BIOMARKERS AND PROGNOSTIC TOOLS FOR PATIENTS WITH DIFFUSE B-CELL LYMPHOMA

Abstract

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

Description

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

[0001] The present invention relates to the diagnosis and therapeutic selection of Diffuse Large B-Cell Lymphomas (DLBCL) and concerns a new in vitro or ex vivo diagnostic method for estimating the survival chances of a patient with DLBCL. The invention also relates to a kit and the use of biomarkers for such in vitro or ex vivo diagnosis. PRIOR TECHNOLOGY

[0002] Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type of non-Hodgkin 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 is a frequent 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 bones, spinal cord, sinuses, testicles, etc.). In such cases, it is referred to as extranodal involvement. When DLBCL develops in the lymph nodes, it manifests itself in particular by an abnormal enlargement of these nodes, which can then be felt by touch.Other symptoms, such as unexplained fever, significant night sweats, and unexplained weight loss, may also occur.

[0003] The diagnosis of DLBCL is based primarily 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 B-cell tumor cells, centroblastic, immunoblastic, or anaplastic (Bonnet et al - Management of diffuse large B-cell lymphoma in 2012 - Swiss Medical Review - ISSN: 1660-9379).

[0004] For more than twenty years, the standard treatment for DLBCL has consisted of a combination of chemotherapy with targeted immunotherapy. This is the "R-CHOP" immunochemotherapy protocol, which combines CHOP polychemotherapy with a therapeutic antibody called rituximab (Rituxan or Mabthera). This treatment has significantly increased survival Patients experience a 60% to 70% remission rate. However, 30% to 40% of patients are refractory or relapse within two years of therapy, which remains a considerable number. At this stage, hematopoietic stem cell transplantation remained the only chance of a cure before the emergence of CAR-T cell therapies, which remain very intensive and expensive. Therefore, there is a real need to be able to identify, as early as possible, this proportion of DLBCL patients at high risk of refractory status to immunochemotherapy, thus enabling more tailored care (for example, by avoiding them undergoing harsh and ineffective chemotherapy). In other words, in order to offer an optimal treatment protocol adapted to each patient with DLBCL, healthcare teams need reliable indicators to estimate the survival prognosis of patients with DLBCL, at the time of diagnosis or as soon as possible.

[0005] To date, the risk of relapse in patients with DLBCL is primarily assessed routinely using a prognostic score called the IPI (International Prognostic Index), which is based on five clinical and 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 evaluated 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 poorer prognosis.These different classifications have allowed for a better description of the various forms of this heterogeneous pathology and will likely have theranostic potential in the future with the emergence of new targeted therapies. However, these different classifications are currently not useful as predictive biomarkers of treatment response at the individual level.

[0006] To date, no reliable and robust method exists to determine, at the time of diagnosis, the survival chances of a patient with DLBCL and to identify, even before the start of R-CHOP treatment, whether the patient will respond favorably or not. Therefore, the search for new indicators to refine the survival chances of patients with DLBCL is necessary to improve existing classifications and thus enable the most appropriate treatment to be offered to each patient.

[0007] In this context, drawing 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 dysregulation at the level of the genome and the epigenome which impact gene expression profiles – have identified Novel biomarkers based on the ectopic expression of one or more genes, the expression of which is associated with the prognosis of patients with DLBCL. The inventors have thus developed a novel method and an associated diagnostic tool enabling the proposal of new biomarkers capable, alone or in combination, of predicting the chances of survival for a patient with DLBCL. The inventors have tested their method on numerous cohorts of patients with DLBCL, validating it for each cohort. Furthermore, the inventors have demonstrated that the survival prediction obtained via this new method represents a reliable and robust indicator, in itself, and can also be used in conjunction with indicators already known in the prior art to refine existing classifications. DESCRIPTION OF THE INVENTION

