BIOMARKERS AND PROGNOSTIC TOOL FOR PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMAS

Abstract

The present invention relates to a method for in vitro or ex vivo diagnosis of the chances of survival of a patient suffering from large-scale diffuse B-cell lymphoma (LBDGC) comprising a step of detecting the expression of at least one of the newly identified LBDGC biomarker genes.

Description

Title of the invention: BIOMARKERS AND PROGNOSTIC TOOL FOR PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMAS FIELD OF THE INVENTION

[0001] The present invention falls within the context of the diagnosis and therapeutic choice of Diffuse Large B-Cell Lymphomas (DLBCL) and relates to a new in vitro or ex vivo diagnostic method for estimating the chances of survival of a patient suffering from DLBCL. The invention also relates to a kit and the use of biomarkers for such in vitro or ex vivo diagnosis. STATE OF THE ART

[0002] Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma (NHL). DLBCL is usually diagnosed after the age of 60 and affects men slightly more often. However, it can also occur in children and young adults. DLBCL represents a common and aggressive form of hematological malignancy with nearly 4,000 new cases per year in France and an increasing incidence. In the majority of cases, DLBCL develops in the lymph nodes. However, it can also affect organs or tissues located outside the lymph nodes (such as the bones, spinal cord, sinuses, testicles, etc.). In such a case, we speak of extra-nodal localization. When DLBCL develops in the lymph nodes, it manifests itself in particular by an abnormal increase in these lymph nodes, which can then be felt to the touch.Other symptoms, such as unexplained fever, severe night sweats, and unexplained weight loss, may also occur.

[0003] The diagnosis of LBDGC is based mainly on the histological analysis of a lymph node biopsy or, more rarely, a bone marrow biopsy. Cytological analysis reveals diffuse invasion by a population of lymphocytic tumor cells of B phenotype, centroblastic, immunoblastic or anaplastic (Bonnet et al - Management of diffuse large B-cell lymphoma in 2012 - Swiss medical journal - ISSN: 1660-9379).

[0004] For more than twenty years, the standard treatment for LBDGC has consisted of a combination of chemotherapy with targeted immunotherapy. This is the "R-CHOP" immunochemotherapy protocol, i.e. the combination of CHOP polychemotherapy and a therapeutic antibody called rituximab (Rituxan or Mabthera). This treatment has significantly increased survival of patients with 60% to 70% remission. However, 30% to 40% of patients are refractory or relapse within two years of therapy, which remains considerable. At this stage, hematopoietic stem cell transplantation remained the only chance of cure before the emergence of CAR-T cell treatments, which remain very heavy and expensive. There is therefore a real need to be able to identify, as early as possible, this portion of patients with LBDGC at high risk of refractory state to immunochemotherapy and thus allow for more appropriate care for their condition (for example, by avoiding them undergoing heavy and ineffective chemotherapy treatment). In other words, in order to propose an optimal treatment protocol adapted to each patient with LBDGC, healthcare teams need reliable indicators to estimate the survival prognosis of the patient with LBDGC, at the time of diagnosis or as soon as possible.

[0005] To date, the risk of relapse in patients with LBDGC is essentially routinely assessed using a prognostic score called IPI (International Prognostic Index) and which is based on 5 clinico-biological parameters (Sehn, Blood, 2007). A molecular classification based on transcriptional profiles (ABC / GCB) and associated with prognosis was also described in 2002 (Rosenwald, NEJM, 2002) and is classically assessed by immunohistochemistry without, for the moment, having any real impact on the treatment offered to patients. More recently, advances in high-throughput sequencing have made it possible to propose new classifications based on mutational profiles (Reddy, Oeil, 2017; Schmitz, NEJM, 2018; Wright Cancer Cell, 2020), with certain groups being associated with a lower prognosis.These different classifications have made it possible to better describe the different forms of this heterogeneous pathology and will probably have theranostic potential in the future with the emergence of new targeted therapies. However, these different classifications are currently of no interest as predictive biomarkers of treatment response on an individual scale.

[0006] To date, no reliable and robust method makes it possible to determine, from the diagnosis of the disease, the chances of survival of a patient suffering from LBDGC and to identify even before the start of R-CHOP treatment whether said patient will respond favorably or not to such treatment. Consequently, the search for new indicators to specify the chances of survival of patients suffering from LBDGC is therefore necessary to allow the existing classifications to be refined and thus to allow the most appropriate treatment to be proposed for each patient.

