Publications by authors named "Catherine Maingonnat"

27 Publications

  • Page 1 of 1

Non-invasive monitoring of diffuse large B-cell lymphoma by cell-free DNA high-throughput targeted sequencing: analysis of a prospective cohort.

Blood Cancer J 2018 08 1;8(8):74. Epub 2018 Aug 1.

INSERM U1245, Centre Henri Becquerel, University of Rouen, Rouen, France.

From a liquid biopsy, cell-free DNA (cfDNA) can provide information regarding basal tumoral genetic patterns and changes upon treatment. In a prospective cohort of 30 diffuse large B-cell lymphomas (DLBCL), we determined the clinical relevance of cfDNA using targeted next-generation sequencing and its correlation with PET scan imaging at the time of diagnosis and during treatment. Using a dedicated DLBCL panel, mutations were identified at baseline for 19 cfDNAs and profiles were consistent with expected DLBCL patterns. Tumor burden-related clinical and PET scan features (LDH, IPI, and metabolic tumor volume) were significantly correlated with the quantity of tumoral cfDNA. Among the four patients presenting additional mutations in their cfDNAs, three had high metabolic tumor volumes, suggesting that cfDNA more accurately reflects tumor heterogeneity than tissues biopsy itself. Mid-treatment, four patients still had basal mutations in their cfDNAs, including three in partial response according to their Deauville scores. Our study highlights the major interests in liquid biopsy, in particular in the context of bulky tumors where cfDNA allows capturing the entire tumoral mutation profile. Therefore, cfDNA analysis in DLBCL represents a complementary approach to PET scan imaging.
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http://dx.doi.org/10.1038/s41408-018-0111-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070497PMC
August 2018

New generation sequencing of targeted genes in the classical and the variant form of hairy cell leukemia highlights mutations in epigenetic regulation genes.

Oncotarget 2018 Jun 22;9(48):28866-28876. Epub 2018 Jun 22.

Normandie Univ, INSERM U1245, Université de Caen, Caen, France.

Classical hairy cell leukemia (HCL-c) is a rare lymphoid neoplasm. mutation, detected in more than 80% of the cases, is described as a driver mutation, but additional genetic abnormalities appear to be necessary for the disease progression. For cases of HCL-c harboring a wild-type gene, the differential diagnosis of the variant form of HCL (HCL-v) or splenic diffuse red pulp lymphoma (SDRPL) is complex. We selected a panel of 21 relevant genes based on a literature review of whole exome sequencing studies (, , , , , , , , , , , , , , , , , , , , and ). We analyzed 20 HCL-c and 4 HCL-v patients. The analysis of diagnostic samples mutations in ( = 18), ( = 4), ( = 3), ( = 2), ( = 2), ( = 2), ( = 2) ( = 1) and ( = 1). was found in 90% (18/20) of HCL-c patients. In HCL-c patients with , other mutations were found in 33% (6/18) of cases. All 4 HCL-v patients had mutations in epigenetic regulatory genes: ( = 2), ( = 1) or ( = 1). The analysis of sequential samples (at diagnosis and relapse) from 5 patients (2 HCL-c and 3 HCL-v), showed the presence of 2 new subclonal mutations ( and ) in one patient and variations of the mutated allele frequency in 2 other cases. In the HCL-v disease, we described new mutations targeting that encode a lysine demethylase protein. This opens new perspectives for personalized medicine for this group of patients.
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http://dx.doi.org/10.18632/oncotarget.25601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034755PMC
June 2018

Non-invasive detection of somatic mutations using next-generation sequencing in primary central nervous system lymphoma.

Oncotarget 2017 Jul;8(29):48157-48168

Department of Hematology, Cancer Center Henri Becquerel, 76000 Rouen, France.

Purpose: Primary central nervous system lymphomas (PCNSL) have recurrent genomic alterations. The main objective of our study was to demonstrate that targeted sequencing of circulating cell-free DNA (cfDNA) released by PCNSL at the time of diagnosis could identify somatic mutations by next-generation sequencing (NGS).

Patients And Methods: PlasmacfDNA and matched tumor DNA (tDNA) from 25 PCNSL patients were sequenced using an Ion Torrent Personal Genome Machine (Life Technologies®). First, patient-specific targeted sequencing of identified somatic mutations in tDNA was performed. Then, a second sequencing targeting MYD88 c.T778C was performed and compared to plasma samples from 25 age-matched control patients suffering from other types of cancer.

Results: According to the patient-specific targeted sequencing, eight patients (32% [95% CI 15-54%]) had detectable somatic mutations in cfDNA. Considering MYD88 sequencing, six patients had the specific c.T778C alteration detected in plasma. Using a control group, the sensitivity was 24% [9-45%] and the specificity was 100%. Tumor volume or deep brain structure involvement did not influence the detection of somatic mutations in plasma.

Conclusion: This pilot study provided evidence that somatic mutations can be detected by NGS in the cfDNA of a subset of patients suffering from PCNSL.
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http://dx.doi.org/10.18632/oncotarget.18325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564634PMC
July 2017

Identification of Somatic Mutations in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type by Massive Parallel Sequencing.

