Publications by authors named "Jean-Philippe Merlio"

94 Publications

MSI-High RAS-BRAF wild-type colorectal adenocarcinomas with MLH1 loss have a high frequency of targetable oncogenic gene fusions whose diagnoses are feasible using methods easy-to-implement in pathology laboratories.

Hum Pathol 2021 May 19;114:99-109. Epub 2021 May 19.

CHU de la Martinique, Service d'anatomie et Cytologie Pathologiques, Fort-de-France, F-97261, France; Univ Brest, Inserm, CHU de Brest, LBAI, UMR1227, Brest, 29200, France. Electronic address:

Targetable kinase fusions are extremely rare (<1%) in colorectal cancers (CRCs), making their diagnosis challenging and often underinvestigated. They have been shown particularly frequently among MSI-High, BRAF/KRAS/NRAS wild-type CRCs with MLH1 loss (MLH1 MSI-High wild-type). We searched for NTRK1, NTRK2, NTRK3, ALK, ROS1, BRAF, RET, and NRG1 kinase fusions in CRCs using methods easy-to-implement in pathology laboratories: immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and fully automated real-time PCR targeted analyses. RNA-sequencing analyses were used for confirmation. Among 84 selected MLH1 deficient (IHC) CRCs cases, MLH1 MSI-High wild-type CRCs consisted first in 19 cases after Idylla™ analyses and finally in 18 cases (21%) after RNA-sequencing (detection of one additional KRASG12D mutation). FISH (and when relevant, IHC) analyses concluded in 5 NTRK1, 3 NTRK3, 1 ALK, 2 BRAF, and 2 RET FISH positive tumors. ALK and NTRK1 rearranged tumors were IHC positive, but pan-TRK IHC was negative in the 3 NTRK3 FISH positive tumors. RNA-sequencing analyses confirmed 12 of 13 fusions with only one false positive RET FISH result. Finally, 12/18 (67%) of MLH1 MSI-High wild-type CRCs contained targetable kinase fusions. Our study demonstrates the feasibility, but also the cost-effectiveness, of a multistep but rapid diagnostic strategy based on nonsequencing methods to identify rare and targetable kinase fusions in patients with advanced CRCs, as well as the high prevalence of these kinase fusions in MLH1 MSI-High wild-type CRCs. Nevertheless, confirmatory RNA-sequencing analyses are necessary in case of low FISH positive nuclei percentage to rule out FISH false-positive results.
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http://dx.doi.org/10.1016/j.humpath.2021.05.006DOI Listing
May 2021

Exploring hTERT promoter methylation in cutaneous T-cell lymphomas.

Mol Oncol 2021 Mar 14. Epub 2021 Mar 14.

Univ. Bordeaux, INSERM, BaRITOn, U1053, F-33000, Bordeaux, France.

Cutaneous T-cell lymphomas (CTCL) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression. We analyzed hTERT promoter methylation status in CTCL cells compared to healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from -650bp to -150bp and a hypomethylated proximal region from -150bp to +150bp. Interestingly, the hypermethylated region matches with the recently named TERT Hypermethylated Oncogenic Region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect on THOR of two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment as well as a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.
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http://dx.doi.org/10.1002/1878-0261.12946DOI Listing
March 2021

Next-Generation Cancer Biomarkers: Extracellular Vesicle DNA as a Circulating Surrogate of Tumor DNA.

Front Cell Dev Biol 2020 2;8:622048. Epub 2021 Feb 2.

Centre Hospitalier Universitaire (CHU) de Toulouse, Toulouse, France.

Extracellular vesicles (EVs) are produced by healthy tissues and tumor cells and are released in various bodily fluids, including blood. They are limited by bilayer phospholipidic membranes, and they carry a rich content in biomolecules. Their release cleanses the cells of their waste or serves as functional local and distant cell-cell communication and molecular exchange particles. This rich and heterogeneous content has been given intense attention in cancer physiopathology because EVs support cancer control and progression. Because of their specific active cargo, they are being evaluated as carriers of liquid biopsy biomarkers. Compared to soluble circulating biomarkers, their complexity might provide rich information on tumor and metastases status. Thanks to the acquired genomic changes commonly observed in oncogenic processes, double-stranded DNA (dsDNA) in EVs might be the latest most promising biomarker of tumor presence and complexity. This review will focus on the recent knowledge on the DNA inclusion in vesicles, the technical aspects of EV-DNA detection and quantification, and the use of EV-DNA as a clinical biomarker.
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http://dx.doi.org/10.3389/fcell.2020.622048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884755PMC
February 2021

Lack of clinical relevance of blood clonality in primary cutaneous marginal zone B-cell lymphoma.

Eur J Dermatol 2021 Feb;31(1):94-96

Dermatology Department, Bordeaux University Hospital, Bordeaux, France, INSERM U1053, UMR BaRITOn, Team 3 - Oncogenesis of cutaneous lymphoma, University of Bordeaux, France.