[0007] In this context, based on the work of researchers at the Institute for Advanced Biosciences (IAB) in Grenoble - who, based on the fact that during oncogenesis, the cell undergoes severe deregulation at the level of the genome and epigenome which impact gene expression profiles, - have identified new biomarkers based on ectopic expression of one or more genes, the expression of which is associated with the prognosis of patients with LBDGC. The inventors have thus developed a novel method and an associated diagnostic tool making it possible to propose new biomarkers capable, alone or in combination, of predicting the chances of survival of a patient with LBDGC. The inventors have tested their method on numerous cohorts of patients with LBDGC, validating it for each of them. In addition, the inventors have demonstrated that the prediction of the chances of survival obtained via this new method represents a reliable and robust indicator, as such, and can also be used in addition to the indicators already known in the prior art in order to refine existing classifications. DESCRIPTION OF THE INVENTION

[0008] The subject of the present invention is therefore a method for in vitro or ex vivo diagnosis of the chances of survival of a patient suffering from large-scale diffuse B-cell lymphoma (LBDGC), comprising a step of detecting the expression of the TRIM9 gene, alone or in combination with the expression of at least one other gene chosen from: DNAJC6, EHHADH, OXTR, OTX1, MT1H, PCDH9 and FOXA3, in a biological sample of said patient.

[0009] Thus, via the detection of an ectopic expression of at least one of the aforementioned genes, in a biological sample, the method of the invention makes it possible to provide a robust indicator, as such, to the care teams regarding the chances of survival of the patient suffering from DLBCL in their care. In addition, this additional indicator can be combined with existing ones (IPI score, ABC / GCB classification), thus reinforcing the robustness of the established prediction. This indicator also has the advantage of being able to be obtained quickly and simply, via technologies already deployed in a large number of medical centers, for example by an RT-qPCR (quantitative PCR after reverse transcription), RT-MLPA (Reverse Transcription Multiplex Ligation-dependent Probe Amplification) or RT-MLPSeq (Reverse Transcription-Multiple Ligation-dependent probe Sequencing) test.Therefore, obtaining this additional indicator via the method according to the invention is inexpensive and can be standardized in the short term, in the laboratories of said medical centers.

[0010] The present invention makes a real contribution to the panel of diagnostic tools for Diffuse Large B-Cell Lymphomas (DLBCL) available to healthcare teams in order to propose the most appropriate therapeutic strategy for each patient suffering from LBDBCL.

[0011] In the context of the invention, the expressions "in vitro diagnosis" and "ex vivo diagnosis" are understood to refer to tests carried out outside the patient, after taking the biological sample from said patient. The tests carried out on biological samples taken from the patient(s) and kept in the laboratory, generally under sterile conditions, without modification over a short period (generally less than 24 hours), are part of an ex vivo diagnosis according to the invention. The tests carried out on such samples and over a longer period of time are generally part of an in vitro diagnosis according to the invention, since they require cell culture steps aimed at keeping the cells of said biological samples taken alive.

[0012] In the context of the invention, the expression "biological sample" refers to a tissue fragment (tumor excision or biopsy) likely to contain lymphocytic tumor cells of B phenotype (also called cancerous B lymphocytes). According to the field of application of the invention and by way of non-exhaustive examples, the biological sample may be any type of tumor biopsy sample such as a tissue biopsy sample (in particular lymph node(s), bone marrow and liver) or a blood sample, in particular a peripheral blood sample.

[0013] In the context of the invention, the expressions “chance(s) of survival”, “prognosis”, “probability(s) of survival” or “prediction(s) of survival” are synonyms and all designate the estimation of the survival of a patient suffering from LBDGC, with regard to indicators specific to this patient.