[0008] The present invention therefore relates to a method for in vitro or ex vivo diagnosis of the chances of survival of a patient with large-scale diffuse B-cell lymphoma (LDBCL), 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, by detecting ectopic expression of at least one of the aforementioned genes in a biological sample, the method of the invention provides healthcare teams with a robust indicator, as such, regarding the survival chances of the DLBCL patient under their care. Furthermore, this additional indicator can be combined with existing indicators (IPI score, ABC / GCB classification), thereby strengthening the robustness of the established prediction. This indicator also has the advantage of being obtainable quickly and easily using technologies already deployed in a large number of medical centers, for example, by RT-qPCR (quantitative reverse transcription PCR), RT-MLPA (Reverse Transcription Multiplex Ligation-dependent Probe Amplification), or RT-MLPSeq (Reverse Transcription-Multiple Ligation-dependent Probe Sequencing).Therefore, obtaining this additional indicator via the process 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 range 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 with DLBCL.

[0011] In the context of the invention, the terms "in vitro diagnostics" and "ex vivo diagnostics" refer to tests performed outside the patient, after the biological sample has been taken from said patient. The tests performed Tests performed on biological samples taken from the patient(s) and kept in the laboratory, generally under sterile conditions, without modification for a short period (generally less than 24 hours), fall within the scope of ex vivo diagnosis according to the invention. Tests carried out on such samples and over a longer period generally fall within the scope of in vitro diagnosis according to the invention, since they require cell culture steps aimed at keeping the cells of said biological samples alive.

[0012] In the context of the invention, the term "biological sample" refers to a tissue fragment (tumor excision or biopsy) likely to contain B-cell tumor lymphocytes (also called cancerous B lymphocytes). Depending on the field of application of the invention, and by way of non-exhaustive example, the biological sample may be any type of tumor biopsy sample such as a tissue biopsy sample (in particular of 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 "survival prediction(s)" are synonyms and all refer to the estimation of the survival of a patient with LBDGC, with regard to indicators specific to that 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 gene expression product. A gene expression product, as defined in the present invention, is any biological molecule resulting from the expression of said gene. For example, the gene expression product may be an RNA transcript or a protein obtained after translation of a transcript by ribosomes. "Transcript" refers to RNA, and in particular messenger RNA (mRNA), resulting from the transcription of the gene. More precisely, 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 expression level of one or more RNA transcripts of the same gene and / or the expression level of one or more isoforms of a protein encoded by the same gene.The quantified expression of the 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 could, for example, be technical background noise that should not be taken into account within the scope of the invention. Conversely, above the reference value, gene expression 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 B-cell phenotype lymphocytes (hereinafter "B lymphocytes"). of a healthy individual. Gene expression detection is performed by any method known to those skilled in the art. In the case of an mRNA transcript, detection can be performed by a direct method, by any method known to those skilled in the art that allows the presence of said transcript in the sample to be determined, or by indirect detection of the transcript after its conversion to DNA by reverse transcription, or after amplification of said transcript, or after amplification of the DNA obtained after conversion of said transcript to DNA. Numerous methods exist for the detection of nucleic acids (see, for example, Kricka et al., Clinical Chemistry, 1999, No. 45(4), pp. 453–458; Relier GH et al., DNA Probes, 2nd ed., Stockton Press, 1993, sections 5 and 6, pp. 173–249).Gene expression can be measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), preferably by quantitative RT-PCR or RT-qPCR (e.g., using the Fluidigm Biomark™ platform), by sequencing (preferably high-throughput sequencing), or by hybridization techniques (e.g., with hybridization microarrays or techniques such as NanoString® nCounter®). Gene expression can also be measured by Reverse Transcription-Multiplex Ligation-dependent Probe Amplification (RT-MPLA).

[0015] In the context of the invention, the term "reference value" refers to the expression level of a given gene in one or more healthy individuals, that is, individuals not affected by DLBCL 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 ​​obtained 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 encoding 8 identified transcripts. Its nucleotide sequence is SEQ ID NO: 1 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with 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 encoding 6 identified transcripts. Its nucleotide sequence is SEQ ID NO: 2 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with 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 encoding 4 identified transcripts. Its nucleotide sequence is SEQ ID NO: 3 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with 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 encoding 6 identified transcripts. Its nucleotide sequence is SEQ ID NO: 4 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 4.