J Invest Dermatol 2017 09 4;137(9):1984-1994. Epub 2017 May 4.

Department of Hematology, Henri Becquerel Comprehensive Cancer Center and Normandie Université, UNIROUEN, INSERM U1245, Team Genomics and biomarkers in lymphoma and solid tumors, Rouen, France. Electronic address:

To determine whether the mutational profile of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) is unique by comparison with other diffuse large B-cell lymphoma subtypes, we analyzed a total cohort of 20 PCLBCL-LT patients by using next-generation sequencing with a lymphoma panel designed for diffuse large B-cell lymphoma. We also analyzed 12 pairs of tumor and control DNA samples by whole-exome sequencing, which led us to perform resequencing of three selected genes not included in the lymphoma panel: TBL1XR1, KLHL6, and IKZF3. Our study clearly identifies an original mutational landscape of PCLBCL-LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1, and CD79B. Other genes involved in B-cell signaling, NF-κB activation, or DNA modeling were found altered, notably TBL1XR1 (33%), MYC (26%) CREBBP (26%), and IRF4 (21%) or HIST1H1E (41%). MYD88 variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of patients. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3, and CIITA. Together, these results show that PCLBCL-LT exhibits a unique mutational landscape, combining highly recurrent hotspot mutations in genes involved in NF-kB and B-cell signaling pathways, which provides a rationale for using selective inhibitors of the B-cell receptor.
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http://dx.doi.org/10.1016/j.jid.2017.04.010DOI Listing
September 2017

Biological and Clinical Relevance of Associated Genomic Alterations in MYD88 L265P and non-L265P-Mutated Diffuse Large B-Cell Lymphoma: Analysis of 361 Cases.

Clin Cancer Res 2017 May 6;23(9):2232-2244. Epub 2016 Dec 6.

Inserm U918, Centre Henri Becquerel, Université de Rouen, IRIB, Rouen, France.

mutations, notably the recurrent gain-of-function L265P variant, are a distinguishing feature of activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL), leading to constitutive NFκB pathway activation. The aim of this study was to examine the distinct genomic profiles of -mutant DLBCL, notably according to the presence of the L265P or other non-L265P MYD88 variants. A cohort of 361 DLBCL cases (94 mutant and 267 wild-type) was submitted to next-generation sequencing (NGS) focusing on 34 genes to analyze associated mutations and copy number variations, as well as gene expression profiling, and clinical and prognostic analyses. Importantly, we highlighted different genomic profiles for MYD88 L265P and MYD88 non-L265P-mutant DLBCL, shedding light on their divergent backgrounds. Clustering analysis also segregated subgroups according to associated genetic alterations among patients with the same mutation. We showed that associated and MYD88 L265P mutations act synergistically to increase NFκB pathway activation, although the majority of MYD88 L265P-mutant cases harbors downstream NFκB alterations, which can predict BTK inhibitor resistance. Finally, although the MYD88 L265P variant was not an independent prognostic factor in ABC DLBCL, associated mutations significantly improved the survival of MYD88 L265P-mutant ABC DLBCL in our cohort. This study highlights the relative heterogeneity of -mutant DLBCL, adding to the field's knowledge of the theranostic importance of mutations, but also of associated alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1922DOI Listing
May 2017

Oncogenic events rather than antigen selection pressure may be the main driving forces for relapse in diffuse large B-cell lymphomas.

Am J Hematol 2017 Jan;92(1):68-76

INSERM U918, Centre Henri Becquerel, Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France.

Little is known on the phylogenetic relationship between diagnostic and relapse clones of diffuse large B-cell lymphoma (DLBCL). We applied high throughput sequencing (HTS) of the VDJ locus of Immunoglobulin heavy chain (IGHV) on 14 DLBCL patients with serial samples, including tumor biopsies and/or peripheral blood mononuclear cells (PBMC). Phylogenetic data were consolidated with targeted sequencing and cytogenetics. Phylogeny clearly showed that DLBCL relapse could occur according either an early or a late divergent mode. These two modes of divergence were independent from the elapsed time between diagnosis and relapse. We found no significant features for antigen selection pressure in complementary determining region both at diagnosis and relapse for 9/12 pairs and a conserved negative selection pressure for the three remaining cases. Targeted HTS and conventional cytogenetics revealed a branched vs. linear evolution for 5/5 IGHV early divergent cases, but unexpected such "oncogenetic" branched evolution could be found in at least 2/7 IGHV late divergent cases. Thus, if BCR signaling is mandatory for DLBCL emergence, oncogenetic events under chemotherapy selection pressure may be the main driving forces at relapse. Finally, circulating subclones with divergent IGHV somatic hypermutations patterns from initial biopsy could be detected in PBMC at diagnosis for 4/6 patients and, for two of them, at least one was similar to the ones found at relapse. This study highlights that oncogenetic intraclonal diversity of DLBCL should be evaluated beyond the scope a single biopsy and represents a rationale for future investigations using peripheral blood for lymphoid malignancies genotyping. Am. J. Hematol. 92:68-76, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajh.24584DOI Listing
January 2017

Detection and prognostic value of recurrent exportin 1 mutations in tumor and cell-free circulating DNA of patients with classical Hodgkin lymphoma.