[0014] In the context of the invention, the expression "detection of the expression of at least one gene" means the quantification of at least one expression product of the gene. The expression product of a gene, within the meaning of the present invention, is any biological molecule resulting from the expression of said gene. For example, the expression product of the gene may be an RNA transcript or a protein obtained after translation of a transcript by the ribosomes. By "transcript", is meant the RNAs, and in particular the messenger RNAs (mRNAs), resulting from the transcription of the gene. More precisely, the transcripts are the RNAs produced by the transcription of a gene followed by post-transcriptional modifications of the pre-RNA forms. In the context of the present invention, it is possible to quantify the level of expression of one or more RNA transcripts of the same gene and / or the level of expression of one or more isoforms of a protein encoded by the same gene.The quantified expression of said detected gene is compared to a reference value. Below this value, the gene is considered not to be expressed, within the meaning of the invention. This may, for example, be technical background noise which should not be taken into consideration within the scope of the invention. Conversely, above the reference value, the expression of the gene is considered to be detected. This is then an ectopic expression, also called out-of-context expression, since the eight genes identified by the inventors are not expressed in lymphocyte cells of B phenotype (hereinafter "B lymphocytes"). of a healthy individual. Detection of gene expression is carried out by any means known to those skilled in the art. In the case of an mRNA transcript, detection may be carried out by a direct method, by any method known to those skilled in the art for determining the presence of said transcript in the sample, or by indirect detection of the transcript after transformation of the latter into DNA by reverse transcription, or after amplification of said transcript or after amplification of the DNA obtained after transformation of said transcript into DNA. Many methods exist for the detection of nucleic acids (see for example Kricka et al., Clinical Chemistry , 1999, No. 45(4), p.453-458; Relier GH et al., DNA Probes, 2nd Ed., Stockton Press , 1993, sections 5 and 6, p.173-249).Gene expression can be measured, in particular, by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably by quantitative RT-PCR or RT-qPCR (for example using Fluidigm's BiomarkTM platform), by sequencing (preferably by high-throughput sequencing) or by hybridization techniques (for example with hybridization microarrays or by techniques such as NanoString® nCounter®). The expression of said genes can also be measured by Reverse Transcription-Multiplex Ligation-dependent Probe Amplification (RT-MPLA).

[0015] In the context of the invention, the expression “reference value” corresponds to the value of the level of expression of a gene considered in one or more healthy individuals, i.e. not suffering from LBDGC and / or not presenting any specific medical condition. This reference value can be determined directly from the analysis of a biological sample from a healthy individual or be the result of several values from several biological samples from the same healthy individual and / or from several healthy individuals.

[0016] In the context of the invention, the DNAJC6 gene refers to the gene known as “DnaJ heat shock protein family (Hsp40) member C6” (Ensembl identifier: ENSG00000185100), located on chromosome 1 (chromosomal location of the gene according to GRCh38 / hg38: chrl: 65,248,219-65,415,871) and currently coding for 8 identified transcripts. It has the nucleotide sequence SEQ ID NO: 1 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 1.

[0017] In the context of the invention, the EHHADH gene refers to the gene known as "enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase" (Ensembl identifier: ENSG00000113790), located on chromosome 3 (chromosomal location of the gene according to GRCh38 / hg38: chr 3: 185,190,624-185,281,990) and currently coding for 6 identified transcripts. It has the nucleotide sequence SEQ ID NO: 2 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 2.

[0018] In the context of the invention, the OXTR gene refers to the gene known as "oxytocin receptor" (Ensembl identifier: ENSG00000180914), located on chromosome 3 (chromosomal location of the gene according to GRCh38 / hg38: chr 3: 8,750,381-8,769,628) and currently coding for 4 identified transcripts. It has the nucleotide sequence SEQ ID NO: 3 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 3.

[0019] In the context of the invention, the OTX1 gene refers to the gene known as "orthodenticle homeobox 1" (Ensembl identifier: ENSG00000115507), located on chromosome 2 (chromosomal location of the gene according to GRCh38 / hg38: chr2: 63,050,057-63,057,836) and currently coding for 6 identified transcripts. It has the nucleotide sequence SEQ ID NO: 4 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 4.

[0020] In the context of the invention, the TRIM9 gene refers to the gene known as “tripartite motif containing 9” (Ensembl identifier: ENSG00000100505), located on chromosome 14 (chromosomal location of the gene according to GRCh38 / hg38: chrl4: 50,975,262-51,096,061) and currently coding for 7 identified transcripts. It has the nucleotide sequence SEQ ID NO: 5 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 5.

[0021] In the context of the invention, the MT1H gene refers to the gene known as "metallothionein 1H" (Ensembl identifier: ENSG00000205358), located on chromosome 16 (chromosomal location of the gene according to GRCh38 / hg38: chrl6: 56,669,814-56,671,129) and currently coding for 2 identified transcripts. It has the nucleotide sequence SEQ ID NO: 6 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 6.