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http://dx.doi.org/10.1684/ejd.2020.3955DOI Listing
February 2021

C6 Ceramide (d18:1/6:0) as a Novel Treatment of Cutaneous T Cell Lymphoma.

Cancers (Basel) 2021 Jan 13;13(2). Epub 2021 Jan 13.

Department of Dermatology, Venerology and Allergology, Goethe University Hospital, 60590 Frankfurt am Main, Germany.

Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.
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http://dx.doi.org/10.3390/cancers13020270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828274PMC
January 2021

, the Elusive Transcription Factor.

Front Genet 2020 18;11:581115. Epub 2020 Dec 18.

Bordeaux University, INSERM U1053 Bordeaux Research in Translational Oncology (BaRITOn), Cutaneous Lymphoma Oncogenesis Team, Bordeaux, France.

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http://dx.doi.org/10.3389/fgene.2020.581115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793725PMC
December 2020

Diagnosis and treatment of lymphomas in the era of epigenetics.

Blood Rev 2021 Jul 17;48:100782. Epub 2020 Nov 17.

Bordeaux University, INSERM U1053 Bordeaux Research in Translational Oncology (BaRITOn), Cutaneous Lymphoma Oncogenesis Team, F-33000 Bordeaux, France. Electronic address:

Lymphomas represent a heterogeneous group of cancers characterized by clonal lymphoproliferation. Over the past decades, frequent epigenetic dysregulations have been identified in hematologic malignancies including lymphomas. Many of these impairments occur in genes with established roles and well-known functions in the regulation and maintenance of the epigenome. In hematopoietic cells, these dysfunctions can result in abnormal DNA methylation, erroneous chromatin state and/or altered miRNA expression, affecting many different cellular functions. Nowadays, it is evident that epigenetic dysregulations in lymphoid neoplasms are mainly caused by genetic alterations in genes encoding for enzymes responsible for histone or chromatin modifications. We summarize herein the recent epigenetic modifiers findings in lymphomas. We focus also on the most commonly mutated epigenetic regulators and emphasize on actual epigenetic therapies.
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http://dx.doi.org/10.1016/j.blre.2020.100782DOI Listing
July 2021

Xenograft and cell culture models of Sézary syndrome reveal cell of origin diversity and subclonal heterogeneity.

Leukemia 2021 Jun 26;35(6):1696-1709. Epub 2020 Oct 26.

Univ. Bordeaux, INSERM, BaRITOn, U1053, F-33000, Bordeaux, France.

Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.
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http://dx.doi.org/10.1038/s41375-020-01068-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179845PMC
June 2021

Circulating Tumor Cell Clusters: United We Stand Divided We Fall.

Int J Mol Sci 2020 Apr 10;21(7). Epub 2020 Apr 10.

Centre Hospitalier Universitaire (CHU) de Toulouse, 31000 Toulouse, France.

The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that "united" CTCs, organized in clusters, resist better and carry stronger metastatic capacities than "divided" single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer.
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http://dx.doi.org/10.3390/ijms21072653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177734PMC
April 2020

Reliable blood cancer cells' telomere length evaluation by qPCR.

Cancer Med 2020 05 6;9(9):3153-3162. Epub 2020 Mar 6.

Bordeaux University, INSERM U1053 Bordeaux Research in Translational Oncology (BaRITOn), Cutaneous Lymphoma Oncogenesis Team, Bordeaux, France.

Background: Telomere shortening is linked to a range of different human diseases, hence reliable measurement methods are needed to uncover such associations. Among the plethora of telomere length measurement methods, qPCR is reported as easy to conduct and a cost-effective approach to study samples with low DNA amounts.

Methods: Cancer cells' telomere length was evaluated by relative and absolute qPCR methods.

Results: Robust and reproducible telomere length measurements were optimized taking into account a careful reference gene selection and by knowing the cancer cells ploidy. qPCR data were compared to "gold standard" measurement from terminal restriction fragment (TRF).

Conclusions: Our study provides guidance and recommendations for accurate telomere length measurement by qPCR in cancer cells, taking advantage of our expertise in telomere homeostasis investigation in primary cutaneous T-cell lymphomas. Furthermore, our data emphasize the requirement of samples with both, high DNA quality and high tumor cells representation.
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http://dx.doi.org/10.1002/cam4.2816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196062PMC
May 2020

Independent prognostic value of ultra-sensitive quantification of tumor pre-treatment T790M subclones in EGFR mutated non-small cell lung cancer (NSCLC) treated by first/second generation TKI, depends on variant allele frequency (VAF): Results of the French cooperative thoracic intergroup (IFCT) biomarkers France project.

Lung Cancer 2020 02 15;140:19-26. Epub 2019 Nov 15.

AP-HP, Hôpital Tenon, Service de Pneumogie, GRC 04 Theranoscan, Sorbonne Université, Paris, France.

