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Title:
PROGNOSIS AND TREATMENT OF CANCERS
Document Type and Number:
WIPO Patent Application WO/2014/154898
Kind Code:
A1
Abstract:
The present invention concerns mutants of "CCAAT/enhancer-binding protein zeta" (also called "CBF2") which do not retain their transactivation capacity, and their use as a biomarker for prognosing survival and/or the response to a treatment of a patient suffering from a cancer. The invention also relates to methods using these mutant proteins for (i) prognosing survival and/or the response to a treatment of a patient suffering from a cancer, and (ii) selecting a patient suffering from cancer in need of being treated with a CCAAT/enhancer- binding protein zeta which retains its transactivation capacity. In a particular embodiment, the patient is suffering from a cancer liable to have a "Microsatellite Instability" phenotype, such as colorectal cancer.

Inventors:
DUVAL ALEX (FR)
BUHARD OLIVIER (FR)
COLLURA ADA (FR)
LAGRANGE ANAÏS (FR)
Application Number:
PCT/EP2014/056373
Publication Date:
October 02, 2014
Filing Date:
March 28, 2014
Export Citation:
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Assignee:
INST NAT SANTE RECH MED (FR)
International Classes:
G01N33/574
Domestic Patent References:
WO2000055174A12000-09-21
WO2008029414A22008-03-13
WO2012127062A12012-09-27
WO1991006629A11991-05-16
WO2000053722A22000-09-14
Foreign References:
US6447997B12002-09-10
EP11305160A2011-02-16
US20070275923A12007-11-29
Other References:
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GUOJUN WU ET AL: "DeltaNp63alpha up-regulates the Hsp70 gene in human cancer.", CANCER RESEARCH, vol. 65, no. 3, 1 February 2005 (2005-02-01), pages 758 - 766, XP055070628, ISSN: 0008-5472
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Attorney, Agent or Firm:
BLOT, Philippe et al. (2 place d'Estienne d'Orves, Paris, FR)
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Claims:
CLAIMS

1 . In vitro use of:

- a mutant CCAAT-binding factor 2 (CBF2) protein devoid of all or part of its transactivation capacity, and/or

- the nucleotide sequence encoding said mutant CBF2, and/or

- a fragment of said mutant CBF2 or of said nucleotide sequence encoding said mutant CBF2,

as a biomarker for prognosing survival and/or the response to a treatment, preferably to a chemotherapy, of a patient suffering from a cancer.

2. The in vitro use according to claim 1 , wherein:

- a mutant heat-shock protein 1 10 (HSP1 10) which:

(1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and

(2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5, and/or

- the nucleotide sequence encoding said mutant HSP1 10 protein, and/or

- a fragment of said mutant HSP1 10 or of said nucleotide sequence encoding said mutant HSP1 10;

is conjointly used as biomarker for prognosing survival and/or the response to a treatment of a patient suffering from a cancer.

3. The in vitro use according to claim 1 or 2, wherein:

- the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10;

is conjointly used as biomarker for prognosing survival and/or the response to a treatment of a patient suffering from a cancer.

4. An in vitro method for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, said method comprising the following steps:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CCAAT-binding factor 2 (CBF2) which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10 (HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5; and

(c) correlating the results of steps (a) and (b), with the prognosis of said patient, thereby deducing the prognosis of said patient.

5. An in vitro method according to claim 4, comprising the additional step between step (b) and step (c) of:

(b') in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10;

the results of steps (a), (b) and (b') being correlated with the prognosis of said patient in step (c). 6. An in vitro method for determining a therapeutic regimen suitable for treating a subject suffering from a cancer liable to have a MSI phenotype, wherein said method comprises or consists of the steps of:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CCAAT-binding factor 2 (CBF2) which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10 (HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5 ; and

(c) deducing and/or selecting a suitable therapeutic regimen for the subject based on the results of steps (a) and (b).

7. An in vitro method according to claim 6, comprising the additional step between steps (b) and (c) of:

(b') in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, the deduction and/or selection of a suitable therapeutic regimen for the subject being based on the results of steps (a), (b) and (b').

8. An in vitro method according any of claims 4 to 6, wherein the method comprises an additional step (b") between steps (b) and (c) or (b) and (b') consisting of determining the presence or the absence of a the gene sequence encoding the wild-type heat-shock protein 1 10 (HSP1 10) in a biological sample of said patient.

9. An in vitro method for determining if a treatment with at least one CCAAT-binding factor 2 (CBF2) protein which retains its transactivation capacity, or a pharmaceutical composition comprising said at least one CBF2 protein which retains its transactivation capacity is suitable for a patient suffering from cancer, comprising or consisting of the following steps:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity;

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in heat-shock protein 1 10 (HSP1 10) gene sequence which leads to expression of a mutant HSP1 10 protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5; and

(c) concluding that a treatment with at least one CBF2 protein which retains its transactivation capacity is suitable when both a genetic variation in the CBF2 gene sequence as defined in step (a) and a genetic variation in the HSP1 10 gene sequence as defined in step (b) are present.

10. An in vitro method according to claim 9, wherein the CBF2 protein which retains its transactivation capacity is a wild-type CBF2 protein.

1 1 . A compound selected from the group consisting of:

a) a CCAAT-binding factor 2 (CBF2) protein with transactivation capacity;

b) a peptido-mimetic of the CBF2 protein of (a); and/or

c) a nucleic acid encoding the CBF2 of (a);

for use in the treatment of a cancer in a patient having both:

i) a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity, and

ii) a genetic variation in HSP1 10 gene sequence which leads to expression of a mutant heat-shock protein 1 10 (HSP1 10) protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5.

12. A combination of:

- at least one compound selected from the group consisting of:

a) a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 , preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 );

b) a peptido-mimetic of the CBF2 protein of (a); and

c) a nucleic acid encoding the CBF2 of (a);

and

- at least one chemotherapeutic agent,

for use in the treatment of a cancer in a patient having both:

i) a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity, and

ii) a genetic variation in HSP1 10 gene sequence which leads to expression of a mutant heat-shock protein 1 10 (HSP1 10) protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5.

13. A kit for :

(i) selecting a patient suffering from a cancer in need of being treated with a CCAAT- binding factor 2 (CBF2) protein which retains its transactivation capacity, and/or

(ii) determining a suitable therapeutic regimen in a subject suffering from cancer, in particular from a cancer liable to have MSI phenotype, and/or

(iii) prognosing survival and/or the response to a treatment of a patient suffering from a cancer, in particular from a cancer liable to have MSI phenotype,

said kit comprising:

- means for determining the presence or the absence of a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity consisting of forward and reverse primers which are able to hybridize to mutated CBF2 nucleotide sequence, and two probes, being able to hybridize specifically to mutated CBF2; and/or

- means for determining the presence or the absence of a CBF2 gene sequence which leads to expression of a wild-type CBF2 protein consisting of forward and reverse primers which are able to hybridize to wild-type CBF2 nucleotide sequence and two probes, being able to hybridize specifically to wild-type CBF2 nucleotide sequence; and/or

- means for determining the presence or the absence of a genetic variation in heat- shock protein (HSP1 10) gene sequence which leads to expression of a mutant HSP1 10 protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5 consisting of forward and reverse primers which are able to hybridize to mutated HSP1 10, and two probes, being able to hybridize specifically to mutated HSP1 10 nucleotide sequence; and/or

- means for determining the presence or the absence of a HSP1 10 gene sequence which leads to expression of a wild-type HSP1 10 protein consisting of forward and reverse primers which are able to hybridize to wild-type HSP1 10, and two probes, being able to hybridize specifically to wild-type HSP1 10; and/or

- means for determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

14. The methods or uses according to any one of claims 1 to 10, wherein the cancer is a cancer liable to have a MSI phenotype.

15. The methods or uses according to claim 14, wherein said cancer is a colorectal cancer with a MSI phenotype.

16. The methods or uses according to any one of claims 1 to 10, 14 or 15, wherein the mutant CBF2 protein devoid of all or part of its transactivation capacity is a CBF2 truncated protein coded by a mutant CBF2 gene sequence wherein the microsatellite repeat of 9 adenosine nucleotides located in exon 2 of the gene encoding CBF2 is deleted of 1 , 4 or 7 adenosine(s), or inserted with 1 , 4, 7, 10 or 13 adenosine(s). 17. The methods or uses according to any one of claims 1 to 10, 14 or 15, wherein the sequence of the mutant CBF2 protein devoid of all or part of its transactivation capacity is sequence SEQ ID NO: 2 or sequence SEQ ID NO: 3.

18. The methods or uses according to any one of claims 1 to 10 or 14 to 17, wherein the mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5, is the mutant HSP1 10DE9 of sequence SEQ ID NO: 6.

19. A compound selected from the group consisting of:

a) a mutant CCAAT-binding factor 2 (CBF2) protein devoid of all or part of its transactivation capacity;

b) a peptido-mimetic of the mutant CBF2 protein of (a);

c) a nucleic acid encoding the mutant CBF2 of (a); and/or

d) an antagonist of wild-type CBF2;

for use in the treatment of a cancer in a patient having both (i) a CBF2 gene sequence which leads to expression of a CBF2 protein with transactivation capacity, and ii) a heat- shock protein 1 10 (HSP1 10) gene sequence which leads to expression of a HSP1 10 protein which exhibits chaperone activity and is capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27).

20. A combination of:

- at least one compound selected from the group consisting of:

a) a mutant CBF2 protein devoid of all or part of its transactivation capacity);

b) a peptido-mimetic of the mutant CBF2 protein of (a);

c) a nucleic acid encoding the mutant CBF2 of (a); and

d) an antagonist of wild-type CBF2;

and

- at least one chemotherapeutic agent,

for use for in the treatment of a cancer in a patient, said patient has both (i) a CBF2 gene sequence which leads to expression of a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 ), and ii) a HSP1 10 gene sequence which leads to expression of a HSP1 10 protein which exhibits chaperone activity and is capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27) (preferably a wild-type HSP1 10, more preferably the HSP1 10 at least 80% identical to SEQ ID NO: 5, more preferably a wild- type HSP1 10 with a sequence SEQ ID NO: 5.

Description:
PROGNOSIS AND TREATMENT OF CANCERS

The present invention concerns mutants of "CCAAT/enhancer-binding protein zeta" (also called "CBF2") which do not retain their transactivation capacity, and their use as a biomarker for prognosing survival and/or the response to a treatment of a patient suffering from a cancer.

The invention also relates to methods using these mutant proteins for (i) prognosing survival and/or the response to a treatment of a patient suffering from a cancer, and (ii) selecting a patient suffering from cancer in need of being treated with a CCAAT/enhancer- binding protein zeta which retains its transactivation capacity.

In a particular embodiment, the patient is suffering from a cancer liable to have a "Microsatellite Instability" phenotype, such as colorectal cancer.

BACKGROUND OF THE INVENTION

Protection from cellular stress is a fundamental function that enables all living organisms to counteract noxious environmental stimuli such as heat, infection, inflammation or toxic agents. Heat shock proteins (also referred to as stress proteins) are a class of functionally related proteins, called chaperone proteins, whose expression is increased when cells are exposed to elevated temperatures or other stress, such as infection, inflammation, exercise, exposure of the cell to toxins (ethanol, arsenic, trace metals and ultraviolet light, among many others), starvation, hypoxia (oxygen deprivation), nitrogen deficiency (in plants), or water deprivation. A crucial aspect of the heat shock response is the rapid and massive production of distinct classes of related proteins conserved in evolution; based on their molecular weight, three families of heat shock proteins (hereinafter abbreviated as "HSPs") are catalogued: 27/40 HSPs (small), 64/74 HSPs (middle), 90/1 10 HSPs (large).

In Human, there are at least eight HSP proteins, including HSP70, HSP90 and HSP1 10 (also named HSP105). It is known that HSP70 and HSP90 are abundantly expressed in cancer cells in a pattern that correlates with poor differentiation, a high proliferative capacity in propensity for metastasis and resistance to various cytotoxic agents.

Besides, it has been demonstrated that HSP1 10 accumulates abnormally in cancer cells and this is believed to enhance their survival. HSP1 10 is especially strongly expressed in colon cancer cells (Kai M et at., Oncol. Rep., 10: 1777-82, 2003) and gene expression profile analysis of primary colorectal cancer (hereinafter abbreviated as "CRC") has linked HSP1 10 expression with metastasis and poor prognosis (Slaby et at., Oncol. Rep., 21 : 1235-41 , 2009). Like other inducible HSPs, HSP1 10 protects the cell against adverse conditions. HSP1 10 not only acts as a nucleotide exchange factor for HSP70 (Andreasson et at., Proc. Natl. Acad. Sci. USA, 105: 16519-24, 2008) but also possesses chaperone anti- aggregation activity. It is approximately four-fold more efficient at binding and stabilizing denatured protein substrates compared to HSC70 and HSP70 (Wang et al., Cancer Res., 63: 5234-5, 2003). Moreover, it is also known that HSP1 10 can act as an inducer of HSP70, thereby playing an important role in the protection of cells against deleterious stress.

Recently, it has been shown that cancers with a "Microsatellite Instability" phenotype (hereinafter abbreviated as "MSI cancers"), more particularly colorectal cancers (hereinafter abbreviated as "CRCs") with a Microsatellite Instability phenotype, universally display mutations in HSP1 10 (Dorard et al., Nat. Med., 17: 1283-9, 201 1 ).

These mutations affect an intronic poly T sequence (a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the HSPH1 gene, i.e. the gene coding the HSP1 10 protein) and result in both HSP1 10 exon 9 skipping and expression of a truncated protein called "HSP1 10DE9".

HSP1 10DE9 constitutes an early marker of MSI CRC patients that predicts disease prognosis and response to treatment, such as chemotherapy (WO 2012/127062). HSP1 10DE9, the first HSPs mutant identified in a cancer so far, completely lacks HSP1 10 anti-apoptotic and chaperone activity. Further, it has been shown that HSP1 10DE9 acts as a dominant negative that binds HSP1 10 and blocks its chaperone and oncogenic properties. In cultured colon cancer cells, mice xenografts and patients, HSP1 10DE9 expression sensitizes tumor cells to chemotherapeutic agents in a dose-dependent manner (Dorard et al., Nat. Med., 17: 1283-9, 201 1 ). Further, MSI CRC patients may be clustered into two groups that displayed large deletions (DT≥5 bp; HSP1 10-Large) or small deletions (0 < DT < 5; MSI HSP1 10-Small) in the T17 microsatellite repeat in HSP1 10 gene: the difference in survival between MSI HSP1 10-Large and HSP1 10-Small groups was significant for patients under chemotherapy (WO 2012/127062).