[0022] In the context of the invention, the PCDH9 gene refers to the gene known as "protocadherin 9" (Ensembl identifier: ENSG00000184226), located on chromosome 13 (chromosomal location of the gene according to GRCh38 / hg38: chrl3: 66,302,834-67,230,445) and currently coding for 6 identified transcripts. It has the nucleotide sequence SEQ ID NO: 7 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 7.

[0023] In the context of the invention, the FOXA3 gene refers to the gene known as “forkhead box A3” (Ensembl identifier: ENSG00000170608), located on chromosome 19 (chromosomal location of the gene according to GRCh38 / hg38: chrl9: 45,863,989-45,873,797) and currently encoding 2 identified transcripts. It has the nucleotide sequence SEQ ID NO: 8 or a sequence exhibiting at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 8.

[0024] Preferably, the subject of the present invention is a method as described above and having the following technical characteristics, taken alone or in combination: - the step of detecting the expression of at least one gene chosen from: DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3 consists of measuring the expression of said genes and, for each gene, comparing said measurement with a reference value of expression of said gene for a healthy individual;

[0025] - the detection of the expression of the genes is carried out via at least one technology chosen from: RT-qPCR, fluorescence in situ hybridization, RNA-SCOPE, high-throughput RNA-seq sequencing, via RT-MLPA and RT-MLPSeq;

[0026] - the detection step comprises detecting the expression of at least two of said genes, preferably at least three of said genes, preferentially at least four of said genes, preferentially at least five of said genes, preferentially at least six of said genes, preferentially at least seven of said genes and even more preferentially eight genes;

[0027] - the detection step comprises detecting the expression of at least the two following genes: DNAJC6 and EHHADH;

[0028] - the detection step comprises detecting the expression of at least the three following genes: DNAJC6, EHHADH and OXTR;

[0029] - the detection step comprises detecting the expression of at least the four following genes: DNAJC6, EHHADH, OXTR and OTX1;

[0030] - the detection step comprises detecting the expression of at least the five following genes: DNAJC6, EHHADH, OXTR, OTX1 and TRIM9;

[0031] - the detection step comprises detecting the expression of at least the six genes following: DNAJC6, EHHADH, OXTR, OTX1, TRIM9 and MT1H;

[0032] - the detection step comprises detecting the expression of at least the seven following genes: DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H and FOXA3;

[0033] - the detection step comprises detecting the expression of the eight genes; and / or

[0034] - the biological sample of the patient is a sample of lymph node of said patient.

[0035] The invention also relates to a kit for in vitro or ex vivo diagnosis. the chances of survival of a patient suffering from diffuse large-scale B-cell lymphoma (DLBCL) comprising means for detecting the expression of at least one gene chosen from: DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3, in a biological sample of said patient.

[0036] The invention also relates to a use of at least one gene among DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3, as a biomarker for the in vitro or ex vivo diagnosis of the chances of survival of a patient suffering from diffuse large-scale B-cell lymphoma (DLBCL), in a biological sample of said patient.

[0037] Advantageously, the use according to the invention is a use of at least two of said genes, preferably at least three of said genes, preferentially at least four of said genes, preferentially at least five of said genes, preferentially at least six of said genes, preferentially at least seven of said genes and even more preferentially eight genes, as a biomarker for the in vitro or ex vivo diagnosis of the chances of survival of a patient suffering from large-scale diffuse B-cell lymphoma (LBDGC), in a biological sample of said patient. Brief description of the drawings

[0038] [Fig-1]: represents the average survival over time of patients with LBDGC and belonging to different cohorts, based on the detection of DNAJC6 gene expression;

[0039] [Fig.2]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested, based on the detection of DNAJC6 or EHHADH gene expression;

[0040] [Fig.3]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested, depending on whether they express 0-1, or 2-3 of the following genes DNAJC6, EHHADH and OXTR;

[0041] [Fig.4]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested, depending on whether they express 0-1, or 2-4 of the following genes DNAJC6, EHHADH, OXTR and OTX1;

[0042] [Fig.5]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested, depending on whether they express 0-2, or 3-5 of the following genes DNAJC6, EHHADH, OXTR, OTX1 and TRIM9;

[0043] [Fig.6]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested, depending on whether they express 0-2, or 3-6 of the following genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9 and MT1H;

[0044] [Fig.7]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested, depending on whether they express 0-2, or 3-7 of the following genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H and PCDH9;

[0045] [Fig.8]: represents the average survival over time of patients with LBDGC and belonging to the different cohorts tested according to whether they express 0-2, or 3-8 of the following genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3.