[0020] In the context of the invention, the TRIM9 gene refers to the gene known as the "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 encoding 7 identified transcripts. Its nucleotide sequence is SEQ ID NO: 5 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with 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 encoding 2 identified transcripts. Its nucleotide sequence is SEQ ID NO: 6 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with 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 encoding 6 identified transcripts. Its nucleotide sequence is SEQ ID NO: 7 or a sequence exhibiting at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with 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 coding for 2 identified transcripts. Its nucleotide sequence is SEQ ID NO: 8 or a sequence showing at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the SEQ ID NO: 8 sequence.

[0024] Preferably, the present invention relates to a process 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 for the expression of said gene in a healthy individual;

[0025] - 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;

[0026] - the detection step includes the detection of 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 of all eight genes;

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

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

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

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

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

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

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

[0034] - the patient's biological sample is a lymph node sample from said patient.

[0035] The invention also relates to a kit for in vitro or ex vivo diagnosis chances of survival of a patient with large-scale diffuse B-cell lymphoma (LDBCL) including means of 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 the 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 with large-scale diffuse B-cell lymphoma (LDBCL), 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, 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, as a biomarker for the in vitro or ex vivo diagnosis of the chances of survival of a patient with large-scale diffuse B-cell lymphoma (LDBCL), in a biological sample of said patient. Brief description of the drawings

[0038] [Fig-1]: represents the mean survival over time of patients with LBDGC and belonging to different cohorts, depending 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, depending on the detection of the expression of the DNAJC6 or EHHADH genes;

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

[0041] [Fig.4]: represents the mean 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 DLBCL belonging to the different cohorts tested, according to whether they express 0-2, or 3-5 of the following genes DNAJC6, EHHADH, OXTR, OTX1 and TRIM9;

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

[0044] [Fig.7]: represents the mean 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 mean survival over time of patients with DLBCL belonging to the different cohorts tested according to whether they express 0-2, or 3-8 the following genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3.

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

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

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

[0049] From among the selected genes, the inventors then proceeded to a step of selecting genes in the NCICCR-DLBCL (TCGA) cohort (also referred to as the "dataset" hereafter) 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 survival prognosis of patients with DLBCL in the Duke 1001 DLBCL cohort (Duke University). To do this, they established an activation threshold for each gene out of the 617, using the bioinformatics method "ectopia", developed by the inventors and published in the international journal BMC Genomics (Jacquet et al. 2023 PMID 37592220).

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

[0052] The inventors thus found that for 89 genes, out of the 617 previously identified genes, aberrant gene activation was significantly associated with a shorter survival probability for patients in the training cohort. The Cox proportional hazards model (a recognized model in this field, 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] During the validation phase, the activation statuses (ON: expressed, OFF: inactivated) of the 89 genes were established in three DLBCL validation cohorts GSE117556A, GSE31312, and GSE181063 available in the public NCBI GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ). The significant impact of aberrant activation (ON status of the gene) on overall patient survival was confirmed in all validation cohorts for the following 8 genes out of the 89 selected candidate genes: DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9, and FOXA3. These 8 genes were selected to create a panel of prognostic biomarkers for DLBCL. For each patient, the number of activated genes from among the 8 genes in this panel is calculated to obtain a prognostic score. This score then allows us to predict the probability of survival for each patient with DLBCL.

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

[0055] Furthermore, the inventors checked for any possible correlation between the expression of these eight genes across all the aforementioned 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 OTX1 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 from 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 selected after the validation phase were used, alone or in various combinations, in multiple cohorts of patients with DLBCL to confirm the robustness of the diagnostic method according to the invention and the associated prognostic tool. These genes, alone or in combination, constitute biomarkers that allow patients with DLBCL to be classified according to their ectopic expression (resulting in an ON activation status) in the biological tissues of patients with DLBCL whose chances of survival are to be estimated. Furthermore, the inventors demonstrated that the greater the number of biomarker genes, among the eight ultimately selected, that exhibit an activated status (ON status), the less favorable the patient's prognosis, as demonstrated by the following results.