Objectives: T790M mutations inEGFR-mutated non-small cell lung cancer (NSCLC) account for nearly 50% of acquired resistance mechanisms to EGFR-TKIs. Earlier studies suggested that tumor T790M could also be detected in TKI-naïve EGFR-mutated NSCLC. The aim of the study is to assess the prevalence and clinical significance of quantification of tumor pre-treatment T790M subclones.

Materials And Methods: We analyzed 366 EGFR-mutated NSCLC patients of the real-life IFCT Biomarkers France study with available pre-treatment formalin-fixed paraffin-embedded (FFPE) tumor DNA before treatment by first/second-generation EGFR-TKI. We used ultra-sensitive Droplet Digital Polymerase Chain Reaction (ddPCR) QX200 (BIO-RAD®, Hercules, CA, USA). All samples were tested in duplicate.

Results: ddPCR identified T790M in 19/240 specimens (8%). T790M-positive and T790M-negative populations were not different for clinical baseline characteristics. T790M Variant Allele Frequency (VAF) was > 0.01% <0.1%, > 0.1% <1%, > 1% <10%, and >10% in five (26.3%), six (31.6%), six (31.6%), and two (10.5%) patients, respectively. T790M VAF was >0.1% in 11/13 (84%) patients with rapid (<3 months) or usual progression (3-20 months) compared to 0/3 with low progression (>20 months) (p = 0.02). In a Cox model, T790M mutation positivity was correlated with overall survival (OS) and progression-free survival (PFS) for 10% > VAF >1% (hazard ratio [HR] = 2.83, 95% confidence interval [CI] 1.13-7.07, p = 0.03; HR=3.62, 95%CI 1.43-4.92, p = 0.007, respectively) and for VAF >10% (HR = 19.14, 95%CI 4.35-84.26, p < 0.001; HR = 17.89, 95%CI 2.21-144.86, p = 0.007, respectively).

Conclusion: Ultra-sensitive detection of tumor T790M mutation concerned 8% of EGFR-mutated TKI-naïve NSCLC patients and has a negative prognostic value only for T790M VAF over 1%.
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http://dx.doi.org/10.1016/j.lungcan.2019.10.013DOI Listing
February 2020

Clinical outcomes of non-small-cell lung cancer patients with BRAF mutations: results from the French Cooperative Thoracic Intergroup biomarkers France study.

Eur J Cancer 2019 07 8;116:86-97. Epub 2019 Jun 8.

Department of Medical Oncology, Thoracic Unit, Gustave Roussy, Villejuif, France. Electronic address:

Introduction: Patients with stage IV non-small-cell lung cancer (NSCLC) and BRAF V600 mutations may benefit from targeted therapies. Chemotherapy outcomes are little known in this population.

Methods: The French Cooperative Thoracic Intergroup (IFCT) Biomarkers France study was a national prospective cohort study aiming to describe the molecular characteristics and clinical outcome of all consecutive NSCLC patients (N = 17,664) screened for molecular alterations. We used this data set to set up a case-control analysis. Cases had stage IV BRAF-mutated (BRAF-MT) NSCLC, whereas controls had NSCLC that was wild-type for EGFR, KRAS, HER2, BRAF, PIK3CA and ALK. Each case was matched for sex, age at diagnosis and smoking status to two controls randomly selected.

Results: Overall, 83 cases with BRAF mutant disease (66.3% V600E) were matched to 166 controls. Five cases received tyrosine kinase inhibition in the first-line and 16 in the second-line. All others were treated with standard chemotherapy. There was no significant difference in first-line and second-line progression-free survival (PFS) between the groups, as well as in the disease control rate, BRAF mutation was not found to be prognostic of overall survival. We found no significant difference in outcome between the treatment types used in first-line or second-line in patients with BRAF-MT disease compared with controls nor between BRAF V600E or non-V600E compared with controls.

Conclusions: BRAF mutation is not a strong prognostic factor in NSCLC. Although taxan-based therapy shows poorest PFS in first-line, no chemotherapy regimen was associated with prognosis.
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http://dx.doi.org/10.1016/j.ejca.2019.04.016DOI Listing
July 2019

Mutations of the B-Cell Receptor Pathway Confer Chemoresistance in Primary Cutaneous Diffuse Large B-Cell Lymphoma Leg Type.

J Invest Dermatol 2019 11 29;139(11):2334-2342.e8. Epub 2019 May 29.

INSERM U1053, Equipe Oncogenèse des lymphomes cutanés, Université de Bordeaux; Service de Biologie des tumeurs, CHU de Bordeaux, Pessac, France. Electronic address:

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P = 0.002 and 51 months, P = 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P = 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.
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http://dx.doi.org/10.1016/j.jid.2019.05.008DOI Listing
November 2019

Primary cutaneous large B-cell lymphomas: relevance of the 2017 World Health Organization classification: clinicopathological and molecular analyses of 64 cases.

