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Contains fulltext : 252063.pdf (Publisher’s version ) (Open Access) Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Contains fulltext : 172708.pdf (Publisher’s version ) (Open Access) Chemotherapeutics, including the platinum compounds oxaliplatin (OXP) and cisplatin (CDDP), are standard care of treatment for cancer. Although chemotherapy has long been considered immunosuppressive, evidence now suggests that certain cytotoxic agents can efficiently stimulate antitumor responses, through the induction of a form of apoptosis, called immunogenic cell death (ICD). ICD is characterized by exposure of calreticulin and heat shock proteins (HSPs), secretion of ATP and release of high-mobility group box 1 (HMGB1). Proper activation of the immune system relies on the integration of these signals by dendritic cells (DCs). Studies on the crucial role of DCs, in the context of ICD, have been performed using mouse models or human in vitro-generated monocyte-derived DCs (moDCs), which do not fully recapitulate the in vivo situation. Here, we explore the effect of platinum-induced ICD on phenotype and function of human blood circulating DCs. Tumor cells were treated with OXP or CDDP and induction of ICD was investigated. We show that both platinum drugs triggered translocation of calreticulin and HSP70, as well as the release of ATP and HMGB1. Platinum treatment increased phagocytosis of tumor fragments by human blood DCs and enhanced phenotypic maturation of blood myeloid and plasmacytoid DCs. Moreover, upon interaction with platinum-treated tumor cells, CD1c(+) DCs efficiently stimulated allogeneic proliferation of T lymphocytes. Together, our observations indicate that platinum-treated tumor cells may exert an active stimulatory effect on human blood DCs. In particular, these data suggest that CD1c(+) DCs are critical mediators of immune responses induced by ICD. 15 p.