Maternal embryonic leucine zipper kinase is a novel target for diffuse large B cell lymphoma and mantle cell lymphoma

Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are among the most aggressive B cell non-Hodgkin lymphomas. Maternal embryonic leucine zipper kinase (MELK) plays a role in cancer cell cycle progression and is associated with poor prognosis in several cancer cell types. In this study, the role of MELK in DLBCL and MCL and the therapeutic potential of MELK targeting is evaluated. MELK is highly expressed in DLBCL and MCL patient samples, correlating with a worse clinical outcome in DLBCL. Targeting MELK, using the small molecule OTSSP167, impaired cell growth and survival and induced caspase-mediated apoptosis in the lymphoma cells. Western blot analysis revealed that MELK targeting decreased the phosphorylation of FOXM1 and the protein levels of EZH2 and several mitotic regulators, such as Cdc25B, cyclin B1, Plk-1, and Aurora kinases. In addition, OTSSP167 also sensitized the lymphoma cells to the clinically relevant Bcl-2 inhibitor venetoclax by strongly reducing Mcl1 levels. Finally, OTSSP167 treatment of A20-inoculated mice resulted in a significant prolonged survival. In conclusion, targeting MELK with OTSSP167 induced strong anti-lymphoma activity both in vitro and in vivo. These findings suggest that MELK could be a potential new target in these aggressive B cell malignancies

Atypical fibrous histiocytoma of the skin with CD30 and p80/ALK1 positivity and ALK gene rearrangement

We report the case of a two patients who presented with a solitary, asymptomatic, angiomatoid nodule on the right thigh. Histopathological finding showed a poorly circumscribed lesion, located in the dermis. The morphological aspect strongly suggested the diagnosis of atypical fibrous histiocytoma (AFH), but surprisingly, the neoplastic cells were diffusely CD30+, with a membrane staining devoid of paranuclear dot. The lesions were tested for p80/ALK1 expression. Surprisingly, we found a diffuse cytoplasmic positivity. Interestingly, using break-apart fluorescent in situ hybridization (FISH), we evidenced an ALK rearrangement in nearly 50% of the neoplastic cells. The expression of CD30 and ALK1 with ALK gene rearrangement raised the possibility of three diagnoses: a primary cutaneous anaplastic large cell lymphoma (ALCL), a cutaneous inflammatory myofibroblastic tumor (IMT), an AFH of the skin associated with ALK gene rearrangement and CD30 positivity. The three hypotheses were discussed and finally, although p80/ALK1 expression and cytogenetic abnormalities in fibrous histiocytoma (FH) are not yet reported to the best of our knowledge, we favored the diagnosis of AFH

The anaphase-promoting complex/cyclosome : a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

BACKGROUND: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. METHODS: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. RESULTS: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. CONCLUSION: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL

Rôle des modulateurs épigénétiques dans la physiopathologie des lymphomes B diffus à grandes cellules

