and 1 μg/ml Hoechst 33342 (Invitrogen) for 10 min, rinsed with Krebs-Ringer solution and fixed with 2% paraformaldehyde for 15 min at room temperature. Cells were examined with a BD Pathway™ 435 High-Content Biolmager workstation (Becton Dickinson, Mountain View, USA) by using an UApo/340 x20/0.75 objective (Olympus, Tokyo, Japan).

Quinacrine flow cytometry

6 10⁵ U20S cells were treated with the cell death inducers for 24 h. After incubation in quinacrine solution (as described above), cells were rinsed and resuspended in PBS containing 1 μg/ml PI. The samples were analyzed by means of a FACS Vantage (Becton Dickinson) and the data was statistically evaluated using the CellQuest Pro software (BD Biosciences). ATP release assays

After cell death induction, extracellular ATP was measured by luciferin-based ENLITEN ATP Assay (Promega, Madison, USA) following the manufacturer's instructions. Intracellular ATP was measured using an ATP Assay kit (Calbiochem, Darmstadt, Germany) based on luciferin-lucif erase conversion following the manufacturer's instructions. For assessment of the chemo luminescent signal, the plates were read in a Fluostar lumino meter (BMG Labtech). Analysis of surface exposed CRT

Cells were treated with the indicated agents for 4 hours and the day after they were collected. For the HaloTag^® staining, cells were incubated for 30 min with HaloTag^® Alexa Fluor^® 488 Ligand, diluted in DMEM medium containing 10% of fetal bovine serum. Then cells were washed and incubated in DMEM medium for 30 min. Thereafter, cells were rinsed with PBS and stained with 1 μg/ml PI (Invitrogen). For CRT immune staining, cells were washed twice with PBS and fixed in 0.25% paraformaldehyde in PBS for 5 min. After washing again twice in cold PBS, cells were incubated for 30 min with primary antibody, diluted in cold blocking buffer (2% fetal bovine serum in PBS), followed by washing and incubation with the Alexa488-conjugated monoclonal secondary antibody in a blocking buffer (for 30 min). Each sample was then analyzed by FACScan (Becton-Dickinson) to identify cell surface CRT. Isotype-matched IgG antibodies were used as controls, and the fluorescent intensity of stained cells was gated on Pi-negative cells. The same staining procedure was applied to U20S CRT-GFP expressing cells grown on coverslips using an Alexa546 coupled secondary antibody before analysis in a Leica TCS SPE confocal microscope (Leica Microsystems, Wetzlar, Germany).

In vivo anti-tumor vaccination

1 10⁶ MCA205 cells, untreated or treated with either OXP, CDDP were injected subcutaneously into 6-week-old female C57BL/6 mice (Janvier, Charles River) into the lower flank, whereas 5 x 10⁵ untreated control cells were inoculated into the contralateral flank 6 days later (Casares et ah, 2005). Tumor growth was evaluated for at least 50 days. All animals were maintained in specific pathogen-free conditions, and all experiments were carried out according to the Federation of European Laboratory Animal Science Association guidelines.

The Ethics Committee of Institut Gustave Roussy approved all the animal experiments. Results

Failure of cisplatin to induce calreticulin (CRT) redistribution from the endoplasmic reticulum (ER) lumen to the cell surface.

To monitor the redistribution of CRT from the ER lumen to peripheral locations close to plasma membrane, inventors generated U20S cells that stably express a CRT-GFP fusion protein (Snapp et al., 2006). Control experiments revealed that this protein was located in the ER lumen, where most of the endogenous CRT resides (not shown). Upon treatment with mitoxanthrone (MTX) or OXP, two immunogenic cell death inducers, CRT-GFP relocates from a preponderantly perinuclear near-to-diffuse location (which is seen in untreated control cells) to a more peripheral granular distribution (Fig. 14A). This increased "granularity", which can be quantified using morphometric image analysis software {Rello-Varona, 2010 #37}, is only observed after treatment of the cells with MTX or OXP, but not after treatment with CDDP (Fig. 14A,B). Nonetheless, CDDP was able to induce chromatin condensation, the morphological hallmark of apoptosis as efficiently as MTX or OXP (percentage values in Fig. 14B). Indeed, pairwise comparisons were always performed at the IC₅₀ of both agents, which was -600 μΜ for OXP and -150 μΜ for CDDP in short-term experiments measuring imminent cell death (as indicated by a loss of the mitochondrial transmembrane potential, ΔΨιη, see below). Surface immunofluorescence staining of CRT (revealed in red) confirmed that a few of the OXP- elicited CRT-GFP granules that were close to the cell surface, actually extruded CRT, which became accessible to a CRT-specific antibody. Again, no immunodetectable CRT was found on the surface of non-permeabilized cells treated with CDDP (Fig. 14 C).

