David Cappellen

Tumour suppressive properties of fibroblast growth factor receptor 2-IIIb in human bladder cancer.

FGFRs (fibroblast growth factor receptors) are encoded by four genes (FGFR1 – 4). Alternative splicing results in various receptor isoforms. The FGFR2-IIIb variant is present in a wide variety of epithelia, including the bladder epithelium. Recently, we have shown that FGFR2-IIIb is downregulated in a subset of transitional cell carcinomas of the bladder, and that this downregulation is associated with a poor prognosis. We investigated possible tumour suppressive properties of FGFR2-IIIb by transfecting two human bladder tumour cell lines, J82 and T24, which have no endogenous FGFR2-IIIb expression, with FGFR2-IIIb cDNA. No stable clones expressing FGFR2-IIIb were isolated with the J82 cell line. For the T24 cell line, stable transfectants expressing FGFR2-IIIb had reduced growth in vitro and formed fewer tumours in nude mice which, in addition, grew more slowly. The potential mechanisms leading to decreased FGFR2-IIIb mRNA levels were also investigated. The 5′ region of the human FGFR2 gene was isolated and found to contain a CpG island which was partially methylated in more than half the cell lines and tumours which do not express FGFR2-IIIb. No homozygous deletion was identified in any of the tumours or cell lines with reduced levels of FGFR2-IIIb. Mutational analysis of the entire coding region of FGFR2-IIIb at the transcript level was performed in 33 bladder tumours. In addition to normal FGFR2-IIIb mRNA, abnormal transcripts were detected in two tumour samples. These abnormal mRNAs resulted from exon skipping which affected the region encoding the kinase domain. Altogether, these results show that FGFR2-IIIb has tumour growth suppressive properties in bladder carcinomas and suggest possible mechanisms of FGFR2 gene inactivation.

Frequent loss of heterozygosity on chromosome 10q in muscle-invasive transitional cell carcinomas of the bladder

Loss of heterozygosity (LOH) on chromosome 10 has been observed in several human cancers including glioblastomas, meningiomas, melanomas and endometrial and prostate carcinomas. We have investigated the incidence of LOH on chromosome 10 in 36 human transitional cell carcinomas (TCCs) of the bladder, three upper urinary tract TCCs and one lymph node metastasis, using a panel of 27 highly polymorphic markers spanning 10p (short arm) and 10q (long arm). Fourteen bladder tumours (39%), the three upper urinary tract tumours and the lymph node metastasis showed LOH for at least one locus on chromosome 10. Remarkably, LOH on chromosome 10 was observed mainly in muscle-invasive (P=0.01) and high grade tumours (P=0.03). For five tumours and the lymph node metastasis, LOH was found at all informative loci, indicating monosomy or isodisomy of chromosome 10. The deletion mapping of the tumours with partial loss delineated two minimal regions of loss on chromosome 10q. One region, the most telomeric, was bounded by markers D10S214 and D10S169 and the other, the most proximal, was bounded by markers D10S222 and D10S531. Our results demonstrate that chromosome 10q LOH is common in muscle-invasive bladder cancers and that two potential tumour suppressor loci, at 10q24.1-q24.3 and 10q26.1-q26.2, may contribute to the malignant progression of these tumours. Localization of the smallest common regions of loss in bladder tumours provides a starting point for the identification of the genes involved.

Tumor homogeneity between primary and metastatic sites for BRAF status in metastatic melanoma determined by immunohistochemical and molecular testing.

BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAFV600 mutations. In order to evaluate BRAF tumor heterogeneity between primary and metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAFV600E antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAFV600E (45.2%), c.1799_1800TG>AA, BRAFV600E2 (3.0%), c.1798_1799GT>AA, BRAFV600K (3.0%), c.1801 A>G, BRAFK601E (1.3%), c.1789_1790CT>TC, BRAFL597S (0.4%), c.1780G>A, BRAFD594N (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAFV600E/E2 mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAFV600E mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAFV600E/E2 mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAFV600E detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.