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma type. DLBCL shows considerable clinical and biological heterogeneity. The international prognostic index (IPI) remains the most used tool to stratify patients in different risk groups but does not reflect DLBCL biological heterogeneity. Therefore, much research is currently focused on the identification of new prognostic markers for more specific patients’ risk stratification and on the development of therapeutic approaches to improve outcome. Epigenetic alterations are involved in lymphoma. Interestingly, epigenetic alterations are reversible and drugs to target some of them have been developed. With the aim to identify new and relevant prognostic factors that allow the stratification of patients with DLBCL we investigated the gene expression profile of 130 epigenetics regulators in two independent cohorts of patients with newly-diagnosed DLBCL homogeneously treated (respectively 233 and 181 cases). Using the Maxstat R function and Benjami-Hochberg multiple testing correction we found that 10 probe sets had a prognostic value for overall survival (OS) including: BRD1, CARM1, BRPF3, CDYL, DNMT3A, DOT1L, HDAC2, PRMT5, SETD8 and SP140. Using multivariate Cox analysis we found that 3 of these genes remained independent prognostic factors: DNMT3A, DOT1L and SETD8. We used these 3 genes to develop a risk score (EpiScore) based on their expression level in the cohort of 233 DLBCL. EpiScore allowed splitting the patients in 3 groups with significant different OS values: group 1 (low risk, low DNMT3A, DOT1L and SETD8 expression), group 2 (intermediate risk, high expression of one of the three genes) and group 3 (high risk, high expression of two or all three genes). EpiScore prognostic value was validated in two other independent cohorts of patients with DLBCL (181 and 69 patients respectively) We then showed that EpiScore was an independent predictor of survival when compared with previously described prognostic factors, such as the IPI, germinal center B cell and activated B cell molecular subgroups, gene expression-based risk score (GERS) and DNA repair score. As gene expression profiling (GEP) is not a technical approach widely performed in routine practise for all DLBCL newly diagnosed we analysed the pattern of expression of the ten epigenetic genes by immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissue sections in a cohort of 65 patients with de novo previously untreated DLBCL. Our results indicate that these epigenetic related proteins are commonly overexpressed in DLBCL compared to reactive lymphoid tissues and may be important for DLBCL pathogenesis. We showed that overexpression of CARM1 and DNMT3A was significantly associated with reduced event free survival. We then designed a new risk score Epi-ImmunoScore (Epi-IS) based on the expression level of CARM1 and DNMT3A by immunohistochemistry. Epi-IS was predictive of OS in DLBCL and allowed splitting patients in two groups (high and low risk). Finally, using gene set enrichment analysis (GSEA) an HDAC gene signature was significantly enriched in the DLBCL samples included in the EpiScore high-risk group and that a significant enrichment of genes encoding for HDAC class II, multiple drug resistance and NOTCH pathways in DLBCL samples with DNMT3A overexpression. According to these data, we compared the response to HDAC inhibitor (SAHA) in DLBCL cell lines with high EpiScore versus low EpiScore and showed that DLBCL cell lines with high EpiScore were significantly more sensitive to SAHA than those low EpiScore. We concluded that EpiScore and Epi-IS, easily evaluated in the routine practice on FFPE tissue sections identified high-risk patients with DLBC. We have also recognized relevant therapeutic targets and identified a number of candidate drugs with potential therapeutic efficiency in DLBCL patients. All these findings may orient future preclinical intervention strategies in DLBCL.Les lymphomes B diffus à grandes cellules (DLBCL) sont le type le plus fréquent de lymphome et montrent une hétérogénéité clinique et biologique. L’index pronostique international (IPI) reste le meilleur outil permettant de stratifier les patients en groupes pronostiques mais n’est pas le reflet de leur hétérogénéité biologique. Les travaux de recherches se sont concentrés sur l’identification de nouveaux marqueurs pronostiques afin de stratifier les patients de manière optimale et de développer de nouvelles thérapeutiques améliorant la survie. Les altérations épigénétiques sont impliquées dans les lymphomes. Ces altérations sont réversibles et des drogues les ciblant existent. Dans le but d’identifier de nouveaux marqueurs pronostiques pertinents permettant de stratifier les patients avec un DLBCL nous avons étudié les données d’expression génique de 130 régulateurs épigénétiques dans 2 cohortes de patients avec un DLBCL au diagnostic (respectivement 233 et 181 cas). La fonction Maxstat R et la procédure de correction statistique Benjami-Hochberg nous ont permis de sélectionner 10 gènes avec un impact pronostique significatif sur la survie globale (OS) : BRD1, CARM1, BRPF3, CDYL, DNMT3A, DOT1L, HDAC2, PRMT5, SETD8 and SP140. Trois gènes conservaient une valeur pronostique indépendante en analyses multivariées : DNMT3A, DOT1L, SETD8. Ces 3 gènes nous ont permis de construire un score pronostique EpiScore basé sur leur niveau d’expression dans la cohorte principale de 233 DLBCL. L’EpiScore permettait de stratifier les patients en 3 groupes d’OS significativement différente : groupe 1 (faible risque, faible expression des 3 gènes), groupe 2 (risque intermédiaire, forte expression d’1 des 3 gènes), groupe 3 (haut risque, forte expression d’au moins 2 des 3 gènes). La valeur pronostique de l’EpiScore a ensuite été validée dans 2 autres cohortes de DLBCL (181 et 69 patients). L’EpiScore conservait sa valeur pronostique indépendante en terme d’OS comparativement aux autres facteurs pronostiques : IPI, sous groupe moléculaire GC versus ABC, gene expression-based risk score (GERS) et le DNA repair score. L’étude des profils d’expression génique a partir d’’ARN n’est pas une technique facilement applicable en routine diagnostique. Nous avons donc évalué les patterns d’expression protéique des 10 gènes initiaux par immunohistochimie sur coupes de paraffine de tumeur de 65 patients avec un DLBCL au diagnostic. Nos résultats montraient que ces modulateurs épigénétiques étaient surexprimés dans les DLBCL comparativement au tissu lymphoïde normal. La surexpression protéique de CARM1 et de DNMT3A était en outre associée une survie sans événement significativement réduite. Nous avons donc établi un nouveau score l’Epi-Immunoscore (Epi-IS) basé sur le niveau d’expression de ces 2 protéines en immunohistochimie. Nous avons montré la valeur prédictive sur l’OS de l’Epi-IS qui permettait de diviser les patients en 2 groupes (faible et haut risque). Finalement en appliquant des analyses en GSEA (gene set enrichment analysis) nous avons observé que les patients EpiScore haut risque avait une signature enrichie en gènes de la voie HDAC et que les échantillons de DLBCL montrant une surexpression de DNMT3A avait des signatures enrichis en gènes des voies HDAC class II, NOTCH et multiple drug resistance. Nous avons donc comparé la réponse aux inhibiteurs des HDAC (HDACi) des lignées cellulaires de DLBCL EpiScore fort versus faible. Les lignées de DLBCL EpiScore fort étaient significativement plus sensibles aux HDCAi que celles EpiScore faible. Nous avons conclu que l’EpiScore et l’Epi-IS, facilement applicable en routine diagnostique sur coupes de paraffine, permettaient d’identifier les patients avec un DLBCL à haut risque. Nous avons également identifié des cibles thérapeutiques pertinentes et un certains de nombres de drogues potentiellement efficaces. Toutes ces données pourraient orienter de futurs essais pré-cliniques dans les DLBCL

