ALBERT

Description: Key Points NR3C1 haploinsufficiency is found in patients with a plasmacytoid dendritic cell neoplasm characterized by very poor clinical outcome. Overexpression of lincRNA-3q is a consistent feature of malignant cells in these patients and can be abrogated by BET protein inhibition.

Description: Introduction & Objectives: Primary Cutaneous Follicle Center Lymphoma (PCFCL) is a very indolent mature B-cell lymphoma that shares germinal center morphology with follicular lymphoma (FL) but lacks the characteristic t(14;18). Unlike FL, immunohistochemistry fails to detect expression of BCL2, CD10, and immunoglobulin in PCFCL. Therefore, we investigated expression of B-cell receptor (BCR) transcripts to gain insight into the immunobiology of PCFCL. Materials & Methods: Full-length heavy and light chain BCR transcripts of 13 histologically confirmed PCFCL were amplified using ARTISAN PCR and sequenced on the PacBio RSII system. BCR from 4 cases were sequenced to a depth of 〉2000 sequences per BCR transcript; the remaining cases to a median depth of 1663 sequences (range: 626-5301). BCR from 51 cases of FL and from peripheral B cells of 12 healthy donors were used as controls. Whole genome sequencing (WGS) and RNAseq were performed on 5 PCFCL on the Illumina HiSeq platform. Results: No PCFCL case carried a t(14;18). In addition to previously described CD79B mutations, an L265P mutation in MYD88 was identified in one case, and two PCFCL carried amplifications in chromosome 2 involving the proto-oncogene REL. ARTISAN PCR demonstrated expression of potentially functional VDJ and VJ genes with heavily mutated V regions (VDJ: 5.9-24.0%; VJ: 4.7-17.9%) in all PCFCL cases, which could be confirmed by RNAseq-based de novo BCR assembly. One PCFCL case expressed IgM, another IgA, and the remaining ten cases expressed IgG. PCFCL VDJ carried relatively long heavy chain CDR3 regions with a median of 19 amino acids (versus 17 in healthy donor PBMCs). In contrast to FL, only minimal intraclonal sequence variation (comparable to the known error rate of the used sequencing method) was observed in PCFCL VDJ and VJ sequences, indicating absence of ongoing somatic hypermutation (SHM). VDJ and/or VJ of 11 PCFCL (85%) carried at least one acquired N-linked glycosylation motifs, six PCFCL (46%) at least two, and one case four such motifs. 75% of acquired N-linked glycosylation motifs were found in different positions than the N-linked glycosylation motifs found in FL BCR (Figure). In contrast, only 17.5% and

Description: The chemokines CXCL9, 10, and 11 exert their action via CXC chemokine receptor-3 (CXCR3), a receptor highly expressed on activated T cells. These interferon γ (IFNγ)–induced chemokines are thought to be crucial in directing activated T cells to sites of inflammation. As such, they play an important role in several chronic inflammatory diseases including ulcerative colitis, multiple sclerosis, artherosclerosis, and delayed-type hypersensitivity reactions of the skin. In this study, we first demonstrate that in COS-7 cells heterologously expressing CXCR3, CXCL11 is a potent activator of the pertussis toxin (PTX)–sensitive p44/p42 mitogen-activated protein kinase (MAPK) and Akt/phosphatidylinositol 3 kinase (PI3K) pathways. Next, we show that these signal transduction pathways are also operative and PTX sensitive in primary human T cells expressing CXCR3. Importantly, abrogation of these signaling cascades by specific inhibitors did not block the migration of T cells toward CXCR3 ligands, suggesting that MAPK and Akt activation is not crucial for CXCR3-mediated chemotaxis of T cells. Finally, we demonstrate that CXCR3-targeting chemokines control T-cell migration via PTX-sensitive, phospholipase C pathways and phosphatidylinositol kinases other than class I PI3Kγ.