Cancers with a "Microsatellite Instability" phenotype are a subset of cancers characterized by a DNA Mismatch Repair (MMR) deficiency, notably because of inactivating alterations of the MMR genes MLH1 , MSH2, MSH6 and PMS2.

The MSI status of a given tumor is classically determined by looking at microsatellite instability in a panel of five genetic microsatellite markers: BAT25, BAT26, NR21 , NR24 and NR27. Tumors with instability at two or more of these markers were defined as being MSI-High (MSI-H), whereas those with instability at one marker or showing no instability were respectively defined as MSI-Low (MSI-L) and Microsatellite Stable (MSS) tumors (Duval and Hamelin, Annales de genetique, 45: 71 -75, 2002).

Tumors with MSI phenotype can occur in the context of rare inherited syndromes such as Lynch syndrome or Constitutional Mismatch-Repair Deficiency (CMMR-D), or can occur sporadically in as many as 10-15% of colorectal, gastric and endometrial cancers and to a lesser extent in many other tumors (Duval and Hamelin, Annales de genetique, 45: 71 -75, 2002).

The Lynch syndrome is due to autosomal mutation in one of the 4 genes MLH1 ,

MSH2, MSH6, PMS2. Patients suffering from Lynch syndrome have a risk of 70% to develop, in their adult years, colon cancer and various cancer affecting endometrium, ovary, stomach, small intestine, liver, superior urinary system, brain and skin.

The CMMR-D/Lynch 3 syndrome is due to biallelic deleterious germline mutations in

MMR genes (Ricciardone et al., Cancer Res., 59: 290-3, 1999; Wang et al., Cancer Res.

59: 294-7, 1999) and is characterized by the development of childhood tumors and a huge clinical spectrum very different from one patient to another. The tumors are mainly lymphomas, leukemias, astrocyte-derived brain tumors and/or very early-onset colorectal tumors.

In sporadic MSI cancers, such as colorectal, gastric or endometrial cancers, the MMR deficiency is due to epigenetic and bi-allelic silencing of MLH1 by de novo methylation of its promoter site in more than 90% of the cases, regardless of the primary site of tumor (Kane et al., Cancer Research 57: 808-81 1 , 1997). Sporadic MSI cancers are not restricted to colorectal, gastric or endometrial cancers but may comprise bladder cancer, urinary tract cancer, ovary cancer, prostate cancer, lymphomas, leukemias, glioblastoma, astrocytoma and neuroblastoma.

Concerning colorectal cancers, it is known that MSI status (i.e. the classification of a tumor as MSI-H or MSI-L/MSS) is predictive of the benefit of adjuvant-based chemotherapy with fluorouracil in stage II and stage III colon cancers (Ribic et al., N. Engl. J. Med. 349:247, 2003), thus providing the medical practitioner with critical information as to patient diagnosis, prognosis, and optimal treatment regimen.

Therefore, it is of particular interest to identify new biomarkers, other than

HSP1 10DE9, useful for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, in particular a cancer liable to have a MSI phenotype, and/or useful for refining a prognosis based on biomarker HSP1 10DE9. DESCRIPTION OF THE INVENTION

The present invention arises from the finding that "CCAAT/enhancer-binding protein zeta" (hereinafter abbreviated as "CBF2") can be use as a biomarker for prognosing survival of patients affected by a cancer, as well as for evaluating their response to a therapy.

Indeed, the inventors have now found that a mutant CBF2 protein devoid of its transactivation capacity (in particular a CBF2 truncated protein coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosine located in exon 2 of the gene encoding CBF2 is deleted of, or inserted with, one adenosine) is a reliable new biomarker for prognosing survival of patients affected by cancer, as well as for prognosing their response to a therapy (more particularly in patients affected by cancer liable to have a MSI phenotype).

This new biomarker is particularly useful for refining the disease prognosis based on i) the status of HSP1 10 (i.e. the presence or the absence of a mutant HSP1 10 protein which does not exhibit chaperone activity and/or is not capable of binding to HSP70 and/or to HSP27, and which is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5; e.g. the mutant protein HSP1 10DE9), and/or ii) the length of thymidine repetition of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

Indeed, it was found in patients who suffer from a colorectal cancer, in particular stage III MSI colorectal cancer, and express HSP1 10DE9 protein, that expression of a CBF2 truncated protein which does not retain its transactivation capacity is indicative of a poor prognosis (i.e. a low rate of relapse-free survival) comparing with patients expressing a wild-type CBF2.

Even more surprisingly, it was found that the length of the thymidine repetition of the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, together with the CBF2 status (i.e. the presence or the absence of a CBF2 truncated protein devoid of its transactivation capacity), allow accurately predicting the disease outcome and the response to a treatment.

As demonstrated in the examples, for patients carrying a large deletion (i.e. a deletion of at least 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, there is no significant influence of CBF2 status on the prognosis (i.e. these patients do not relapse).

In contrast, in patients carrying a small deletion (i.e. a deletion of less than 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, there is a significant influence of CBF2 status on the rate of relapse-free survival. Indeed, as demonstrated in the examples (e.g. (see Figure 14), patients expressing a wild-type CBF2 and carrying a small deletion of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 have a better prognosis than patients expressing a CBF2 truncated protein devoid of its transactivation capacity and carrying a small deletion of the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

Besides, whatever the CBF2 status, patients carrying a large deletion (i.e. a deletion of at least 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 have a better prognosis than patients who carry a small deletion (i.e. less than 5 thymidines) of the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

Similarly, in MSI colorectal cancer, for stage II and stage III patients who express both HSP1 10DE9 protein and a CBF2 truncated protein without transactivation capacity who received chemotherapy, the relapse-free survival is worse in patients with a small deletion (i.e. less than 5 thymidines) of the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 compared to patients with a large deletion (i.e. a deletion of at least 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

Definitions

As used herein, "biological sample" may consist of a blood sample, a serum sample, a plasma sample, a feces sample or a biopsy of a cancer. Preferably the biological sample may consist of a biopsy of an adenoma or a primary cancer. Still more preferably the biological sample may consist of a biopsy of an adenoma or a primary cancer selected from the group consisting of colorectal cancer, stomach cancer, endometrial cancer, bladder cancer, urinary tract cancer, ovary cancer, prostate cancer, lymphomas, leukemias, glioblastoma, astrocytoma and neuroblastoma. Preferably, the biological sample is a biopsy of a colorectal cancer.

The term "wild-type CBF2" (or wild type CCAT-binding factor 2) refers to the CCAAT/enhancer-binding protein zeta (also abbreviated as "CEBPZ", and also known as "CCAAT-box-binding transcription factor", "CCAAT-binding factor" (abbreviated as "CBF"), "NOC1 " and "HSP-CBF"). An amino acid sequence of wild-type CBF2 is shown as SEQ ID NO: 1 (Swiss-Prot accession number Q03701 ). The term "CBF2" encompasses the protein of SEQ ID NO: 1 (full-length and mature isoforms) as well as homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

The wild-type CBF2 is a transcription factor which binds to the CCAAT box (a cis- acting element) which is present in heat shock genes, in particular in gene sequences coding for HSP70, HSP40 and HSP1 10. CBF2 is a transcriptional activator which has been shown to be involved in HSP70 expression. Indeed, expression of CBF2 in COS cells increased the activity of the human HSP70 promoter in a CCAAT-dependent way. GAL4 fusion experiments further pinpointed two regions important for transcriptional activation: the acidic N-terminal domain and a central part highly conserved across species (Lum LS et a!., Mol. Cell. Biol. 10: 6709-17, 1990).

In the context of the invention, the expressions " mutant CBF2 protein devoid of all or part of its transactivation capacity" and " mutant CBF2 protein which has lost all or part of its transactivation capacity" are used interchangeably and refer to a CBF2 protein which cannot increase the transcriptional activity of the human HSP1 10 promoter in a CCAAT- dependent way, or which increases the transcription activity in a lesser extent compared with a wild-type CBF2.

Methods for assessing whether a mutant does not increase the transcriptional activity of the human HSP1 10 promoter in a CCAAT-dependent way are well known to the one skilled in the art.

For instance, as disclosed in the examples of the application, the transcriptional activity may be tested as follows:

- co-transfecting cells with i) an expression vector comprising HSP1 10 containing CCAAT boxes (or comprising HSP40 promoter, this promoter being highly homologous to that of HSP1 10) operably linked to a reporter gene (for instance Luciferase), and ii) the mutant CBF2 protein to be tested or a wild-type CBF2; and

- comparing expression of the reporter gene in cells expressing the mutant CBF2 protein to be tested with that of cells expressing wild-type CBF2.

When reporter gene expression is lower in cells expressing the mutant CBF2 protein to be tested, then the mutant CBF2 protein does not increase the transcriptional activity of the human HSP1 10 promoter in a CCAAT-dependent way and is considered as being devoid of all or part of its transactivation capacity.

"Mutant CBF2 proteins devoid of all or part of its transactivation capacity" include truncated proteins lacking the nuclear localization signal corresponding to amino acids 943 to 947 of SEQ ID NO: 1 and/or the hydrophobic domain corresponding to amino acids 500 to 650 of SEQ ID NO: 1 that represents a conserved domain involved in protein- protein interaction among transcription factors.

Preferably, the "mutant CBF2 protein devoid of all or part of its transactivation capacity" is a CBF2 truncated protein coded by a mutant CBF2 gene sequence wherein the microsatellite repeat of 9 adenosine nucleotides located in exon 2 of the gene encoding CBF2 is deleted of 1 , 4 or 7 adenosine(s), or inserted with 1 , 4, 7, 10, 13 etc adenosine(s)). More preferably, the mutant CBF2 protein is coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosine is deleted of one adenosine (to make the reading easier, hereinafter this CBF2 truncated protein will be called "CBF2 del-1 " or "CBF2 -1 "), or by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosine is inserted with one adenosine (to make the reading easier, hereinafter this CBF2 truncated protein will be called "CBF2 lns+1 " or "CBF2 +1 ").

Most preferably, the mutant CBF2 protein is a CBF2 truncated proteins which lacks the nuclear localization signal and the hydrophobic domain is chosen from the group consisting of:

- a mutant CBF2 protein coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosines located in exon 2 is deleted of one adenosine: this CBF2 truncated protein comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO: 2, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2.

- a mutant CBF2 protein coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosines located in exon 2 is inserted with one adenosine: this CBF2 truncated protein comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO: 3, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3.

Advantageously, the mutated mutant CBF2 protein comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 3. As used herein, the term "microsatellite repeat of 9 adenosine nucleotides located in exon 2 of the gene encoding CBF2" refers to the adenosines repetition comprised between position 10 and position 18 of the sequence of SEQ ID NO: 4: ACTTGTGTCAAAAAAAAAGATGTTGAATCA 5 (corresponding to the sequences that spans nucleotides 1450-1458 of mRNA sequence with NCBI accession number NM_005760.2). The term "wild-type HSP1 10" (or wild-type heat shock protein 1 10) refers to the heat-shock 1 10kDa protein. An amino acid sequence of wild-type HSP1 10 is shown as SEQ ID NO: 5 (Swiss-Prot accession number Q92598). The term "wild-type HSP1 10" encompasses the protein of SEQ ID NO: 5 (full-length and mature isoforms) as well as homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

The expression "a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat- shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5" has been previously defined in application WO 2012/127062. A particularly preferred example of such a mutant HSP1 10 protein is HSP1 10DE9 (SEQ ID NO: 6) (also disclosed in WO 2012/127062). As used herein, the term "microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding heat-shock protein 1 10 (HSP1 10)" refers to the thymidine repetition comprised between positions 73 and 89 of SEQ ID NO:7:

GAAAACCCTGTCCATCCATTGGAATTGAGTTTTATATTAAAAGATGACTGGGAAGTGT TCATGTGCTCATGATTTTTTTTTTTTTTTTTAAGTGTGCAATACTTTCCCCTTTCCCCGG CATTTAAAGTTAGAGAATTTTCCGTCACAGATGCAGTTCCTTTTCC.

By "chaperone activity", it is meant, without limitations, the ability to allow protein folding, protein complex formation, transmembrane transport of proteins and targeting some of them to lysosomal degradation. The skilled in the art can easily determine whether the mutated HSP1 10 possess a chaperone activity. For example, the chaperone activity may be determined using a protein thermolability assay, as described in the paragraph entitled "HSP1 10 chaperone activity" of Example 1 of application WO 2012/127062.

The term "binding" refers to a specific binding, as opposed to a non-specific binding.

The skilled in the art can easily determine whether the mutated HSP1 10 is capable of binding to HSP70, HSP27 and/or wild-type HSP1 10. For example, capacity of mutated HSP1 10 to bind to HSP70, HSP27 and/or wild-type HSP1 10 may be determined by immunoprecipitation assays as described in the paragraph entitled "Immunoprecipitation and Western-blotting" of Example 1 of application WO 2012/127062. Preferably, the mutant HSP1 10 protein lacks the domain consisting of amino acids 381 to 858 of SEQ ID NO: 5. In another preferred embodiment, the mutated HSP1 10 protein is encoded by an mRNA lacking exon 9. Exon 9 of the gene coding for HSP1 10 is for example described between positions 1536 and 1642 in the NCBI Reference sequence NM 006644.2 (published version at December 27, 2010).

More preferably, the mutated HSP1 10 protein comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO: 6, still more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6. Most preferably, the mutated HSP1 10 protein comprises or consists of SEQ ID NO: 6.

In the frame of the present application, the percentage of identity is calculated using a global alignment (i.e., the two sequences are compared over their entire length). Methods for comparing the identity and homology of two or more sequences are well known in the art. The « needle » program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk world wide web site. The percentage of identity in accordance with the invention is preferably calculated using the EMBOSS::needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

Protein comprising or consisting of an amino acid sequence "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may comprise mutations such as deletions, insertions and/or substitutions compared to the reference sequence. In case of substitutions, the substitution preferably corresponds to a conservative substitution as indicated in the table below.