Immunofluorescence detection of CRT requires several washing steps that might perturb the integrity of cells. To avoid this problem, inventors generated a chimeric protein that contains CRT in its N-terminus and the HaloTag^® moiety in its C-terminus followed by the KDEL endoplasmic reticulum retention signal (Fig. 14D). This construct can be detected with commercially available HaloTag^® ligands, which are either cell-permeable (as exemplified by HaloTag Alexa Fluor 488, green fluorescence) or cell-impermeable (as exemplified by HaloTag^® TMR Ligand, red fluorescence) (Fig. 14E). The CRT-HaloTag^® fusion protein underwent a similar intracellular redistribution (detected by staining with HaloTag^® Alexa Fluor® 488) as did CRT-GFP when the cells were treated with MTX (not shown) or OXP. Moreover, U20S cells expressing the CRT-HaloTag^® fusion protein did not stain with the cell-impermeable HaloTag^® ligand, unless they were treated with immunogenic cell death inducers such as OXP (Fig. 14F). Again, CDDP failed to induce the surface exposure of CRT-HaloTag^®, as determined by fluorescence microscopy (Fig. 14F) or cytofluorometric analysis of HaloTag^® TMR Ligand-stained cells (Fig. 14G). In conclusion, CDDP is unable to induce CRT exposure in conditions in which it does induce nuclear apoptosis.

Failure of cisplatin to elicit ER stress.

When used at their IC₅₀, CDDP and OXP had a comparable potency in inducing nuclear apoptosis (not shown). Moreover, both agents were able to induce mitochondrial perturbations that were assessed by two different methods. First, inventors determined the ΔΨιη dissipation by means of the ΔΨιη-βεηβίίίνε fluorochrome DiOC₆(3) (Fig. 15A,B). Second, they measured the relocation of a Bax-GFP fusion protein (von Haefen et al., 2004) from a diffuse to a punctate (presumably mitochondrial) pattern (Fig. 15C,D). Both CDDP and OXP induced similar mitochondrial perturbations (Fig. 15).

Moreover, both agents induced a similar release of ATP, which is one of the obligatory signals linked to immunogenic cell death (Ghiringhelli et al, 2009). This result was obtained using two different methods, namely staining of the cells with the ATP-sensitive fluorochrome quinacrine (Fig. 15A,B), or by measuring the residual ATP content within the cells (Fig. 16C) or the ATP secreted into the supernatant by means of a luciferase- based assay (Fig. 16D). Thus, both agents lead to similar perturbations in energy metabolism.

However, OXP and CDDP ware rather different in their capacity to elicit the redistribution of G3BP-GFP (Tourriere et al, 2003) or LC3-GFP (Kabeya et al, 2000) from a diffuse to a punctiform distribution, which indicates the formation of stress granules (Fig. 17A,B) or of autophagosomes (Fig. 17C,D), respectively. This difference was particularly remarkable at early time points. The formation of stress granules and autophagosomes is subordinated to the mandatory phosphorylation of eIF2cc, which is also required for the redistribution of CRT to the cell surface (Obeid et ah, 2007b). Indeed, OXP was much more efficient than CDDP in inducing eIF2cc phosphorylation on serine 51, as determined by means of a phospho-neoepitope-specific antibody (Fig. 17E). Accordingly, the activating phosphorylation of PERK, the principal eIF2cc kinase elicited by chemotherapeutic agents (Panaretakis et al. , 2009), was detectable shortly after treatment with OXP but not CDDP (Fig.l7E). Altogether, these results suggest that CDDP is much less efficient in inducing an ER stress response than OXP. Identification of thapsigargin as an agent that reestablishes CRT relocalization in response to cisplatin.

