Genetic and epigenetic insights into cutaneous T-cell lymphoma.

Primary cutaneous T-cell lymphomas (CTCLs) constitute a heterogeneous group of non-Hodgkin T-cell lymphomas that present in the skin. In recent years, significant progress has been made in the understanding of the pathogenesis of CTCLs. Progress in CTCL classifications combined with technical advances, in particular next-generation sequencing, enabled a more detailed analysis of the genetic and epigenetic landscape and transcriptional changes in clearly defined diagnostic entities. These studies not only demonstrated extensive heterogeneity between different CTCL subtypes but also identified recurrent alterations that are highly characteristic for diagnostic subgroups of CTCLs. The identified alterations, in particular, involve epigenetic remodeling, cell cycle regulation, and the constitutive activation of targetable oncogenic pathways. In this respect, aberrant JAK-STAT signaling is a recurrent theme; however, it is not universal for all CTCLs and has seemingly different underlaying causes in different entities. A number of the mutated genes identified are potentially actionable targets for the development of novel therapeutic strategies. Moreover, these studies have produced an enormous amount of information that will be critically important for the further development of improved diagnostic and prognostic biomarkers that can assist in the clinical management of patients with CTCL. In the present review, the main findings of these studies in relation to their functional impact on the malignant transformation process are discussed for different subtypes of CTCLs.

Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring.

Deregulation of JAK2 signaling underlies primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma.

Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare variant of cutaneous T-cell lymphoma with an aggressive clinical course and a very poor prognosis. Until now, neither a systematic characterization of genetic alterations driving pcAECyTCL has been performed, nor effective therapeutic regimes for patients have been defined. Here, we present the first highresolution genetic characterization of pcAECyTCL by using wholegenome and RNA sequencing. Our study provides a comprehensive description of genetic alterations (i.e., genomic rearrangements, copy number alterations and small-scale mutations) with pathogenic relevance in this lymphoma, including events that recurrently impact genes with important roles in the cell cycle, chromatin regulation and the JAKSTAT pathway. In particular, we show that mutually exclusive structural alterations involving JAK2 and SH2B3 predominantly underlie pcAECyTCL. In line with the genomic data, transcriptome analysis uncovered upregulation of the cell cycle, JAK2 signaling, NF-κB signaling and a high inflammatory response in this cancer. Functional studies confirmed oncogenicity of JAK2 fusions identified in pcAECyTCL and their sensitivity to JAK inhibitor treatment. Our findings strongly suggest that overactive JAK2 signaling is a central driver of pcAECyTCL, and consequently, patients with this neoplasm would likely benefit from therapy with JAK2 inhibitors such as Food and Drug Adminstration-approved ruxolitinib.

Whole-genome profiling of primary cutaneous anaplastic large cell lymphoma.

Primary cutaneous anaplastic large cell lymphoma (pcALCL), a hematological neoplasm caused by skin-homing CD30+ malignant T cells, is part of the spectrum of primary cutaneous CD30+ lymphoproliferative disorders. To date, only a small number of molecular alterations have been described in pcALCL and, so far, no clear unifying theme that could explain the pathogenetic origin of the disease has emerged among patients. In order to clarify the pathogenetic basis of pcALCL, we performed high-resolution genetic profiling (genome/transcriptome) of this lymphoma (n=12) by using whole-genome sequencing, whole-exome sequencing and RNA sequencing. Our study, which uncovered novel genomic rearrangements, copy number alterations and small-scale mutations underlying this malignancy, revealed that the cell cycle, T-cell physiology regulation, transcription and signaling via the PI-3-K, MAPK and G-protein pathways are cellular processes commonly impacted by molecular alterations in patients with pcALCL. Recurrent events affecting cancer-associated genes included deletion of PRDM1 and TNFRSF14, gain of EZH2 and TNFRSF8, small-scale mutations in LRP1B, PDPK1 and PIK3R1 and rearrangements involving GPS2, LINC-PINT and TNK1. Consistent with the genomic data, transcriptome analysis uncovered upregulation of signal transduction routes associated with the PI-3-K, MAPK and G-protein pathways (e.g., ERK, phospholipase C, AKT). Our molecular findings suggest that inhibition of proliferation-promoting pathways altered in pcALCL (particularly PI-3-K/AKT signaling) should be explored as potential alternative therapy for patients with this lymphoma, especially, for cases that do not respond to first-line skin-directed therapies or with extracutaneous disease.