In a preferred embodiment, the protein comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence only differs from the reference sequence by conservative substitutions.

In another preferred embodiment, the protein comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a naturally-occurring allelic variant of the reference sequence.

In still another preferred embodiment, the protein comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence corresponds to a homologous sequence derived from another non- human mammalian species than the reference sequence.

The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Said cancer may be a sporadic or an hereditary cancer. Said cancer may be an adenoma (i.e. a benign tumor, which may evolve and become malignant) or a malignant tumor (i.e. a primary tumor or a metastatic tumor). Examples of cancer include, but are not limited to, sarcomas, carcinomas, leukemias, lymphomas, germ cells tumors and blastomas.

The cancer may be selected from the group consisting of prostate cancer (e.g. prostate adenocarcinoma), lung cancer (e.g. squamous cellular carcinoma), breast cancer (e.g. infiltrated ductal carcinoma), ovary cancer (e.g. serous papillary carcinoma), uterus cancer (squamous cellular carcinoma), brain cancer (e.g. astrocytoma), colon cancer (e.g. colon adenocarcinoma), colorectal cancer, rectal cancer (e.g. rectal adenocarcinoma), cancer of the striated muscle (e.g. rhabdomyosarcoma), thyroid cancer, testicular cancer. In a most preferred embodiment, the cancer is selected from the group consisting of lung cancer, prostate cancer, ovary cancer, uterus cancer, brain cancer, colon cancer, colorectal cancer, rectal cancer and cancer of the striated muscle, bladder cancer, liver cancer, kidney cancer, thyroid cancer.

In a preferred embodiment said cancer is a cancer liable to have a MSI phenotype".

"A cancer liable to have a MSI phenotype" refers to a sporadic or hereditary cancer in which microsatellite instability may be present (MSI) or absent (MSS). Detecting whether microsatellite instability is present may for example be performed by genotyping microsatellite markers, such as BAT25, BAT26, NR21 , NR24 and NR27, e.g. as described in Buhard et at., J Clin Oncol 24 (2), 241 (2006) and in European patent application No. EP 1 1 305 160.1 . A cancer is defined as having a MSI phenotype if instability is detected in at least 2 microsatellite markers. On the contrary, if instability is detected in one or no microsatellite marker, then said cancer has a MSS phenotype.

Examples of cancers liable to have a MSI phenotype include adenoma or primary tumors, such as colorectal cancer (also called colon cancer or large bowel cancer), stomach cancer, endometrial cancer, bladder cancer, urinary tract cancer, ovary cancer, prostate cancer, lymphomas, leukemia, glioblastoma, astrocytoma and neuroblastoma. Preferably, the cancer is a colorectal cancer. Still preferably, the cancer is a stage II or stage III colorectal cancer. More preferably, the cancer is a stage II or stage III MSI colorectal cancer.

By "stage II colorectal cancer", it is meant tumor with no lymph node spreading and no distant invasion (American Joint Comittee on Cancer" 2010, Chap. 14. p. 173-201 ).

By "stage III colorectal cancer", it is meant it is meant tumor with lymph node spreading (American Joint Comittee on Cancer" 2010, Chap. 14. p. 173-201 ).

A sporadic cancer liable to have a MSI phenotype may refer to a cancer due to somatic genetic alteration of one of the Mismatch Repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. For example, a sporadic cancer liable to have a MSI phenotype can be a cancer due to de novo bi-allelic methylation of the promoter of MLH1 gene.

An hereditary cancer liable to have a MSI phenotype may refer to a cancer that occurs in the context of Lynch syndrome or Constitutional Mismatch-Repair Deficiency (CMMR-D).

A patient suffering from Lynch syndrome is defined as a patient with an autosomal mutation in one of the 4 genes MLH1, MSH2, MSH6, PMS2.

A patient suffering from CMMR-D is defined as a patient with a germline biallelic mutation in one of the 4 genes MLH1, MSH2, MSH6, PMS2.

As used herein, the patient is a human or a non-human mammal, in particular a rodent, a feline, a canine, a bovine or an ovine mammal. In a preferred embodiment, the patient is a human patient. More particularly, the patient is a child, an adult, a man or a woman.

By "treatment", it is meant, without limitation, a chemotherapeutic treatment and/or a radiotherapeutic treatment and/or a surgical treatment. As used herein, the term "treatment" encompasses therapeutic methods, e.g. aiming at curing, improving the condition of the patient and/or extending the lifespan of the patient suffering from the cancer. It also encompasses prophylactic methods such as methods aiming at preventing the appearance or the spreading of metastases, as well as methods aiming at preventing a relapse.

By "chemotherapeutic treatment' is meant a treatment using at least one anti-cancer agent (e.g. an antiproliferative / neoplasic drug). The anti-cancer agent may be chosen from the group consisting of:

- an alkylating agent (e.g. Cyclophosphamide, Chlorambucil and Melphalan); - an antimetabolite (e.g. Methotrexate, Cytarabine, Fludarabine, 6-

Mercaptopurine and 5- Fluorouracil); - an antimitotic (e.g. Vincristine, Paclitaxel (Taxol), Vinorelbine, Docetal and Abraxane);

- a topoisomerase inhibitor (e.g. Doxorubicin, Irinotecan, Platinum derivatives, Cisplatin, Carboplatin, Oxaliplatin);

- a hormonal therapy (e.g. Tamoxifen);

- an aromatase inhibitor (e.g. Bicalutamide, Anastrozole, Examestane and Letrozole);

- a signaling inhibitor (e.g. Imatinib (Gleevec), Gefitinib and Erlotinib);

- a monoclonal antibody (e.g. Rituximab, Trastuzumab (Herceptin) and Gemtuzumab ozogamicin);

- a biologic response modifier such as Interferon-alpha;

- a differentiating agent (e.g. Tretinoin and Arsenic trioxide); and/or

- an agent that block blood vessel formation (antiangiogenic agents) (e.g.

Bevicizumab, Serafinib and Sunitinib).

The chemotherapeutic treatment may for example be performed with an alkylating agent, such as e.g. oxaliplatin. The chemotherapeutic treatment may also be an adjuvant chemotherapeutic treatment, e.g. a chemotherapeutic treatment using 5-fluorouracil agent.

In some embodiments, the chemotherapeutic treatment may combine at least 2, 3,

4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 anti-cancer agents as defined above, such as e.g. a combination of oxaliplatin, 5-fluorouracil (5-FU) and folinic acid (i.e. the FOLFOX treatment), a combination of folinic acid, 5-fluorouracil and irinocetan (i.e. the FOLFIRI treatment), or a combination of 5-fluorouracil and folinic acid (i.e. the FUFOL or LV5FU2 treatment).

The expression "homozygous genetic variation" means that the same genetic variation is present on both alleles of the gene carrying said genetic variation.

The expression "heterozygous genetic variation" means on the contrary that said genetic variation is only present on one allele of the gene carrying said genetic variation.

The expression "effective amount" is intended to mean an amount sufficient to treat the cancer. It will be appreciated that this amount will vary with the effectiveness of therapeutic agent(s) employed, with the nature of any carrier used, with the seriousness of the disease and the age of the patient. The determination of appropriate amounts for any given composition is within the skill in the art, through standard series of tests designed to assess appropriate therapeutic levels. By "patient in need" is meant an individual suffering from a cancer, or an individual that is in remission after having suffered from cancer.

In the frame of the present invention, the individual preferably is a human individual. However, the veterinary use of the polypeptides and drugs according to the present invention is also contemplated. The individual may thus also correspond to a non-human individual, preferably a non-human mammal.

The term "treating" is meant to encompass both therapeutic and prophylactic methods, i.e. a method aiming at curing, improving the condition and/or extending the lifespan of an individual suffering from the cancer. It also refers to methods aiming at preventing the appearance or the spreading of metastases, as well as methods aiming at preventing a relapse.

The term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.

In particular, the antibody according to the invention may correspond to a polyclonal antibody, a monoclonal antibody (e.g. a chimeric, humanized or human antibody), a fragment of a polyclonal or monoclonal antibody or a diabody.

In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (V L ) and a constant domain (C L ). The heavy chain includes four domains, a variable domain (V H ) and three constant domains (C H 1 , C H 2 and C H 3, collectively referred to as C H ). The variable regions of both light (V L ) and heavy (V H ) chains determine binding recognition and specificity to the antigen.

The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). They refer to amino acid sequences which, together, define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1 , L-CDR2, L-CDR3 and H-CDR1 , H-CDR2, H-CDR3, respectively. Therefore, an antigen-binding site includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework regions (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species, as defined by Kabat et at., 1991 (Kabat et at., 1991 , Sequences of Proteins Of Immunological Interest, National Institute of Health, Bethesda, Md). As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular, 90%, 95% or 100%) to the framework region of naturally occurring human antibody.

The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition, that is directed against a specific antigen and which may be produced by a single clone of B cells or hybridoma, or by recombinant methods.

A "humanized antibody" is a chimeric, genetically engineered, antibody in which the CDRs from a mouse antibody ("donor antibody") are grafted onto a human antibody ("acceptor antibody"). Thus, a humanized antibody is an antibody having CDRs from a donor antibody and variable region framework and constant regions from a human antibody. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab') 2 , Fab', Fd, dAb, dsFv, scFv, sc(Fv) 2 , CDRs, diabodies and multi-specific antibodies formed from antibodies fragments.

The term "Fab" denotes an antibody monovalent fragment having a molecular weight of about 50,000 and antigen binding activity, and consisting of the V L , V H , C L and C H 1 domains.

The Fv fragment is the N-terminal part of the Fab fragment and consists of the variable portions of one light chain and one heavy chain.

The term "F(ab') 2 " refers to an antibody bivalent fragment having a molecular weight of about 100,000 and antigen binding activity, which comprises two Fab fragments linked by a disulfide bridge at the hinge region.

The term "Fab"' refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab') 2 fragment.

The term "Fd" refers to an antibody fragment consisting of the V H and C H 1 domains. The term "dAb" (Ward et al., 1989 Nature 341 :544-546) refers to a single variable domain antibody, i.e. an antibody fragment which consists of a V H or V L domain. A single chain Fv ("scFv") polypeptide is a covalently linked V H ::V L heterodimer which is usually expressed from a gene fusion including V H and V L encoding genes linked by a peptide-encoding linker. "dsFv" is a V H ::V L heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv) 2 .

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a V H domain connected to a V L domain in the same polypeptide chain (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding sites.

Antibodies according to the invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. The antibodies of this invention can be obtained by producing and culturing hybridomas.

As used herein the term "peptidomimetic" refers to peptide-like structures which have non-amino acid structures substituted but which mimic the chemical structure of a peptide and retain the functional properties of the peptide. Peptidomimetics may be designed in order to increase peptide stability, bioavailability, solubility, etc.

Detailed description of the invention

Methods and uses for prognosing survival and/or the response to a treatment of a patient suffering from a cancer

In a first aspect, the invention relates to the use of a mutant CCAT-binding factor 2 (CBF2) protein devoid of all or part of its transactivation capacity, and/or the nucleotide sequence encoding said mutant CBF2 (i.e. gene sequence and/or mRNA) and/or a fragment of said mutant CBF2 or of said nucleotide sequence encoding said mutant CBF2, as a biomarker for prognosing survival and/or the response to a treatment (preferably a chemotherapy) of a patient suffering from a cancer, in particular from a cancer liable to have MSI phenotype.

In an embodiment, the first aspect of the invention is directed to the use of:

i) a mutant CBF2 protein devoid of all or part of its transactivation capacity and/or the nucleotide sequence encoding said mutant CBF2, and/or a fragment of said mutant CBF2 or of said nucleotide sequence encoding said mutant CBF2; and

ii) a mutant heat-shock protein 1 10 (HSP1 10) which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat- shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5, and/or the nucleotide sequence encoding said mutant HSP1 10 protein, and/or a fragment of said mutant HSP1 10 or of said nucleotide sequence encoding said mutant HSP1 10;

as biomarkers for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, in particular from a cancer liable to have MSI phenotype.

Also, the present invention relates to the in vitro use of a mutant CCAT-binding factor 2 (CBF2) protein as defined above, wherein:

- a mutant heat-shock protein 1 10 (HSP1 10) which:

(1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and

(2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5, and/or

- the nucleotide sequence encoding said mutant HSP1 10 protein, and/or

- a fragment of said mutant HSP1 10 or of said nucleotide sequence encoding said mutant HSP1 10;

is conjointly used as biomarker for prognosing survival and/or the response to a treatment of a patient suffering from a cancer.

In an embodiment, the first aspect of the invention is directed to the use of:

i) a mutant CBF2 protein devoid of all or part of its transactivation capacity and/or the nucleotide sequence encoding said mutant CBF2, and/or a fragment of said mutant CBF2 or of said nucleotide sequence encoding said mutant CBF2; and

ii) a HSP1 10 mutant protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5, and/or the nucleotide sequence encoding said mutant HSP1 10 protein, and/or a fragment of said mutant HSP1 10 or of said nucleotide sequence encoding said mutant HSP1 10; and/or

iii) the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10;

as biomarkers for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, in particular from a cancer liable to have MSI phenotype.

Also, the present invention relates to the in vitro use of a mutant CCAT-binding factor 2 (CBF2) protein, optionally conjointly used with a mutant heat-shock protein 1 10 (HSP1 10) as defined above, wherein: - the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10;

is conjointly used as biomarker for prognosing survival and/or the response to a treatment of a patient suffering from a cancer.

In some embodiment, the presence or the absence of a wild-type HSP1 10 protein and/or of the nucleotide sequence encoding said wild-type HSP1 10 protein is also determined.

In some embodiment, the presence or the absence of a wild-type CBF2 protein and/or of the nucleotide sequence encoding said wild-type CBF2 protein is also determined.