To identify compounds that might restore the defective CRT exposure pathway in tumor cells responding to CDDP, inventors conducted a high-content screen. This screen was based on the utilization of the ICCB library whose 480 components were individually tested for their capacity to stimulate the redistribution of CRT-GFP in U20S cells that were either left untreated (not shown) or cultured for 4 h in the presence of 150 μΜ CDDP (Fig. 18A). When the results obtained in the absence and in the presence of CDDP were plotted for each compound individually, one single agent, THAPS, was identified as being particularly efficient in inducing CRT-GFP granularity in the presence (but not in the absence) of CDDP (Fig. 18B). This result was confirmed in several independent determinations on U20S cells expressing CRT-GFP (Fig. 18C,D). Moreover, THAPS was capable of inducing the redistribution of CRT-HaloTag^® (Fig. 19A), as well as that of endogenous CRT (Fig. 19B), as determined using the cell-impermeable CRT-HaloTag^® ligand or antibodies recognizing CRT, respectively. While THAPS alone (in the absence of CDDP) was comparably inefficient in inducing CRT-GFP granularity (Fig. 18D), CRT- HaloTag^® exposure (Fig. 19A) or native CRT exposure (Fig. 19B), it was highly efficient in the presence of CDDP. Very similar results were obtained in additional cell lines, including human cervical carcinoma HeLa cells (not shown), mouse Lewis lung carcinoma cells (Fig. 19F), colorectal carcinoma CT26 cells (Fig. 19D), and methylcholanthrene- induced MCA205 fibrosarcoma cells (Fig. 19E). Of note, THAPS exhibited no major cytotoxic effects and did not increase the toxicity of CDDP in any of these cellular models, as exemplified for U20S cells in which inventors monitored ΔΨιη (Fig. 20A), intracellular ATP content (Fig. 20B,C,D) and extracellular ATP release (Fig. 20E).

Confirming the strong correlation between CRT exposure and immunogenicity, CDDP- treated MCA204 cells were inefficient in inducing a protective anticancer immune response when injected subcutaneous ly into immunocompetent B6B157 mice one week before rechallenge with live tumor cells, in conditions in which OXP -treated MCA205 cells readily induce such a tumor-protective response (which precludes the growth of live MCA205 cells). However, the vaccine of dying cells, generated in the presence of CDDP combined with THAPS, elicited an effective anticancer immune response in vivo (Fig. 19F).

In conclusion, THAPS can reestablish the defective CRT exposure and associated immunogenicity of CDDP-induced cell death.

Conclusions

In contrast to other cytotoxic agents including anthracyclins and OXP, CDDP fails to induce immunogenic tumor cell death that would allow the stimulation of an anticancer immune reponse and hence amplify its therapeutic efficacy. This failure to induce immunogenic cell death can be attributed to CDDP's incapacity to elicit the translocation of CRT from the lumen of the ER to the cell surface. The previous results show that, in contrast to OXP, CDDP is unable to activate the protein kinase-like ER kinase (PER )- dependent phosphorylation of the eukaryotic translation initiation factor 2a (eIF2a). Accordingly, CDDP also failed to stimulate the formation of stress granules and macroautophagy, two processes that only occur after eIF2cc phosphorylation. Using a screening method allowing the following of the voyage of CRT from the ER lumen to the cell surface, inventors identified in particular THAPS, an inhibitor of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) as a molecule that on its own does not stimulate CRT exposure, yet endows CDDP with the capacity to do so. Such a molecule is identified, in the context of the present invention, as a compensatory molecule. Thus, the combination of THAPS and CDDP effectively induced the translocation of CRT to the plasma membrane, as well as immunogenic cell death, while each agent alone was inefficient. Altogether, these results underscore the contribution of the ER stress response to the immunogenicity of cell death, in particular the ER Ca²⁺ fluxes for the translocation of CRT to the cell surface.