Histopathology 2019 Jun 25;74(7):1067-1080. Epub 2019 Apr 25.

Pathology Department, University Hospital of Bordeaux, Hôpital Haut-Lévêque, Bordeaux, France.

Aims: We applied the 2017 World Health Organization (WHO) classification criteria to categorise a series of 64 primary cutaneous large B-cell lymphomas (PCLBCLs), containing a majority (≥80%) of large cells and a proliferative rate of ≥40%, raising the problem of the differential diagnosis between PCLBCL, leg type (PCLBCL-LT) and primary cutaneous follicle centre lymphoma, large cell (PCFCL-LC). The aims were to determine the reproducibility and prognostic relevance of the 2017 WHO criteria.

Methods And Results: Morphology and phenotype identified 32 PCLBCLs-LT and 25 PCFCLs-LC; seven cases (11%) remained unclassified. Morphology was less reproducible than immunophenotype. Pertinent markers for the differential diagnosis were MUM1, FOXP1, CD10, and IgM. bcl-2 and bcl-6 were expressed by both PCFCLs-LC and PCLBCLs-LT at substantial levels. Neither Ki67 expression nor p63 expression was of diagnostic value. MYD88 was found to be mutated only in PCLBCLs-LT (n = 22, 69%). According to Hans/Hans modified algorithms, 23 of 25 PCFCLs-LC had germinal centre (GC) status, and the 32 PCLBCLs-LT had non-GC status. Overall survival was poorer for PCLBCLs-LT than PCFCLs-LC (P = 0.0002). Non-GC cases had poorer overall survival than GC cases (P = 0.0007). In PCLBCLs-LT, MYC expression was associated with cutaneous relapses (P = 0.014). When GC/non-GC status was applied to unclassified cases, only a single case remained discordant.

Conclusions: Our results support the 2017 WHO classification criteria for PCLBCL diagnosis. The Hans modified algorithm using CD10 and MUM1 distinguished PCFCLs-LC from PCLBCLs-LT with optimal diagnostic value without requiring bcl-6 immunolabelling (poorly reproducible). Rare unclassified cases may constitute a provisionally heterogeneous subgroup for which GC/non-GC status (relevant for prognosis) may guide therapeutic decisions.
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http://dx.doi.org/10.1111/his.13832DOI Listing
June 2019

Calcium Independent Effect of Orai1 and STIM1 in Non-Hodgkin B Cell Lymphoma Dissemination.

Cancers (Basel) 2018 Oct 26;10(11). Epub 2018 Oct 26.

Department of Life and Health Sciences, University of Bordeaux, F-33076 Bordeaux, France.

Ca release-activated Ca channels, composed of Orai1 and STIM1 (stromal interaction molecule 1) proteins, are the main Ca entry mechanism in lymphocytes. Their role in cell migration and metastasis is demonstrated in solid cancers but it remains elusive in malignant hemopathies. Diffuse large B cell lymphoma (DLBCL) is characterized by the dissemination of neoplastic B cells throughout the organism which is under the control of chemokines such as Stromal Derived Factor 1 (SDF-1) and its receptor CXCR4. CXCR4 activation triggers a complex intracellular signaling including an increase in intracellular Ca concentration whose role is still unclear. Using pharmacological and genetic approaches, we revealed that STIM1 and Orai1 were responsible for Ca influx induced by SDF-1. Furthermore, we provide in vitro and in vivo evidence that they are necessary for basal or SDF-1-induced DLBCL cell migration which is independent of Ca entry. We identify that they act as effectors coupling RhoA and ROCK dependent signaling pathway to MLC2 phosphorylation and actin polymerization. Finally, we revealed an alteration of Orai1 and STIM1 expression in extra-nodal DLBCL. Thus, we discovered a novel Ca-independent but Orai1 and STIM1-dependent signaling pathway involved in basal and CXCR4 dependent cell migration, which could be relevant for DLBCL physiopathology.
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http://dx.doi.org/10.3390/cancers10110402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267368PMC
October 2018

SATB1 Is a Pivotal Epigenetic Biomarker in Cutaneous T-Cell Lymphomas.

J Invest Dermatol 2018 08;138(8):1694-1696

INSERM U1053, Bordeaux Research in Translational Oncology University Bordeaux, Bordeaux, France; Tumor Bank and Tumor Biology Laboratory, Centre Hospitalier Universitaire de Bordeaux, Pessac, France. Electronic address:

The SATB1 protein has been the focus of two recent studies of cutaneous T-cell lymphomas. Fredholm et al. observed a stage-related decrease of SATB1 expression in epidermotropic cutaneous T-cell lymphomas. SATB1 was negatively regulated by STAT5 through microRNA-155, which in turn triggered enhanced expression of T helper type 2 cytokines such as IL-5 and IL-9. In parallel, Sun et al. found that SATB1 expression was up-regulated by promoter demethylation in a subset of cutaneous anaplastic lymphoma and was associated with T helper type 17 polarization in patients with better therapeutic responses.
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http://dx.doi.org/10.1016/j.jid.2018.04.018DOI Listing
August 2018

A Single-Arm Phase II Trial of Lenalidomide in Relapsing or Refractory Primary Cutaneous Large B-Cell Lymphoma, Leg Type.