High Frequency and Clinical Prognostic Value of MYD88 L265P Mutation in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg-Type

IMPORTANCE The activating mutation of MYD88 L265P is a frequent feature of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT), reported in up to 69% of the cases. Whether patients with MYD88 mutation display specific clinical and evolutive features has not been evaluated. OBJECTIVE To identify the clinical characteristics associated with MYD88 mutation, confirm its high prevalence, and evaluate its effect on prognosis in patients with PCLBCL-LT. DESIGN, SETTING, AND PARTICIPANTS A retrospective multicenter study was conducted using the medical records of patients from dermatology departments belonging to the French Study Group for Cutaneous Lymphomas. Sixty-one patients with a diagnosis of PCLBCL-LT made between 1988 and 2010 who were available for molecular study were included. Of these, 58 patients displaying interpretable results constituted the study group. Median follow-up was 33 months, and 39 patients (67%) were monitored until death. MAIN OUTCOMES AND MEASURES Clinical features (age, sex, number of skin lesions, tumor stage, and location as leg vs elsewhere), MYD88 mutation (allele-specific TaqMan polymerase chain reaction assay), treatment regimen, and outcome were recorded. Baseline characteristics and outcome were compared according to the status of MYD88. RESULTS The median age of the patients was 79 years, and 59% were female. Skin lesions were located on the leg in 76% of the cases. Thirty-four of 58 patients (59%) harbored the MYD88 L265P mutation. Patients had similar clinical characteristics at presentation regardless of their MYD88 status, except that those harboring the MYD88 mutation were older (P = .006) and had more frequent involvement of the leg (P = .008). Patients harboring the MYD88 mutation had 3- and 5-year-specific survival rates of 65.7% and 60.2% vs 85.4% and 71.7% in patients with the wild-type allele. The MYD88 mutation was significantly associated with shorter disease-specific survival in univariate (P = .03) and multivariate (odds ratio, 3.01; 95% CI, 1.03-8.78; P = .04) analysis. There was no significant difference between the groups in their treatment regimens. Considering overall survival, in univariate (P = .002) and multivariate (odds ratio, 2.94; 95% CI, 1.18-7.30; P = .02) analysis, MYD88 L265P mutation was an independent adverse prognostic factor. CONCLUSIONS AND RELEVANCE This study confirms the high prevalence of MYD88 L265P mutation in PCLBCL-LT and shows its association with shorter survival. The clinical effect of MYD88 mutation activating the nuclear factor-κB pathway supports the use of targeted therapies at the time of relapse after conventional therapies.

MYD88 Somatic Mutation Is a Genetic Feature of Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type

Abbreviations: ABC, activated B cell; DLBCL, diffuse large B-cell lymphoma; GC, germinal center; PCBCL, primary cutaneous B-cell lymphoma; PCFCL, primary cutaneous follicle center lymphoma; PCDLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; PCMZL, primary cutaneous marginal zone lymphoma

Multiple genetic alterations in primary cutaneous large B-cell lymphoma, leg type support a common lymphomagenesis with activated B-cell-like diffuse large B-cell lymphoma