Haematologica 2016 09 13;101(9):1094-101. Epub 2016 Jun 13.

Department of Hematology, Centre Henri Becquerel, Rouen, France INSERM U918, Centre Henri Becquerel, University of Rouen, Rouen, France

Classical Hodgkin lymphoma is one of the most common lymphomas and shares clinical and genetic features with primary mediastinal B-cell lymphoma. In this retrospective study, we analyzed the recurrent hotspot mutation of the exportin 1 (XPO1, p.E571K) gene, previously identified in primary mediastinal B-cell lymphoma, in biopsies and plasma circulating cell-free DNA from patients with classical Hodgkin lymphoma using a highly sensitive digital PCR technique. A total of 94 patients were included in the present study. This widely expressed XPO1 E571K mutation is present in one quarter of classical Hodgkin lymphoma patients (24.2%). Mutated and wild-type classical Hodgkin lymphomas were similar regarding the main clinical features. Patients with a detectable XPO1 mutation at the end of treatment displayed a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation in plasma cell-free DNA (2-year progression-free survival: 57.1%, 95% confidence interval: 30.1-100% versus 2-year progression-free survival: 90.5%, 95% confidence interval: 78.8-100%, respectively, P=0.0601). To conclude, the detection of the XPO1 E571K mutation in biopsy and plasma cell-free DNA by digital PCR may be used as a novel biomarker in classical Hodgkin lymphoma for both diagnosis and minimal residual disease, and pinpoints a crucial role of XPO1 in classical Hodgkin lymphoma pathogenesis. The detection of somatic mutation in the plasma cell-free DNA of patients represents a major technological advance in the context of liquid biopsies and noninvasive management of classical Hodgkin lymphoma.
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http://dx.doi.org/10.3324/haematol.2016.145102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5060026PMC
September 2016

Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma.

Am J Hematol 2016 09 4;91(9):923-30. Epub 2016 Jul 4.

Departement of Hematology, Inserm U955 Team 09, APHP Hospital Henri Mondor, Créteil, France.

Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein. Am. J. Hematol. 91:923-930, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajh.24451DOI Listing
September 2016

HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis and is down-regulated by both deletion and epigenetic alterations.

Leuk Res 2016 06 11;45:90-100. Epub 2016 Apr 11.

Henri Becquerel Center, INSERM U918, Institut de Recherche et d'Innovation Biomédicale (iRiB) Rouen, France.

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1, HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in many human tumors. Here, we report HACE1 as a candidate tumor suppressor gene down-regulated by a combination of deletion and epigenetic mechanisms. HACE1 deletions were observed in 40% of B-cell lymphoma tumors. Hypermethylation of the HACE1 promoter CpG177 island was found in 60% (68/111) of cases and in all tested B-cell lymphoma lines. Using HDAC inhibitors, we observed predominantly inactive chromatin conformation (methylated H3 histones H3K9me2) in HACE1 gene promoter region. We demonstrated in Ramos and Raji cells that down-regulation of HACE1 expression was associated with a significant decrease in apoptosis and an accumulation of cells in the S and G2/M phases. Our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and can be downregulated by deacetylation of its promoter region chromatin, which makes HACE1 a potential target for HDAC inhibitors.
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http://dx.doi.org/10.1016/j.leukres.2016.04.007DOI Listing
June 2016

Digital PCR for quantification of recurrent and potentially actionable somatic mutations in circulating free DNA from patients with diffuse large B-cell lymphoma.

Leuk Lymphoma 2016 09 17;57(9):2171-9. Epub 2016 Feb 17.

a Department of Hematology , Centre Henri Becquerel , Rouen , France ;

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments.
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http://dx.doi.org/10.3109/10428194.2016.1139703DOI Listing
September 2016

Next-Generation Sequencing in Diffuse Large B-Cell Lymphoma Highlights Molecular Divergence and Therapeutic Opportunities: a LYSA Study.

Clin Cancer Res 2016 06 27;22(12):2919-28. Epub 2016 Jan 27.

Inserm U918, Centre Henri Becquerel, Université de Rouen, IRIB, Rouen, France.

Purpose: Next-generation sequencing (NGS) has detailed the genomic characterization of diffuse large B-cell lymphoma (DLBCL) by identifying recurrent somatic mutations. We set out to design a clinically feasible NGS panel focusing on genes whose mutations hold potential therapeutic impact. Furthermore, for the first time, we evaluated the prognostic value of these mutations in prospective clinical trials.

Experimental Design: A Lymphopanel was designed to identify mutations in 34 genes, selected according to literature and a whole exome sequencing study of relapsed/refractory DLBCL patients. The tumor DNA of 215 patients with CD20(+)de novo DLBCL in the prospective, multicenter, and randomized LNH-03B LYSA clinical trials was sequenced to deep, uniform coverage with the Lymphopanel. Cell-of-origin molecular classification was obtained through gene expression profiling with HGU133+2.0 Affymetrix GeneChip arrays.

