Method for Generating a Profile of the DNA Repair Capabilities of Tumour Cells and the Uses Thereof

Abstract

The invention relates to a method for generating a profile of DNA repair capacities of tumor cells and uses thereof for cancer prognosis, choice, monitoring and/or the prediction of the therapeutic efficacy of a cancer treatment in a patient, and also for screening anticancer drugs. The invention also relates to a reference library comprising profiles of DNA repair capacities for various subtypes of a cancer, obtained by the method of the invention, and to uses thereof for the classification of cancers.

Claims

1-11. (canceled)

12. An in vitro method for generating a profile of DNA repair capacities of tumor cells, comprising at least the following steps: a) incubating a tumor cell extract comprising a DNA repair activity with at least two damaged DNA molecules comprising distinct DNA lesions and at least two different labeled nucleotides; b) measuring the quantity of each labeled nucleotide incorporated in each damaged DNA molecule resulting from the activity of the repair enzymes present in said cellular extract in step a); c) determining, from the values measured in step b), at least one of the following parameters: c1) the enzymatic signature of the DNA repair; c2) the contribution of the repair of each DNA lesion independently for each labeled nucleotide compared to the total repair; and c3) the relative rate of incorporation of each labeled nucleotide for at least two DNA lesions and independently for each DNA lesion; and d) establishing the profile of DNA repair capacities of said tumor cells based on the parameters determined in step c).

13. The method according to claim 12, wherein said tumor cells are isolated from samples of different previously characterized tumors, and at least one reference library comprising reference profiles of various subtypes of a cancer including at least the cancer subtypes of the patients to be tested are obtained.

14. The method according to claim 12, wherein the tumor is a metastatic melanoma for which the mutational status of the BRAF and NRAS genes is determined.

15. The method according to claim 12, wherein step a) is performed with: at least one first DNA molecule comprising a single type of lesion repaired by the base excision repair system selected from the group consisting of: 8-oxo-G; abasic sites; Ethenobases; thymine and/or cytosine glycols, and at least one second DNA molecule comprising a type of lesion repaired by the nucleotide excision repair system chosen from photoproducts.

16. The method according to claim 12, wherein said damaged DNA molecules are supercoiled plasmids.

17. The method according to claim 12, wherein the different labeled nucleotides comprise at least labeled dGTP and dCTP nucleotides.

18. The method according to according to claim 12, wherein the profile of step d) comprises: the enzymatic signature of the DNA repair, the contribution of the repair of each lesion independently for each labeled nucleotide compared to the total repair; and the relative rate of incorporation of each labeled nucleotide for at least two DNA lesions, independently for each DNA lesion.

19. The method according to claim 12, wherein the profile of step d) is compared with a reference library.

20. A method for cancer prognosis in a patient, comprising generating a profile of DNA repair capacities of the patient tumor cells according to the method of claim 12 and establishing the prognosis of the patient based on the profile of DNA repair capacities of the patient.

21. A method for the choice, monitoring and/or prediction of therapeutic efficacy of a cancer treatment in a patient, comprising generating a profile of DNA repair capacities of the patient tumor cells according to the method of claim 12 and administering a cancer treatment to the patient based on the profile of DNA repair capacities of the patient.

22. A reference library containing metastatic melanoma samples of mutated BRAF, mutated NRAS, or non-mutated for BRAF and NRAS and the profiles of repair capacities of the various subtypes of said cancer obtained by the method according to claim 12.

23. A method for classification of a cancer of a defined type, comprising at least the following steps, starting from a patient tumor sample: i) establishing the profile of DNA repair capacities of tumor cells isolated from said sample according to the method of claim 12; ii) comparing the profile of repair capacities obtained in the preceding step with a reference library containing profiles of repair capacities of various subtypes of said cancer type; and iii) determining the cancer subtype affecting the patient by similarity of the profile of repair capacities obtained from the patient with a reference profile of a cancer subtype from the reference library.

24. The method according to claim 13, wherein said cancer subtypes guide the treatment choice for the patients.

25. The method according to claim 15, wherein the Ethenobases are Etheno-guanines and/or Etheno-Adenines.

26. The method according to claim 15, wherein the photoproducts comprise a mixture of cyclobutane type pyrimidine dimers and pyrimidines-pyrimidiones (6-4).