J Invest Dermatol 2018 09 27;138(9):1982-1989. Epub 2018 Mar 27.

INSERM U1053, Bordeaux Research in Translational Oncology, Team 3 oncogenesis of cutaneous lymphomas, Université de Bordeaux, Bordeaux, France; Tumor Bank and Tumor Biology Laboratory, CHU Bordeaux, Pessac, France.

Although the combination of rituximab and polychemotherapy has improved prognosis of primary cutaneous diffuse large B-cell lymphoma, leg type, the advanced age of patients limits therapeutic options in relapsing/refractory cases. A multicenter, single-arm, phase II trial was conducted to assess the benefits and safety of lenalidomide in refractory/relapsing primary cutaneous diffuse large B-cell lymphoma, leg type. The primary endpoint was the 6-month overall response rate. Secondary endpoints were 12-month overall response rate, overall and specific survival, duration of response, progression-free survival, safety, and identification of prognostic factors. Among the 19 patients included, the 6-month overall response rate was 26.3% (90% confidence interval [CI] = 11-47.6), including four complete responses and one partial response. At 12 months, there were still two complete responses and one partial response. Median progression-free survival was 4 months. Median overall and specific survivals were 19.4 and 23.8 months, respectively. Reduced doses tended to be associated with higher 6-month overall response rate and progression-free survival. Absence of the MYD88 mutation was associated with a higher overall response under treatment (80.0% vs. 33.3%; P = 0.05). The most common grade 3 adverse events were hematologic. Two grade 5 adverse events occurred (sepsis and pulmonary embolism). Lenalidomide at reduced doses may allow prolonged responses in a few patients and represents a therapeutic option in relapsing/refractory primary cutaneous diffuse large B-cell lymphoma, leg type.
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http://dx.doi.org/10.1016/j.jid.2018.03.1516DOI Listing
September 2018

Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas.

Mod Pathol 2018 08 26;31(8):1332-1342. Epub 2018 Mar 26.

INSERM U1053, University of Bordeaux, 33000, Bordeaux, France.

In nodal diffuse large B-cell lymphoma, the search for double-hit with MYC and BCL2 and/or BCL6 rearrangements or for dual expression of BCL2 and MYC defines subgroups of patients with altered prognosis that has not been evaluated in primary cutaneous large B-cell lymphoma. Our objectives were to assess the double-hit and dual expressor status in a cohort of 44 patients with primary cutaneous large B-cell lymphoma according to the histological subtype and to evaluate their prognosis relevance. The 44 cases defined by the presence of more than 80% of large B-cells in the dermis corresponded to 21 primary cutaneous follicle centre lymphoma with large cell morphology and 23 primary cutaneous diffuse large B-cell lymphoma, leg type. Thirty-one cases (70%) expressed BCL2 and 29 (66%) expressed MYC. Dual expressor profile was observed in 25 cases (57%) of either subtypes (n = 6 or n = 19, respectively). Only one primary cutaneous follicle centre lymphoma, large-cell case had a double-hit status (2%). Specific survival was significantly worse in primary cutaneous diffuse large B-cell lymphoma, leg type than in primary cutaneous follicle centre lymphoma, large cell (p = 0.021) and for the dual expressor primary cutaneous large B-cell lymphoma group (p = 0.030). Both overall survival and specific survival were worse for patients belonging to the dual expressor primary cutaneous diffuse large B-cell lymphoma, leg type subgroup (p = 0.001 and p = 0.046, respectively). Expression of either MYC and/or BCL2 negatively impacted overall survival (p = 0.017 and p = 0.018 respectively). As the differential diagnosis between primary cutaneous follicle centre lymphoma, large cell and primary cutaneous diffuse large B-cell lymphoma, leg type has a major impact on prognosis, dual-expression of BCL2 and MYC may represent a new diagnostic criterion for primary cutaneous diffuse large B-cell lymphoma, leg type subtype and further identifies patients with impaired survival. Finally, the double-hit assessment does not appear clinically relevant in primary cutaneous large B-cell lymphoma.
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http://dx.doi.org/10.1038/s41379-018-0041-7DOI Listing
August 2018

PD-L1 and PD-L2 Are Differentially Expressed by Macrophages or Tumor Cells in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type.

Am J Surg Pathol 2018 03;42(3):326-334

INSERM U1053, Team 3 Oncogenesis of Cutaneous Lymphomas.