Results: The Lymphopanel was informative for 96% of patients. A clear depiction of DLBCL subtype molecular heterogeneity was uncovered with the Lymphopanel, confirming that activated B-cell-like (ABC), germinal center B-cell like (GCB), and primary mediastinal B-cell lymphoma (PMBL) are frequently affected by mutations in NF-κB, epigenetic, and JAK-STAT pathways, respectively. Novel truncating immunity pathway, ITPKB, MFHAS1, and XPO1 mutations were identified as highly enriched in PMBL. Notably, TNFAIP3 and GNA13 mutations in ABC patients treated with R-CHOP were associated with significantly less favorable prognoses.

Conclusions: This study demonstrates the contribution of NGS with a consensus gene panel to personalized therapy in DLBCL, highlighting the molecular heterogeneity of subtypes and identifying somatic mutations with therapeutic and prognostic impact. Clin Cancer Res; 22(12); 2919-28. ©2016 AACRSee related commentary by Lim and Elenitoba-Johnson, p. 2829.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2305DOI Listing
June 2016

Whole exome sequencing of relapsed/refractory patients expands the repertoire of somatic mutations in diffuse large B-cell lymphoma.

Genes Chromosomes Cancer 2016 Mar 26;55(3):251-67. Epub 2015 Nov 26.

INSERM U918, Centre Henri Becquerel, Rouen, France.

Despite the many efforts already spent to enumerate somatic mutations in diffuse large B-cell lymphoma (DLBCL), previous whole-genome and whole-exome studies conducted on patients of mixed outcomes failed at characterizing the 30% of patients who will relapse or resist current immunochemotherapies. To address this issue, we performed whole-exome sequencing of normal/tumoral DNA pairs in 14 relapsed/refractory (R/R) patients subclassified by full-transcriptome arrays (six activated B-cell like, three germinal center B-cell like, and five primary mediastinal B-cell lymphomas), from the LNH-03 LYSA clinical trial program. Aside from well-known DLBCL features, gene and pathway level recurrence analyses proposed several interesting leads including TBL1XR1 and activating mutations in IRF4 or in the insulin regulation pathway. Sequencing-based copy number analysis defined 23 short recurrently altered regions involving genes such as REL, CDKN2A, HYAL2, and TP53. Moreover, it highlighted mutations in genes such as GNA13, CARD11, MFHAS1, and PCLO as associated with secondary variant allele amplification events. The five primary mediastinal B-cell lymphomas (PMBL), while unexpected in a R/R cohort, showed a significantly higher mutation rate (P = 0.003) and provided many insights on this classical Hodgkin lymphoma related subtype. Novel genes such as XPO1, MFHAS1, and ITPKB were found particularly mutated, along with various cytokine-based signaling pathways. Among these analyses, somatic events in the NF-κB pathway were found preponderant in the three DLBCL subtypes, confirming its major implication in DLBCL aggressiveness and pinpointing several new candidate genes.
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http://dx.doi.org/10.1002/gcc.22328DOI Listing
March 2016

Immunohistochemical and genomic profiles of diffuse large B-cell lymphomas: implications for targeted EZH2 inhibitor therapy?

Oncotarget 2015 Jun;6(18):16712-24

INSERM U918, Centre Henri Becquerel, Université de Rouen, IRIB, Rouen, France.

Enhancer of Zeste Homolog 2 (EZH2) plays an essential epigenetic role in Diffuse Large B Cell Lymphoma (DLBCL) development. Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, most notably affecting tyrosine 641 (Y641), inducing hyper-trimethylation of H3K27 (H3K27me3). Novel EZH2 inhibitors are being tested in phase 1 and 2 clinical trials but no study has examined which patients would most benefit from this treatment. We evaluated the immunohistochemical (IHC) methylation profiles of 82 patients with DLBCL, as well as the mutational profiles of 32 patients with DLBCL using NGS analysis of a panel of 34 genes involved in lymphomagenesis. A novel IHC score based on H3K27me2 and H3K27me3 expression was developed, capable of distinguishing patients with wild-type (WT) EZH2 and patients with EZH2 Y641 mutations (p = 10-5). NGS analysis revealed a subclonal EZH2 mutation pattern in EZH2 mutant patients with WT-like IHC methylation profiles, while associated mutations capable of upregulating EZH2 were detected in WT EZH2 patients with mutant-like IHC methylation profiles. IHC and mutational profiles highlight in vivo hyper-H3K27me3 and hypo-H3K27me2 status, pinpoint associated activating mutations and determine EZH2 mutation clonality, maximizing EZH2 inhibitor potential by identifying patients most likely to benefit from treatment.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599301PMC
http://dx.doi.org/10.18632/oncotarget.3154DOI Listing
June 2015

CD70: A Potential Target in Breast Cancer?

J Cancer 2014 22;5(9):761-4. Epub 2014 Oct 22.

1. Department of Medical Oncology, Centre Henri Becquerel, Rouen, France; ; 3. INSERM U918, Centre Henri Becquerel, IRIB, Rouen, France.