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

[0059] Across all cohorts, the expression levels of the different genes were measured using RNA-seq (RNA sequencing) or Affymetrix-type RNA microarray 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, RT-MLPA, or RT-MLPSeq. In clinical practice, RT-qPCR, RT-MLPA, or RT-MLPSeq tests offer 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. 10

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

[0062] - from the detection of DNAJC6 gene expression (groups “Activated” on the [Fig. 1] and

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

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

[0065] For each of the cohorts, it is noted that patients in the "Not activated" group, for whom no ectopic expression is detected, have better chances of survival than 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 than biomarkers ([Fig.2])

[0067] In this test, performed on all the cohorts described above, the patients in each cohort were differentiated according to:

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

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

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

[0071] For each of the cohorts, it is noted 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 above, the patients in each cohort were differentiated according to:

[0074] - from the 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 on [Fig.3]),

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

[0077] For each of the cohorts, it is noted that patients in group 0-1, for whom the expression of at most one of the three genes is detected, have better chances of survival than 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 EHHADH DNAJC6 _ OXTR _ genes and OTX1 as a biomarker ([Fig.4])

[0079] In this test, performed on all the cohorts described above, the patients in each cohort were differentiated according to:

[0080] - from the 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 on the [Fig.4],

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

[0083] For each of the cohorts, it is noted that patients in group 0-1, for whom the expression of at most one of the four genes is detected, have better chances of survival than 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 EHHADH DNAJC6 _ OXTR genes OTX1 and TRIM9 as biomarkers ([Fig. 5D

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

[0086] - from the 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 on [Fig.5]),

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

[0089] For each of the cohorts, it is noted that patients in group 0-2, for whom the expression of a maximum of 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 EHHADH DNAJC6 _ OXTR genes OTX1, TRIM9 and MT1H as biomarkers ([Fig.6])

[0091] In this test, performed on all the cohorts described above, the patients in each cohort were differentiated according to:

[0092] - from the 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 on [Fig.6]),

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

[0095] For each of the cohorts, it is noted 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 EHHADH DNAJC6 _ OXTR genes OTX1, TRIM 9, MT 1H and FOX A3 as biomarkers ([Fig.7])

[0097] In this test, performed on all the cohorts described above, the patients in each cohort were differentiated according to:

[0098] - from the 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] - from 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 on [Fig.7]),

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

[0101] For each of the cohorts, it is noted that patients in group 0-2, for whom the expression of a maximum of 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 the genes DNAJC6, EHHADH, OXTR, OTX1, TRIM9, MT1H, PCDH9 and FOXA3 as biomarkers ([Fig. 8D

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

[0104] - from the 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 on [Fig.8]),

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

[0107] For each of the cohorts, it is noted that patients in group 0-2, for whom the expression of a maximum of 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 with 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] In addition, the data presented also demonstrate that the robustness of this indicator is enhanced as the number of the eight biomarker genes (whose expression is detected) used increases.

[0110] Finally, this indicator provided by the diagnostic method according to the invention and by the associated prognostic tool being complementary and distinct from 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 with large-scale diffuse B-cell lymphoma (LDBCL).

Claims

Demands

1. A method for in vitro or ex vivo diagnostic assessment of the survival chances of a patient with diffuse large-scale B-cell lymphoma (DLBCL), comprising a step of detecting the expression of the TRIM9 gene, in a biological sample of said patient.

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

3. A method according to any one of the preceding claims, wherein 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. A method according to any one of the preceding claims, wherein the detection of gene expression is carried out via at least one technology selected 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 detection step comprises the detection of 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. A method according to claim 5, wherein the detection step comprises the detection of the expression of the eight genes.

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

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