[0046] The present invention is illustrated in a non-limiting manner by the following examples. EXAMPLES OF ACHIEVEMENT

[0047] Example 1: identification and validation of the eight biomarker genes

[0048] The inventors established a list of genes exhibiting specific or predominant expression in certain human tissues and not expressed or weakly expressed in B lymphocytes of healthy patients. More than 2000 genes were retained at this stage.

[0049] Among the genes selected, the inventors then carried out a step of selecting genes in the cohort (also called “dataset” hereinafter) NCICCR-DLBCL (TCGA) for which ectopic activation in B lymphocytes was observed in more than 10% of cases. 617 genes were selected at this step.

[0050] In the next step, the inventors identified candidate genes capable of individually predicting the vital prognosis of patients with LBDGC in the Duke 1001 DLBCL cohort (Duke University). To do this, they established an activation threshold for each gene among the 617, using the bioinformatics method "ectopy", developed by the inventors and published in the international journal BMC Genomics (Jacquet et al. 2023 PMID 37592220).

[0051] The “ectopy” method aims to identify ectopic activations of normally silent genes from cancer transcriptomic data sets, to evaluate their impact on the probability of patient survival and to determine the most robust candidate biomarkers.

[0052] The inventors thus found that for 89 genes, among the 617 genes previously identified, aberrant activation of the gene was significantly associated with a shorter survival probability of patients in the training cohort. The Cox proportional hazard model (a model recognized in this field and described in the publication "Analysis of survival data. Cox & Oakes. 1984. ISBN 978-0-412-24490-2") was used in the survival analysis to identify candidate genes.

[0053] In the validation phase, the activation statuses (ON: expressed, OFF: extinguished) of the 89 genes were established in three LBDGC validation cohorts GSE117556A, GSE31312 and GSE181063 available in the NCBI GEO public database (https: / / www.ncbi.nlm.nih.gov / geo / ). The significant impact of aberrant activation (gene ON status) on overall patient survival was confirmed in all validation cohorts for the following 8 genes among the 89 candidate genes retained: DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3. These 8 genes were retained to create a panel of prognostic biomarkers for LBDGC. For For each patient, the number of activated genes among the 8 genes in this panel is calculated to obtain a prognostic score. This score thus obtained makes it possible to predict the probability of survival for each patient suffering from LBDGC.

[0054] Finally, this panel of prognostic biomarkers of LBDGC was tested in three entirely independent cohorts (GSE10846, GSE117556B and the aggregation of cohorts GSE53786, GSE32918 and GSE87371- available in the public NCBI GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ), which were not used for the identification of the 8 biomarker genes finally retained. The inventors demonstrated that the number of genes whose ectopic expression was detected is significantly associated with the probability of overall survival in all test cohorts. The inventors have thus demonstrated the robustness and reliability of this new panel of prognostic biomarkers of LBDGC for the diagnosis of the chances of survival of patients with diffuse large B-cell lymphomas.

[0055] Furthermore, the inventors checked for any possible correlation between the expressions of these eight genes in all the mentioned datasets. They confirmed that these expressions were not correlated. Indeed, no expression of these eight genes is correlated in their tissue of origin, namely: the brain for the DNAJC6, TRIM9 and PCDH9 genes, the liver for the EHHADH and F0XA3 genes, the peritoneum - retroperitoneum for the EHHADH and MT1H genes, the skin for the 0TX1 gene and the breast and uterus for the OXTR gene. This step is necessary to confirm that none of these genes is redundant in terms of predictive impact.

[0056] Finally, the inventors have demonstrated that the indicator provided by the diagnostic method according to the invention and the associated prognostic tool is distinct and complementary to existing indicators (IPI score, ABC / GCB classification) using multivariate survival analysis methods (Cox model). Example 2: Results

[0057] The eight biomarker genes retained after the validation phase were used, alone or in various combinations, in multiple cohorts of patients suffering from LBDGC, in order to confirm the robustness of the diagnostic method according to the invention and the associated prognostic tool. The latter form, alone or in combinations, biomarkers making it possible to classify patients suffering from LBDGC according to their ectopic expression (resulting in an ON activation status) in the biological tissues of patients suffering from LBDGC whose chances of survival are to be estimated. In addition, the inventors have demonstrated that the greater the number of biomarker genes, among the eight biomarker genes ultimately retained, which have the activated status (ON status), the less favorable the patient's vital prognosis is, as demonstrated by the following results.