CD70 is a co-stimulatory molecule involved in the immune response and also in cancer development and progression. Recent studies show that high CD70 expression in cancer cells may inhibit the anti-tumor response. Furthermore, CD70 expression has been reported as a predictive marker of resistance to chemotherapy in ovarian cancers. Some in vitro studies have shown that CD70 expression is epigenetically down-regulated through hypermethylation of its promoter during tumoral progression. This study evaluated the level of CD70 expression in surgical samples of breast invasive tumors and determined its correlation with CD70 promoter methylation. Twenty "luminal A" and 20 "basal-like" frozen samples from early breast tumors were retrospectively selected. CD70 expression was evaluated by quantitative real-time PCR. Total DNA was bisulfite-treated, and methylation levels of 5 consecutive CG sites present in the proximal region (-464, -421) of the promoter were assessed by pyrosequencing analysis. Statistical analyses were performed using the Mann-Whitney test. The median relative CD70 expression level was 0.37 and was significantly higher in the basal-like group (0.78 [0.24-31.7]) compared to the luminal A group (0.25 [0.03-1.83], p=0.0001). The median methylation level was 61%, with no significant difference between the basal-like (63%) and luminal A (58%) groups. No correlation was found between CD70 expression and CD70 methylation level. In this study, higher CD70 expression was observed in the basal-like group, but this expression was not related to promoter methylation. The higher expression in the poor-prognosis subgroup of patients makes CD70 a potential target for emerging anti-CD70 therapies.
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http://dx.doi.org/10.7150/jca.10360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216800PMC
November 2014

Activating somatic mutations in diffuse large B-cell lymphomas: lessons from next generation sequencing and key elements in the precision medicine era.

Leuk Lymphoma 2015 May 9;56(5):1213-22. Epub 2014 Oct 9.

INSERM U918, Centre Henri Becquerel, Rouen University and IRIB , Rouen , France.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30-40% of newly diagnosed non-Hodgkin lymphomas. Historically, DLBCL has been thought to involve recurrent translocations of the immunoglobulin heavy (IGH) locus and the deregulation of rearranged oncogenes. Whole exome sequencing (WES) of more than 200 DLBCLs has completely redefined the genetic landscape of the disease by identifying recurrent single nucleotide variants and providing new therapeutic opportunities in DLBCL molecular subtypes. Some of these somatic mutations target genes that play a crucial role in B-cell function (B cell receptor [BCR] signaling, nuclear factor κB [NF-κB] pathway, Toll-like receptor [TLR] signaling and phosphatidylinositol 3-kinase [PI3K] pathway), immunity, cell cycle/apoptosis or chromatin modification. In this review, following an overview of the somatic mutations reported in DLBCL, we focus on activating and clustered mutations targeting genes including MYD88, CD79A/B, EZH2 and CARD11 and discuss their clinical and therapeutic relevance in the precision medicine era.
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http://dx.doi.org/10.3109/10428194.2014.941836DOI Listing
May 2015

Targetable activating mutations are very frequent in GCB and ABC diffuse large B-cell lymphoma.

Genes Chromosomes Cancer 2014 Feb 5;53(2):144-53. Epub 2013 Nov 5.

Inserm U918, Centre Henri Becquerel, Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France.

Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy that can be divided in two major subgroups, germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Activating mutations of genes involved in the BCR and NF-κB pathways (CD79A, CD79B, MYD88, and CARD11) or in epigenetic regulation (EZH2) have been recently reported, preferentially in one of the two DLBCL subtypes. We analyzed the mutational status of these five recurrently mutated genes in a cohort of 161 untreated de novo DLBCL. Overall, 93 mutations were detected, in 61 (38%) of the patients. The L265P MYD88 mutation was the most frequent MYD88 variant (n = 18), observed exclusively in the ABC subtype. CD79A/CD79B ITAM domains were targeted in ABC DLBCL (12/77; 16%), whereas CARD11 mutations were equally distributed in the two subtypes. The EZH2 Y641 substitution was found almost exclusively in the GCB subgroup (15/62; 24%). Twenty cases (12%) displayed two activating mutations, including the most frequent CD79/MYD88 variants combination (n = 8) which is observed exclusively in the ABC subtype. When considering only ABC DLBCL patients treated by rituximab plus chemotherapy, the presence of an activating NF-κB mutation was associated with an unfavorable outcome (3-years OS 26% for mutated cases versus 67% for the cases without mutations, P = 0.0337). Our study demonstrates that activating and targetable mutations are observed at a very high frequency in DLBCL at the time of diagnosis, indicating that sequencing of a limited number of genes could help tailor an optimal treatment strategy in DLBCL.
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http://dx.doi.org/10.1002/gcc.22126DOI Listing
February 2014

Interim positron emission tomography scan associated with international prognostic index and germinal center B cell-like signature as prognostic index in diffuse large B-cell lymphoma.

Leuk Lymphoma 2012 Jan 1;53(1):34-42. Epub 2011 Aug 1.

UMR INSERM U918, Centre Henri Becquerel, Rouen, France.