Description: Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is a malignancy of mature, skin-homing T cells. Sézary syndrome (Sz) is often considered to represent a leukemic phase of MF. In this study, the pattern of numerical chromosomal alterations in MF tumor samples was defined using array-based comparative genomic hybridization (CGH); simultaneously, gene expression was analyzed using microarrays. Highly recurrent chromosomal alterations in MF include gain of 7q36, 7q21-7q22 and loss of 5q13 and 9p21. The pattern characteristic of MF differs markedly from chromosomal alterations observed in Sz. Integration of data from array-based CGH and gene-expression analysis yielded several candidate genes with potential relevance in the pathogenesis of MF. We confirmed that the FASTK and SKAP1 genes, residing in loci with recurrent gain, demonstrated increased expression. The RB1 and DLEU1 tumor suppressor genes showed diminished expression associated with loss. In addition, it was found that the presence of chromosomal alterations on 9p21, 8q24, and 1q21-1q22 was associated with poor prognosis in patients with MF. This study provides novel insight into genetic alterations underlying MF. Furthermore, our analysis uncovered genomic differences between MF and Sz, which suggest that the molecular pathogenesis and therefore therapeutic requirements of these cutaneous T-cell lymphomas may be distinct.

Description: Background The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In app. 10% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CARD11. Recent evidence suggests that autoantigen recognition by the BCR sustains the viability of ABC DLBCL in vitro (Young, PNAS 2015), although the functional relationship to CARD11 mutations remains unresolved. Antigen-independent, constitutively active BCR signaling is a fundamental oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We therefore explored the hypothesis that autonomous BCR signaling akin to CLL could be operative in ABC DLBCL and would represent an alternative but functionally complementary oncogenic mechanism to activating CARD11 mutations in ABC DLBCL lymphomagenesis. Methods BCR transcripts were identified from archived fresh-frozen biopsies of 14 histologically confirmed, CD10-negative, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased isolation of full-length BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR were identified by single molecule sequencing on the PacBio platform. All biopsies were screened for mutations in CARD11 and CD79 by Sanger sequencing. To test for autonomous BCR signaling activity, murine triple KO (TKO) cells were transduced with functional DLBCL BCR (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon restoring functionality of the BCR signalling cascade with tamoxifen as calcium flux by flow cytometry and robust cellular proliferation. Results Re-examination by immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6 was possible in 12 cases and identified three cases as GCB-type DLBCL. Nine cases were classified as non-GCB DLBCL, including one primary CNS DLBCL and three leg-type primary cutaneous DLBCL (LT DLBCL). One non-GCB DLBCL was found to carry a potentially activating CARD11 M212K mutation (Table). BCR of the three GCB DLBCL served as controls and lacked an autonomous BCR signal. In contrast, TKO cells transduced with five of the seven remaining DLBCL BCR (71%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. In accordance with our hypotheses, the CARD11-mutated non-GCB DLBCL case lacked autonomous BCR signaling. This case and the second, non-GCB DLBCL case lacking autonomous BR activity that carried wild-type CARD11 both expressed a BCR with the IGHV4-34 allele. Unexpectedly, the three LT DLBCL also did not exhibit autonomous BCR signaling. Conclusion Our data demonstrate autonomous BCR activity in a dominant fraction of non-GCB DLBCL. In striking similarity to CLL, these results point to an oncogenic role of a structurally normal BCR in non-GCB DLBCL that does not require binding of external autoantigens. Consistent with our hypotheses, antigen-independent signaling of a structurally normal BCR signaling cascade is a compelling candidate alternative mechanism to activating mutations in signal-transducing components of the BCR pathway. Both mechanisms may therefore induce the characteristic gene expression signature of ABC DLBCL. In addition, this finding readily explains the higher likelihood of ABC DLBCL with wild-type CD79 and CARD11 to respond to BCR signaling inhibition by BTK inhibitors (Wilson, Nat Med 2015). In contrast, LT DLBCL, which otherwise resembles ABC DLBCL, does not exhibit autonomous BCR signaling, suggesting yet another oncogenic mechanism that may explain its dismal prognosis. Finally, the non-GCB DLBCL lacking both autonomous BCR signaling and a potentially activating CARD11 mutation expressed the inherently autoreactive IGHV4-34 allele, opening the possibility that strong autoantigen stimulation might substitute for autonomous BCR signaling activity, thereby offering an alternative explanation for the BCR signaling pathway addiction in IGHV4-34-expressing cases. Table. Table. Disclosures Vermeer: Innate Pharma: Other: Investigator in a clinical trial.