27. The method according to claim 16, wherein the supercoiled plasmids are immobilized on a solid support.

28. The method according to claim 17, wherein the different labeled nucleotides comprise labeled dCTP, dGTP, dATP and dUTP nucleotides.

Description

[0201] FIG. 1 shows the repair signatures of various DNA lesions by metastatic melanoma extracts, in presence of various labeled nucleotides. A. dCTP. B. dGTP. C. dATP. D. dUTP. 8oxoG: 8-oxo-guanine. AbaS: abasics sites. CPD: Cyclobutane type pyrimidine dimers CPD-64: a mixture of cyclobutane type pyrimidine dimers (CPD) and pyrimidines-pyrimidiones (6-4) (6-4 photoproducts or 6-4PP). Etheno: mixture of Etheno-guanines (Etheno-G) and Etheno-adenines (Etheno-A). Glycols: Mixture of thymine and cytosine glycols.

[0202] FIG. 2 shows the classification of repairs signatures by repair similarity. A. All data together. B. dCTP. C. dGTP. D. dATP. E. dUTP.

[0203] FIG. 3 shows the contribution from each repair pathway to the total repair for each of the labeled nucleotides. A. dCTP. B. dGTP. C. dATP. D. dUTP. X: not tested.

[0204] FIG. 4 shows the relative incorporation rate of each labeled nucleotide for the repair of each of the lesions. A. 8oxoG: 8-oxo-guanine. B. AbaS: abasic sites. C. CPD: Cyclobutane type pyrimidine dimers D. CPD-64: a mixture of cyclobutane type pyrimidine dimers (CPD) and pyrimidines-pyrimidiones (6-4) (6-4 photoproducts or 6-4PP). E. Glycols: Mixture of thymine and cytosine glycols. F. Etheno: mixture of Etheno-guanines (Etheno-G) and Etheno-adenines (Etheno-A). *: only dCTP and dGTP were used.

[0205] FIG. 5 shows the correlation between survival (abscissa) and intensity of the repair signal (ordinate) by mutation group and by dNTP. A. WT-dCTP. B. NRAS-dCTP. C. BRAF-dCTP. D. WT-dGTP. E. NRAS-dGTP. F. BRAF-dGTP. G. WT-dATP. H. BRAF-dATP. I. WT-dUTP. J. BRAF-dUTP.

EXAMPLE: PRODUCTION OF REPAIR SIGNATURES AND REPAIR PROFILES FROM METASTATIC MELANOMA SAMPLES

1. Material and Methods

1.1 Patients

[0206] The samples (node or tumor) are from patients with a known metastatic melanoma subtype (BRAF; NRAS; WT (not mutated for BRAF and NRAS)) whose clinical data are presented in Table I.

[0207] The various treatment types administered to the study patients are shown in Table II below.

TABLE-US-00001 TABLE II Treatment Administered to the Patients Name Type DERMA Vaccine with melanoma specific antigen 3 MAGE-A3 antigen-specific cancer immunotherapeutic (ASCI) Ipilimumab anti CTLA-4 monoclonal antibody Nivolumab anti-PDL-1 monoclonal antibody Pembrolizumab anti PD-1 monoclonal antibody Dabrafenib mutated BRAF enzyme inhibitor Vemurafenib muted BRAF enzyme inhibitor Trametinib MEK enzyme inhibitor RadioT Radiation therapy

1.2 Sample Preparation

[0208] The tumor or lymph node samples are collected by surgical resection and then prepared as follows.

