Thibault Kervarrec

H3F3 mutation status of giant cell tumors of the bone, chondroblastomas and their mimics: a combined high resolution melting and pyrosequencing approach

Behjati et al recently described recurrent mutations of H3F3 genes in giant cell tumors of the bone and chondroblastomas. Both these entities belong to the spectrum of giant cell-rich bone lesions, often presenting a diagnostic challenge for the pathologist. Our aim was to investigate the value of searching for H3F3 mutations in the diagnosis of giant cell tumors of the bone and giant cell-rich chondroblastomas. Two hundred eighty-one bone lesion samples, including 170 giant cell tumors of the bone, 26 chondroblastomas and 85 other giant cell-rich and/or epiphyseal tumors, were analyzed. Mutation status was determined using first high resolution melting screening and then mutation profiling pyrosequencing. Mutational status was compared with clinical data and, for giant cell tumors of the bone, with p63 immunostaining status. As histone methylation changes have been reported in association with H3F3 mutations, the methylation status of lysine 37 was investigated. H3F3A and H3F3B were found in 85% of giant cell tumors of the bone and 88% of chondroblastomas. In addition to the major G35W mutation, we found two rare H3F3A mutations: one G35R and one G35V. Among the other tumors studied, we only found H3F3A gene mutations in two cases of ‘dedifferentiated chondrosarcoma mimicking giant cell tumor of the bone’. A H3F3B mutation was also observed in one case of dedifferentiated chondroblastoma. P63 expression in giant cell tumors of the bone seems to be associated with H3F3 gene mutations (P=0.004). H3F3 mutations did not correlate with clinical data, outcome or methylation changes in Lysin 37. In conclusion, H3F3 mutations are sensitive and specific markers of giant cell tumors of the bone and chondroblastomas. High resolution melting and pyrosequencing procedures are high-performance tools in this context. Determination of H3F3 mutation will allow reclassification of some entities belonging to the spectrum of giant cell-rich lesions.

Cutaneous Rosai-Dorfman Disease Located on the Breast: Rapid Effectiveness of Methotrexate After Failure of Topical Corticosteroids, Acitretin and Thalidomide.

An erythematous cutaneous plaque on the breast with no signs of infection requires a skin biopsy to rule out dermal extension of an underlying mammary adenocarcinoma, primary skin tumour (e.g. angiosarcoma or malignant lymphoma) (1), or inflammatory lymphoedema of the breast (2). It can more rarely reveal location on the skin of systemic disease. Rosai-Dorfman disease (RDD), also known as sinus histiocytosis with massive lymphadenopathy, is a benign proliferative disorder of histiocytes first described in 1965 by Destombes on nodal biopsy (3). It was subsequently recognised as a distinct clinicopathologic entity by Rosai & Dorfman (4). Cases of RDD located solely on the skin have rarely been reported, and thus treatment is not well-codified (5). We report a case of RDD on the breast which showed rapid partial response to methotrexate.

Interstitial granulomatous dermatitis occurring in a patient with SAPHO syndrome one month after starting leflunomide, and subsequently disappearing with ustekinumab.

Interstitial granulomatous dermatitis (IGD) is a rare skin condition, which in half of the cases is associated with rheumatoid arthritis, lupus erythematosus or other inflammatory diseases [1]. Drug causality has been suspected [2-5]. We report a case of IGD occurring during SAPHO syndrome treated for one month with leflunomide (Arava®). Leflunomide was withdrawn with no resolution of IGD, but with exacerbation of rheumatism. Ustekinumab (Stelara®) was started and was associated with the disappearance [...]

Interest of variations in miR-152 and miR-122 in a series of hepatocellular carcinomas related to HCV infection

Hepatocellular carcinoma (HCC) is a common outcome of chronic hepatitis C virus (HCV) infection and constitutes the main burden of this disease. The molecular mechanisms underlying the development of HCC are multiple and might involve certain microRNA (miR). As discordant results have been reported concerning the detection of expression of miR‐152 and miR‐122 in HCC, our aim was to measure the levels of both miRs in serum and liver samples.

Merkel cell carcinomas infiltrated with CD33+ myeloid cells and CD8+ T cells are associated with improved outcome

Background: Merkel cell carcinoma (MCC) is a rare tumor of the skin that has an aggressive behavior. Immunity is the main regulator of MCC development, and many interactions between lymphocytes and tumor cells have been proven. However, the impact of tumor‐infiltrating myeloid cells needs better characterization. Objective: To characterize tumor‐infiltrating myeloid cells in MCC and their association with other immune effectors and patient outcome. Methods: MCC cases were reviewed from an ongoing prospective cohort study. In all, 103 triplicate tumor samples were included in a tissue microarray. Macrophages, neutrophils, and myeloid‐derived suppressor cells were characterized by the following markers: CD68, CD33, CD163, CD15, CD33, and human leukocyte antigen‐DR. Associations of these cell populations with programmed cell death ligand 1 expression, CD8 infiltrates, and vascular density were assessed. Impact on survival was analyzed by log‐rank tests and a Cox multivariate model. Results: The median density of macrophages was 216 cells/mm2. CD68+ and CD33+ macrophage densities were associated with CD8+ T‐cell infiltrates and programmed cell death ligand 1 expression. In addition, MCC harboring CD8+ T cell infiltrates and brisk CD33+ myeloid cell infiltrates were significantly and independently associated with improved outcomes (recurrence‐free and overall survival). Limitations: Sampling bias and the retrospective design were potential study limitations. Conclusion: Infiltration of CD33+ myeloid cells and CD8+ T lymphocytes defines a subset of MCC associated with improved outcome.