Whole-genome analysis uncovers recurrent IKZF1 inactivation and aberrant cell adhesion in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematological malignancy with a poorly understood pathobiology and no effective therapeutic options. Despite a few recurrent genetic defects (eg, single nucleotide changes, indels, large chromosomal aberrations) have been identified in BPDCN, none are disease-specific, and more importantly, none explain its genesis or clinical behavior. In this study, we performed the first high resolution whole-genome analysis of BPDCN with a special focus on structural genomic alterations by using whole-genome sequencing and RNA sequencing. Our study, the first to characterize the landscape of genomic rearrangements and copy number alterations of BPDCN at nucleotide-level resolution, revealed that IKZF1, a gene encoding a transcription factor required for the differentiation of plasmacytoid dendritic cell precursors, is focally inactivated through recurrent structural alterations in this neoplasm. In concordance with the genomic data, transcriptome analysis revealed that conserved IKZF1 target genes display a loss-of-IKZF1 expression pattern. Furthermore, up-regulation of cellular processes responsible for cell-cell and cell-ECM interactions, which is a hallmark of IKZF1 deficiency, was prominent in BPDCN. Our findings suggest that IKZF1 inactivation plays a central role in the pathobiology of the disease, and consequently, therapeutic approaches directed at reestablishing the function of this gene might be beneficial for patients.

Array-based CGH of primary cutaneous CD8+ aggressive EPIDERMO-tropic cytotoxic T-cell lymphoma.

Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare provisionally categorized cutaneous lymphoma characterized by an aggressive course. Its pathogenesis and molecular mechanisms are still unknown, and only two individual cases have so far been molecularly characterized. The aim of this study was to define the pattern of numerical chromosomal alterations in tumor samples taken from 20 patients with pcAECyTCL at the time of diagnosis by means of array-comparative genomic hybridization (a-CGH). a-CGH detected numerous genomic aberrations in all the patients and, putting these together as a whole, they affected all the chromosomes. However, no specific profile of recurrent copy number alterations (CNAs) was found. Most of the gains involved regions previously described in other aggressive cutaneous lymphomas such as 7q, 8q24.3, and 17q, whereas the most significant CNA was the loss of 9p21.3 (CDKN2A-CDKN2B), which has already been found in a variety of malignant tumors and is associated with aggressive cutaneous T-cell lymphomas. In brief, CGH analysis revealed a large number of CNAs with only few recurring regions that probably do not represent driving events. The genomic instability found in this aggressive variant of cutaneous lymphoma may therefore be a secondary event but, at the time of the diagnosis of pcAECyTCL, the genomic integrity of tumor cells is already compromised.

Genomic analysis reveals recurrent deletion of JAK-STAT signaling inhibitors HNRNPK and SOCS1 in mycosis fungoides.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). Causative genetic alterations in MF are unknown. The low recurrence of pathogenic small-scale mutations (ie, nucleotide substitutions, indels) in the disease, calls for the study of additional aspects of MF genetics. Here, we investigated structural genomic alterations in tumor-stage MF by integrating whole-genome sequencing and RNA-sequencing. Multiple genes with roles in cell physiology (n = 113) and metabolism (n = 92) were found to be impacted by genomic rearrangements, including 47 genes currently implicated in cancer. Fusion transcripts involving genes of interest such as DOT1L, KDM6A, LIFR, TP53, and TP63 were also observed. Additionally, we identified recurrent deletions of genes involved in cell cycle control, chromatin regulation, the JAK-STAT pathway, and the PI-3-K pathway. Remarkably, many of these deletions result from genomic rearrangements. Deletion of tumor suppressors HNRNPK and SOCS1 were the most frequent genetic alterations in MF after deletion of CDKN2A. Notably, SOCS1 deletion could be detected in early-stage MF. In agreement with the observed genomic alterations, transcriptome analysis revealed up-regulation of the cell cycle, JAK-STAT, PI-3-K and developmental pathways. Our results position inactivation of HNRNPK and SOCS1 as potential driver events in MF development.

Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN.

RNA-seq analysis of Lgr6+ stem cells and identification of an Lgr6 isoform.

We studied Lgr6+ stem cells in experimental UV carcinogenesis in hairless mice. For further characterization through RNA-seq, these stem cells were isolated by FACS from transgenic hairless mice bearing an EGFP-Ires-CreERT2 reporter cassette inserted into exon 1 of the Lgr6 gene (purity confirmed by human ERT2 expression). Between Lgr6/EGFP+ and Lgr6/EGFP- basal cells (Tg/wt), 682 RNAs were differentially expressed, indicating stemness and expression of cancer-related pathways in Lgr6/EGFP+ cells. We discovered that suspected "Lgr6 null" mice (Tg/Tg) expressed RNA of an Lgr6 isoform (delta-Lgr6, lacking 74 N-terminal aa) which could be functional and explain the lack of a phenotype.

Molecular advances in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a group of malignancies derived from skin-homing T cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common CTCL variants. In recent years, the genetic landscape of SS/MF has been characterized using genome-wide nextgeneration sequencing approaches. These studies have revealed that genes subjected to oncogenic mutations take part in cell cycle regulation, chromatin modification, Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling, T-cell receptor (TCR)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and microtubule associated protein kinase (MAPK) signaling, which suggests that deregulation of these cellular processes underlies lymphomagenesis. These studies provide the groundwork for functional and clinical studies that will lead to better risk assessment and more effective therapeutic approach in CTCL patients.

"Next top" mouse models advancing CTCL research.

This review systematically describes the application of in vivo mouse models in studying cutaneous T-cell lymphoma (CTCL), a complex hematological neoplasm. It highlights the diverse research approaches essential for understanding CTCL's intricate pathogenesis and evaluating potential treatments. The review categorizes various mouse models, including xenograft, syngeneic transplantation, and genetically engineered mouse models (GEMMs), emphasizing their contributions to understanding tumor-host interactions, gene functions, and studies on drug efficacy in CTCL. It acknowledges the limitations of these models, particularly in fully replicating human immune responses and early stages of CTCL. The review also highlights novel developments focusing on the potential of skin-targeted GEMMs in studying natural skin lymphoma progression and interactions with the immune system from onset. In conclusion, a balanced understanding of these models' strengths and weaknesses are essential for accelerating the deciphering of CTCL pathogenesis and developing treatment methods. The GEMMs engineered to target specifically skin-homing CD4+ T cells can be the next top mouse models that pave the way for exploring the effects of CTCL-related genes.

Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients.

BACKGROUND: The risk of developing cutaneous squamous cell carcinoma (SCC) is markedly increased in organ transplant recipients (OTRs) compared to the normal population. Next to sun exposure, the immunosuppressive regimen is an important risk factor for the development of SCC in OTRs. Various gene mutations (e.g. TP53) and genetic alterations (e.g. loss of CDKN2A, amplification of RAS) have been found in SCCs. The aim of this genome-wide study was to identify pathways and genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs). METHODS: To perform the analysis in an isogenic background, RNA and DNA were isolated from SCC, AK and normal (unexposed) epidermis (NS) from each of 13 OTRs. Samples were subjected to genome-wide expression analysis and genome SNP analysis using Illumina's HumanWG-6 BeadChips and Infinium II HumanHap550 Genotyping BeadChips, respectively. mRNA expression results were verified by quantitative PCR. RESULTS: Hierarchical cluster analysis of mRNA expression profiles showed SCC, AK and NS samples to separate into three distinct groups. Several thousand genes were differentially expressed between epidermis, AK and SCC; most upregulated in SCCs were hyperproliferation related genes and stress markers, such as keratin 6 (KRT6), KRT16 and KRT17. Matching to oncogenic pathways revealed activation of downstream targets of RAS and cMYC in SCCs and of NFκB and TNF already in AKs. In contrast to what has been reported previously, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles. CONCLUSION: Vast differences in gene expression profiles exist between SCC, AK and NS from immunosuppressed OTRs. Moreover, several pathways activated in SCCs were already activated in AKs, confirming the assumption that AKs are the precursor lesions of SCCs. Since the drastic changes in gene expression appeared unlinked to specific genomic gains or losses, the causal events driving SCC development require further investigation. Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs. Further study is required to identify the mechanisms of early activation of NFκB and TNF, and to establish whether these pathways offer a feasible target for preventive intervention among OTRs.