This experiment also allowed the identification of other compensatory molecules (in particular microtubules destabilizers), identified in the description part, which are also able to induce an immunogenic cell death.

Example 4: Restoration of the immunogenicity of conventional treatment-induced cancer cell death The protocol described in example 3 has been applied in example 4 to screen other compounds (figure 21) from the US drug collection library from MS discovery (US 090917A). These compounds have been screened at ΙμΜ.

Example 5:

Inventors attempted to generate a molecular parameter signature of a pathological complete response (pCR) from two datasets of gene-expression arrays in neoadjuvant (before any surgery step) anthracycline treated-breast cancer patient cohorts (cohorts respectively herein identified HOUSTON FEC and IGGO FEC). One dataset of gene- expression arrays in neoadjuvant taxane treated-breast cancer patients was used as negative control (cohort IGGO TET). Inventors extracted a set of 43 genes from cohort HOUSTON FEC and 53 genes from cohort IGGO FEC/TET implicated in the "calreticulin" pathway from the global sets of genes identified previously in the description (using respectively 22 283 probes and 61 359 probes) to construct the molecular parameter signature.

Inventors performed a molecular classifier development analysis based on a supervised learning classification technique (Support Vector Machines - SVM) (Figure 22). Leave- one-out cross-validation (LOOCV) was used to estimate the prediction accuracy of the rule determined on the training set (the previously mentioned 43 and 53 genes). One sample is left out, and the remaining samples are used to build the prediction rule, which is then used to classify the left-out sample. Then, they performed, in one hand, univariate analyses with contingency tables: the statistical significance of the discrimination between pCR and non- pCR patients was assessed by Fisher's exact test. In the other hand, multivariate methods, such as logistic regression and ROC analyses, were performed to validate the independency of the molecular parameter signature as compared to classical clinical factors [age at diagnosis, hormone receptors, tumor grade, tumor size (pT), node status (pN)] (Figure 22).

To determine the optimal molecular classifier in each cohort, inventors classified the genes from the highest to the lowest significant based on a non parametric Mann- Whitney test between pCR and non-pCR groups, and they proceed to a systematic approach by testing the discrimination potential of decreasing sets of genes (from n=43/53 to 3) (Figure 23). The best prediction rules were obtained with a molecular parameter signature based on 3 genes (HOUSTON FEC LOOCV, p=0.0005; IGGO FEC LOOCV, p=0.002; IGGO TET LOOCV, p=0.43) (Figure 23). In multivariate analyses using logistic regression, the "Calreticulin" molecular classifiers based on the 3 most significant genes of each cohort were retained as the sole independent prognostic factors for pCR, except for the cohort IGGO TET (negative control) (Figure 24 A). ROC analyses revealed the ability of the 3 genes based - « Calreticulin » molecular classifiers to discriminate significantly pCR patients from non-pCR patients, except for the cohort IGGO TET where the predictive value of the model was based only on the pT factor (Figure 24B). In order to identify the best molecular classifier in common between the two anthracycline treated cohorts, they compared the most discriminant genes between these two cohorts. The classifier was constructed with the 3 candidate genes located in the grey areas (Figure 25). Univariate analyses based on non-pCR vs pCR contingency tables and multivariate analyses revealed that the common classifier based on the CCR1, EIF2AK2 and DNAJC10 gene expressions ("CALR pathway" signature) was able to predict accurately the response of a human subject having a tumor to anthracyclines (Figure 26).

Inventors then attempted to optimize the molecular classifier by integrating host genetic parameters, such as single nucleotide polymorphisms, in the algorithm. Multivariate analyses in HOUSTON FEC cohort revealed that the association of a MTHFR SNP (rsl801 133) with the "CALR pathway" signature improved the prediction accuracy of the molecular classifier (Figure 27). This MTHFR SNP was the most discriminant SNP between pCR and non pCR groups among a set of 384 selected SNPs located in immune candidate genes (Table 1). The interpretation of results revealed in particular that patients carrying the mutated allele of MTHFR SNP (rsl801133) associated with an over- expression of CCR1 and EIF2AK2 genes, and an under-expression of DNAJC10 gene, have higher chance to respond to anthracyc lines than patients carrying the wild-type allele of MTHFR SNP associated with an under-expression of CCR1 and EIF2AK2 genes, and an over-expression of DNAJC10. These results demonstrate that tumour parameters, such as gene expression signatures, and host (the subject having a tumor) genetic parameters, such as SNPs, constitutes a powerful combination usable to predict or assess the response of a subject to a treatment of cancer, in particular to anthracyclines.