In the context of the invention, the expression "a fragment" when referring to a mutant protein (CBF2 or HSP1 10), or to the corresponding nucleotide sequence, refers to a fragment from said mutant protein or from said corresponding nucleotide sequence which is specifically found in the mutant protein, or in the corresponding nucleotide sequence, and which is not present in the wild-type protein, or in the wild-type nucleotide sequence. The fragment thus relates to a polypeptide or its corresponding nucleotide sequence shorter than the mutant protein or its corresponding nucleotide sequence and which amino acid chain or corresponding nucleotide sequence consists of consecutive amino acids or corresponding consecutive nucleotides that may comprise for example at least 15, 20, 50, 100, 200 or 250 consecutive amino acids or corresponding consecutive nucleotides found in the mutant protein, or in its corresponding nucleotide sequence, and which are not present in the wild-type protein, or in the wild-type nucleotide sequence. In a patient who does not express a mutant HSP1 10 protein as defined above but expresses a wild-type HSP1 10 protein, the presence of a mutant CBF2 protein devoid of all or part of its transactivation capacity and/or the presence of the nucleotide sequence encoding said mutant CBF2 tends to be indicative of a good prognosis of survival and/or of response to the treatment.

In patients with both (i) a nucleotide sequence encoding a mutant HSP1 10 protein which does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5 (the mutant HSP1 10 protein being preferably HSP1 10DE9), and (ii) a small deletion (i.e. a deletion of less than 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, those with a wild-type CBF2 have a better prognosis (survival and/or of response to the treatment compared) than patients expressing a CBF2 truncated protein devoid of all or part of its transactivation capacity and carrying a small deletion of the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

Besides, whatever the CBF2 status, in patients carrying a nucleotide sequence encoding a mutant HSP1 10 protein which does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5 (the mutant HSP1 10 protein being preferably HSP1 10DE9), a large deletion (i.e. a deletion of at least 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 is indicative of a better prognosis of survival and/or of response to the treatment compared with patients who carry a small deletion (i.e. less than 5 thymidines) of the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

By "use as a biomarker" is meant an in vitro use, wherein mutated CBF2 and HSP1 10 may be detected e.g. using ligands, antibodies, probes and/or primers, and wherein the length of the thymidine deletions in the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 may be detected by genotyping or sequencing.

In a second aspect, the invention relates to an in vitro method for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, said method comprising a step consisting of determining the presence or the absence of a genetic variation in the gene sequence encoding CBF2 which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity in a biological sample of said patient.

In an embodiment of the second aspect, the invention relates to an in vitro method for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, said method comprising the following steps:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CBF2 which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and/or (b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10 (HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5 ; and/or

(b') optionally, in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10; and/or

(c) correlating the results of steps (a), (b), and/or (b'), with the prognosis of said patient, thereby deducing the prognosis of said patient.

Also, the present invention relates to an in vitro method for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, said method comprising the following steps:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CCAAT-binding factor 2 (CBF2) which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10 (HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5; and

(c) correlating the results of steps (a) and (b), with the prognosis of said patient, thereby deducing the prognosis of said patient.

In an embodiment, the in vitro method as defined above thus comprises the additional step between step (b) and step (c) of:

(b') in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10;

the results of steps (a), (b) and (b') being correlated with the prognosis of said patient in step (c). In another embodiment, the second aspect of the invention relates to an in vitro method for prognosing survival and/or the response to a treatment of a patient suffering from a cancer, said method comprising or consisting of the following steps:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CBF2 which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and/or

(a') in a biological sample of said patient, determining the presence or the absence of a the gene sequence encoding the wild-type CBF2; and/or

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10

(HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5 ; and/or

(b") in a biological sample of said patient, determining the presence or the absence of a the gene sequence encoding the wild-type heat-shock protein 1 10 (HSP1 10); and/or

(c) optionally, in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10; and/or

(d) correlating the results of steps (a), (b), and/or (c) where appropriate, with the prognosis of said patient, thereby deducing the prognosis of said patient.

In some embodiments, steps (a), (a'), (b), (b'), (b") and/or (c) where appropriate, are carried out in the same biological sample.

In a particular embodiment of the first and second aspects of the invention according to any one of the embodiments previously disclosed, the use and the method are carried out to prognose (predict) the response of a patient suffering from a cancer to a treatment, preferably to a chemotherapeutic treatment.

More preferably, when the patient suffers from a colorectal cancer, the treatment is a chemotherapeutic treatment using at least an alkylating agent such as e.g. oxaliplatin, or a treatment with 5-fluorouracil, or the FOLFOX treatment, or the FUFOL treatment.

In a first implementation of the first and second aspects of the invention according to any one of the embodiments, said patient is a patient suffering from a stage II or stage III colorectal cancer with MSI phenotype, and preferably suffering from a stage III colorectal cancer with MSI phenotype. In a second implementation of the first and second aspects of the invention according to any one of the embodiments or according to the first implementation, said patient is a patient that received a chemotherapeutic treatment, such as an adjuvant chemotherapeutic treatment.

In a preferred implementation of the first and second aspects of the invention according to any one of the embodiments or according to the first or the second implementation, said patient is a patient suffering from a stage II or stage III colorectal cancer with MSI phenotype, more preferably a patient who received an adjuvant chemotherapeutic treatment.

As shown in the examples, in patients with (i) a genetic variation in the gene sequence encoding a mutant HSP1 10 as defined in step (b) (preferably a genetic variation which leads to the expression of HSP1 10DE9 protein), and (ii) a small deletion (i.e. a deletion of less than 5 thymidines) of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, those with a genetic variation in the gene sequence encoding a mutant CBF2 as defined in step (a) have a poorer prognosis as compared with patients who do not have a genetic variation in the gene sequence encoding CBF2 as defined in step (a) (preferably patients who have gene sequence encoding wild-type CBF2).

Besides, the presence in the biological sample from a patient of a genetic variation in the gene sequence encoding CBF2 as defined in step (a) and the absence in the biological sample of a genetic variation in the gene sequence encoding HSP1 10 as defined in step (b) (and preferably the presence of a gene sequence encoding wild-type HSP1 10) indicate a better prognosis of the patient as compared with a patient who does not have a genetic variation in the gene sequence encoding CBF2 as defined in step (a) (preferably a patient who has gene sequence encoding wild-type CBF2) and does not have a genetic variation in the gene sequence encoding HSP1 10 as defined in step (b) (preferably a patient who has gene sequence encoding wild-type HSP1 10).

Further, whatever the status of the gene sequence encoding CBF2 (i.e. wild-type sequence or presence of a genetic variation in the gene sequence encoding CBF2 as defined in step (a)), in patients with a genetic variation in the gene sequence encoding HSP1 10 as defined in step (b) (preferably a genetic variation which leads to the expression of HSP1 10DE9 protein), a small thymidine deletion (i.e. a deletion of less than 5 thymidines, for instance 3 or 4 thymidines), is indicative of bad prognosis of survival and/or of response to the treatment, and a large thymidine deletion (i.e. a deletion of at least 5 thymidines, e.g. 5 to 8 thymidines), is indicative of good prognosis of survival and/or of response to the treatment.

In the context of the invention, a genetic variation may be homozygous or heterozygous.

The presence or the absence of a genetic variation in the gene sequences encoding CBF2 and HSP1 10 as defined in steps (a) and (b) may be determined at polynucleotide sequence level (i.e. gene sequence or mRNA level) and/or at protein level. The analysis may be a qualitative analysis or a quantitative measure.

Numerous methods allowing determining the presence of a genetic variation in a biological sample are well known from the one skilled in the art.

For determining genetic variation at protein level, these methods include, without being limited to, immunochemistry, Elisa and Western blotting assays carried out with antibodies specifically detecting the mutated protein or the wild-type protein.

The level of expression of the protein may be, for instance, determined using immunological detection methods such as an ELISA assay. The methods involve an antibody which binds to the protein, for example a monoclonal or polyclonal antibody, an antibody variant or fragments such as a single chain antibody, a diabody, a minibody, a single chain Fv fragment (sc(Fv)), a Sc(Fv)2 antibody, a Fab fragment or a F(ab')2 fragment, or a single domain antibody. Such antibodies are well known in the art and are commercially available. They may also notably be obtained by immunization of animals (for example rabbits, rats or mice) with the protein of interest. Antibodies may be used to determine protein expression in a range of immunological assays including competitive and non-competitive assay systems using techniques such as western blotting, immunohistochemistry/ immunofluorescence (i.e protein detection on fixed cells or tissues), radioimmunoassay such as RIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays, ECLIA (electrochemiluminescence immunoassay) and protein A immunoassays. Such assays are routine and well known to the person skilled in the art (Ausubel et al (1994) Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York).

For determining genetic variation at gene sequence level, these methods include, without being limited to, sequencing (e.g. automated sequencing, microsequencing and pyrosequencing), hybridization methods with DNA probes specific of said genetic variation (e.g. comparative genomic hybridization (CGH), matrix-CGH, array-CGH, oligonucleotide arrays and representational oligonucleotide microarray (ROMA)), high-throughput technologies for genotyping, and amplification methods such as quantitative polymerase chain reaction (qPCR) or a polymerase chain reaction (PCR) followed by sequencing, microsequencing, pyrosequencing or RFLP, ligase chain reaction (LCR), Amplification Refractory Mutation System (ARMS), and High Resolution Melting analysis (HRM PCR).

For determining genetic variation at mRNA level, these methods include, without being limited to, Northern blotting, a polymerase chain reaction (PCR) (e.g. reverse- transcription PCR (RT-PCR) and quantitative reverse-transcription PCR (QRT-PCR)), amplification methods such as QRT-PCR or QRT-PC followed by sequencing, microsequencing, pyrosequencing, transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), methods such as QRT-PCR or QRT-PC for creating a DNA copy (cDNA) followed by Amplification Refractory Mutation System (ARMS) or High Resolution Melting analysis (HRM PCR).

The amplification refractory mutation system (ARMS) is an amplification strategy in which a polymerase chain reaction (PCR) primer is designed in such a way that it is able to discriminate among templates that differ by a single nucleotide residue. ARMS has also been termed allele-specific PCR or PCR amplification of specific alleles (PASA). Thus, an ARMS primer can be designed to amplify a specific member of a multi-allelic system while remaining refractory to amplification of another allele that may doffer by as little as a single base from the former. The main advantage of ARMS is that the amplification step and the diagnostic steps are combined, in that the presence of an amplified product indicates the presence of a particular allele and vice versa. For routine diagnosis, this characteristic of ARMS means that it is a very time-efficient method.

High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double- stranded DNA samples. HRM analysis is performed on double stranded DNA samples. Typically polymerase chain reaction (PCR) will be used prior to HRM analysis to amplify the DNA region in which the mutation of interest lies. Essentially the PCR process turns a tiny amount of the region of DNA of interest into a large amount, so the quantities are large enough for better analysis. In the tube there are now many of copies of the region of DNA of interest. This region that is amplified is known as the amplicon. After the PCR process the HRM analysis begins. The process is simply a precise warming of the amplicon DNA from around 50 ° C up to around 95 ° C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart. The secret of HRM is to monitor this process happening in real-time. This is achieved by using a fluorescent dye. The dyes that are used for HRM are known as intercalating dyes and have a unique property. They bind specifically to double-stranded DNA and when they are bound they fluoresce brightly. In the absence of double stranded DNA they have nothing to bind to and they only fluoresce at a low level. At the beginning of the HRM analysis there is a high level of fluorescence in the sample because of the billions of copies of the amplicon. But as the sample is heated up and the two strands of the DNA melt apart, presence of double stranded DNA decreases and thus fluorescence is reduced. The HRM machine has a camera that watches this process by measuring the fluorescence. The machine then simply plots this data as a graph known as a melt curve, showing the level of fluorescence versus the temperature. The melting temperature of the amplicon at which the two DNA strands come apart is entirely predictable. It is dependent on the sequence of the DNA bases. If you two samples from two different people are compared, they should give exactly the same shaped melt curve. However if one of the people has a mutation in the amplified DNA region, then this will alter the temperature at which the DNA strands melt apart. So now the two melt curves appear different. The difference may only be tiny, perhaps a fraction of a degree, but because the HRM machine has the ability to monitor this process in "high resolution", it is possible to accurately document these changes and therefore identify if a mutation is present or not.

In a preferred embodiment, the absence or the presence of a genetic variation in the gene sequences encoding CBF2 and HSP1 10 as defined in steps (a) and (b) is determined by sequencing the gene sequence encoding CBF2 (or at least the gene sequence fragment where the genetic variation is supposed to be) and/or the gene sequence encoding HSP1 10 (or at least the portion of the gene sequence fragment where the genetic variation is supposed to be), or by sequencing the cDNA (or at least the cDNA sequence fragment where the genetic variation is supposed to be) obtained after carrying out an RT-PCR on mRNA(s) of CBF2 and /or of HSP1 10.

In another preferred embodiment, the absence or the presence of a genetic variation in the gene sequence encoding CBF2 and/or of a gene sequence encoding a wild-type CBF2 protein is (are) determined by sequencing, using pairs of primers specific for CBF2 exon 2. In particular, for carrying out this embodiment, the forward primer may have the sequence 5'-CCATGAAGAAAGTGAATTGG-3' (SEQ ID NO: 8), the reverse primer may have the sequence 5'- TCCCTTACTTTGTCATCACC -3' (SEQ ID NO: 9).

In another preferred embodiment, the absence or the presence of a genetic variation in the gene sequence encoding HSP1 10 and/or of a gene sequence encoding a wild-type HSP1 10 protein is (are) determined by sequencing, using a pair of primers specific for HSP1 10 intron 8. In particular, for carrying out this embodiment, the forward primer may have the sequence 5'-CCCTGTCCATCCATTGGAATTGA-3' (SEQ ID NO: 10), the reverse primer may have the sequence 5'-GGAACTGCATCTGTGACGGAA-3' (SEQ ID NO: 1 1 ).

In another preferred embodiment, the absence or the presence of a genetic variation in the gene sequences encoding CBF2 and HSP1 10 as defined in steps (a) and (b) is determined by QRT-PCR using a forward primer, a reverse primer and a probe, which can be used to detect the presence of the genetic variation.