Role of epigenetic modifiers in diffuse large B cell lymphoma pathophysiology

Les lymphomes B diffus à grandes cellules (DLBCL) sont le type le plus fréquent de lymphome et montrent une hétérogénéité clinique et biologique. L’index pronostique international (IPI) reste le meilleur outil permettant de stratifier les patients en groupes pronostiques mais n’est pas le reflet de leur hétérogénéité biologique. Les travaux de recherches se sont concentrés sur l’identification de nouveaux marqueurs pronostiques afin de stratifier les patients de manière optimale et de développer de nouvelles thérapeutiques améliorant la survie. Les altérations épigénétiques sont impliquées dans les lymphomes. Ces altérations sont réversibles et des drogues les ciblant existent. Dans le but d’identifier de nouveaux marqueurs pronostiques pertinents permettant de stratifier les patients avec un DLBCL nous avons étudié les données d’expression génique de 130 régulateurs épigénétiques dans 2 cohortes de patients avec un DLBCL au diagnostic (respectivement 233 et 181 cas). La fonction Maxstat R et la procédure de correction statistique Benjami-Hochberg nous ont permis de sélectionner 10 gènes avec un impact pronostique significatif sur la survie globale (OS) : BRD1, CARM1, BRPF3, CDYL, DNMT3A, DOT1L, HDAC2, PRMT5, SETD8 and SP140. Trois gènes conservaient une valeur pronostique indépendante en analyses multivariées : DNMT3A, DOT1L, SETD8. Ces 3 gènes nous ont permis de construire un score pronostique EpiScore basé sur leur niveau d’expression dans la cohorte principale de 233 DLBCL. L’EpiScore permettait de stratifier les patients en 3 groupes d’OS significativement différente : groupe 1 (faible risque, faible expression des 3 gènes), groupe 2 (risque intermédiaire, forte expression d’1 des 3 gènes), groupe 3 (haut risque, forte expression d’au moins 2 des 3 gènes). La valeur pronostique de l’EpiScore a ensuite été validée dans 2 autres cohortes de DLBCL (181 et 69 patients). L’EpiScore conservait sa valeur pronostique indépendante en terme d’OS comparativement aux autres facteurs pronostiques : IPI, sous groupe moléculaire GC versus ABC, gene expression-based risk score (GERS) et le DNA repair score. L’étude des profils d’expression génique a partir d’’ARN n’est pas une technique facilement applicable en routine diagnostique. Nous avons donc évalué les patterns d’expression protéique des 10 gènes initiaux par immunohistochimie sur coupes de paraffine de tumeur de 65 patients avec un DLBCL au diagnostic. Nos résultats montraient que ces modulateurs épigénétiques étaient surexprimés dans les DLBCL comparativement au tissu lymphoïde normal. La surexpression protéique de CARM1 et de DNMT3A était en outre associée une survie sans événement significativement réduite. Nous avons donc établi un nouveau score l’Epi-Immunoscore (Epi-IS) basé sur le niveau d’expression de ces 2 protéines en immunohistochimie. Nous avons montré la valeur prédictive sur l’OS de l’Epi-IS qui permettait de diviser les patients en 2 groupes (faible et haut risque). Finalement en appliquant des analyses en GSEA (gene set enrichment analysis) nous avons observé que les patients EpiScore haut risque avait une signature enrichie en gènes de la voie HDAC et que les échantillons de DLBCL montrant une surexpression de DNMT3A avait des signatures enrichis en gènes des voies HDAC class II, NOTCH et multiple drug resistance. Nous avons donc comparé la réponse aux inhibiteurs des HDAC (HDACi) des lignées cellulaires de DLBCL EpiScore fort versus faible. Les lignées de DLBCL EpiScore fort étaient significativement plus sensibles aux HDCAi que celles EpiScore faible. Nous avons conclu que l’EpiScore et l’Epi-IS, facilement applicable en routine diagnostique sur coupes de paraffine, permettaient d’identifier les patients avec un DLBCL à haut risque. Nous avons également identifié des cibles thérapeutiques pertinentes et un certains de nombres de drogues potentiellement efficaces. Toutes ces données pourraient orienter de futurs essais pré-cliniques dans les DLBCL.Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma type. DLBCL shows considerable clinical and biological heterogeneity. The international prognostic index (IPI) remains the most used tool to stratify patients in different risk groups but does not reflect DLBCL biological heterogeneity. Therefore, much research is currently focused on the identification of new prognostic markers for more specific patients’ risk stratification and on the development of therapeutic approaches to improve outcome. Epigenetic alterations are involved in lymphoma. Interestingly, epigenetic alterations are reversible and drugs to target some of them have been developed. With the aim to identify new and relevant prognostic factors that allow the stratification of patients with DLBCL we investigated the gene expression profile of 130 epigenetics regulators in two independent cohorts of patients with newly-diagnosed DLBCL homogeneously treated (respectively 233 and 181 cases). Using the Maxstat R function and Benjami-Hochberg multiple testing correction we found that 10 probe sets had a prognostic value for overall survival (OS) including: BRD1, CARM1, BRPF3, CDYL, DNMT3A, DOT1L, HDAC2, PRMT5, SETD8 and SP140. Using multivariate Cox analysis we found that 3 of these genes remained independent prognostic factors: DNMT3A, DOT1L and SETD8. We used these 3 genes to develop a risk score (EpiScore) based on their expression level in the cohort of 233 DLBCL. EpiScore allowed splitting the patients in 3 groups with significant different OS values: group 1 (low risk, low DNMT3A, DOT1L and SETD8 expression), group 2 (intermediate risk, high expression of one of the three genes) and group 3 (high risk, high expression of two or all three genes). EpiScore prognostic value was validated in two other independent cohorts of patients with DLBCL (181 and 69 patients respectively) We then showed that EpiScore was an independent predictor of survival when compared with previously described prognostic factors, such as the IPI, germinal center B cell and activated B cell molecular subgroups, gene expression-based risk score (GERS) and DNA repair score. As gene expression profiling (GEP) is not a technical approach widely performed in routine practise for all DLBCL newly diagnosed we analysed the pattern of expression of the ten epigenetic genes by immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissue sections in a cohort of 65 patients with de novo previously untreated DLBCL. Our results indicate that these epigenetic related proteins are commonly overexpressed in DLBCL compared to reactive lymphoid tissues and may be important for DLBCL pathogenesis. We showed that overexpression of CARM1 and DNMT3A was significantly associated with reduced event free survival. We then designed a new risk score Epi-ImmunoScore (Epi-IS) based on the expression level of CARM1 and DNMT3A by immunohistochemistry. Epi-IS was predictive of OS in DLBCL and allowed splitting patients in two groups (high and low risk). Finally, using gene set enrichment analysis (GSEA) an HDAC gene signature was significantly enriched in the DLBCL samples included in the EpiScore high-risk group and that a significant enrichment of genes encoding for HDAC class II, multiple drug resistance and NOTCH pathways in DLBCL samples with DNMT3A overexpression. According to these data, we compared the response to HDAC inhibitor (SAHA) in DLBCL cell lines with high EpiScore versus low EpiScore and showed that DLBCL cell lines with high EpiScore were significantly more sensitive to SAHA than those low EpiScore. We concluded that EpiScore and Epi-IS, easily evaluated in the routine practice on FFPE tissue sections identified high-risk patients with DLBC. We have also recognized relevant therapeutic targets and identified a number of candidate drugs with potential therapeutic efficiency in DLBCL patients. All these findings may orient future preclinical intervention strategies in DLBCL