Material and Methods: Cohort F. Andre (IGR/Houston) (described in Lancet Oncol. Submitted).

Patients for gene expression analysis and metagene predictor validation have been selected from a database of 591 patients who received preoperative anthracyclines- based, taxanes- free chemotherapy at the Institut Gustave Roussy between 1987 and 2003. Inclusion criteria consisted of (1) pathologic complete response (pCR) defined as the absence of any invasive cancer or isolated tumor cells in the breast after completion of chemotherapy and (2) availability of frozen, pre-treatment samples in the institutional tumor bank for molecular analysis. Twenty six cases were identified and 26 additional cases were selected as controls. The controls included tumors that were resistant to chemotherapy defined as less than 75% clinical response and residual invasive disease (RD) present at the time of pathologic exam after chemotherapy, and were matched for Endoplasmic Reticulum (ER)- expression. A further double-checking of clinical characteristics revealed that one patient with pCR actually received 2 cycles of docetaxel in addition to 4 courses of FEC (anthracyclines). This patient was retained in the analysis. The study was approved by the local IRB; all patients signed informed consent for tumor banking and future molecular analysis of their tissues.

Cohort R.Iggo/H. Bonnefoi (Bonnefoi et al, 2007) EORTC

Breast cancer patients treated in neoadjuvant (before any surgical step) FEC versus TET (anthracyclines versus taxanes) Data basis on tumor profiling (microarrays) described in Lancet Oncol 2007 and available online. The microarray analyses, such as SVMs (Brown et al., 2000) and non parametric Mann- Whitney test, were performed with the MEV software version 4.5 (Saeed et al, 2006; Saeed et al, 2003). For multivariate logistic regression and ROC (Receiver operating characteristic) analyses, the SPSS 18.0 software was used. The Fisher's exact test was performed with the StatEL software (ad Science, France).

In clinical databases, when a category of an ordinal variable had too few observations in databases, these observations were pooled with a consecutive category (tumor size Tl and T2, node status Nl and N2 and grade 1 and 2). Missing values for grade, pN and SNPs were assigned to a separate category to avoid a decrease in the sample size in the logistic regression analysis.

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Claims

An in vitro method of assessing the sensitivity of a subject having a tumor to a treatment of cancer, which method comprises a step of determining the ability of the subject and/or of the tumor to induce an anticancer immune response, the inability of at least one of the subject and of the tumor to induce an anticancer immune response being indicative of a resistance of the subject to the treatment of cancer.

The method according to claim 1 , wherein the treatment of cancer is a conventional immunogenic treatment of cancer selected from a chemotherapy using a drug selected from an anthracyclin, a platin, and an antimitotic agent; and radiotherapy.

The method according to anyone of claims 1 or 2, wherein the presence, in the subject, of an alteration leading to the abnormal expression of an immune gene selected from a gene encoding aryl hydrocarbon receptor (AHR) and a gene encoding methylene tetrahydro folate reductase (MTHFR or NADPH), determines the inability of the subject to induce an anticancer immune response.

The method of claim 3, wherein the alteration is a single nucleotide polymorphism (SNP) selected from the list comprising rsl0250822 (SEQ ID NO:3), rsl 1505406 (SEQ ID NO: 4), rsl476080 (SEQ ID NO: 5), rsl7779352 (SEQ ID NO: 6), rs2066853 (SEQ ID NO: 7), rs2074113 (SEQ ID NO: 8), rs2158041 (SEQ ID NO: 9), rs2282885 (SEQ ID NO: 10), rs34938955 (SEQ ID NO: 11), rs35225673 (SEQ ID NO: 12), rs4986826 (SEQ ID NO: 13), rs713150 (SEQ ID NO: 14), rs7796976 (SEQ ID NO: 15), rs7811989 (SEQ ID NO: 16) and rsl801133 (SEQ ID NO: 194), and said alteration is indicative of a subject being unable to induce an anticancer immune response.