In particular, for carrying out a QRT-PCR for HSP1 1 0, the forward primer may have the sequence 5'-GCTACACGAATTCCAGCTGTGA-3' (SEQ ID NO: 12), the reverse primer may have the sequence 5'-GAGCAGCATGGTTTCGACTAAA-3' (SEQ ID NO: 13), and a probe (e.g. a fluorescent probe) which specifically hybridizes to a sequence comprising the genetic variation. When the mutant HSP1 1 0 protein is HSP1 10delE9, the probe may have the sequence 5'-ATGTGCATTACAGTGTTC-3' (SEQ ID NO: 14).

In another preferred embodiment, the absence or the presence of a genetic variation in the gene sequence encoding CBF2 as defined in steps (a) is determined by carrying out a RT-PCR to obtain a cDNA fragment, and then the so obtained cDNA is sequenced. The RT-PCR is performed according to method well known to one of ordinary skill in the art. For sequencing the cDNA, the pair of primers used may be a pair of primers specific for CBF2 exon 2. In particular, the forward primer may have the sequence 5'- CCATGAAGAAAGTGAATTGG-3' (SEQ ID NO: 8), the reverse primer may have the sequence 5'- TCCCTTACTTTGTCATCACC -3' (SEQ ID NO: 9).

For the QRT-PCR, preferably, the probes are labelled with at least one fluorescent label or dye. The fluorescent dye can be a wide variety of dyes known in the art, including 6-FAM™, VIC ® , TET™, NED™, Cy3 ® , Cy5 ® , HEX, TAMRA, DABCYL, BHQ™, DDQ, etc.

More preferably, the probes are labelled with a reporter dye and a quencher dye. Still more preferably, the probes are labelled at their 5' end with the reporter dye and at their 3' end with the quencher dye. The reporter dye may be a fluorescent dye, which can be for instance 6-FAM™, VIC ® , TET™, NED™, Cy3 ® , Cy5 ® , HEX, etc. The quencher dye may be a non-fluorescent dye (e.g. MGB™) or a fluorescent dye, which can be for instance TAMRA, DABCYL, BHQ™, DDQ, etc.

In a preferred embodiment, the QRT-PCR comprises an initial denaturation step followed by cycles of denaturation-annealing-elongation steps.

The initial denaturation step may be performed under heating conditions ranging from 90 ° C to 1 05° C, during 15 sec to 15 min. Prefeably, the heating conditions range from 92 °C to 102°C, more preferably from 95 °C to 10°C, still more preferably the heating conditions are at 95°C. Preferably, the initial denaturation step is performed during 1 min to 15 min, more preferably during 8 min to 12 min, still more preferably during 5 min to 10 min, and still more preferably the initial denaturation step is performed during 10 min.

In a preferred embodiment, the initial denaturation step is performed at 95 °C during

10 min.

Each cycle of denaturation-annealing-elongation step includes a denaturation phase under heating conditions, followed by an annealing phase performed under conditions which allow the hybridization of the primers and the probe to the sequence to be amplified, and an elongation phase performed under conditions which allow the polymerase to synthesize an extension product from each primer that is annealed to the sequence to be amplified.

The denaturation phase may be performed between 90 'Ό to 105°C, preferably 92 °C to 100°C, more preferably between 94 °C to 98 °C, dung 10 sec to 4 min, preferably during 10 sec to 2 min, more preferably during 15 sec to 1 min.

The annealing phase may be performed between 35 °C and 70 °C, preferably between 40 °C to 65 °C, more preferably between 45°Cto 60 °C, still more preferably between 50°C to 60 °C, during 10 sec to 2 min, prefeably during 20 sec to 1 ,5 min, more preferably during 30 sec to 1 min.

The elongation phase may be performed between 40 °C and 80 °C, preferably between 50 °C to 75 °C, more preferably between 55°Cto 65 °C, still more preferably between 58°C to 62 °C, during 10 sec to 5 min, prefeably during 20 sec to 3 min, more preferably during 30 sec to 1 min, still more preferably during 30 sec to 45 sec.

In a preferred embodiment, the denaturation phase is performed at 95°C during 15 sec, the annealing phase and the elongation phase are combined and performed at 60 °C during 1 min. The denaturation-annealing-elongation step may be repeated during 30 to 60 cycles, preferably during 35 to 50 cycles, more preferably during 40 to 45 cycles. Still more preferably, the denaturation-annealing-elongation step is repeated during 40 cycles.

In a particularly preferred embodiment, the thermal cycling conditions of the QRT- PCR comprised an initial denaturation step is performed at 95°C during 10 min, and 40 cycles consisting of a denaturation phase at 95°C curing 15 sec, and an elongation phase at 60 °C during 1 min.

The size of the deletion may be determined by determining the length of the thymidine repetition of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, i.e. the number of thymidines in the repetition.

As used herein, determining the length of the thymidines repetition of the microsatellite repeat of 17 thymidines located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 is equivalent to determining the length of the thymidines deletion within said microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

Determining the length of the thymidine repetition by may be performed by methods known by the skilled in the art. For instance, it may be performed by genotyping. Genotyping the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 may be performed by methods known by the skilled in the art. For instance, genotyping as described in paragraph "Mutation analysis" of Example 1 of application WO 2012/127062 or as described in paragraph "Diagnostic method according to the invention" of application WO 2012/127062. Genotyping the microsatellite repeat of 17 thymidine nucleotides may be performed by methods known by the skilled in the art. For instance, genotyping the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 may be performed by amplifying the said microsatellite repeat and isolating the amplification products by capillary electrophoresis. Amplification of said microsatellite repeat can be performed using conventional polymerase chain reaction (PCR) techniques as described in paragraph "Mutation analysis" of Example 1 of application WO 2012/127062. Suitable methods may also include Amplification Refractory Mutation System (ARMS), and High Resolution Melting analysis (HRM PCR).

In a specific implementation of the first aspect of the invention, according to any one of the embodiments disclosed, the forward primer and the reverse primer for genotyping the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 have respectively the sequences 5'-CCCTGTCCATCCATTGGAATTGA-3' (SEQ ID NO: 10) and 5'- GGAACTGCATCTGTGACGGAA-3' (SEQ ID NO: 1 1 ) as described in paragraph "Mutation analysis" of Example 1 of application WO 2012/127062.

Method for determining a suitable therapeutic regimen

The method according to the second aspect of the invention as disclosed above can be used to determine and/or select the therapeutic regimen suitable for treating a subject suffering from a cancer liable to have a MSI phenotype. Wild-type HSP1 10 is involved in tumour resistance to treatments (e.g. chemotherapeutic treatment), while mutant HSP1 10 proteins as defined above, in particular HSP1 10DE9, play an important role in tumour sensitivity to treatments. Further, it has been shown that small thymidine deletions {i.e. deletions of less than 5 thymidines) in the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 correlate with a low expression of HSP1 10DE9, while large thymidine deletions {i.e. deletions of at least 5 thymidines) in the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 correlate with a high expression of HSP1 10DE9. Besides, since CBF2 is a translation activator of HSP1 10, determining the status of CBF2 allows predicting the level of expression of HSP1 10 (wild-type or mutant HSP1 10): in patients who express a mutant CBF2 protein devoid of all or part of its transactivation capacity, expression of HSP1 10 (mutant or wild-type) is low compared to patients expressing wild- type CBF2. Therefore, determining the status of CBF2, HSP1 10 and the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 allows a therapeutic regimen suitable for treating a subject suffering from a cancer to be determined.

Thus, in a third aspect, the invention also relates to an in vitro method for determining a therapeutic regimen suitable for treating a subject suffering from a cancer liable to have a MSI phenotype, wherein said method comprises or consists of the steps of :

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CBF2 which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10 (HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type

HSP1 10 protein of SEQ ID NO: 5 ; and

(b') optionally, in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, and

(c) deducing and/or selecting a suitable therapeutic regimen for the subject based on the results of steps (a), (b) and/or (b'). Also, the present invention relates to an in vitro method for determining a therapeutic regimen suitable for treating a subject suffering from a cancer liable to have a MSI phenotype, wherein said method comprises or consists of the steps of:

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding CCAAT-binding factor 2 (CBF2) which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity; and

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the gene sequence encoding the heat-shock protein 1 10 (HSP1 10) which leads to expression of a mutant HSP1 10 protein which (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild- type HSP1 10 protein of SEQ ID NO: 5 ; and

(c) deducing and/or selecting a suitable therapeutic regimen for the subject based on the results of steps (a) and (b).

In an embodiment, the in vitro method as defined above thus comprises the additional step between steps (b) and (c) of:

(b') in a biological sample of said patient, determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10,

the deduction and/or selection of a suitable therapeutic regimen for the subject being based on the results of steps (a), (b) and (b').

In a preferred embodiment, the method according to the third aspect of the invention comprises an additional step between steps (a) and (b) (i.e. a step (a')) consisting of determining the presence or the absence of a the gene sequence encoding the wild-type CBF2; and/or an additional step between steps (b) and (c) or between (b) and (b') (i.e. a step (b")) consisting of determining the presence or the absence of a the gene sequence encoding the wild-type heat-shock protein 1 10 (HSP1 10) in a biological sample of said patient.

The invention also pertains to the use of the status of CBF2 (i.e. wild-type or mutant CBF2 as defined in step (a)), and/or of HSP1 10 (i.e. wild-type or mutant HSP1 10 as defined in step (b)) and/or of the microsatellite repeat of 17 thymidines localized in the splicing acceptor site of intron 8 of the gene encoding HSP1 10, as biomarker(s) for determining a suitable therapeutic regimen. In some embodiments, steps (a), (a'), (b), (b'), (b") and/or (c) where appropriate, are carried out in the same biological sample.

In some embodiments, said cancer liable to have a MSI phenotype may be a colorectal cancer, preferably a stage II or stage I II colorectal cancer.

A "suitable therapeutic regimen" may refer to surgery, chemotherapy and/or radiotherapy suitable for treating a subject suffering from a cancer liable to have a MSI phenotype. Preferably, a suitable therapeutic regimen is a chemotherapy treatment.

The presence of a genetic variation in the gene sequence encoding CBF2 which leads to expression of a mutant CBF2 protein devoid of all or part of its transactivation capacity (preferably on both alleles) and the presence of gene sequence coding for wild- type HSP1 10 (preferably on both alleles) indicate that a suitable therapeutic regimen may be a chemotherapeutic treatment with a single agent, e.g. 5-fluorouracil or oxaliplatin when the patient suffers from a colorectal cancer.

The presence of a genetic variation in the gene sequence encoding CBF2 as defined in step (a) (on one or on both alleles), the presence of genetic variation in the gene sequence encoding HSP1 10 as defined in step (b), and the presence of a small thymidine deletion (i.e. a deletion of less than 5 thymidines, for instance 3 or 4 thymidines) in the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 indicate that a suitable therapeutic regimen may be a chemotherapeutic treatment that combines at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 agents (for instance, when the patient suffers from a colorectal cancer, a combination of oxaliplatin, 5-fluorouracil and folinic acid, i.e. the FOLFOX treatment, or a combination of 5-fluorouracil and folinic acid, i.e. the FUFOL or LV5FU2 treatment).

The presence of a genetic variation in the gene sequence encoding CBF2 as defined in step (a) (on one or on both alleles), the presence of genetic variation in the gene sequence encoding HSP1 10 as defined in step (b), and the presence of a large thymidine deletion (i.e. a deletion of at least 5 thymidines, for instance 7 or 8 thymidines) in the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 indicate that a suitable therapeutic regimen may be a chemotherapeutic treatment that combines at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 agents (for instance, when the patient suffers from a colorectal cancer, a combination of oxaliplatin, 5-fluorouracil and folinic acid, i.e. the FOLFOX treatment, or a combination of 5-fluorouracil and folinic acid, i.e. the FUFOL or LV5FU2 treatment). The presence of a genetic variation in the gene sequence encoding HSP1 10 as defined in step (b) (preferably on both alleles), the presence of gene sequence coding for wild-type CBF2 (preferably on both alleles), and the presence of a large thymidine deletion in the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 indicate that a suitable therapeutic regimen may be a chemotherapeutic treatment with a single agent (e.g. 5-fluorouracil or oxaliplatin when the patient suffers from a colorectal cancer). In contrast, the presence of a genetic variation in the gene sequence encoding HSP1 10 as defined in step (b) (preferably on both alleles), the presence of gene sequence coding for wild-type CBF2 (preferably on both alleles), and the presence of a small thymidine deletion in the microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10 indicate that a suitable therapeutic regimen may be a chemotherapeutic treatment that combines at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 agents (for instance, when the patient suffers from a colorectal cancer, a combination of oxaliplatin, 5-fluorouracil and folinic acid, i.e. the FOLFOX treatment, or a combination of 5-fluorouracil and folinic acid, i.e. the FUFOL or LV5FU2 treatment).

Determining the presence or the absence of genetic variations in the gene sequence encoding CBF2 and HSP1 10, the presence of the absence of a gene sequence coding for wild-type HSP1 10 and CBF2 proteins, and the length of the thymidine repetition may be performed as disclosed above.

Method for determining if a treatment with a CBF2 protein which retains its transactivation capacity is suitable for a patient suffering from cancer

In a fourth aspect, the invention relates to an in vitro method for determining if a treatment with at least one CBF2 protein which retains its transactivation capacity, or a pharmaceutical composition comprising said at least one CBF2 protein which retains its transactivation capacity is suitable for a patient suffering from cancer, comprising or consisting of the following steps :

(a) in a biological sample of said patient, determining the presence or the absence of a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity;

(b) in a biological sample of said patient, determining the presence or the absence of a genetic variation in HSP1 10 gene sequence which leads to expression of a mutant HSP1 10 protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat- shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5; and

(c) concluding that a treatment with at least one CBF2 protein which retains its transactivation capacity is suitable when both a genetic variation in the CBF2 gene sequence as defined in step (a) and a genetic variation in the HSP1 10 gene sequence as defined in step (b) are present.

In this aspect of the invention, the CBF2 protein which retains its transactivation capacity is preferably a wild-type CBF2 protein.

In an embodiment, the patient undergoes or is intended to undergo a chemotherapeutic treatment. Preferably, when the patient suffers from a colorectal cancer, the treatment is a chemotherapeutic treatment using at least an alkylating agent such as e.g. oxaliplatin, or a treatment with 5-fluorouracil, or the FOLFOX treatment, or the FUFOL treatment.