[0058] For each dataset (cohort) tested, patients were separated into two groups according to their activation status of the chosen gene(s). The difference in survival probabilities between the two established groups was calculated by the Cox model. The inventors proved that the number of activated genes is significantly correlated with the survival probability of patients in all the considered datasets (Cox p-value < 0.05), thus demonstrating the robust prediction of the survival chances of patients with diffuse B-cell lymphomas on a large scale via the method according to the invention and the associated tool.

[0059] In all cohorts, the expression levels of the different genes were measured by RNA-seq (for RNA sequencing) or Affymetrix-type RNA chip technologies. However, the inventors also verified and confirmed that their method and the associated tool could be advantageously standardized on other technologies, such as RT-qPCR or RT-MLPA or RT-MLPSeq methods. In clinical practice, RT-qPCR, RT-MLPA or RT-MLPSeq tests have several advantages over high-throughput RNA-seq sequencing. They are significantly less expensive, standardizable and widely available in hospital laboratories.

[0060] 2.1 _: Use of the DNAJC6 gene as a biomarker ([Fig. 1 0

[0061] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0062] - detection of the expression of the DNAJC6 gene (“Activated” groups on the [Fig.l]) and

[0063] - the absence of detection of the expression of this gene (“Not activated” groups) on [Fig.l]),

[0064] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0065] It is noted for each of the cohorts that the patients in the “Not activated” group for whom no ectopic expression is detected have better chances of survival than the patients in the “Activated” group for whom gene expression is detected.

[0066] 2. 2 _: Use of a combination of the EHHADH _ and DNAJC6 genes as as biomarkers ([Fig.2])

[0067] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0068] - detection of the expression of the DNAJC6 and / or EHHADH genes (groups 1-2 on [Fig.2]) and

[0069] - the absence of detection of the expression of these genes (groups 0 in [Fig.2]),

[0070] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0071] It is noted for each of the cohorts that patients in group 0 for whom no ectopic expression is detected have better chances of survival than patients in group 1-2 for whom the expression of at least one of the two genes is detected.

[0072] 2, 3: Use of a combination of the EHHADH DNAJC6 and OXTR genes in as biomarkers ([Fig. 3 ])

[0073] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0074] - detection of the expression of at least two of the following genes: DNAJC6, EHHADH and OXTR (groups 2-3 in [Fig.3]) and

[0075] - the absence of detection of the expression of this gene or the detection of the expression of one of these three genes (groups 0-1 in [Fig.3]),

[0076] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0077] It is noted for each of the cohorts that the patients in group 0-1 for whom at most the expression of one of the three genes is detected have better chances of survival than the patients in group 2-3 for whom the expression of at least two of the three genes is detected.

[0078] 2, 4: Use of a combination of the genes EHHADH DNAJC6 _ OXTR _ and OTX1 as biomarkers ([Fig.4])

[0079] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0080] - detection of the expression of at least two of the following genes EHHADH, DNAJC6, OXTR and OTX1 (groups 2-4 in [Fig.4]) and

[0081] - the absence of detection of the expression of this gene or the detection of the expression of one of these four genes (groups 0-1 in [Fig.4],

[0082] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0083] It is noted for each of the cohorts that the patients in group 0-1 for whom at most the expression of one of the four genes is detected have better chances of survival than the patients in group 2-4 for whom the expression of at least two of the four genes is detected.

[0084] 2, 5: Use of a combination of the genes EHHADH DNAJC6 _ OXTR OTX1 and TRIM9 as biomarkers ([Fig.5D

[0085] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0086] - detection of the expression of at least three of the following genes EHHADH, DNAJC6, OXTR, OTX1 and TRIM9 (groups 3-5 in [Fig.5]) and

[0087] - the absence of detection of the expression of this gene or the detection of the expression of one or two of these five genes (groups 0-2 in [Fig.5]),

[0088] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0089] It is noted for each of the cohorts that patients in group 0-2 for whom the expression of at most two of the five genes is detected have better chances of survival than patients in group 3-5 for whom the expression of at least three of the five genes is detected.