a. Cellular Dissociation

[0209] The cellular dissociation is performed starting from a biopsy of metastatic melanoma lymph nodes or tumors. The biopsy, about 5 mm.sup.3, is placed in a 100 mm diameter petri dish with a few drops of RPMI-1640 Glutamax (Life Technologies). It is then cut in fragments using a scalpel. These fragments are transferred to a 15 mL centrifuge tube containing 5 mL of digestion medium (1 mg/mL Collagenase D (Roche), 50 IU/mL DNase I (Roche), RPMI-1640 Glutamax (Life Technologies)). The tube containing the fragments is incubated 30 minutes at 37 C. in an oven and the fragments are returned to suspension every 10 minutes using a 10 mL pipette. At the end of the incubation, a volume of 2.5 mL of digestion medium stored at room temperature is added. The fragments are again incubated for 30 minutes at 37 C. in an oven and returned to suspension every 10 minutes using a 10 mL pipette. At the end of incubation, the tube is centrifuged at 1200 RPM for 10 minutes at 4 C. The supernatant is eliminated and the pellet is taken up in 10 mL of cold PBS-EDTA buffer (10 mM EDTA (Sigma), 1X D-PBS (Life Technologies)). The cells are then deposited on a 70 um cellular sieve (Dutcher), positioned on a 50 mL centrifuged tube stored in ice. The 15 mL tube is rinsed by adding an additional 5 mL of cold PBS-EDTA buffer and the residual fragments are passed through the sieve. A sample is taken in order to count the cells. The 50 mL tube is centrifuged at 1200 RPM for 10 minutes at 4 C. The supernatant is eliminated and the pellet is taken up in an Albumin/DMSO (90% Human Albumin, 10% DMSO (Sigma)) cryopreservation solution at a rate of 410.sup.6 cells per cryo-tube. The cells are progressively frozen at 80 C. in a freezer container and then transferred to liquid nitrogen for long-term storage.

b. Preparation of Nuclear Extracts

[0210] The tumor cells are thawed at ambient temperature, and then centrifuged at 500 RCF (Relative Centrifugal Force) for five minutes of 4 C. The supernatant is eliminated and the cells washed with 1 mL of cold PBS and then re-centrifuged at 500 RCF for five minutes at 4 C. The cells are taken up in 1 mL of buffer A (10 mM HEPES pH 7.8, 1.5 mM MgCl.sub.2, 10 mM KCl, 0.02% Triton X-100, 0.5 mM DTT, 0.5 mM PMSF (phenylmethylsulfonyl fluoride) and incubated for 10 minutes in ice. Each tube is vortexed for 30 seconds. The cells are centrifuged five minutes at 2300 RCF, the supernatant is eliminated, and then the cells are taken up and 30 L of buffer B (10 mM HEPES pH 7.8, 1.5 mM MgCl.sub.2, 400 mM KCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 100 L antiproteases 0.7 X (Complete-mini, Roche). After 20 minutes incubation over ice, lysis of the nuclear membrane is done by two freeze/thaw cycles in liquid nitrogen at 180 C. and 4 C. The tubes are centrifuged at 16,000 RCF for 10 minutes at 4 C. The supernatant is distributed in 10 L aliquot portions. A fraction of the supernatant is collected for proteomic assay by using the micro-BCA method (Interchim). The aliquot portions undergo rapid freezing over 30 seconds in liquid nitrogen at 180 C. and then are stored at 80 C.

1.3 Analysis of DNA Enzymatic Repair Activities

[0211] The analysis was done on chips comprising lesioned plasmids, prepared as previously described (Millau et al., Prunier et al., op. cit.).

[0212] The reaction mixture is prepared for each dNTP-biotin, so as to get DNA repair enzymatic signatures in the presence of various dNTP labeled with biotin. The reaction lasted three hours at 30 C. in reaction chambers filled with 12 L of medium made up of 4.8 L of 5X buffer (200 mM Hepes KOH pH 7.8, 35 mM MgCl.sub.2, 2.5 mM DTT, 1.25 M of each unlabeled dNTP (Perkin Elmer), 17% Glycerol, 50 mM phosphocreatine (Sigma), 10 mM EDTA, 250 g/mL creatine phosphokinase, 0.5 mg/mL BSA, 0.5 L ATP 100 mM (Amersham) and 1.25 M of dNTP-biotin (Perkin Elmer)), in the presence of nuclear extracts at a final concentration of 0.2 mg/mL, with the solution topped off to 24 L with H.sub.2). Each sample is tested on two chips (in two reaction chambers) for each of the dNTP-biotin.

[0213] After incubation, the slides are rinsed twice for three minutes with PBS/Tween 0.05%, and then twice for three minutes with MilliQ water. The slides are incubated in a 225 ng/mL bath of Streptavidine-Cy5, in the presence of 0.1 mg/mL of BSA for 30 minutes at 30 C. The slides are rinsed twice for three minutes with PBS/Tween 0.05%, and then twice for three minutes with MilliQ water. Finally, the slides are centrifuged for 30 seconds and then dried for 10 minutes at 30 C. The slides are scanned at 635 nm (Innoscan Innopsys) for quantification of the fluorescence. The fluorescence value of the control plasmid (no lesions) is subtracted from the total intensity value obtained for each lesion. The data are normalized as described in Millau et al., op. cit. The data are expressed in Fluorescence Intensity (Arbitrary Units) and standard deviation for each sample and each of the dNTP tested.