In some embodiments, the genetic variation in the gene sequence encoding

HSP1 10 leads to expression of mutant HSP1 10DE9 (SEQ ID NO: 6) and/or the genetic variation in the gene sequence encoding CBF2 leads to expression of a mutant CBF2 chosen from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 3. Method for treating a patient suffering from a cancer

In a fifth aspect, the invention relates to a method for treating a patient suffering from a cancer, the patient has both i) a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity, and ii) a genetic variation in HSP1 10 gene sequence which leads to expression of a mutant HSP1 10 protein, said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat- shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5, said method comprises the administration to the patient of an effective amount of at least one compound selected from the group consisting of:

a) a CCAT-binding factor 2 (CBF2) protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 , preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to

SEQ ID NO: 1 );

b) a peptido-mimetic of the CBF2 protein of (a); and

c) a nucleic acid encoding the CBF2 of (a). In a sixth aspect, the invention also relates to at least one compound selected from the group consisting of:

a) a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 , preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 );

b) a peptido-mimetic of the CBF2 protein of (a); and

c) a nucleic acid encoding the CBF2 of (a);

for use in the treatment of a cancer in a patient, said patient has both i) a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity, and ii) a genetic variation in heat-shock protein 1 10 (HSP1 10) gene sequence which leads to expression of a mutant HSP1 10 protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5.

In particular, the invention also relates to a combination of:

- at least one compound selected from the group consisting of:

a) a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 , preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 );

b) a peptido-mimetic of the CBF2 protein of (a); and

c) a nucleic acid encoding the CBF2 of (a);

and

- at least one chemotherapeutic agent,

for use in the treatment of a a cancer in a patient, said patient has both i) a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity, and ii) a genetic variation in heat-shock protein 1 10 (HSP1 10) gene sequence which leads to expression of a mutant HSP1 10 protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5.

Preferably, the treatment combines at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 chemotherapeutic agents, as, for example, oxaliplatin, 5-fluorouracil and folinic acid, i.e. the FOLFOX treatment, or a combination of 5-fluorouracil and folinic acid, i.e. the FUFOL or LV5FU2 treatment.

In a preferred embodiment of the above recited method and use for treating a patient suffering from a cancer according to the fifth and sixth aspects of the invention, the patient was identified by the method for selecting a patient suffering from a cancer in need of being treated with a CBF2 protein which retains its transactivation capacity according to the fourth aspect of invention disclosed above, or was previously identified/diagnosed by the method or use of the first or second aspect of the invention according to any one of the embodiments and implementations.

In an implementation of the use and method according to any one of the embodiments of the fifth and sixth aspects of the invention, the cancer has a MSI phenotype. In a seventh aspect, the invention relates to a method for treating a patient suffering from a cancer, the patient has both (i) a CBF2 gene sequence which leads to expression of a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 ), and ii) a HSP1 10 gene sequence which leads to expression of a HSP1 10 protein which exhibits chaperone activity and is capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27) (preferably a wild-type HSP1 10, more preferably the HSP1 10 at least 80% identical to SEQ ID NO: 5, more preferably a wild-type HSP1 10 with a sequence SEQ ID NO: 5), said method comprises the administration to the patient of an effective amount of at least one compound selected from the group consisting of:

a) a mutant CBF2 protein devoid of all or part of its transactivation capacity);

b) a peptido-mimetic of the mutant CBF2 protein of (a);

c) a nucleic acid encoding the mutant CBF2 of (a); and

d) an antagonist of wild-type CBF2. In an eighth aspect, the invention also relates to at least one compound selected from the group consisting of:

a) a mutant CBF2 protein devoid of all or part of its transactivation capacity);

b) a peptido-mimetic of the mutant CBF2 protein of (a);

c) a nucleic acid encoding the mutant CBF2 of (a); and

d) an antagonist of wild-type CBF2;

for use for in the treatment of a cancer in a patient, said patient has both (i) a CBF2 gene sequence which leads to expression of a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 ), and ii) a HSP1 10 gene sequence which leads to expression of a HSP1 10 protein which exhibits chaperone activity and is capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27) (preferably a wild-type HSP1 10, more preferably the HSP1 10 at least 80% identical to SEQ ID NO: 5, more preferably a wild- type HSP1 10 with a sequence SEQ ID NO: 5.

In particular, the invention also relates to a combination of:

- at least one compound selected from the group consisting of:

a) a mutant CBF2 protein devoid of all or part of its transactivation capacity);

b) a peptido-mimetic of the mutant CBF2 protein of (a);

c) a nucleic acid encoding the mutant CBF2 of (a); and

d) an antagonist of wild-type CBF2;

and

- at least one chemotherapeutic agent,

for use for in the treatment of a cancer in a patient, said patient has both (i) a CBF2 gene sequence which leads to expression of a CBF2 protein with transactivation capacity (preferably a wild-type CBF2, more preferably the CBF2 at least 80% identical to SEQ ID NO: 1 ), and ii) a HSP1 10 gene sequence which leads to expression of a HSP1 10 protein which exhibits chaperone activity and is capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27) (preferably a wild-type HSP1 10, more preferably the HSP1 10 at least 80% identical to SEQ ID NO: 5, more preferably a wild- type HSP1 10 with a sequence SEQ ID NO: 5.

Preferably, the treatment combines at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 chemotherapeutic agents, as, for example, oxaliplatin, 5-fluorouracil and folinic acid, i.e. the FOLFOX treatment, or a combination of 5-fluorouracil and folinic acid, i.e. the FUFOL or LV5FU2 treatment.

In a preferred embodiment of the above recited method and use for treating a patient suffering from a cancer according to the seventh and eighth aspects of the invention, the patient was previously identified/diagnosed by the method or use of the first or second aspect of the invention according to any one of the embodiments and implementations.

In a preferred embodiment of the seventh and eighth aspects of the invention, the mutant CBF2 protein devoid of all or part of its transactivation capacity is a CBF2 truncated protein coded by a mutant CBF2 gene sequence wherein the microsatellite repeat of 9 adenosine nucleotides located in exon 2 of the gene encoding CBF2 is deleted of 1 , 4 or 7 adenosine(s), or inserted with 1 , 4, 7, 10, 13 etc adenosine(s)). More preferably, the mutant CBF2 protein is coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosine is deleted of one adenosine (to make the reading easier, hereinafter this CBF2 truncated protein will be called "CBF2 del-1 " or "CBF2 -1 "), or by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosine is inserted with one adenosine (to make the reading easier, hereinafter this CBF2 truncated protein will be called "CBF2 lns+1 " or "CBF2 +1 ").

Most preferably, the mutant CBF2 protein is a CBF2 truncated proteins which lacks the nuclear localization signal and the hydrophobic domain is chosen from the group consisting of:

- a mutant CBF2 protein coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosines located in exon 2 is deleted of one adenosine: this CBF2 truncated protein comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO: 2, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 2.

- a mutant CBF2 protein coded by a mutant CBF2 gene sequence wherein the microsatellite sequence of nine adenosines located in exon 2 is inserted with one adenosine: this CBF2 truncated protein comprises or consists of an amino acid sequence at least 80% identical to SEQ ID NO: 3, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 3.

As used herein, the term "antagonist" refers to an agent (i.e. a molecule) which inhibits or blocks the transactivation capacity of CBF2.

The antagonists of the present invention may act by trapping CBF2 in the cytoplasm, so that CBF2 cannot move to the nucleus of the cells. Such an antagonist may be an antibody anti-CBF2.

Other example of antagonist of the present invention may be a mutant of CBF2 which can move into the nucleus and bind to HSP1 10 promoter but which as not the capacity of increasing the transcriptional activity of the HSP1 10 promoter. This antagonist may for instance interfere with wild-type CBF2 binding to HSP1 10 promoter.

Antagonists according to the present invention also include molecules which reduce or prevent the expression of CBF2, such as a nucleic acid molecule interfering specifically with CBF2 expression. Such nucleic acid is for example, an antisense oligonucleotide comprising a single-stranded polynucleotide sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences, an interfering RNA (iRNA), or a ribozyme. Said nucleic acid can have a sequence from 15 to 50 nucleotides, preferably from 15 to 30 nucleotides.

Antisense or sense oligonucleotides comprise fragments of the targeted polynucleotide sequence encoding CBF2. Such a fragment generally comprises at least about 14 nucleotides, typically from about 14 to about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a nucleic acid sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res., 1988, 48:2659), and van der Krol et al. (BioTechniques, 1988, 6:958).

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block or inhibit protein expression by one of several means, including enhanced degradation of the mRNA by RNAse H, inhibition of splicing, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of proteins. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L)-lysine. Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid by any gene transfer method, including, for example, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or lipofection, or by using gene transfer vectors such as Epstein-Barr virus or adenovirus.

Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleic acid by formation of a conjugate with a ligand-binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand-binding molecule does not substantially interfere with the ability of the ligand-binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.

Additional methods for preventing expression of the CBF2 include RNA interference (RNAi). RNAi is carried out through the use of an interfering RNA (iRNA) which is capable of down-regulating the expression of the targeted protein. As used herein, the term "iRNA" encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single- stranded RNA (ssRNA), micro-RNA (miRNA) and short hairpin RNA (shRNA) molecules, specific for pre-mRNA or mRNA of CBF2. Short hairpin RNAs (shRNA) will be cleaved by the cellular machinery into siRNA specific for pre-mRNA or mRNA of said receptors or said ligands (Paddison et al., Genes & Development, 16: 948-958, 2002

RNA interference, designate a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-translational level.

In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length. In vivo, dsRNA introduced into a cell is cleaved into a mixture of short dsRNA molecules called siRNA. siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided. The chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of siRNA. In a preferred embodiment, the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, preferably about 20-30 base nucleotides.

RNAi can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the RNAi, including modifications that make the RNAi resistant to nuclease digestion.

RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (WO 00/53722), or in combination with a cationic peptide (US 2007275923). They may also be administered in the form of their precursors or encoding DNAs.

Ribozymes can also function as inhibitors of the expression of the CBF2 receptor for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of CBF2 mRNA sequence are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

The antagonists according to the invention are capable of inhibiting or eliminating the functional transactivation of the human HSP1 10 promoter in vivo and/or in vitro. The antagonist may inhibit the functional transactivation of the human HSP1 10 promoter by at least about 10%, preferably by at least about 30%, preferably by at least about 50%, preferably by at least about 70, 75 or 80%, still preferably by 85, 90, 95, or 100%.

In an implementation of the use and method according to any one of the embodiments of the seventh and eighth aspects of the invention, the cancer has a MSS phenotype.

The nucleic acid encoding the CBF2 as defined in item (c) of the fifth and sixth aspects of the invention, and the nucleic acid encoding the mutant CBF2 as defined in item (c) of the seventh and eighth aspects of the invention, can be used in a gene therapy treatment. These nucleic acids are preferably present in a vector, preferably operably linked to a promoter and under the control a terminator and/or an enhancer allowing its expression. The vector may for example correspond to a viral vector such as an adenoviral or a lentiviral vector.

The compounds as defined in items (a) to (c) of the fifth and sixth aspects of the invention, as well as the compounds as defined in items (a) to (d) of the seventh and eighth aspects of the invention, used in the above recited method or use for treating a patient suffering from a cancer are preferably provided in a pharmaceutically acceptable carrier, excipient or diluent which is not prejudicial to the patient to be treated.

Pharmaceutically acceptable carriers and excipient that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a- tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

As appreciated by skilled artisans, compositions are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral route, including for instance intramuscular, subcutaneous, intravenous, intraperitoneal or local intratumoral injections. The oral route can also be used, provided that the composition is in a form suitable for oral administration, able to protect the active principle from the gastric and intestinal enzymes.

Further, as previously indicated the amount of compounds as defined in items (a) to (c) used in the above recited method or use for treating a patient suffering from a cancer is a therapeutically effective amount.

The exact amount of compounds as defined in items (a) to (c) of the fifth and sixth aspects of the invention, and the compounds as defined in items (a) to (d) of the seventh and eighth aspects of the invention, to be used and the composition to be administered will vary according to the age and the weight of the patient being treated, the stage of the cancer to be treated, the mode of administration, the frequency of administration as well as the other ingredients in the composition which comprises the antihypertensive drugs. Such concentrations can be routinely determined by those of skilled in the art. The amount of compounds as defined in items (a) to (c) of the fifth and sixth aspects of the invention, and the compounds as defined in items (a) to (d) of seventh and eighth aspects of the invention, actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual anti-cancer agents administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, as well as the possibility of co-usage with other agents, etc.

The total dose required for each treatment may be administered by multiple doses or in a single dose.

Depending on the intended route of delivery, the compounds may be formulated as liquid (e.g., solutions, suspensions), solid (e.g., pills, tablets, suppositories) or semisolid (e.g., creams, gels) forms. Kits according to the invention

A ninth aspect of the invention provides kits that are useful in the above methods and use of the invention.

Such kits comprise:

- means for determining the presence or the absence of a genetic variation in the

CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity; and/or

- means for determining the presence or the absence of a CBF2 gene sequence which leads to expression of a wild-type CBF2 protein; and/or

- means for determining the presence or the absence of a genetic variation in

HSP1 10 gene sequence which leads to expression of a mutant HSP1 10 protein, said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5; and/or

- means for determining the presence or the absence of a HSP1 10 gene sequence which leads to expression of a wild-type HSP1 10 protein; and/or

- means for determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10.

In some embodiments, said means for determining the presence or the absence of wild-type / mutated HSP1 10 and wild-type / mutated CBF2 as defined above may be forward primers, reverse primers and/or probes, and/or an antibody that specifically recognizes the wild-type and/or the mutated protein(s).