Description: Mycosis fungoides and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) derived from mature CD4-positive skin-homing T cells with Sézary representing the most aggressive leukemic form of the disease. To investigate the pathogenic mechanisms of Sézary syndrome and aggressive CTCLs, we performed whole exome sequencing of matched tumor and normal DNA samples from 42 CTCL cases, including 26 Sézary syndrome. Copy number analysis revealed a distinctive pattern of somatic copy number alterations in Sézary syndrome including highly prevalent recurrent chromosomal deletions involving the TP53, RB1, PTEN, DNMT3A and CDKN1B tumor suppressor genes. Moreover, exome-wide somatic mutation analysis identified a broad spectrum of somatic mutations in key genes involved in epigenetic regulation (TET2, CREBBP, MLL2, MLL3, BRD9, SMARCA4 and CHD3) and signaling, including mutations in MAPK1, BRAF, CARD11 and PRKG1. In this context, we identified three mutations involving position E322 in MAPK1 (p.Glu322Ala and p.Glu322Lys). Functional characterization of these recurrent alleles showed increased ERK1/2 activation upon expression of both MAPK1 E322K and E322A. In addition we identified two mutations (p.Ser615Phe and p.Ser626Lys) in CARD11, an important mediator of NFκB activation. Notably, and in contrast with diffuse B-cell lymphomas where CARD11 mutations are primarily located in the coiled-coil domain, CARD11 alleles present in CTCL cases are located in the linker domain region of the protein. Functional characterization of CTCL associated CARD11 S615F and CARD11 E626K linker mutations revealed markedly increased NFκΒ activity in reporter assays, which was further enhanced upon TCR activation. Finally, we also noted the presence of two closely located mutations (p.Glu17Lys and p.Arg21Gln) in the N-terminal dimerization domain of the cGKIβ protein, in Sézary syndrome samples. cGKIβ, PRKG1 gene, is a cGMP-binding protein implicated in nitric oxide signaling. cGKIβ signaling in T-cells antagonizes TCR activation-induced interleukin 2 release and proliferation. Structural and functional characterization of the two PRKG1 mutations found in Sézary syndrome revealed impaired dimerization and decreased cGKIβ-mediated activation after stimulation with the cGMP analog 8-CPT-cGMP. Moreover, analysis of the effects of cGKIβ E17K and cGKIβ R21Q expression in JURKAT T cells revealed increased NFAT activity after PMA plus ionomycin stimulation, supporting a positive role for these mutations in enhancing the TCR signaling response. In all, these results mechanistically involve increased MAPK-ERK, NFκB and NFAT signaling in the pathogenesis of CTCLs. To analyze the therapeutic potential of targeting these oncogenic signaling pathways in CTCL we performed analysis of MAPK, JNK and NFκB signaling in CTCL cell lines. Therapeutically, inhibition of NFκB signaling with Mi-2 and bortezomib was broadly and highly active across all cell lines tested, while MEK1/2 inhibition with U0126 and inhibition of NFAT with FK506 showed more modest antitumor effects. Collectively, our findings provide new insights into the genetics of Sézary syndrome and CTCL and support the development of personalized therapies targeting key oncogenically activated signaling pathways for the treatment of these diseases. Disclosures No relevant conflicts of interest to declare.

Description: In the European Organization for Research and Treatment of Cancer (EORTC) classification 2 types of primary cutaneous large B-cell lymphoma (PCLBCL) are distinguished: primary cutaneous follicle center cell lymphomas (PCFCCL) and PCLBCL of the leg (PCLBCL-leg). Distinction between both groups is considered important because of differences in prognosis (5-year survival 〉 95% and 52%, respectively) and the first choice of treatment (radiotherapy or systemic chemotherapy, respectively), but is not generally accepted. To establish a molecular basis for this subdivision in the EORTC classification, we investigated the gene expression profiles of 21 PCLBCLs by oligonucleotide microarray analysis. Hierarchical clustering based on a B-cell signature (7450 genes) classified PCLBCL into 2 distinct subgroups consisting of, respectively, 8 PCFCCLs and 13 PCLBCLsleg. PCLBCLs-leg showed increased expression of genes associated with cell proliferation; the proto-oncogenes Pim-1, Pim-2, and c-Myc; and the transcription factors Mum1/IRF4 and Oct-2. In the group of PCFCCL high expression of SPINK2 was observed. Further analysis suggested that PCFCCLs and PCLBCLs-leg have expression profiles similar to that of germinal center B-cell–like and activated B-cell–like diffuse large B-cell lymphoma, respectively. The results of this study suggest that different pathogenetic mechanisms are involved in the development of PCFCCLs and PCLBCLs-leg and provide molecular support for the subdivision used in the EORTC classification.