Socs1-knockout in skin-resident CD4+ T cells in a protracted contact-allergic reaction results in an autonomous skin inflammation with features of early-stage mycosis fungoides.

Recent detailed genomic analysis of mycosis fungoides (MF) identified suppressor of cytokine signaling 1 (SOCS1), an inhibitor of JAK/STAT signaling, as one of the frequently deleted tumor suppressors in MF, and one-copy deletion of SOCS1 was confirmed in early-stage MF lesions. To better understand the functional role of SOCS1 in the genesis of MF, we used a genetically engineered mouse model emulating heterozygous SOCS1 loss in skin resident CD4+ T cells. In these mice an experimentally induced contact-allergic reaction was maintained for 20 weeks. Ten weeks after discontinuing contact-allergic challenges, only the skin with locally one-copy deletion of Socs1 in CD4+ T cells still showed high numbers of CD3+/CD4+ Socs1 k.o. cells in the dermis (p ＜ 0.0001) with prevalent Stat3 activation (p ＜0.001). And in one out of 9 mice, this had progressed to far more dramatic increases, including the thickened epidermis, and with an explosive growth of Socs1 k.o. T cells in circulation; indicative of cutaneous lymphoma. Hence, we show that Socs1 mono-allelic loss in CD4+ T cells locally in protractedly inflamed skin results in autonomous skin inflammation with features of early-stage MF.

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides.

The miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C-ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor-stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C-ALCL and BID. The up-regulation of miR-155, miR-27b, miR-30c and miR-29b in C-ALCL was validated by miRNA-Q-PCR on independent study groups. Additionally, the miRNA expression profiles of C-ALCL were compared with those of tumor-stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA-Q-PCR demonstrated statistically significantly differential expression of miR-155, miR-27b, miR-93, miR-29b and miR-92a between tumor-stage MF and C-ALCL. This study, the first describing the miRNA expression profile of C-ALCL, reveals differences with tumor-stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.

A novel mouse model for Sézary syndrome using xenotransplantation of Sézary cells into immunodeficient RAG2(-/-) γc(-/-) mice.

Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2(-/-) γc(-/-) mice that are completely devoid of T-, B- and NK-cell activity. Injection of the SS cell line SeAx led to long-term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT-78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2(-/-) γc(-/-) mice results in long-term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.

In vivo modelling of cutaneous T-cell lymphoma: The role of SOCS1.

Introduction: Mycosis fungoides (MF), the most common type of Cutaneous T cell Lymphoma (CTCL), is characterized by an inflamed skin intermixed with proliferating malignant mature skin-homing CD4+ T cells. Detailed genomic analyses of MF skin biopsies revealed several candidate genes possibly involved in genesis of these tumors and/or potential targets for therapy. These studies showed, in addition to common loss of cell cycle regulator CDKN2A, activation of several oncogenic pathways, most prominently and consistently involving JAK/STAT signaling. SOCS1, an endogenous inhibitor of the JAK/STAT signaling pathway, was identified as a recurrently deleted gene in MF, already occurring in the earliest stages of the disease. Methods: To explore the mechanisms of MF, we create in vivo mouse models of autochthonous CTCLs and these genetically engineered mouse models (GEMMS) can also serve as valid experimental models for targeted therapy. We describe the impact of allelic deletion of Socs1 in CD4 T cells of the skin. To achieve this, we crossed inducible Cre-transgenic mice in the CD4 lineage with transgenic mice carrying floxed genes of Socs1. We first determined optimal conditions for Socs1 ablation with limited effects on circulating CD4 T-cells in blood. Next, we started time-course experiments mimicking sustained inflammation, typical in CTCL. FACS analysis of the blood was done every week. Skin biopsies were analyzed by immunocytochemical staining at the end of the experiment. Results: We found that the Socs1 knockout transgenic group had thicker epidermis of treated skin compared with the control group and had more CD3 and CD4 in the skin of the transgenic group compared to the control group. We also noted more activation of Stat3 by staining for P-Stat3 in Socs1 knockout compared to wt CD4+T cells in the skin. The results also indicated that single copy loss of Socs1 in combination with sustained inflammation is insufficient to start a phenotype resembling early stage mycosis fungoides within eight weeks in these mice. Conclusion: In sum, we developed and optimized an autochthonous murine model permitting selective knockout of Socs1 in skin infiltrating CD4 T-cells. This paves the way for more elaborate experiments to gain insight in the oncogenesis of CTCL.