As checkpoint molecules' inhibition may represent a therapeutic option in relapsing cases, we assessed programmed death ligands' (PD-L1/PD-L2) expression in a series of 29 primary cutaneous diffuse large B-cell lymphoma, leg-type (PCDLBCL-LT) cases. Double immunostaining for either PD-L1 or PD-L2 was associated either with PAX5 staining to evaluate tumor cells or with CD68 or CD163 staining for macrophages. The microenvironment of PCDLBCL-LT was characterized by immunostainings for CD3 (tumor-infiltrating lymphocytes), FOXP3 (regulatory T cells), programmed cell death-1, and CD33 (myeloid-derived suppressor cells). The 9p24.1 locus encoding for PD-L1/PD-L2 was evaluated by fluorescence in situ hybridization. A PD-L1 expression was observed in all cases. However, double staining with PD-L1/PAX5 identified only 1 case harboring PD-L1 expression by tumor cells. All cases displayed PD-L1 expression by numerous immune cells, characterized as CD68 CD163 M2 macrophages. A normal fluorescence in situ hybridization pattern was observed in 21 of 26 cases. Three cases (11.5%) harbored a low polysomy status including the case with PD-L1 expression by tumor cells. Interestingly, 2 cases (7.7%) exhibited a PD-L1/PD-L2 locus break-apart pattern, and PD-L2 expression by tumor cells was observed. PD-L2 expression by tumor cells was not observed in the 24 cases without 9p24.1 rearrangement. Treating patients with relapsing PCDLBCL-LT by using immune checkpoint inhibitors may have an indirect effect through immune cells, except in rare cases with 9p24.1 rearrangement leading to PD-L2 expression by tumor cells. Reprogramming tumor-associated macrophages with anticancer therapies is appealing in such lymphoma subtypes wherein M2 macrophages represent the majority of immune cells.
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http://dx.doi.org/10.1097/PAS.0000000000000983DOI Listing
March 2018

Expression of TFH Markers and Detection of RHOA p.G17V and IDH2 p.R172K/S Mutations in Cutaneous Localizations of Angioimmunoblastic T-Cell Lymphomas.

Am J Surg Pathol 2017 Dec;41(12):1581-1592

*Pathology Department, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris †INSERM U955 équipe 9, Institut Mondor de Recherche Biomédicale ‡Paris Est Creteil University (UPEC) ¶Biological Immunology Department, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris #Dermatology department, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris **Hematology Department, Lymphoid hemopathy Unit, Henri Mondor Hospital, Assistance Publique-Hôpitaux de Paris, Créteil ††Dermatology Department, Saint-André Hospital, CHU de Bordeaux ‡‡INSERM U1053, Bordeaux Research in Translational Oncology, Team 3 Oncogenesis of Cutaneous Lymphomas, Bordeaux University, Bordeaux §Pathoogy department, Haut-Lévèque Hospital, CHU de Bordeaux §§Tumor Biology Department, Haut-Lévèque Hospital, CHU de Bordeaux, Pessac, France ∥Pathology Department Prof Lima Basto, Lisboa, Portugal.

Skin biopsies of 41 angioimmunoblastic T-cell lymphoma patients were retrospectively analyzed for the expression of follicular helper T-cell (TFH) markers, Epstein-Barr virus (EBV), and the presence of RHOA (p.G17V) and IDH2 (p.R172K/S) mutations using allele-specific polymerase chain reaction. We categorized cases into 4 distinctive patterns: (1) low-density lymphocytic perivascular infiltrates (n=11), (2) dense perivascular infiltrates with atypical cells and occasional inflammatory cells (n=13), (3) diffuse infiltrates reminiscent of angioimmunoblastic T-cell lymphoma (n=4), or (4) other aspects (n=13). Two EBV and 2 plasmacytoid lymphoproliferative disorders were seen. We observed variable expression of TFH markers (CD10 [50%], BCLB6 [84%], PD1 [94%], CXCL13 [84%], and ICOS [97.5%]), and EBV B-blasts (26%). A TFH phenotype was identified in 82% and 73%, respectively, of cases with the most challenging patterns 1 and 2. TFH markers and EBV can thus help for diagnosis and are detected in samples with low-density infiltrates. We found RHOA G17V and IDH2 R172K/S mutations in the skin in 14/18 (78%) and 3/16 (19%) cases, respectively. The RHOA G17V mutation was identified in a proportion of biopsies with patterns 1 and 2, which represent a diagnostic challenge. The RHOA G17V mutation was detected both in the skin and lymph node (LN) biopsies in 7/9 (64%) cases, and in only the skin or the LN of 1 sample each. The frequency of RHOA G17V mutation was similar to that reported in LNs. It may represent a sensitive diagnostic marker in the skin, helpful in cases with low-density infiltrates.
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http://dx.doi.org/10.1097/PAS.0000000000000956DOI Listing
December 2017

Molecular analysis of immunoglobulin variable genes supports a germinal center experienced normal counterpart in primary cutaneous diffuse large B-cell lymphoma, leg-type.