EGFR Expression and KRAS and BRAF Mutational Status in Intestinal-Type Sinonasal Adenocarcinoma

Accumulation of molecular alterations, including EGFR overexpression and mutations in KRAS and BRAF, contribute to colorectal carcinogenesis. Since intestinal-type adenocarcinoma (ITAC) of the nasal cavity and paranasal sinus has morphologic and phenotypic features that are usually indistinguishable from colorectal cancer (CRC), it is likely that both tumor types share equivalent genetic alterations. Data from a series of 43 patients treated surgically for ITAC in Montpellier, France between November 1998 and December 2012 were collected. Tumors were characterized for mutations in KRAS and BRAF as well as EGFR overexpression. Kaplan-Meier survival curves were constructed using overall survival as the primary end points. Patient survival was analyzed using the hazards ratio. Twenty seven tumors (63%) showed EGFR positivity and 30% exhibited a high expression level (+2/+3). KRAS mutations were detected in 43% of cases. BRAF mutations were identified in 3.6% of specimens. Patients with age superior to 60 years, metastatic status, and KRAS mutations had significant overall survival values (p = 0.026, p = 0.001 and p = 0.03, respectively). Our results indicate that KRAS mutations and EGFR expression are frequent in ITAC and that KRAS mutations predict good patient prognosis in ITAC. Finally, EGFR directed molecular treatments could be investigated in a subset of patients affected by ITAC

Sarcomes des fosses nasales et des sinus (Une étude clinicopathologique de 48 cas)