Cell-of-origin classification using the Hans and Lymph2Cx algorithms in primary cutaneous large B-cell lymphomas.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) and primary cutaneous follicle center lymphoma with a diffuse population of large cells (PCFCL-LC) are both primary cutaneous B-cell lymphomas with large-cell morphology (CLBCL) but with different clinical characteristics and behavior. In systemic diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), gene-expression profiling (GEP) revealed two molecular subgroups based on their cell-of-origin (COO) with prognostic significance: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. This study investigated whether COO classification is a useful tool for classification of CLBCL. For this retrospective study, 51 patients with PCDLBCL-LT and 15 patients with PCFCL-LC were analyzed for their COO according to the immunohistochemistry-based Hans algorithm and the NanoString GEP-based Lymph2Cx algorithm. In PCFCL-LC, all cases (100%) classified as GCB by both Hans and Lymph2Cx. In contrast, COO classification in PCDLBCL-LT was heterogeneous. Using Hans, 75% of the PCDLBCL-LT patients classified as non-GCB and 25% as GCB, while Lymph2Cx classified only 18% as ABC, 43% as unclassified/intermediate, and 39% as GCB. These COO subgroups did not differ in the expression of BCL2 and IgM, mutations in MYD88 and/or CD79B, loss of CDKN2A, or survival. In conclusion, PCFCL-LC uniformly classified as GCB, while PCDLBCL-LT classified along the COO spectrum of DLBCL-NOS using the Hans and Lymph2Cx algorithms. In contrast to DLBCL-NOS, the clinical relevance of COO classification in CLBCL using these algorithms has limitations and cannot be used as an alternative for the current multiparameter approach in differentiation of PCDLBCL-LT and PCFCL-LC.

Genetic Stability of Driver Alterations in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type and Their Relapses: A Rationale for the Use of Molecular-Based Methods for More Effective Disease Monitoring.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is a rare, aggressive cutaneous lymphoma with a 5-year disease-specific survival of only ~55%. Despite high response rates to initial immune-polychemotherapy, most patients experience a disease relapse. The genetic evolution of primary and relapsed/refractory disease has only scarcely been studied in PCDLBCL-LT patients. Therefore, in this retrospective cohort study, 73 primary/pre-treatment and relapsed/refractory biopsies of 57 patients with PCDLBCL-LT were molecularly characterized with triple FISH and targeted next-generation sequencing for 52 B-cell-lymphoma-relevant genes, including paired analysis in 16 patients. In this cohort, 95% of patients harboured at least one of the three main driver alterations (mutations in MYD88/CD79B and/or CDKN2A-loss). In relapsed/refractory PCDLBCL-LT, these oncogenic aberrations were persistently present, demonstrating genetic stability over time. Novel alterations in relapsed disease affected mostly CDKN2A, MYC, and PIM1. Regarding survival, only MYC rearrangements and HIST1H1E mutations were statistically significantly associated with an inferior outcome. The stable presence of one or more of the three main driver alterations (mutated MYD88/CD79B and/or CDKN2A-loss) is promising for targeted therapies addressing these alterations and serves as a rationale for molecular-based disease monitoring, improving response evaluation and early identification and intervention of disease relapses in these poor-prognostic PCDLBCL-LT patients.

Oncogenomic analysis of mycosis fungoides reveals major differences with Sezary syndrome.

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.