1.4 Statistical Analyses

[0214] The Fluorescence Intensity values obtained are centered-reduced, meaning that their average is brought to 0 and the standard deviation to 1.

[0215] The data are then analyzed by hierarchical classification such as described in the Forestier et al publication, by using the Euclidean distance (PLoS One. 2012; 7:e51754).

2. Results

2.1 Repair Signatures

[0216] The method according to the invention was implemented on metastatic melanoma samples (lymph nodes or tumors) for patients with known BRAF and NRAS gene mutation status (Table I). The repair reactions were done with 0.2 mg/mL of nuclear protein extract in duplicate for three hours at 30 C. The fluorescence intensities were measured with a scanner in order to quantify the level of incorporation of each triphosphate nucleoside in each plasmid. The fluorescence intensities obtained with the control plasmid are subtracted (I.sub.X1I.sub.c).

Repair Signatures with the Various DNTP

[0217] The different labeled nucleotides reveal different distribution value profiles for a single lesion for each mutation group (FIGS. 1A to 1D) allowing to thus get more precise information on the specific distribution mechanisms.

[0218] BRAF and NRAS mutations affect the DNA repair signature at different levels (overall and specific activities). In particular, the samples with the mutated BRAF gene show a very low repair activity compared to wild-type.

Classification of Repair Signatures, All Data Combined

[0219] The data are then centered-reduced (average to 0; standard deviation to 1) for each lesion, so as to allow unbiased classification.

[0220] Next, a hierarchical classification is done with Euclidean distance. The samples, in sufficient number, serve both to make up the reference library comprising the classification of the repair signatures depending on the mutations (e.g. BRAF, NRAS), and to determine the differences of each of the samples compared to the theoretical class thereof.

[0221] The samples are classified by repair signature similarity. The results show that the various dNTPs determine different classes up to a few exceptions (FIG. 2A). This indicates that each dNTP provides distinct and complementary information about the samples.

[0222] In particular, when all the results are considered and classified together, it is seen that the samples analyzed with dCTP remain grouped but separated by mutation group. This shows that the BRAF and NRAS mutations exert a dominant effect on the DNA repair signature.

[0223] This confirms that the analysis by dCTP shows that mutations in the signaling pathways (MAP Kinase) impact the DNA repair signature. This information is important and places a particular role for the analysis by dCTP, especially if the treatment applied is the targeted therapy (guided by the BRAF and NRAS mutations).

[0224] When dGTP is used, the WT are on one side, and the mBRAF and mNRAS are in another class. Two categories of mBRAF samples are identified: a first group has a weak repair activity and the second group, combined with the mNRAS, has a high repair activity (FIG. 2C).

[0225] The results obtained with dATP and especially dUTP are more fragmented.

[0226] The individual analyses by dNTP provide additional information (FIGS. 2B, C, D, E).

Classification of Repair Signatures Obtained with Labeled dCTP

[0227] The use of dCTP discriminates the mutated BRAF from other sample categories (FIG. 2B). All the BRAF samples are in a shared class. It is however separated into two subclasses, which distinguishes two categories of mutated BRAF samples (Class 1.1 containing 1507 and 1514; and Class 1.2 containing 1405, 1502 and 1401).

[0228] Class 1.1 corresponds to patients who have the longest survival.

[0229] The samples 1404_WT and 1407_WT are classified together (FIG. 2B).

[0230] In contrast, the sample 1506_WT is classified with the majority of mutated BRAF samples (FIG. 2B). It therefore has a profile similar to the mutated BRAF samples from Class 1.1 (1507 and 1514) and not to the WT samples.

[0231] The mutated NRAS samples are in a shared group (FIG. 2B). However the sample 1402_NRAS is different; it is part of a specific profile (FIG. 2B) which has a very low survival.

Classification of Repair Signatures Obtained with Labeled dGTP

[0232] For dGTP, as for dCTP, the samples 1404_WT and 1407_WT are classified together which confirms the similarity of their profile (FIG. 2C). Similarly, 1506_WT is classified separately, with the mutated BRAF samples (1502, 1507 and 1514), (FIG. 2C).