Description: Introduction : Primary cutaneous T cell lymphomas (CTCL) represent 70% of all cutaneous lymphomas. The most frequent is mycosis fungoides/Sézary syndrome (MF/ SS). In this entity, few high resolution cytogenetic studies have been performed. Our aim was to analyze chromosomal abnormalities in MF tumoral stage by array comparative genomic hybridization (ArrayCGH) and to describe potential candidate genes related to this disease. Patients and methods : Forty-one patients (22 males/19 females) with MF tumor stage were included from centres collaborating in the EORTC Cutaneous Lymphoma Group. DNA was extracted from frozen tissues containing more than 70% of tumor cells. ArrayCGH tecnhology was performed to detect genomic imbalances (gains/losses) using the Humane Genome CGH Microarray Kit 44B (Agilent Techologies). This array consists on 44.000 oligo probes of 60 bp covering all the human genome with a mean resolution of 50–100 Kb. CGH-Analytics 3.2.25 and InSilicoArray CGH (http://isacgh.bioinfo.cipf.es) was used for array analysis and to define SORIs (Smallest Overlapping Region of Imbalance). Results: Genomic abnormalities were observed in thirty-two cases (76%). Losses (62.36%) were more frequently detected than gains (37.64%). The mean chromosomal imbalances per case were 3.5 gains (0–14) and 5.6 losses (0–30). The minimal common regions altered were gains of 7q33.3 (55%), 17q21.1q21.3 (42.5%) and 8q24.21. Regarding to the losses, 9p21.3 (42,5%), 17p13.1 (42,5%) and 10p11.22 (17,5%) were the most frequent detected. SORIs and potential candidates genes from the most frequently altered regions are summarized in the following table. Type of change Cytoband First gene start (Kbp) size (Mb) Frequency Candidate genes Loss 9p21.3 MTAP 21795 0,2 42,50% MTAP, CDKN2A, CDKN2B Loss 17p13.1 DULLARD 7094 1,01 27,5% TP53 Loss 10p11.22 chr10:031132968 31132 1,5 17,5% TCF8 Gain 7q33.3 BG495318 135521 14,1 55% BRAF Gain 17q21.1q21.3 SMARCE1 36050 4,7 42,5% STAT5A/STATB Gain 8q24.21 M13930 128816 0,75 32,5% C-MYC Conclusions : Our results have shown high recurrent chromosomal abnormalities. Moreover, arrayCGH technology has allowed us to define in detail new common regions and to describe potential candidate genes in MF tumor stage as STAT5A/STA5B (17q21.2), BRAF (7q33) and TCF8 (10p11.22). Our findings are similar to those recently published by Vermeer et al in Sézary Syndrome, which lead to confirm the relationship between these two entities. Deletion of 9p21.3 (CDKN2A, CDKN2B, MTAP genes) and 17p13.1 (TP53) are concordance to previous studies.

Description: CD4+CD56+ hematodermic neoplasm (CD4+CD56+HN) is an aggressive hematopoietic malignancy with distinct clinicopathologic and immunophenotypic features that commonly involve the skin, bone marrow, and blood. Differentiation from cutaneous myelomonocytic leukemia (c-AML) may be exceedingly difficult and requires extensive phenotyping. The molecular mechanisms involved in the development of CD4+CD56+HN are largely unresolved. Moreover, recurrent chromosomal alterations have not yet been precisely defined in CD4+CD56+HN and c-AML. In the present study an integrated genomic analysis using expression profiling and array-based comparative genomic hybridization (CGH) was performed on lesional skin biopsy samples of patients with CD4+CD56+HN and c-AML. Our results demonstrate that CD4+CD56+HN and c-AML show distinct gene-expression profiles and distinct patterns of chromosomal aberrations. CD4+CD56+HN is characterized by recurrent deletion of regions on chromosome 4 (4q34), chromosome 9 (9p13-p11 and 9q12-q34), and chromosome 13 (13q12-q31) that contain several tumor suppressor genes with diminished expression (Rb1, LATS2). Elevated expression of the oncogenes HES6, RUNX2, and FLT3 was found but was not associated with genomic amplification. We noted high expression of various plasmacytoid dendritic-cell (pDC)–related genes, pointing to the cell of origin of this malignancy.