[(18)F]-fluorodeoxyglucose positron emission tomography (FDG-PET) imaging is essential to optimize the initial staging and to predict the prognosis of diffuse large B-cell lymphoma (DLBCL). To assess the relationship between the germinal center B cell-like/activated B cell-like (GCB/ABC) classification and PET scan features in DLBCL, 57 cases treated with rituximab and a cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP)/CHOP-like regimen were analyzed. The expression profile of 18 GCB/ABC related genes and five genes coding for glucose transporters (GLUTs) was determined from frozen tissues using DASL (cDNA-mediated Annealing, Selection, Ligation and extension) technology. According to the gene expression profile (GEP), 30 cases of DLBCL were classified as GCB subtype (2-year progression-free survival [PFS] 76%) and 27 cases as ABC subtype (2-year PFS 51%, p = 0.03). Using a semiquantitative assessment of the decrease in standard uptake value (SUV) at interim PET performed after 3-4 cycles of chemotherapy, we defined fast (n = 36) and slow (n = 9) metabolic responders. In multivariate analysis, GCB/ABC subtype, age-adjusted international prognostic index (aaIPI) and slow/fast metabolic response were independent variables that predicted outcome. A score incorporating aaIPI, fast/slow metabolic response and GCB/ABC classification was used to define two groups with highly significantly distinct outcomes. Our study suggests that the combination of GEP, aaIPI and interim PET more accurately predicts DLBCL prognosis and is therefore suitable for tailoring therapeutic strategies.
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http://dx.doi.org/10.3109/10428194.2011.600482DOI Listing
January 2012

PVRL2 is translocated to the TRA@ locus in t(14;19)(q11;q13)-positive peripheral T-cell lymphomas.

Genes Chromosomes Cancer 2007 Nov;46(11):1011-8

Groupe d'Etude des Proliférations Lymphoides (GPL), European Institute for Peptides Research (IFRMP23), Centre Henri Becquerel, Rouen, France.

Very few recurrent chromosomal abnormalities have been identified in T-cell non-Hodgkin lymphomas. These involve the TRA@/TRD@ gene at chromosome band 14q11 in up to 15% of cases. We recently reported a novel and recurrent translocation, t(14;19)(q11;q13), in peripheral T-cell lymphoma (PTCL). Fluorescence in situ hybridization analysis performed in three cases suggested an involvement of the TRA@/TRD@ locus at 14q11 and of a region telomeric to BCL3 on 19q13. We now report the molecular cloning of these translocations. Sequence analysis confirmed the involvement of the TRA@/TRD@ and indicated that the breakpoints were located mainly in the TRAJ region. On chromosome 19, our results revealed a new clustering of breakpoints outside the region involved in t(14;19)(q32;q13)-positive B-cell malignancies. Remarkably, all three breaks were located downstream or within the PVRL2 gene, in a small 10.3 kb interval, suggesting a nonrandom location of the breakpoints. For two patients, a high mRNA expression of both PVRL2 and BCL3 was found. In conclusion, we identified PVRL2 as a new recurrent partner gene of the TRA@ locus in PTCL. These results suggest that both BCL3 and PVRL2 may participate in the pathogenesis of these PTCLs, but further studies should be undertaken to investigate the precise role of these genes.
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http://dx.doi.org/10.1002/gcc.20490DOI Listing
November 2007

Hyaluronectin modulation of lung metastasis in nude mice.

Eur J Cancer 2006 Dec 23;42(18):3253-9. Epub 2006 Aug 23.

Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.

Hyaluronectin (HN) is a glycoprotein with a high affinity to hyaluronic acid (HA) and known to be a component of the extracellular matrix of tumours. Clinical studies have shown that a low level of HN correlates to tumours with poor prognosis, whereas a high level of HN correlates to tumours with good prognosis. We previously demonstrated in vitro that hyaluronidase activity, which promotes tumour progression and metastatic spread by degradation of HA into angiogenic oligosaccharides, was inhibited or promoted by HN, according to the level of HN-expression. This raises the question of the role played by HN in cancer, and particularly if high and low levels of HN-expression could trigger opposite effects on tumour growth and/or metastatic spread. To address this issue, we used a model of spontaneous lung fluorescent metastases that we characterised previously. We stably transfected the human HN cDNA into fluorescent H460MGFP cells and selected two clones characterised by different levels of HN-expression: HN110 and HN704, with a high and a low level of HN-expression, respectively. In vitro, we demonstrated that HN704 cell migration was significantly increased. Inoculation of clones to nude mice had no significant effect on tumour growth, but clearly revealed opposite effects on metastatic spread: HN110 significantly decreased the number of fluorescent metastases whereas HN704 significantly increased it. We also analysed HN, HA and hyaluronidase contents in sera and tumours. These results demonstrate that HN can play a role as either a suppressor or promoter of metastatic spread.
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http://dx.doi.org/10.1016/j.ejca.2006.06.012DOI Listing
December 2006

Expression of HYAL2 mRNA, hyaluronan and hyaluronidase in B-cell non-Hodgkin lymphoma: relationship with tumor aggressiveness.