[0233] All the mutated BRAF and mutated NRAS samples are classified in a single group, separated into two subgroups (FIG. 2C). The samples 1405_BRAF and 1401_BRAF are close to mutated NRAS in this classification (FIG. 2C).

[0234] dGTP distinguishes between the WT samples for BRAF and NRAS and the mutated samples for BRAF and NRAS; by using this nucleotide, the samples having activated pathways can be distinguished from samples having inactivated signaling pathways.

Classification of Repair Signatures Obtained with Labeled dATP

[0235] For dATP, as for dCTP and dGTP, the samples 1404_WT and 1407_WT are classified together which confirms the similarity of their profile (FIG. 2D).

[0236] All the mutated BRAF are classified in the same group, which again includes 1506_WT, confirming that the sample has a profile similar to the mutated BRAF samples (FIG. 2D).

[0237] The use of dATP does not discriminate between the two WT (1404 and 1506) of the NRAS (1505 and 1511) except for 1408_NRAS which shows a specific profile (FIG. 2D).

Classification of Repair Signatures Obtained with Labeled dUTP

[0238] For dUTP, as for dCTP, dGTP and dATP, the samples 1404_WT and 1407_WT are classified together which confirms the similarity of their profile (FIG. 2E).

[0239] The use of dUTP reveals a similarity of the samples 1404_WT and 1407_WT with 1505_NRAS (FIG. 2E).

[0240] The set of the other samples is in a class divided in two, where the mutation groups are mixed but where 1506_WT is found with the mutated BRAF and in particular 1507_BRAF, 1502_BRAF and 1401_BRAF (FIG. 2E).

[0241] As with dGTP, the 1405_BRAF sample is close to mutated NRAS samples in this classification made from the dUTP data (FIG. 2E).

[0242] In conclusion, despite the recognized genomic diversity of metastatic melanomas, the method according to the invention brings out repair type signatures for each of the mutations in the BRAF or NRAS genes and in the group not comprising mutations in these genes.

[0243] By using various labeled dNTPs, profile similarities can be identified which cannot be determined on the basis of mutations alone and classes can be established beyond the classification by mutation type, which is not sufficient for predicting the response to therapies. A combined analysis of the results obtained with the various dNTPs reveals unexpected information and brings out new subgroups.

[0244] The dNTP by dNTP classifications identify mismatching patients for the classification of subtypes by the detection of mutations and consequently identifies dysfunctions in the regulation of DNA repair by the signaling pathways. This information can be used in the case of prescriptions for targeted therapy, for which it is the mutations in the key genes like BRAF and NRAS which guide the prescription.

[0245] The various repair profiles observed in each subtype of melanoma (WT, mutated BRAF, NRAS) are compared with the repair profiles from the reference library in order to determine the profiles which correspond to patients responding to the targeted therapies. This comparison is thus used to administer the right treatment to the right group of patients.

2.2 Contribution of Each Repair Pathway to the Total Repair

[0246] For the calculation of the contribution, the negative data are adjusted to 0, specifically ((I.sub.X1-I.sub.c)=0, if negative). This parameter qualifies the specific repair activities relative to each other for a single sample, and identifies the missing activities.

[0247] By comparison with the reference library made up of cancer types and/or subtypes, it identifies specific repair activities out of line (increased or reduced or absent) compared to the others.

dCTP Label

[0248] The analysis of the contribution of each repair pathway to the total repair, for the repair reactions done with dCTP, brings out a difference between the mutated BRAF samples and the other categories, mainly for the repair of 8oxoG and the Ethenobases (Etheno), (FIG. 3A). The BRAF samples have very specific profiles compared to the other NRAS or WT samples.

[0249] As it relates to the 8oxoG, for 4 out of 5 samples, the relative incorporation of dCTP is either weaker than for the other samples (for 1405, 1502 and 1401) or zero (for 1514).

[0250] It is the same for the incorporation of dCTP for the repair of Etheno (reduced for 1405 and 1502, zero for 1514).

[0251] Inversely for the sample 1507, the incorporation of dCTP is increased compared to the other samples for the repair of 8oxoG and Etheno and relatively weak for Glycols; which distinguishes this mutated BRAF sample from the others.

dGTP Label

[0252] For the contribution analysis, the dGTP label serves to distinguish the WT samples from the other samples essentially in the level of incorporation thereof for the repair of 8oxoG, which is in general higher (FIG. 3B).