J Dermatol Sci 2017 Nov 26;88(2):238-246. Epub 2017 Jul 26.

UMR CNRS 7276, Univ. Limoges,2 avenue Martin Luther King, 87042 Limoges, France.

Background: Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-).

Objectives: As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin.

Methods: Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model.

Results: A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch μ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression.

Conclusion: These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell.
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http://dx.doi.org/10.1016/j.jdermsci.2017.07.008DOI Listing
November 2017

EGFR tyrosine kinase inhibitors chemotherapy in wild-type pre-treated advanced nonsmall cell lung cancer in daily practice.

Eur Respir J 2017 08 10;50(2). Epub 2017 Aug 10.

Service d'Oncologie Multidisciplinaire et Innovations Thérapeutiques, Assistance Publique Hôpitaux de Marseille, Aix-Marseille Université, Marseille, France

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are approved for second-line treatment of wild-type (-wt) nonsmall cell lung cancer (NSCLC). However, results from randomised trials performed to compare EGFR-TKIs with chemotherapy in this population did not show any survival benefit. In the era of immunotherapy, many drugs are approved for second-line treatment of wt NSCLC and there is a need to reassess the role of EGFR-TKIs in this setting.The Biomarkers France study is a large nationwide cohort of NSCLC patients tested for mutations. We used this database to collect clinical, biological, treatment and outcome data on wt patients who received second-line treatment with either EGFR-TKIs or chemotherapy.Among 1278 patients, 868 received chemotherapy and 410 received an EGFR-TKI. Median overall survival and progression-free survival were longer with chemotherapy than with an EGFR-TKI. Overall survival was 8.38 4.99 months, respectively (hazard ratio 0.70, 95% CI 0.59-0.83; p<0.0001) and progression-free survival was 4.30 2.83 months, respectively (hazard ratio 0.66, 95% CI 0.57-0.77; p<0.0001).This study is helpful to guide a multiline treatment strategy for wt NSCLC patients. Immunotherapy is approved for second-line treatment. For third-line treatment, chemotherapy results in longer overall survival and progression-free survival, and should be preferred to EGFR-TKIs.
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http://dx.doi.org/10.1183/13993003.00514-2017DOI Listing
August 2017

Assessment of BRAF mutation in pulmonary Langerhans cell histiocytosis in tissue biopsies and bronchoalveolar lavages by droplet digital polymerase chain reaction.

Virchows Arch 2018 Feb 15;472(2):247-258. Epub 2017 Jul 15.

Laboratory of Tumor Biology and Tumor Biobank, CHU Bordeaux, Pessac, F-33604, Bordeaux, France.

The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF and MAP2K1 mutations in patients with PCLH is now considered for such assessment, the aim of our study was to evaluate digital droplet polymerase chain reaction (ddPCR) in PCLH diagnosis. We retrospectively analyzed BRAF detection in a cohort of 42 PCLH tissues and 18 bronchoalveolar lavages (BALs) by ddPCR, immunohistochemistry, high-resolution melting PCR (HRM), and next-generation sequencing (NGS). The presence of BRAF mutation was assessed by at least two concordant techniques to further evaluate specificity and sensitivity of each method. The BRAF mutation prevalence was detected in 18 out of 41 cases by ddPCR, 10 out of 36 cases by HRM PCR, and 16 out of 31 cases by NGS. BRAF immunohistochemistry sensitivity was 94%, and specificity was 79%. HRM PCR sensitivity was only 59%, and specificity was 100%. NGS sensitivity and specificity were 100% for interpretable cases (n = 31), but in 11 cases, this technique was non-contributive. The analysis of BAL samples by ddPCR revealed a BRAF mutation both in tissue and in BAL samples in one patient, a wild-type status both in tissue and in BAL samples in two patients, and a wild-type BRAF status in BAL and a BRAF mutation in tissue samples in four patients. The study supports the usefulness of ddPCR for BRAF status assessment in either tissue or BAL samples to increase the accuracy of PLCH diagnosis.
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http://dx.doi.org/10.1007/s00428-017-2185-0DOI Listing
February 2018

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

TP53 alterations in primary and secondary Sézary syndrome: A diagnostic tool for the assessment of malignancy in patients with erythroderma.

PLoS One 2017 16;12(3):e0173171. Epub 2017 Mar 16.

INSERM U1053, Bordeaux Research in Translational Oncology University Bordeaux, Bordeaux, France.