In some embodiments, the kits comprise forward and reverse primers which are able to hybridize to mutated HSP1 10 and wild-type HSP1 10, and two probes, being able either to hybridize specifically to mutated HSP1 10 nucleotide sequence, or to wild-type HSP1 10 nucleotide sequence as described above. In some embodiments, the kits comprise forward and reverse primers which are able to hybridize to mutated CBF2 nucleotide sequence and wild-type CBF2 nucleotide sequence, and two probes, being able either to hybridize specifically to mutated CBF2, or to wild-type CBF2 as described above. In some embodiments, the kits comprise forward and reverse primers which are able to hybridize to mutated HSP1 10 nucleotide sequence and wild-type HSP1 10 nucleotide sequence, and two probes, being able either to hybridize specifically to mutated HSP1 10 nucleotide sequence, or to wild-type HSP1 10 nucleotide sequence as described above, and or forward and reverse primers which are able to hybridize to mutated CBF2 nucleotide sequence and wild-type CBF2 nucleotide sequence, and two probes, being able either to hybridize specifically to mutated CBF2 nucleotide sequence, or to wild-type CBF2 nucleotide sequence as described above.

Also, the present invention also relates to a kit for:

(i) selecting a patient suffering from a cancer in need of being treated with a CCAAT- binding factor 2 (CBF2) protein which retains its transactivation capacity, and/or

(ii) determining a suitable therapeutic regimen in a subject suffering from cancer, in particular from a cancer liable to have MSI phenotype, and/or

(iii) prognosing survival and/or the response to a treatment of a patient suffering from a cancer, in particular from a cancer liable to have MSI phenotype,

said kit comprising:

- means for determining the presence or the absence of a genetic variation in the CBF2 gene sequence which leads to expression of a CBF2 protein devoid of all or part of its transactivation capacity consisting of forward and reverse primers which are able to hybridize to mutated CBF2 nucleotide sequence, and two probes, being able to hybridize specifically to mutated CBF2; and/or

- means for determining the presence or the absence of a CBF2 gene sequence which leads to expression of a wild-type CBF2 protein consisting of forward and reverse primers which are able to hybridize to wild-type CBF2 nucleotide sequence and two probes, being able to hybridize specifically to wild-type CBF2 nucleotide sequence; and/or

- means for determining the presence or the absence of a genetic variation in heat- shock protein (HSP1 10) gene sequence which leads to expression of a mutant HSP1 10 protein, wherein said mutant HSP1 10 protein (1 ) does not exhibit chaperone activity and/or is not capable of binding to heat-shock protein 70 (HSP70) and/or to heat-shock protein 27 (HSP27), and (2) is capable of binding to a wild-type HSP1 10 protein of SEQ ID NO: 5 consisting of forward and reverse primers which are able to hybridize to mutated HSP1 10, and two probes, being able to hybridize specifically to mutated HSP1 10 nucleotide sequence; and/or

- means for determining the presence or the absence of a HSP1 10 gene sequence which leads to expression of a wild-type HSP1 10 protein consisting of forward and reverse primers which are able to hybridize to wild-type HSP1 10, and two probes, being able to hybridize specifically to wild-type HSP1 10; and/or - means for determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10. - Means for determining the presence of the absence of a mutated HSP110 or a wild-type HSP1 10:

The forward and reverse primers hybridize respectively to a nucleotide sequence (e.g. SEQ ID NO: 15) encoding the mutated HSP1 10DE9 (SEQ ID NO: 6) or to a nucleotide sequence (e.g. SEQ ID NO: 16) encoding the wild-type HSP1 10 (SEQ ID NO: 5) or to the corresponding reverse sequences under high stringency and specific hybridization conditions.

Furthermore, the probe hybridizing to mutated HSP1 10 nucleotide sequence according to the invention specifically hybridizes to the nucleotide sequence encoding the mutated HSP1 10DE9 and the probe hybridizing to wild-type HSP1 10 nucleotide sequence according to the invention specifically hybridizes to the nucleotide sequence encoding the wild-type HSP1 10.

The forward primer for determining the presence of the absence of a mutated HSP1 10 or a wild-type HSP1 10 may have a nucleotide sequence comprising, or consisting of, SEQ ID NO: 12 or a sequence differing from sequence SEQ ID NO: 12 by one or two nucleotide substitution(s).

The reverse primer according to the invention may have a nucleotide sequence comprising, or consisting of, SEQ ID NO: 13 or a sequence differing from SEQ ID NO: 13 by one or two nucleotide substitution(s).

The probe specifically hybridizing to mutated HSP1 10 may have a nucleotide sequence comprising, or consisting of, sequence SEQ ID NO: 14 or a sequence differing from sequence SEQ ID NO: 14 by one or two nucleotide substitution(s).

The probe specifically hybridizing to wild-type HSP1 10 may have a nucleotide sequence comprising, or consisting of, sequence 5'-TACAGTGTGCAATACTT-3' (SEQ ID NO: 17) or a sequence differing from sequence SEQ ID NO: 17 by one or two nucleotide substitution(s).

In some embodiments, the invention provides a kit comprising an antibody that specifically recognizes a mutated HSP1 10 protein and an antibody that specifically recognizes a wild-type HSP1 10 as described above.

In some embodiments, said antibody that specifically recognizes a mutated HSP1 10 protein and said antibody that specifically recognizes a wild-type HSP1 10 are identical. For example, said antibody may be the Ab24503 antibody from Abeam. In some embodiments, said antibody that specifically recognizes a mutated HSP1 10 protein and said antibody that specifically recognizes a wild-type HSP1 10 are different. For example, said antibody that specifically recognizes a mutated HSP1 10 protein is able to recognize a mutated HSP1 10 protein of sequence SEQ ID NO: 6, and said antibody that specifically recognizes a wild-type HSP1 10 may be the SPA-1 101 antibody from Stressgen.

- Means for determining the presence of the absence of a mutated CBF2 or a wild-type CBF2:

The forward and reverse primers hybridize respectively to a nucleotide sequence (e.g. SEQ ID NO: 18) encoding the mutated CBF2-1 (SEQ ID NO: 2), to the nucleotide sequence (e.g. SEQ ID NO: 19) encoding the mutated CBF2+1 (SEQ ID NO: 3) and to the nucleotide sequence (e.g. SEQ ID NO: 20) encoding the wild-type CBF2 (SEQ ID NO: 1 ) or to the corresponding reverse sequences under high stringency and specific hybridization conditions.

The forward primer for determining the presence of the absence of a mutated CBF2 (in particular CBF2-1 and CBF2+1 ) or a wild-type CBF2 may have a nucleotide sequence comprising, or consisting of, SEQ ID NO: 8 or a sequence differing from sequence SEQ ID NO: 8 by one or two nucleotide substitution(s).

The reverse primer according to the invention may have a nucleotide sequence comprising, or consisting of, SEQ ID NO: 9 or a sequence differing from SEQ ID NO: 9 by one or two nucleotide substitution(s). - Means for determining the length of thymidine repetitions of a microsatellite repeat of 17 thymidine nucleotides located in the splicing acceptor site of intron 8 of the gene encoding HSP1 10:

The forward and reverse primers which can be used to detect the length of the thymidine deletions in the microsatellite repeat hybridize to the sequence SEQ ID NO: 7 or to the sequence reverse to sequence SEQ ID NO: 7 under high stringency and specific hybridization conditions.

In some embodiments, said means for detecting the length of the thymidine deletion in the microsatellite repeat may be a forward primer and a reverse primer as defined hereabove.

The forward primer according to the invention may have a nucleotide sequence comprising, or consisting of, 5'-CCCTGTCCATCCATTGGAATTGA-3' (SEQ ID NO: 10) or a sequence differing from sequence SEQ ID NO: 14 by one or two nucleotide substitution(s).

The reverse primer according to the invention may have a nucleotide sequence comprising, or consisting of, 5'-GGAACTGCATCTGTGACGGAA-3' (SEQ ID NO: 1 1 ) or a sequence differing from SEQ ID NO: 15 by one or two nucleotide substitution(s).

The forward and reverse primers and probes according to the invention are preferably 15 to 30 nucleotides long, more preferably 17 to 25 nucleotides long. In particular, said forward and reverse primers and probe may, independently from each other, be 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotide long.

Preferably, the probe is labelled. For instance, the probe can be labelled in a covalent or non-covalent manner with a wide variety of labels known in the art, including hapten labels (e.g. biotin), mass tag labels (e.g. stable isotope labels), radioactive labels, metal chelate labels, luminescent label (e.g. fluorescent, phosphorescent and chemiluminescent labels), etc.

Preferably, the probe is labelled with at least one fluorescent label or dye. The fluorescent dye can be a wide variety of dyes known in the art, including 6-FAMTM, VIC®, TETTM, NEDTM, Cy3®, Cy5®, HEX, TAMRA, DABCYL, BHQTM, DDQ, etc.

More preferably, the probe is labelled with a reporter dye and a quencher dye. Still more preferably, the probe is labelled at its 5' end with the reporter dye and at its 3' end with the quencher dye. The reporter dye may be a fluorescent dye, which can be for instance 6-FAMTM, VIC®, TETTM, NEDTM, Cy3®, Cy5®, HEX, etc. The quencher dye may be a non-fluorescent dye (e.g. MGBTM) or a fluorescent dye, which can be for instance TAMRA, DABCYL, BHQTM, DDQ, etc.

In a more preferred embodiment, the probe specifically hybridizing to mutated

HSP1 10 is labelled with the reporter dye VICTM at its 5' end and with the quencher dye TAMRA at its 3' end and the probe specifically hybridizing to wild-type HSP1 10 is labelled with the reporter dye 6-FAMTM at its 5' end and with the quencher dye TAMRA at its 3' end.

The antibodies used in the kits can be labeled with detectable compound such as fluorophores or radioactive compounds. For example, the antibody specifically binding to the wild-type and/or mutant HSP1 10 protein(s) and/or wild-type and/or mutant CBF2 protein(s) may be labeled with a detectable compound. Alternatively, when the kit comprises an antibody, the kit may further comprise a secondary antibody, labeled with a detectable compound, which binds to an unlabelled antibody specifically binding to wild- type and/or mutant HSP1 10 protein(s) and/or wild-type and/or mutant CBF2 protein(s).

The conditions of temperature and ionic strength determine the "stringency" of the hybridization. High stringency hybridization conditions correspond to a high Tm between 60 °C to 75 °C. Hybridization requires that the two raicleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.

According to an embodiment, the kits according to the invention comprise, in addition to the means for determining the presence of a genetic variation in the CBF2 gene sequence and/or in the HSP1 10 gene sequence, to the means for determining the presence of wild-type CBF2 gene sequence and/or HSP1 10 gene sequence, and/or to the means for determining the length of thymidine repetitions, in separate containers or in the same container, at least one element chosen from the group consisting of:

- a control sample comprising a known concentration of wild-type HSP1 10 protein;

- a control sample comprising a known concentration of mutant HSP1 10 protein as disclosed above;

- a control sample comprising a known concentration of wild-type CBF2 protein; and

- a control sample comprising a known concentration of mutant CBF2 protein as disclosed above.

Further, the kits may comprise instructions for the use of said kits (i) in selecting a patient suffering from a cancer in need of being treated with a CBF2 protein which retains its transactivation capacity, and/or (ii) in determining a suitable therapeutic regimen in a subject suffering from cancer (in particular from a cancer liable to have MSI phenotype), and/or (iii) in prognosing survival and/or the response to a treatment of a patient suffering from a cancer (in particular from a cancer liable to have MSI phenotype).

The means for measuring the expression level of HGF may also include reagents such as e.g. reaction, hybridization and/or washing buffers. The means may be present, e.g., in vials or microtiter plates, or be attached to a solid support such as a microarray as can be the case for primers and probes.

In a preferred implementation of the first to the sixth, and ninth aspects of the invention, according to any one of the embodiments and implementations disclosed, the genetic variation in the gene sequence encoding HSP1 10 leads to expression of mutant HSP1 10DE9 (SEQ ID NO: 6) and/or the genetic variation in the gene sequence encoding CBF2 leads to expression of a mutant CBF2 chosen from the group consisting of SEQ ID NO: 2 or SEQ ID NO: 3. All the sequences with the accession numbers given in the application are those present in the cited database at the date of filing. All references cited herein, including journal articles or abstracts, published patent applications, issued patents or any other references, are entirely incorporated by reference herein, including all data, tables, figures and text presented in the cited references.

The invention will be further illustrated by the following figure and examples. However, these examples and figure should not be interpreted in any way as limiting the scope of the present invention. Brief description of the figures

Figure 1 illustrates the mutation frequency in 18 genes containing a coding microsatellite sequence.

Figure 2 depicts the gene sequences coding for wild-type CBF2 and mutated CBF2 proteins which lack their NLS (nuclear localization signal) and hydrophobic domain. CBF2 mutations have been found in MSI colorectal tumors. Frameshift mutations affecting the A 9 microsatellite in exon 2 cause premature stop codon formation and truncation of the protein. Sequences of the aberrant C-terminal tails are indicated (including 2 or 1 1 aminoacids, respectively).

Figure 3 illustrates the expression of CBF2 wmRNAs in series of 35 (A) and 20 (B) MSI primary colorectal tumors.

Figure 4 illustrates the analysis of the transcriptional activity of CBF2.

A) Transfection of HCT1 16 cells with a CCAAT-HSP40/HSP1 10 promoter containing vector fused to the luciferase gene. The data refer to the average fold activation of luciferase after 30h post transfection.

B) Quantification of HSP110 gene expression in two groups of MSI primary colorectal tumors (i.e. CBF2 mutated or CBF2 wt).

C) Quantification of endogenous HSP1 10, HSP1 10DE9 and HSP70 mRNAs in HCT1 16 cells transiently transfected with CBF2 (after 48h). ( * P < .05; *** P < .001 ).

Figure 5 illustrates a correlation analysis between CBF2, HSP1 10 and HSP70 expression. The expression of CBF2 and HSP1 10 are significantly correlated when measured in a large series of MSS CRCs (Left panel). We found no evidence for a correlation between CBF2 and HSP70 expression in the same conditions (Right panel).