Dans le tractus naso-sinusien, une grande variété de sarcomes a été observée, mais chaque entité a été décrite séparément. Nos objectifs étaient d'obtenir des informations épidémiologiques sur les sarcomes des fosses nasales et des sinus de l'adulte, et de définir des critères pronostiques de ces tumeurs. 48 cas de sarcomes des fosses nasales et des sinus, de l'adulte âgé d'au moins 18 ans, ont été inclus. Pour chaque patient le type histologique était déterminé ainsi que le grade de la tumeur. Les survies globales (OS), sans récidive locale (LRFS) et sans métastase (MFS) ont été déterminées. 9 variables ont été étudiées pour leur valeur pronostique. Les types histologiques de sarcomes des fosses nasales et des sinus les plus fi'équemment rencontrés étaient les suivants: rhabdomyosarcomes alvéolaires (RMSA) (33.3%), rhabdomyosarcomes embryonnaires (RMSE) (14.6%), sarcomes inclassés (14.6%), léiomyosarcomes (l2.5%).L'OS, la MSF et la LRFS à 5 ans étaient de 62.3%,73% et 88.8%. L'histotype était le facteur prédictifle plus significatif, avec un pronostique plus péjoratif pour le groupe des rhabdomyosarcomes (RMS).Les facteurs prédictifs d'une réponse incomplète étaient le sous type RMS et l'absence de chirurgie. Il n'existait pas de différence pronostique significative entre RMSA et RMSE. Nos résultats montrent que l'histotype est le facteur pronostique le plus significatif des sarcomes des fosses nasales et des sinus, avec un pronostic péjoratif associé aux RMS, sans différence entre les RMSA et RMSE. La chirurgie quelque soit la qualité des berges d'exérèse est associée à une rémission complète et devrait donc toujours être envisagée devant un sarcome nasosinusien.MONTPELLIER-BU Médecine UPM (341722108) / SudocSudocFranceF

Indolent cytotoxic T cell lymphoproliferation associated with nodular regenerative hyperplasia: a common liver lesion in the context of common variable immunodeficiency disorder

International audiencePatients with common variable immunodeficiency disorder (CVID) are subject to lymphoproliferative disorders and predisposed to lymphoma. Some patients may also develop liver lesions. The purpose of this study was to define clinical and histopathological features of patients with CVID presenting with liver lesions suspicious of lymphoma. Four CVID cases corresponding to these criteria were retrieved from our files. Liver biopsy specimens were subjected to morphologic, immunophenotypic and molecular analysis. All patients presented with hepatosplenomegaly and two furthermore with lymphadenopathy. The clinical working diagnosis in the four cases was lymphoma. All liver biopsies revealed nodular regenerative hyperplasia (NRH), associated with mild to marked sinusoid lymphocytic infiltrate consisting of "activated" cytotoxic T cells (CD8+, Tia1+, granzyme B+, TCRβF1+, CD56-). EBER was negative in all cases. T cell clonality was found in one of the two interpretable cases. All patients had an indolent course and clinical symptoms regressed with immunoglobulin replacement. This study suggests that indolent proliferation in the liver sinusoid of cytotoxic T cell associated with NRH is a specific liver lesion in the context of CVID. In CVID patients clinically suspected of lymphoma, pathologists should avoid a misdiagnosis of aggressive T cell lymphoma with a risk of over treatment

Spatial and temporal homogeneity of T-cell receptor gamma chain rearrangements in mycosis fungoides: A next-generation sequencing analysis

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Primary Cutaneous Gamma-Delta T-cell Lymphoma: Not an Aggressive Disease in All Cases

International audiencePrimary cutaneous gamma-delta T-cell lymphoma (PCGDTCL) is a rare form of primary cutaneous lymphoma characterized by a clonal proliferation of mature γ/δ T lymphocytes. PCGDTCL is usually considered an aggressive and rapidly evolving malignancy. Contrasting with this patter, we hereby report 2 unusual cases with a protracted indolent evolution