Differentiating Merkel cell carcinoma of lymph nodes without a detectable primary skin tumor from other metastatic neuroendocrine carcinomas: The ELECTHIP criteria

Background: Merkel cell carcinoma (MCC) can present as a cutaneous tumor or a lymph node metastasis without a primary tumor. MCC presenting without a primary tumor (MCCWOPT) can be misinterpreted on histologic examination as lymph node metastasis (LNM) from another neuroendocrine carcinoma (LNMNEC). However, this distinction is crucial for therapeutic management. Objective: To determine the discriminative criteria for the differential diagnosis of MCCWOPT, LNM from cutaneous MCC, and LNMNECs. Methods: Clinical, morphologic, and immunohistochemical data (expression of cytokeratins AE1, AE3, 7, 19, and 20; chromogranin A, synaptophysin, thyroid transcription factor‐1 [TTF‐1]), as well as the presence of Merkel cell polyomavirus (by immunohistochemistry and PCR) were compared in patients with MCCWOPT (n = 17), LNM from a cutaneous MCC (n = 11), and LNMNEC (n = 20; 8 lung, 7 thyroid, 3 digestive tract, 2 other). Results: MCC (including MCCWOPT and LNM from a cutaneous MCC) differed from LNMNEC by 7 discriminative criteria: 1) elderly age, 2) location of the tumor, 3) extent of the disease, 4) cytokeratin expression, 5) TTF‐1 expression, 6) histologic type, and 7) Merkel cell polyomavirus detection, summarized under the acronym ELECTHIP. All MCC patients had ≥5 of the ELECTHIP criteria, whereas all patients with LNMNEC (except 1) had <3 criteria. Limitations: The discriminant ability of the ELECTHIP criteria should be validated in a second independent set. Conclusion: MCCWOPT can be distinguished from other LNMNEC by the ELECTHIP criteria.

Detection of the Merkel cell polyomavirus in the neuroendocrine component of combined Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. The main etiological agent is Merkel cell polyomavirus (MCPyV), detected in 80% of cases. About 5% of cases, called combined MCC, feature an admixture of neuroendocrine and non-neuroendocrine tumor cells. Reports of the presence or absence of MCPyV in combined MCC are conflicting, most favoring the absence, which suggests that combined MCC might have independent etiological factors and pathogenesis. These discrepancies might occur with the use of different virus identification assays, with different sensitivities. In this study, we aimed to determine the viral status of combined MCC by a multimodal approach. We histologically reviewed 128 cases of MCC and sub-classified them as “combined” or “conventional.” Both groups were compared by clinical data (age, sex, site, American Joint Committee on Cancer [AJCC] stage, immunosuppression, risk of recurrence, and death during follow-up) and immunochemical features (cytokeratin 20 and 7, thyroid transcription factor 1 [TTF1], p53, large T antigen [CM2B4], CD8 infiltrates). After a first calibration step with 12 conventional MCCs and 12 cutaneous squamous cell carcinomas as controls, all eight cases of combined MCC were investigated for MCPyV viral status by combining two independent molecular procedures. Furthermore, on multiplex genotyping assay, the samples were examined for the presence of other polyoma- and papillomaviruses. Combined MCC differed from conventional MCC in earlier AJCC stage, increased risk of recurrence and death, decreased CD8 infiltrates, more frequent TTF1 positivity (5/8), abnormal p53 expression (8/8), and frequent lack of large T antigen expression (7/8). With the molecular procedure, half of the combined MCC cases were positive for MCPyV in the neuroendocrine component. Beta papillomaviruses were detected in 5/8 combined MCC cases and 9/12 conventional MCC cases. In conclusion, the detection of MCPyV DNA in half of the combined MCC cases suggests similar routes of carcinogenesis for combined and conventional MCC.

Multiple “halo nevi” occurring during pembrolizumab treatment for metastatic melanoma

A female in her 30s, with a previous history of acral melanoma (T1aN0M0) treated by excision, presented in 2015 with homolateral ilioinguinal lymph node metastasis. She underwent complete lymph node dissection but presented unresectable regional relapse (T1aN3M0) 3 months later. No BRAF, CKIT, or NRAS mutations were found in tumoral tissue, and the treatment was pembrolizumab (2 mg/kg every 3 weeks). Eight weeks later, an achromic halo appeared around a preexisting nevus of the right

Merkel cell carcinoma and cellular cytotoxicity: sensitivity to cellular lysis and screening for potential target antigens suitable for antibody-dependent cellular cytotoxicity

The recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpoint inhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further.

Étude historique de la visualisation des protéines

Resume L’histoire de la visualisation des proteines est inseparable de celle de la biologie structurale qui etudie la structure et l’organisation spatiale des macromolecules biologiques. Les anciens avaient initialement imagine les proteines comme des noyaux organiques azotes entoures d’une copule minerale. La determination a l’echelle atomique de la structure 3D des proteines fait appel a des techniques d’approche biophysiques elaborees dans la premiere moitie du vingtieme siecle. La cristallographie par diffraction des rayons X permet d’obtenir une carte de densite electronique. La spectroscopie par resonance magnetique nucleaire (RMN) definit une carte des distances inter-protons et des angles de torsion du squelette de la proteine. La cryomicroscopie electronique donne une image directe de la molecule saisie dans son milieu aqueux. Ces trois familles de techniques evoluent de maniere spectaculaire pour grandir en precision, en definition et en analyse spatiale et temporelle. Aujourd’hui ces methodes convergent grâce a des bases de donnees interactives et des outils bioinformatiques de modelisation dynamique vers la definition de modeles de liaisons « proteines-ligands », la decouverte d’inhibiteurs pharmacologiques, la prediction de structure macromoleculaire et une comprehension accrue des maladies du repliement anormal des proteines.