[0090] 2, 6: Use of a combination of the genes EHHADH DNAJC6 _ OXTR OTX1, TRIM9 and MT1H as biomarkers ([Fig.6])

[0091] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0092] - detection of the expression of at least three of the following genes EHHADH, DNAJC6, OXTR, OTX1, TRIM9 and MT1H (groups 3-6 in [Fig.6]) and

[0093] - the absence of detection of the expression of these genes or the detection of the expression of one or two of these six genes (groups 0-2 in [Fig.6]),

[0094] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0095] It is noted for each of the cohorts that patients in group 0-2 for whom the expression of at most two of the six genes is detected have better chances of survival than patients in group 3-6 for whom the expression of at least three of the six genes is detected.

[0096] 2, 7: Use of a combination of the genes EHHADH DNAJC6 _ OXTR OTX1, TRIM 9, MT 1H and FOX A3 as biomarkers ([Fig.7])

[0097] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0098] - detection of the expression of at least three of the following genes EHHADH, DNAJC6, OXTR, OTX1, TRIM9, MT1H and FOXA3 (groups 3-7 in [Fig.7]) and

[0099] - the absence of detection of the expression of these genes or the detection of the expression of one or two of these seven genes (groups 0-2 in [Fig.7]),

[0100] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0101] It is noted for each of the cohorts that patients in group 0-2 for whom the expression of at most two of the seven genes is detected have better chances of survival than patients in group 3-7 for whom the expression of at least three of the seven genes is detected.

[0102] 2.8: Use of a combination of genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3 as biomarker ([Fig.8D

[0103] In this test, carried out on all the cohorts described previously, the patients of each cohort were differentiated according to:

[0104] - detection of the expression of at least three of the following genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3 (groups 3-8 in [Fig.8]) and

[0105] - the absence of detection of the expression of these genes or the detection of the expression of one to two of these eight genes (groups 0-2 in [Fig.8]),

[0106] in their respective biological samples (these genes being known to be expressed only ectopically in B lymphocytes).

[0107] It is noted for each of the cohorts that patients in group 0-2 for whom the expression of at most two of the eight genes is detected have better chances of survival than patients in group 3-8 for whom the expression of at least three of the eight genes is detected. 2.9#: Conclusions

[0108] It is clear from these experimental data that a method, a kit and a use according to the invention make it possible to provide an indicator as to the chances of survival of patients suffering from LBDGC which is 1 / additional and complementary to those already available to medical teams, 2 / robust and reliable as such and 3 / able to be obtained quickly and according to technologies already deployed in a large number of medical centers, for example by an RT-qPCR test or an RT-MLPA or MLPSeq test.

[0109] Furthermore, the data presented also demonstrate that the robustness of this indicator is enhanced the greater the number of the eight biomarker genes (whose expression is detected) used.

[0110] Finally, this indicator provided by the diagnostic method according to the invention and by the associated prognostic tool being complementary and distinct from the existing indicators (IPI score, ABC / GCB classification), its combination with the latter makes it possible to further refine the determination of the chances of survival of patients suffering from large-scale diffuse B-cell lymphoma (LBDGC).

Claims

Claims

1. Method for in vitro or ex vivo diagnosis of the chances of survival of a patient suffering from large-scale diffuse B-cell lymphoma (LBDGC), comprising a step of detecting the expression of the TRIM9 gene, in a biological sample of said patient.

2. Method according to the preceding claim, the detection step further comprises the detection of the expression of at least one gene chosen from: DNAJC6, EHHADH, OXTR, 0TX1, MT1H, PCDH9 and F0XA3

3. Method according to any one of the preceding claims, in which the step of detecting the expression of the gene(s) consists of measuring the expression of said genes and, for each gene, comparing said measurement with a reference value of expression of said gene for a healthy individual.

4. Method according to any one of the preceding claims, wherein the detection of gene expression is carried out via at least one technology chosen from: RT-qPCR, fluorescence in situ hybridization, RNA-SCOPE, high-throughput RNA-seq sequencing, via RT-MLPA and RT-MLPSeq.

5. A method according to any one of the preceding claims, wherein the detecting step comprises detecting the expression of at least two of said genes, preferably at least three of said genes, preferably at least four of said genes, preferably at least five of said genes, preferably at least six of said genes, preferably at least seven of said genes and even more preferably all eight genes.

6. The method of claim 5, wherein the detecting step comprises detecting the expression of the eight genes.

7. A method according to any preceding claim, wherein the biological sample of the patient is a lymph node sample of said patient.

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