The method according to claim 1, wherein the step of determining the ability of the tumor to induce an anticancer immune response consists in verifying the expression by tumor cells of a tumor sample of the subject, of an immunogenic cell death marker selected from a protein allowing or enhancing CRT exposure at the surface of tumor cells, and a protein expressed during the endoplasmic reticulum (ER) stress response and/or during the macroautophagic response of the subject's immune system.

6. An in vitro method of assessing the sensitivity of a subject having a tumor to a treatment of cancer according to claim 1, which method comprises a step of detecting the presence of an anticancer immune response of the subject undergoing the therapeutic treatment of cancer, the absence of an anticancer immune response being indicative of a resistance of the subject to the therapeutic treatment of cancer.

7. The method according to anyone of claims 1 or 2, wherein the presence, in a tumor sample of the subject, of a genetic alteration leading to the abnormal expression of a gene selected from CCRl, EIF2AK2, DNAJC10, PDIA3, EIF2A, PPP1CB, IKBKB, PPP1CC, and BAX, determines the inability of the subject to induce an anticancer immune response.

8. The method according to claim 7, wherein the abnormal expression is a downregulation of the expression of CCRl, a downregulation of the expression of EIF2AK2, an upregulation of the expression of DNAJC10, and/or an upregulation of the expression of PDIA3.

9. The method according to claim 7, wherein the abnormal expression is a downregulation of the expression of CCRl, a downregulation of the expression of EIF2AK2, and an upregulation of the expression of DNAJC10.

10. The method according to claim 7, wherein the abnormal expression is a downregulation of the expression of CCRl, a downregulation of the expression of EIF2AK2, and an upregulation of the expression of PDIA3.

11. The method according to claim 9 or 10, further comprising a step of controlling, in a tumor or blood sample of the subject, the presence of a single nucleotide polymorphism (SNP) selected from the list of SNP according to claim 4; the detection of at least one of:

i. an abnormal expression of the proteins encoded by (i) a gene encoding CCR1, (ii) a gene encoding EIF2AK2, and (iii) a gene encoding DNAJC10 or PDIA3, and

ii. an alteration in the gene encoding MTHFR,

being indicative of a resistance of the subject to the therapeutic treatment of cancer.

12. The method according to claim 11, wherein the alteration in the gene encoding MTHFR is a single nucleotide polymorphism (SNP) corresponding to rsl801133 (SEQ ID NO: 194).

13. A kit to detect the abnormal expression of a gene selected from CCR1, EIF2Ak2, DNAJC10, PDIA3, EIF2A, PPP1CB, IKBKB, PPP1CC, BAX and combinations thereof, in a tumor sample of the subject, the kit comprising (i) at least one pair of primers and (ii) at least one fluorescent probe allowing the quantitative detection of the expression of a gene selected from CCR1, EIF2Ak2, DNAJC10, PDIA3, EIF2A, PPP1CB, IKBKB, PPP1CC, BAX, and (iii) a leaflet providing the control quantitative expression values corresponding to at least one of said genes in a control population.

14. A kit to detect the abnormal expression of a gene selected from ^H ? and MTHFR, in a tumor, blood or serum sample of the subject, the kit comprising (i) at least one pair of primers, and (ii) at least two differently labelled probes, the first probe recognizing the wild-type allele and the second probe recognizing the mutated allele of a gene selected from ^H ? and MTHFR.

15. A kit comprising:

a. (i) at least one pair of primers, (ii) at least one fluorescent probe allowing the quantitative detection of the expression of a gene selected from CCR1, EIF2Ak2, DNAJC10, PDIA3, EIF2A, PPP1CB, IKBKB, PPP1CC and BAX and (iii) a leaflet providing the control quantitative expression values corresponding to at least one of said genes in a control population; and (i) at least one pair of primers, and (ii) at least two differently labelled probes, the first probe recognizing the wild-type allele and the second probe recognizing the mutated allele of a gene selected from ^Hi? and MTHFR.