[0253] Inversely, the mutated BRAF are distinguished from other samples by a lower dGTP incorporation level with 8oxoG (FIG. 3B).

[0254] With this label, note that the Glycols are not repared for 1506_WT, which distinguishes this sample from two other WT (FIG. 3B).

[0255] It is also noted that with this label there are no repairs of the CPD-64 for 1402_NRAS (FIG. 3B), which has a very low survival.

dATP Label

[0256] For the two BRAF samples where it is measurable (1502 and 1401), the contribution of dATP to the repair of CPD-64 is very small compared to other samples (FIG. 3C)._This label is not incorporated by the 1507_BRAF sample, whatever the lesion considered (FIG. 3C).

dUTP Label

[0257] This label is not incorporated by the sample 1507_BRAF, whatever the lesion considered (FIG. 3D). The contribution of dUTP to the repair of CPD-64 is zero for the BRAF 1401 sample (FIG. 3D).

[0258] In conclusion, the calculations of the contribution bring out specific profiles which characterize the samples independently from the fluorescence intensity. In this way the relative importance of each repair pathway can be qualitatively compared against all the functional repair activities assayed at the same time.

[0259] Despite the recognized genomic diversity of metastatic melanomas, repair type profiles can be associated with each of the mutations of the BRAF or NRAS genes and in the group not comprising mutations in these genes. Thus it is necessary to take this information into consideration when seeking to identify all of the repair defects.

2.3 Shows the Relative Incorporation Rate of Each Nucleotide for the Repair of Each of the Lesions

[0260] For this calculation it is necessary to form a combination of values that were obtained independently by performing repair reactions in the presence of different dNTPs.

[0261] For each sample assayed, the contribution of each labeled dNTP to the resulting total fluorescence is examined for the set of four dNTPs or two dNTPs (in some examples). The resulting information supplements the repair signature profile and the contribution profile. The samples 1514_BRAF and 1402_NRAS were characterized solely with labeled dCTP and labeled dGTP. All the other samples were characterized with the four labeled dNTP. The results are shown in FIGS. 4A to 4F.

[0262] dGTP is the majority nucleotide incorporated for the repair of 8oxoG (FIG. 4A). For the sample 1505_NRAS, it is noted that other nucleotides are relatively significantly incorporated, which indicates the involvement of alternative repair pathways, or errors of the polymerases acting in the patch repair and distinguishes this sample (FIG. 4A).

[0263] The sample 1507_BRAF is distinguished from other samples for the repair of abasic sites (FIG. 4B).

[0264] If no defect in the repair is present, for the repair of a given lesion, an identical contribution of each dNTP is expected. In fact, for a given lesion, the repair systems, because of their specificity, should be identical between samples. However, disparities between the samples are observed, which discloses defects of the repair. For each mutation group, the profiles not conforming to the expected profile can signal alterations of one or more repair mechanisms which leads to the incorporation of some nucleotides in the place of the expected ones, and supports the appearance of mutations.

2.4 Correlation between the Repair Signature and Patient Survival

[0265] The capacity of the enzymatic repair signature to predict the survival was analyzed from reduced, centered data combining the patient deceased 18 months after collection of the tumor sample (1514) and patients not deceased after 24 months (M24); patients lost from sight were not incorporated in the study. Four survival groups were defined:

[0266] M3: deceased 3 months post-collection

[0267] M7: deceased 7 months post-collection

[0268] M9: deceased 9 months post-collection

[0269] M18: deceased 18 months post-collection

[0270] M24: survived at least 24 months post-collection.

Results are to be distinguished according to the mutation group considered:

[0271] WT Patients for BRAF and NRAS- dCTP: A very poor survival is associated with very low DNA repair capacities (FIG. 5A). The most discriminating repair measurements are CPD-64, Glycols, AbaS and CPD (FIG. 5A).

[0272] dGTP: Just as for dCTP, a very poor survival is associated with very low DNA repair capacities (FIG. 5D). The most discriminating repair measurements are 8oxoG, CPD CPD-64 and AbaS (FIG. 5D).

[0273] dATP: As with the preceding nucleotides, a very poor survival is associated with very low DNA repair capacities (FIG. 5G). Two lesions are discriminating: Etheno and AbaS (FIG. 5G).