Int J Cancer 2005 Jan;113(2):207-12

INSERM EMI 9906-IFR23, Molecular Biology Laboratory, Department of Hematology, Centre Henri-Becquerel, Rue d'Amiens, 76038 Rouen Cedex, France.

Hyaluronidases and their substrate, hyaluronan (HA), were mainly explored in solid tumors but rarely in hematologic malignancies. While HA involvement was demonstrated in invasion and metastasis in most cases of solid tumors, the role of hyaluronidases in cancer progression remains controversial. One of the hyaluronidases, HYAL2, is suspected to be involved in the first step of HA degradation. In this work, HYAL2 mRNA, HA and total hyaluronidases expression were examined in lymphoma tissue extracts and correlated to the lymphoma subtype. Real-time RT-PCR was performed to evaluate HYAL2 mRNA. HA and hyaluronidase were assayed by enzyme-linked sorbent assay. Our results showed that HYAL2 mRNA expression was correlated to lymphoma diagnosis (p = 6 x 10(-3)) and was significantly lower in high-grade lymphoma, i.e., diffuse large B-cell diffuse lymphomas (DLBCLs). Several forms of hyaluronidase were detected by zymography and total hyaluronidase activity detected in tissue extracts was not significantly different according to tumor grade. HA levels also correlated to lymphoma subtype (p = 1 x 10(-5)) and were higher in DLBCLs. Moreover, HYAL2 mRNA and HA expressions were inversely correlated (p = 0.035). HYAL2 gene is localized on chromosome 3p21, which contains candidates tumor suppressor genes. Our results suggest that HYAL2 may have a prognostic significance in lymphomas and an antioncogenic activity. Conversely, HA overexpression in high-grade lymphomas is in favor of its involvement in tumor development and could provide a useful target for lymphoma therapy using HA-binding peptides.
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http://dx.doi.org/10.1002/ijc.20562DOI Listing
January 2005

Biodistribution of injected tritiated hyaluronic acid in mice: a comparison between macromolecules and hyaluronic acid-derived oligosaccharides.

In Vivo 2004 Mar-Apr;18(2):181-7

Laboratoire d'Oncologie Moléculaire, Centre Henri-Becquerel, rue d'Amiens, 76000 Rouen, France.

Background: Hyaluronan (HA) has been reported to bind specifically and with high affinity to various cell types and to directly modify cell behaviour. In a previous report we demonstrated that both high molecular weight molecules (HA(H)) and HA-derived oligosaccharides were efficient at triggering terminal differentiation of acute myeloid leukemia (AML) blasts, in vitro, through CD44 ligation.

Materials And Methods: To explore the possibility of using HA for a differentiation therapy in AML, we investigated whether intravenous injection of tritiated HA(H) and/or HA-derived oligosaccharides (HA10-20) into mice accumulated in bone marrow, the main site of AML cell proliferation.

Results: The present work showed that the level of HA in bone marrow: 1) was maximum 5 hours after injection of either HA(H) or HA10-20; 2) was about 40 times higher after HA(H) than after HA10-20 injection. The amount of HA in bone marrow (5.8% ID/g) was two-fold higher than in serum, indicating that it was not due to circulating blood. Finally, using chromatographic analysis, we showed that about 34% of tritiated HA present in bone marrow 5 hours after HA(H) injection displayed a size higher or equal to HA10.

Conclusion: After a single injection of macromolecular hyaluronan in mouse bone marrow we obtained a concentration of oligosaccharides close to the one shown to trigger AML cell differentiation in vitro. A part of the oligosaccharides had a size higher than or equal to the minimal one required to interact with HA receptors.
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November 2004

Hyaluronidase in sera of tumour-bearing nude mice.

Biomarkers 2003 May-Aug;8(3-4):333-8

Department of Molecular Oncology, Centre Henri-Becquerel, Rue d'Amiens, 76000 Rouen, France.

Cancer cell lines often secrete hyaluronidase, suggesting that this enzyme could be used as a marker of growing tumours. We have measured hyaluronidase in the sera of non-grafted mice and mice grafted with human tumour-derived hyaluronidase-secreting H460M and SA87 cells or non-secreting CB 193 cells. Mouse serum hyaluronidase was measured at pH 3.8 using the enzyme-linked sorbent assay (ELSA) technique by reference to human serum whose activity at pH 3.8 was determined by the Reissig technique. The serum hyaluronidase in non-grafted mice ranged from 310-520 mU l(-1) (mean+/-SD 432+/-70 mU l(-1), median 440 mU l(-1)). Hyaluronidase increased in the sera of tumour-bearing mice grafted with H460M cells or with SA87 cells, but not in the sera of mice grafted with CB 193 cells. Serum hyaluronidase activity in H460M or SA87 tumour-bearing mice correlated with the tumour mass, increased with time, and decreased after tumour removal. Zymography detected two different hyaluronidase forms in the sera of non-grafted mice: type 1 had only one hyaluronidase band and type 2 had five different bands. In both types, enzyme augmentation in tumour-bearing mice correlated with the presence of an additional enzyme band that was not seen in normal sera and that migrated as the cancer cell enzyme did; there was no augmentation of the normal isoform(s). These results show that serum hyaluronidase can be used to follow the development of tumours in mice grafted with hyaluronidase-secreting cells.
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http://dx.doi.org/10.1080/1354750031000120143DOI Listing
June 2004

Human monocytes synthesize hyaluronidase.

