Pierre-Julien Viailly

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

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 AACR. See related commentary by Lim and Elenitoba-Johnson, p. 2829

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

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.

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

Abstract 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.

Kinetics, prognostic and predictive values of ESR1 circulating mutations in metastatic breast cancer patients progressing on aromatase inhibitor

Purpose To assess the prognostic and predictive value of circulating ESR1 mutation and its kinetics before and after progression on aromatase inhibitor (AI) treatment. Patients and methods ESR1 circulating D538G and Y537S/N/C mutations were retrospectively analyzed by digital droplet PCR after first-line AI failure in patients treated consecutively from 2010 to 2012 for hormone receptor-positive metastatic breast cancer. Progression-free survival (PFS) and overall survival (OS) were analyzed according to circulating mutational status and subsequent lines of treatment. The kinetics of ESR1 mutation before (3 and 6 months) and after (3 months) AI progression were determined in the available archive plasmas. Results Circulating ESR1 mutations were found at AI progression in 44/144 patients included (30.6%). Median follow-up from AI initiation was 40 months (range 4-94). The median OS was decreased in patients with circulating ESR1 mutation than in patients without mutation (15.5 versus 23.8 months, P=0.0006). The median PFS was also significantly decreased in patients with ESR1 mutation than in patients without mutation (5.9 vs 7 months, P=0.002). After AI failure, there was no difference in outcome for patients receiving chemotherapy (n = 58) versus non-AI endocrine therapy (n=51) in patients with and without ESR1 mutation. ESR1 circulating mutations were detectable in 75% of all cases before AI progression, whereas the kinetics 3 months after progression did not correlate with outcome. Conclusion ESR1 circulating mutations are independent risk factors for poor outcome after AI failure, and are frequently detectable before clinical progression. Interventional studies based on ESR1 circulating status are warranted.

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

Purpose: MYD88 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 MYD88-mutant DLBCL, notably according to the presence of the L265P or other non-L265P MYD88 variants. Experimental Design: A cohort of 361 DLBCL cases (94 MYD88 mutant and 267 MYD88 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. Results: 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 MYD88 mutation. We showed that associated CD79B 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 CD79B mutations significantly improved the survival of MYD88 L265P–mutant ABC DLBCL in our cohort. Conclusions: This study highlights the relative heterogeneity of MYD88-mutant DLBCL, adding to the fields knowledge of the theranostic importance of MYD88 mutations, but also of associated alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy. Clin Cancer Res; 23(9); 2232–44. ©2016 AACR.

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

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.

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

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.

Somatic mutations of cell-free circulating DNA detected by targeted next-generation sequencing and digital droplet PCR in classical Hodgkin lymphoma

Lucile Bessi, Pierre-Julien Viailly, Elodie Bohers, Philippe Ruminy, Catherine Maingonnat, Philippe Bertrand, NasrinSarafan Vasseur, Ludivine Beaussire, Marie Cornic, Pascaline Etancelin, Vincent Camus, Jean-Michel Picquenot, Herv e Tilly, Aspasia Stamatoullas and Fabrice Jardin INSERM U1245, Centre Henri Becquerel and Rouen University, Rouen, France; Department of Pathology, Centre Henri Becquerel, Rouen, France; Department of Clinical Hematology, Centre Henri Becquerel, Rouen, France

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

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.

Abstract 4810: Exome sequencing of refractory diffuse large B-cell lymphomas highlights candidate genes for targeted resequencing

As the most common lymphoid malignancy, Diffuse Large B-Cell Lymphoma (DLBCL) has largely benefited from immunochemotherapy combinations developed in the past decade. However 30% to 40% of patients still do not respond to treatment, or relapse in a few months. The mechanisms involved in short term treatment failure are poorly understood, and biomarkers to predict refractoriness in newly diagnosed patients are still lacking. To address this issue, 14 normal / tumoral pairs of exomes from patients who progressed or relapsed within 12 months were sequenced on a HiSeq2500 platform. Somatic events were called using VarScan2, and significantly mutated pathways were highlighted by PathScan (False Discovery Rate, FDR \textless 0.001). Genes from 175 pathways were found to be more mutated than expected according to the background mutation rate observed throughout the exomes (2.68 non synonymous mutations per megabase, ranging from 0.69 to 4.66), including genes encoding histone monomers (HIST1H1/2) and transcription regulators (CREBBP, EP300, TP53), members and relatives of the NFKB signaling pathway (MYD88, TNFAIP3, NFKBIA/E), or other signaling cascades (IRF4, CIITA, SOCS1). To pinpoint genes and pathways unusually enriched in such refractory patients, a published series of 39 DLBCL genome pairs was used (Morin et al, Blood 2013). Variant annotation and filtering was reapplied to enforce target region consistency, and Fisher tests were conducted independently in the two series. 22 of the 175 pathways selected by PathScan were found to be enriched in refractory patients (FDR \textless 0.01) but not in the comparative series (FDR \textgreater 0.5). One of the most relevant was “IL4 mediated signaling events”, which includes genes such as STAT6, IRF4 or SOCS1. These 3 genes, along with 31 other genes frequently mutated in DLBCL, were sequenced in a series of 216 DLBCLs with an orthogonal method (PGM sequencer). Although the survival impact of IL4 related gene mutations could not be confirmed, their prevalence (17.5%) was far above what was previously described in DLBCL. Molecular profiling by Affymetrix U133+2 arrays revealed a strong association with the Primary Mediastinal B-Cell lymphoma (PMBL) subtype (Fisher p = 5e-10), a molecular subtype observed in 4/14 and 22/216 patients of the refractory and extended series respectively. We propose here an exhaustive description of mutations found in 14 exomes from refractory DLBCL patients, compared to 39 previously published genomes. The extension to 216 patients enrolled in the LNH03 LYSA (LYmphoma Study Association) clinical trial program using targeted resequencing and transcriptomic arrays allowed us to refine these results, and highlight the high prevalence of somatic mutations in IL4 signaling related genes in PMBLs. Citation Format: Sylvain Mareschal, Pierre-Julien Viailly, Philippe Bertrand, Elodie Bohers, Jean-Philippe Jais, Martin Figeac, Thierry J. Molina, Fabienne Desmots, Thierry Fest, Gilles Salles, Corinne Haioun, Herve Tilly, Karen Leroy, Fabrice Jardin. Exome sequencing of refractory diffuse large B-cell lymphomas highlights candidate genes for targeted resequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2015-4810