Recent massive parallel sequencing data have evidenced the genetic diversity and complexity of Sézary syndrome mutational landscape with TP53 alterations being the most prevalent genetic abnormality. We analyzed a cohort of 35 patients with SS and a control group of 8 patients with chronic inflammatory dermatoses. TP53 status was analyzed at different clinical stages especially in 9 patients with a past-history of mycosis fungoides (MF), coined secondary SS. TP53 mutations were only detected in 10 patients with either primary or secondary SS (29%) corresponding to point mutations, small insertions and deletions which were unique in each case. Interestingly, TP53 mutations were both detected in sequential unselected blood mononuclear cells and in skin specimens. Cytogenetic analysis of blood specimens of 32 patients with SS showed a TP53 deletion in 27 cases (84%). Altogether 29 out of 35 cases exhibited TP53 mutation and/or deletion (83%). No difference in prognosis was observed according to TP53 status while patients with secondary SS had a worse prognosis than patients with primary SS. Interestingly, patients with TP53 alterations displayed a younger age and the presence of TP53 alteration at initial diagnosis stage supports a pivotal oncogenic role for TP53 mutation in SS as well as in erythrodermic MF making TP53 assessment an ancillary method for the diagnosis of patients with erythroderma as patients with inflammatory dermatoses did not display TP53 alteration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173171PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354275PMC
September 2017

Characteristics and Outcomes of Patients with Lung Cancer Harboring Multiple Molecular Alterations: Results from the IFCT Study Biomarkers France.

J Thorac Oncol 2017 06 9;12(6):963-973. Epub 2017 Feb 9.

Toulouse University Hospital, Paul Sabatier Toulouse University, Toulouse, France. Electronic address:

Introduction: Little is known about the prevalence, prognosis, and response to treatment of advanced NSCLC harboring multiple genomic alterations.

Methods: The French Biomarkers France database, which includes 17,664 patients, was used. The prevalence of multiple alterations, their associations, their impact on prognosis (overall survival [OS]), and their response to targeted or conventional treatments (progression-free survival [PFS] and objective response rate) were assessed and compared with those of patients harboring single or no mutation.

Results: We identified 162 patients (0.9%) with double alterations and three with triple mutations. Multiple molecular alterations preferentially involved KRAS (67.3%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA) (53.3%), and EGFR (42.4%). Patients with multiple alterations were more likely to be male (56.4%), be never-smokers (25.8 versus 34.7%, p < 0.001), and exhibit adenocarcinomas (83.6%). OS did not differ between single and multiple alterations. Patients with EGFR/KRAS and EGFR/PIK3CA mutations experienced worse PFS than did patients with only EGFR mutations (7.1 and 7.1 versus 14.9 months, p = 0.02 and 0.002, respectively). Concomitant mutations in patients harboring anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement bore little impact on OS (17.7 versus 20.3 months, p = 0.57) or PFS (10.3 versus 12.1 months, p = 0.93). Patients harboring KRAS mutations plus another alteration had an OS time (13.4 versus 11.2 months, p = 0.28), PFS time (6.4 versus 7.2 months, p = 0.78), and objective response rate under first-line chemotherapy (41.7% versus 37.2%) similar to those of patients harboring KRAS mutations only.

Conclusions: With almost 1% of patients harboring multiple alterations, the dogma of mutually exclusive mutations should be reconsidered. Although double mutations do not decrease OS, they do alter PFS under first-line treatment for patients with EGFR mutations. Among limited numbers of patients, therapies targeting the dominant oncogene seem to usually remain active.
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http://dx.doi.org/10.1016/j.jtho.2017.02.001DOI Listing
June 2017

Evaluation of a fast and fully automated platform to diagnose and mutations in formalin-fixed and paraffin-embedded non-small cell lung cancer samples in less than one day.

J Clin Pathol 2017 Jun 2;70(6):544-549. Epub 2017 Feb 2.

Department of Pathology, CHRU Brest, Brest, France.

Aims: Searching for and mutations within non-small cell lung carcinoma (NSCLC) samples remains time-consuming and can delay treatment choices in patients with acute deterioration. We evaluated the performances of the fully automated Idylla platform to quickly detect these mutations in NSCLC samples.

Methods: We used the Idylla Mutation Assay and the Idylla Mutation Test to analyse 18 formalin-fixed paraffin-embedded NSCLC tumour samples with known and mutation status according to next-generation sequencing (NGS) and droplet digital PCR (ddPCR) for mutations.

Results: Idylla assays identified and activating mutations in 4 and 10 NSCLC samples, respectively. resistance mutations were identified in only 1 sample using Idylla but in 4 and 14 samples using NGS and ddPCR, respectively. No false-positive result was noted with Idylla assays. Mutation written report was obtained after 130 min ( assays) to 140 min ( assays).

Conclusions: The Idylla platform is an interesting ancillary first-line fast and fully automated tool to detect and mutations in NSCLC samples allowing rapid treatment choices in patients with acute deterioration.
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http://dx.doi.org/10.1136/jclinpath-2016-204202DOI Listing
June 2017

Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene.

Genes (Basel) 2016 Dec 17;7(12). Epub 2016 Dec 17.

Service de Biologie des Tumeurs, Centre Hospitalier Universitaire de Bordeaux, Hôpital du Haut Lévêque, Pessac F-33604, France.

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the gene. gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.
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http://dx.doi.org/10.3390/genes7120133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192509PMC
December 2016