Figures 6 and 7 illustrates the ability or non ability of wild type CBF2 and CBF2 mutants to activate endogenous expression of HSP1 10 ( Fig. 6) and HSP70 (Fig. 7)

Figure 8 illustrates that in MSI CRC cells displaying HSP1 10 T17 deletions, CBF2 transact! vates HSP1 10DE9

Figure 9 shows that after 24 hours, HSP1 10 begins to be degraded in HCT1 16 whereas it is not the case of the HSP1 10DE9

Figures 10 and 11 shows the effect of inhibition of CBF2 on CRC cells proliferation using ShRNA in clones displaying small HSP1 10 T17 deletion status (Fig.10) and large HSP1 10 T17 deletion status (Fig.11 )

Figures 12 and 13 shows the effect of inhibition of CBF2 using ShRNA on cell death with TRAIL in clones displaying small HSP1 10 T17 deletion status (Fig.12) and large HSP1 10 T17 deletion status (Fig.13)

Figure 14 depicts a Survival analysis in stage II and stage III MSI colorectal cancer patients according to the size of deletions in the HSP110 T 17 DNA repeat. Patients were classified into four groups according to the size of deletion in the T 17 (DT > 5 bp, T 11 ; T 10 , T 9 for MSI HSP110-Large patients; 0< DT < 5, T 17 to T 12 for MSI HSP770-Small patients) and CBF2 mutation status.

EXAMPLE 1 : Materials and Methods

Tumor samples, cell lines. CRC cell lines were purchased from American Type Culture Collection (see Http://www.cephb.fr/fr/qaccc/). Primary tumors and normal colonic tissues were obtained from patients undergoing surgery. The MSI status was determined as described (18).

Mutation analysis. Tumor DNA from sample was extracted using QIAmp DNA Tissue Kit (Qiagen). Specific primers for HSP110 intron 8 (forward primer of sequence 5'- CCCTGTCCATCCATTGGAATTGA-3' (SEQ ID NO: 10) and reverse primer of sequence 5'-GGAACTGCATCTGTGACGGAA-3' (SEQ ID NO: 1 1 )) and CBF2 exon 2 (forward primer of sequence 5'-CCATGAAGAAAGTGAATTGG-3' (SEQ ID NO: 8) and reverse primer of sequence 5'- TCCCTTACTTTGTCATCACC -3' (SEQ ID NO: 9)) were designed using e-primer3 (https://bioweb.pasteur.fr/seganal/interfaces/eprimer3.html) . PCR reaction were done in a final volume of 20 μΙ containing 100 ng od genomic DNA, 0.15 μΜ of each primer and 1 unit of HotStarTaq DNA polymerase (Qiagen). The thermal cycling condition comprised an initial denaturation step at 94°C for 10 minutes and 40 cycles at 94°C for 30 s and 57°C for 30 s. Fluorescent PCR products were run on ABI3130 Genetic Analyzer (Applied Biosystems).

Plasmids, transfection, luciferase activity and fluorescence. EGFP-CBF2 wt and mutants were constructed by Proteogenix. Transfection were done using turbofect reagent (Fermentas) in 6-well plate. Luciferase assay was performed as described (8) for measurement of luciferase activity, Dual-Luciferase assay system (Promega) was used. HCT1 16 cells grown in 24-well plate were transfected using turbofect transfection reagent according the manufacturer's instructions with EGFP-CBF2, EGFP-CBF2+1 , EGFP- CBF2-1 or empty vector EGFP-C2. Live acquisitions were done two day after transfections were recorded with inverted fluorescence microscope (Olympus 1X81 ).

Real-Time quantitative RT-PCR analysis of HSP110 (wild-type and mutant).

Total RNA was isolated using RNeasy minikit (Qiagen). For quantitative RT-PCR, we used the Applied SDS Biosystems analysis software. Primers and internal probes were designed using Primer Express (Applied Biosystems) and synthesized by Applied Biosystems: the forward primer has the sequence 5'-GCTACACGAATTCCAGCTGTGA-3' (SEQ ID NO: 12), the reverse primer has the sequence 5'- GAGCAGCATGGTTTCGACTAAA-3' (SEQ ID NO: 13), the probe which specifically hybridizes to the sequence encoding HSP1 10delE9 comprising the genetic variation has the sequence 5'-ATGTGCATTACAGTGTTC-3' (SEQ ID NO: 14) labelled with the reporter dye VIC™ at its 5' end and with the quencher dye TAMRA at its 3' end, the probe which specifically hybridizes to the sequence encoding wild-type HSP1 10 has the sequence 5'- 6-TACAGTGTGCAATACTT-3' (SEQ ID NO: 17) labelled with the reporter dye 6-FAM™ at its 5' end and with the quencher dye TAMRA at its 3' end.

The thermal cycling conditions comprised an initial denaturation step at 95°C, 10 min, 40 cycles at 95 °C 15 sec, and 60 °C, 1 min.

Genomic and gene expression arrays and analysis. Gene expression analysis using arrays was carried out on the IGBMC microarray platform (Strasbourg, France). Total RNA was amplified, labeled and hybridized to Affymetrix Human Genome U133 plus2 GeneChips following the manufacturer's protocol (Affymetrix, Santa Clara, CA). The chips were scanned with the Affymetrix GeneChip Scanner 3000 and raw intensities were quantified from subsequent images using GCOS 1 .4 software (Affymetrix). Data were normalized using the Robust Multi-array Average method and implemented in the R package affy (Irizarry RA et al., Nucleic Acids Res., 31 : e15, 2003). Patients. Patients included those who underwent curative surgical resection of histologically proven MMR-deficient CRC and for whom tumor tissue could be retrieved from tumor collection. Survival curves were calculated according to the Kaplan-Meier method with an end-point at 5 years. Differences between curves were assessed using the log-rank test. Univariate and multivariate associations for outcome were performed using the Cox regression model.

Statistical analysis. All statistical analyses were stratified according to clinical centers in order to satisfy the sample independence assumption and to take into account the potential heterogeneity of different centers. For the analysis of associations with patient outcome, RFS was used and this was defined as the time from surgery to the date of first recurrence (relapse, or death from CRC) or last contact. RFS was considered as censored when the patient was alive without relapse at last contact. Survival curves were obtained according to the method of Kaplan and Meier and differences between survival distributions were assessed by log-rank test using an endpoint of five years. Univariate and multivariate models were computed using Cox proportional-hazards regression. For multivariate analyses, only those variables with information available for all sample groups were included in models. Survival analyses were performed using the R package survival. For all analyses, P values of less than 0.05 were considered to indicate statistical significance.

EXAMPLE 2: Mutations of CBF2 are observed with those of other target genes for microsatellite instability in CRC

Mutations in CBF2 and 17 other target genes {ATR, BAX, BLM, GRB14, GRK4, IGF2R, MBD4, MSH3, MSH6, RAD50, RECQL, RIZ, CDX2, TCF4, TFDP2, TGFBR2, SLC35F5) ave been extensively characterized with the aim to have a precise idea of their overall frequencies in these malignancies. The search was undertaken by PCR and fluorescence genotyping in a multicentric cohort including several hundreds of patients with primary colon tumor whose MSI phenotype was prospectively identified at diagnosis (Figure 1 ). CBF2 mutations were detected in 27,5% of tumor samples (F = 109/396). Mutations were either 1 bp deletion or insertion causing protein truncation (Figure 2). mutant CBF2 proteins (i.e. del-1 or lns+1 ) lost important functional domains consequently, e.g. a nuclear localization signal (NLS) and a hydrophobic domain that represent a conserved domain involved in protein-protein interaction among transcription factors (Figure 2) (Lum LS et al., Mol. Cell. Biol., 10: 6709-17, 1990). EXAMPLE 3: Mutant CBF2 mRNAs are degraded in primary tumors.

Expression of CBF2 mRNA has been quantified in two independents series containing 35 and 20 MSI primary colorectal tumors using gene expression arrays. CBF2 mRNA were significantly more expressed in CBF2 wild-type tumors compared to CRC displaying CBF2 mutations (P < .05; Figure 3A and B).

EXAMPLE 4: The mutant CBF2 protein has lost its transactivation capacity

The transactivation ability of wild type and mutant CBF2 proteins have been compared using an HSP40 promoter (highly homologous to the one of HSP110) containing CCAAT boxes (Luciferase assay). HCT1 16 colon cancer cells were co- transfected with CBF2 (CBF2 wt , CBF2 m/ins+ , CBF2 m/de ) and HSP40/LUC expression vector. The results clearly demonstrate that mutant CBF2s have lost their transactivation ability (P < .001 ; Figure 4A).

Besides, the inventors quantified the expression of HSP110 mRNA in a series of 35

MSI primary colorectal tumors that displayed the CBF2 mutation or not using gene expression arrays. Expectedly, HSP110 mRNA was found to be significantly more expressed in CBF2 wild-type tumors compared to CRC displaying CBF2 mutations (P < 0.05; Figure 4B).

Using quantitative RT-PCR, an increased expression of the endogenous HSP110 chaperone (by more than 2 folds) was observed following transient transfection of HCT1 16 cells with a CBF2 wt vector (Figure 4C).

EXAMPLE 5: CBF2 and HSP110 expression are correlated

In a large series of MSS CRCs (that are MMMR-proficient and thus do not display mutations in CBF2 nor in HSP110), the expression of CBF2, HSP1 10 and HSP70 were determined. A highly significant correlation between CBF2 and HSP1 10 expressions at the mRNA level was observed (P = 4.8x10 "14 ; Figure 5, left panel). In contrast, there was no significant correlation between CBF2 and HSP70 expression (Figure 5, right panel).

EXAMPLE 6: Wild type CBF2 and CBF2 mutants ability or non ability to activate endogenous expression of HSP110 and HSP70

As shown in Figures 6 and 7, it has been shown that wild type CBF2 but not CBF2 mutants is able to activate endogenous expression of HSP1 10 and HSP70. In addition, in MSI CRC cells displaying HSP1 10 T17 deletions, it has been demonstrated that CBF2 transactivates both HSP1 10 and HSP1 10DE9 isoforms, expectedly (see Figure 8).

EXEMPLE 7: CBF2 mutations silencing leads to modify the expression ratio of

HSP110 and HSP110DE9 in tumor cells and increases resistance to apoptosis of MSI CRC cells with small HSP110 mutations

It has been shown by the inventors that in MSI CRC cells, CBF2 mutations always occur within the context of HSP1 10DE9 expression due to HSP1 10 T17 deletions.

Of interest, it was observed increased stability of HSP1 10DE9 mutant compared to

HSP1 10wt when inhibiting protein synthesis with cycloheximide (see Figure 9). This indicates the transactivation by CBF2 of a mutated HSP1 10 allele leads to increase the HSP1 10DE9/HSP1 10wt protein ratio in MSI colon tumors (chemosensitizing effect).

In addition, inhibition of CBF2 using ShRNA showed to lead to the proliferation of CRC cells. This functional impact was mainly observed in clones displaying large HSP1 10 T17 deletion and high expression of HSP1 10DE9 (see Figures 10 and 11 ).

Inhibition of CBF2 using ShRNA showed to inhibit cell death following treatment with TRAIL. This functional impact was mainly observed in clones displaying large HSP1 10 T17 deletion and low expression of HSP1 10DE9 (see Figures 12 and 13).

EXEMPLE 8: The mutant CBF2 proteins that have no more NLS have lost their capacity to reach the nucleus in HCT116 colon cancer cells.

Using GFP-expression vector, the inventors investigated the capacity of CBF2 to reach the nucleus. Expectedly, mutant CBF2s showed an aberrant cellular localization (e.g. restricted to the cytosol) compare to CBF2wt (data not shown).

EXAMPLE 7: CBF2 mutations are clinically relevant in patients with MSI CRC

The inventors next investigated the putative clinical relevance of CBF2 mutations in a cohort of 337 patients who underwent curative surgical resection of histologically proven stage ll-lll MSI CRC for whom clinical data and appropriate tissue material were available. In patients with stage III tumors (N = 100), CBF2 mutations were associated with worse relapse-free survival (RFS) in univariate (HR, 0.47 [95% confidence interval (17), 0.22-1 ], Log-rank P=.047) and multivariate analyses (HR, 0.47 [95% CI, 0.22-0.99], P=.048) (see Table 1 below). No survival difference was apparent for stage II CRC patients (see Table 1 below). Nevertheless, stage II and stage III patients who received chemotherapy and CBF2 mutations showed worse RFS compared to wild type CBF2 patients (see Table 1 below).

Of interest, it was observed that such a clinical impact of CBF2 in MSI CRC was likely to depend on the status of the mutational status of HSP1 10 chaperone (Figure 14 and Table 1 below) when stage III MSI CRC patients were clustered into 4 groups according to large (DT >5 bp; HSPH OLarge) or small (0 < DT < 5; HSP1 10 Small) somatic HSP1 10 T 7 deletions and CBF2 mutations status in tumor DNA. Among the 18 target genes for MSI whose mutational status was determined in MSI CRCs (Figure 1 ), CBF2 was the only one whose mutations were clinically relevant (data not shown).

Table 1 : Association of clinical annotations to HSP1 10 status and CBF2 mutation in MSI colorectal cancer patients in univariate and multivariate Cox analyses

UN IVARIATE ANALYSIS MULTIVARIATE ANALYSIS

Annotation Value

Modality Model Modality model

TNM3 Patients n.event H.R. 95%C.I. P value P value n H.R. 95%C.I. P value P value

(Wald) (Logrank) (Wald) (Logrank)

HSP1 10 wt 104 32 5,1 1 .2-21 0,028 0,015 100 5 1 .2-21 0,03

7,10E-03 CBF2 wt 100 31 0,5 0.22-1 0,051 0,047 100 0,47 0.22-0.99 0,048

UN IVARIATE ANALYSIS MULTIVARIATE ANALYSIS

Annotation Value

Under chemotherapy modality Model modality model Patients n n.event H.R. 95%C.I. P value P value n H.R. 95%C.I. P value P value

(Wald) (Logrank) (Wald) (Logrank)

HSP1 10 wt 74 18 7,9 1 -61 0,048 0,020 69 7,5 0.97-58 0,054

6,40E-03 CBF2 wt 69 17 0,3 0.12-0.93 0,036 0,029 69 0,34 0.12-0.93 0,036

UN IVARIATE ANALYSIS MULTIVARIATE ANALYSIS

Annotation Value

Modality Model Modality Model

TNM2 Patients n n.event H.R. 95%C.I. P value P value n H.R. 95%C.I. P value P value

(Wald) (Logrank) (Wald) (Logrank)

HSP1 10 wt 233 38 0,7 0.35-1 .5 0,37 0,37 214 0,74 0.35-1 .6 0,45 0,45 CBF2 wt 214 34 0,9 0.44-1 .9 0,79 0,79