Br J Haematol 2002 Oct;119(1):199-203

Laboratory of Molecular Oncology, Centre Henri-Becquerel, Rouen, France.

The involvement of hyaluronic acid (HA) oligosaccharides and blood-derived mononuclear cells in inflammatory processes prompted us to determine whether peripheral blood mononuclear cells (PBMC) possess hyaluronidase activity. PBMC were incubated with macromolecular-tritiated HA at pH 3.8 and supernatants were analysed by size exclusion chromatography to reveal digestion of HA. This digestion was due to the CD14-positive (CD14+), adherent, non-specific esterase-positive, subpopulation of PBMC. Hyaluronidase activity (72 kDa) was found in aqueous and non-ionic detergent PBMC extracts but not in the medium in which the cells had been cultured. These results indicate that hyaluronidase is, at least in part, linked to the membrane rather than excreted. Hence, monocytes have one or more hyaluronidases that can generate a pool of active HA fragments within tissues. Hyaluronidase activity was also found in 3/3 myelomonocytic lineage leukaemias but not in 3/3 lymphoblastic leukaemias.
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http://dx.doi.org/10.1046/j.1365-2141.2002.03733.xDOI Listing
October 2002

Hyaluronidase is more elevated in human brain metastases than in primary brain tumours.

Anticancer Res 2002 Jul-Aug;22(4):2423-7

Laboratoire d'Oncologie Moléculaire, Centre Henri-Becquerel, Rouen, France.

Background: Hyaluronidase is hypothesised to play a role in cancer invasion and metastasis formation.

Materials And Methods: Hyaluronidase activity was investigated at pH 3.8 in extracts of 30 human brain tumours (17 glioblastomas and 13 brain metastases of carcinomas) and in cancer cell cultures with the ELSA method and zymography.

Results: In brain metastases, hyaluronidase activities were significantly higher than in glioma extracts (9.16 +/- 4.48 mU/g vs 4.25 +/- 5.74) which was not explained by serum hyaluronidase contamination. Serum hyaluronidase of tumour patients' sera was within the normal values determined in 28 matched blood donors'sera (33.8 +/- 11 U/l). The maximum hyaluronidase/albumin (U/g) ratio was 0.9, below which the hyaluronidase content of tumours was below the maximum value calculated from the albumin content of the tumour extract and could not be considered as local production by tumour cells. The hyaluronidase content and hyaluronidase/albumin ratio of metastasis extracts was significantly higher than in glioma extracts and patients' sera, whereas no significant difference was found between the ratios of glioma extracts and sera. The production of hyaluronidase was studied in cell extracts and in culture media of 3 human glioma-derived cell lines and of the brain metastasis-derived cell line SA87. Hyaluronidase activity of the metastasis-derived cell line SA87 was 100 to 1000-fold that of glioma cell lines.

Conclusion: These results suggest that hyaluronidase is associated with the more aggressive cancer cells and is directly or indirectly involved in brain metastasis phenotype.
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September 2002

Importance of hyaluronan length in a hyaladherin-based assay for hyaluronan.

Anal Biochem 2002 Mar;302(2):285-90

Department of Molecular Oncology, Centre Henri-Becquerel, Rue d'Amiens, Rouen 76000, France.

Specific hyaladherin-based assays have been set up to measure the concentration of hyaluronan in biological fluids. Hyaluronectin (HN; a hyaladherin extracted from ovine brain) binds to hyaluronan (HA) that must be 10 units (HA10) or more long. It was therefore of interest to determine whether HN would continue to bind to HA10 in full-length HA since conformational changes might mask potential binding sites. We used the enzyme-linked sorbent assay (ELSA) to assay HA and hyaluronan-derived oligosaccharides, with different standard HAs, and the results were compared to results obtained with the carbazole technique. Oligosaccharide length was calculated from the ratio glucuronic acid/reducing N-acetylglucosamine in fractions of hyaluronidase-digested macromolecular hyaluronan prepared by chromatography; the size of the HA12 oligosaccharide was confirmed by matrix-assisted laser desorption ionization mass spectrometry. During the digestion of macromolecular HA with hyaluronidase, the binding of HN to HA first increased and then decreased as shown using the ELSA. The concentration of HA fragments of HA60 and below was overestimated when intact macromolecular HA was used as the reference for the ELSA, while the concentration of HA100 and above was underestimated when HA10 was used as the reference. The binding of HN to HA20, HA40, and HA60 saccharides was consistent with binding to multiples of HA10 sites. In conclusion, the level of HN binding is determined by the conformation of HA, which may mask binding sites. Hence, calibration HA used in the ELSA must be adapted to the size of HA to assay.
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http://dx.doi.org/10.1006/abio.2001.5557DOI Listing
March 2002