Primary cutaneous large B-cell lymphoma, leg type has been individualized from nodal diffuse large B-cell lymphoma. The objective of this study was to screen primary cutaneous large B-cell lymphoma, leg type for genetic alterations recently described in nodal diffuse large B-cell lymphoma. Skin biopsies from 23 patients were analyzed for IRF4, BCL2, BCL6, and MYC expression. FISH testing was performed for BCL2, BCL6, MYC with separation probes and for CDKN2A and PRDM1/BLIMP1 deletion. Multiple sequential FISH analyses with up to six probes were performed to define samples with multiple cytogenetic alterations. MYD88 mutations were studied by Sanger sequencing. All cases but one displayed at least one genetic alteration (96%). Nine patients exhibited a single genetic mutation and 12 combined several alterations (52%). We observed a split for BCL2, BCL6, or MYC in 1/23, 6/23, and 3/23 of cases, respectively. No double-hit lymphoma was observed. CDKN2A deletion was detected by FISH in only 5/23 cases. BLIMP1 and/or 6q deletion was observed at a higher rate in 10/20 of cases. No correlation between rearrangement and immunohistochemical expression was found for BCL2 or MYC. FISH tracking of sequential hybridizations showed that several alterations were carried by the same nuclei. The p.L265P MYD88 mutation was found in 11/18 (61%) of cases. Contrary to most cutaneous lymphomas that rarely harbor primary genetic alteration of their nodal histological equivalent, primary cutaneous large B-cell lymphoma, leg type seems to be a ‘cutaneous counterpart’ of activated B-cell-like diffuse large B-cell lymphoma with a similar cytogenetic profile and a high rate of MYD88 oncogenic L265P mutation. This also suggests a common lymphomagenesis with NF-κB activation, strong IRF4 expression and terminal B-cell differentiation blockage. Our data support the use of therapies targeting NF-κB, as most patients displayed disease progression and resistance to conventional therapies.

Assessment of diagnostic criteria between primary cutaneous anaplastic large‐cell lymphoma and CD30‐rich transformed mycosis fungoides; a study of 66 cases

Transformed mycosis fungoides (TMF) large cells may express CD30 antigen, and because of this, the differential diagnosis between CD30‐rich TMF and primary cutaneous anaplastic large‐cell lymphoma (cALCL) may be difficult, and especially in distinguishing cALCL associated with MF vs. CD30‐rich TMF.

MYD88 somatic mutation is a diagnostic criterion in primary cutaneous large B-cell lymphoma

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. MYD88 somatic mutation is a diagnostic criterion in primary cutaneous large B-cell lymphoma Sarah Menguy, Audrey Gros, Anne Pham-Ledard, Maxime Battistella, Nicolas Ortonne, François Comoz, Brigitte Balme, Vanessa Szablewski, Laurence Lamant, Agnès Carlotti, et al.

Molecular alterations and tumor suppressive function of the DUSP22 (Dual Specificity Phosphatase 22) gene in peripheral T-cell lymphoma subtypes

Monoallelic 6p25.3 rearrangements associated with DUSP22 (Dual Specificity Phosphatase 22) gene silencing have been reported in CD30+ peripheral T-cell lymphomas (PTCL), mostly with anaplastic morphology and of cutaneous origin. However, the mechanism of second allele silencing and the putative tumor suppressor function of DUSP22 have not been investigated so far. Here, we show that the presence, in most individuals, of an inactive paralog hampers genetic and epigenetic evaluation of the DUSP22 gene. Identification of DUSP22-specific single-nucleotide polymorphisms haplotypes and fluorescence in situ hybridization and epigenetic characterization of the paralog status led us to develop a comprehensive strategy enabling reliable identification of DUSP22 alterations. We showed that one cutaneous anaplastic large T-cell lymphomas (cALCL) case with monoallelic 6p25.3 rearrangement and DUSP22 silencing harbored exon 1 somatic mutations associated with second allele inactivation. Another cALCL case carried an intron 1 somatic splice site mutation with predicted deleterious exon skipping effect. Other tested PTCL cases with 6p25.3 rearrangement exhibited neither mutation nor deletion nor methylation accounting for silencing of the non-rearranged DUSP22 allele, thus inactivated by a so far unknown mechanism. We also characterized the expression status of four DUSP22 splice variants and found that they are all silenced in cALCL cases with 6p25.3 breakpoints. We finally showed that restoring expression of the physiologically predominant isoform in DUSP22-deficient malignant T cells inhibits cellular expansion by stimulating apoptosis and impairs soft agar clonogenicity and tumorigenicity. This study therefore shows that DUSP22 behaves as a tumor suppressor gene in PTCL.

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

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 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 NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 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 NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.