[0274] dUTP: As with the preceding nucleotides, a very poor survival is associated with very low DNA repair capacities (FIG. 5I). Two lesions are discriminating: Glycols and AbaS (FIG. 5I).

Mutated BRAF Patients

[0275] dCTP: Survival is inversely proportional to some DNA repair activities (FIG. 5C). In particular, an inverse linear relation is observed between the CPD-64 repair level and survival. For patients deceased 3 or 9 months post-collection, the repair level is greater than for patients deceased or still living at 18 or 24 months for CPD-64, AbaS, CPD, and Glycols (FIG. 5C).

[0276] dGTP: An inverse linear relationship is observed between the repair of some DNA repair activities, in particular AbaS, and survival (FIG. 5F). For patients deceased 3 or 9 months post-collection, the repair level is greater than for patients deceased or still living at 18 or 24 months for AbaS, 8oxoG, CPD, and CPD-64 (FIG. 5F).

[0277] dATP: An inverse linear relation is observed between the AbaS, Etheno repair level and survival (FIG. 5H). For the patient's deceased 3 months post-collection, the repair level is greater than for patients deceased or still living at 24 months for AbaS, Etheno, CPD and CPD-64 (FIG. 5H).

[0278] dUTP: An inverse linear relation is observed between the Glycol, AbaS repair level and survival (FIG. 5J). For patients deceased 3 months post-collection, the repair level is greater than for patients deceased or still living at 24 months for Glycols, AbaS, CPD, and 8oxoG.

Mutated NRAS Patients

[0279] dCTP: A proportional relationship is observed between survival and DNA repair level for CPD-64 and Glycols, and an inverse relationship is observed for Etheno and 8oxoG (FIG. 5B).

[0280] dGTP: A proportional relationship is observed between survival and DNA repair level for CPD-64 and AbaS, and an inverse relationship is observed for Etheno and 8oxoG (FIG. 5E).

TABLE-US-00002 TABLE III Correlation between Intensity of the Repair Signal and Patient Survival WT BRAF NRAS dCTP Glycols Correl Glycols Anti Glycols Correl CPD-64 CPD-64 Correl CPD-64 dGTP CPD Correl CPD-64 Anti CPD-64 Correl 8oxoG AbaS Correl AbaS CPD-64 dATP Etheno Correl Etheno Anti NA AbaS AbaS Correl dUTP Glycol Correl Glycol Anti NA AbaS AbaS Correl CPD *Correl: Positive correlation Anti-Correl: Negative correlation NA: not-applicable

[0281] This study shows that the repair signature predicts patient survival; some activities are more predictive than others (FIG. 5). Interestingly, the relation between repair signal intensity and patient survival varies depending on the group of mutations (Tableau III).

TABLE-US-00003 TABLE I Clinical Data about the Patients Tested Sample Initial Treatment Sample Gender Page PhotoType type Mutation treatment at M3 1401 M 42 III. Scapular BRAF Derma Vemurafenib node-Skin-trunk 1402 M 32 II Node-trunk NRAS Ipilimumab Pembrolizumab Pembrolizumab 1404 F 62 III. Node-sub WT Ipilimumab clavicular 1405 M 50 II Arm BRAF Vemurafenib M3 metastases Dabrafenib 1407 M 69 III. Cerebral WT Radiation Radiation metastases therapy therapy + Ipilimumab 1408 M 60 III. Sub-clavicular NRAS node 1502 F 50 III. Cervical BRAF node 1505 F 83 II Inguinal NRAS node 1506 M 78 II Node WT M3 1507 M 46 III. Abdominal BRAF node 1511 M 64 III. Inguinal NRAS node 1514 M 50 III. Node BRAF Treatment Treatment Treatment Treatment Sample at M6 at M9 at M18 at M24 Death 1401 Nivolumab M9 M9 1402 M3 1404 Nivolumab M7 M7 1405 M3 1407 Nivolumab Nivolumab Nivolumab Nivolumab Not deceased at M24 1408 Nivolumab Nivolumab Pembrolizumab Ipilimumab Not deceased at M24 1502 Lost from view M3 1505 Lost from view M3 1506 M3 1507 Thyroid cancer Not deceased at M24 1511 Pembrolizumab Pembrolizumab Pembrolizumab Nivolumab Not deceased at M24 1514 Dabrafenib + RadioT M18 trametinib