T-cell prolymphocytic leukemia (T-PLL) is an aggressive malignancy with a median survival of the patients of less than two years. Besides characteristic chromosomal translocations, frequent mutations affect the ATM gene, JAK/STAT pathway members, and epigenetic regulators. We here performed a targeted mutation analysis for 40 genes selected from a RNA sequencing of 10 T-PLL in a collection of 28 T-PLL, and an exome analysis of five further cases. Nonsynonymous mutations were identified in 30 of the 40 genes, 18 being recurrently mutated. We identified recurrently mutated genes previously unknown to be mutated in T-PLL, which are SAMHD1, HERC1, HERC2, PRDM2, PARP10, PTPRC, and FOXP1. SAMHD1 regulates cellular deoxynucleotide levels and acts as a potential tumor suppressor in other leukemias. We observed destructive mutations in 18% of cases as well as deletions in two further cases. Taken together, we identified additional genes involved in JAK/STAT signaling (PTPRC), epigenetic regulation (PRDM2), or DNA damage repair (SAMHD1, PARP10, HERC1, and HERC2) as being recurrently mutated in T-PLL. Thus, our study considerably extends the picture of pathways involved in molecular pathogenesis of T-PLL and identifies the tumor suppressor gene SAMHD1 with ~20% of T-PLL affected by destructive lesions likely as major player in T-PLL pathogenesis.

The pathogenesis of ocular adnexal marginal zone lymphomas of mucosa-associated lymphatic tissue-type (OAML) is still poorly understood. We analyzed 63 cases of such lymphomas for non-synonymous mutations in 24 candidate genes by amplicon sequencing. We validated frequent mutations in the NF-κB regulators MYD88, TNFAIP3 and TNIP1 in OAML, but also identified recurrent mutations in several additional components of the NF-κB pathway, including BCL10 and NFKBIA. Overall, 60% of cases had mutations in at least one component of NF-κB signaling, pointing to a central role of its genetic deregulation in OAML pathogenesis. Mutations in NOTCH1 and NOTCH2 were each found in 8% of cases, indicating a pathogenetic function of these factors in OAML. KMT2D was identified as the first epigenetic regulator with mutations in OAML, being mutated in 22% of cases. Mutations in MYD88 were associated with an inferior disease-free survival. Overall, we identified here highly recurrent genetic lesions in components of the NF-κB pathway, of NOTCH1 and NOTCH2 as well as KMT2D in OAML and thereby provide major novel insights into the pathogenesis of this B cell malignancy.

T-cell prolymphocytic leukaemia (T-PLL) is an aggressive leukaemia. The primary genetic alteration in T-PLL are the inv(14)(q11q32)/t(14;14)(q11;q32) leading to TRD/TRA-TCL1A fusion, or the t(X;14)(q28;q11) associated with TRD/TRA-MTCP1 fusion. However, additional cooperating abnormalities are necessary for emergence of the full neoplastic phenotype. Though the pattern of secondary chromosomal aberrations is remarkably conserved, targets of the changes are largely unknown. We analysed a cohort of 43 well-characterized T-PLL for hotspot mutations in the genes JAK3, STAT5B and RHOA. Additionally, we selected a subset of 23 T-PLL cases for mutational screening of 54 genes known to be recurrently mutated in T-cell and other haematological neoplasms. Activating mutations in the investigated regions of the JAK3 and STAT5B genes were detected in 30% (13/43) and 21% (8/39) of the cases, respectively, and were mutually exclusive. Further, we identified mutations in the genes encoding the epigenetic regulators EZH2 in 13% (3/23), TET2 in 17% (4/23) and BCOR in 9% (2/23) of the cases. We confirmed that the JAK-STAT pathway is a major mutational target, and identified epigenetic regulators recurrently mutated in T-PLL. These findings complement the mutational spectrum of secondary aberrations in T-PLL and underscore the potential therapeutical relevance of epigenetic regulators in T-PLL.

T-cell prolymphocytic leukemia (T-PLL) is a rare and poor-prognostic mature T-cell leukemia. Recent studies detected genomic aberrations affecting JAK and STAT genes in T-PLL. Due to the limited number of primary patient samples available, genomic analyses of the JAK/STAT pathway have been performed in rather small cohorts. Therefore, we conducted-via a primary-data based pipeline-a meta-analysis that re-evaluated the genomic landscape of T-PLL. It included all available data sets with sequence information on JAK or STAT gene loci in 275 T-PLL. We eliminated overlapping cases and determined a cumulative rate of 62.1% of cases with mutated JAK or STAT genes. Most frequently, JAK1 (6.3%), JAK3 (36.4%), and STAT5B (18.8%) carried somatic single-nucleotide variants (SNVs), with missense mutations in the SH2 or pseudokinase domains as most prevalent. Importantly, these lesions were predominantly subclonal. We did not detect any strong association between mutations of a JAK or STAT gene with clinical characteristics. Irrespective of the presence of gain-of-function (GOF) SNVs, basal phosphorylation of STAT5B was elevated in all analyzed T-PLL. Fittingly, a significant proportion of genes encoding for potential negative regulators of STAT5B showed genomic losses (in 71.4% of T-PLL in total, in 68.4% of T-PLL without any JAK or STAT mutations). They included DUSP4, CD45, TCPTP, SHP1, SOCS1, SOCS3, and HDAC9.Overall, considering such losses of negative regulators and the GOF mutations in JAK and STAT genes, a total of 89.8% of T-PLL revealed a genomic aberration potentially explaining enhanced STAT5B activity. In essence, we present a comprehensive meta-analysis on the highly prevalent genomic lesions that affect genes encoding JAK/STAT signaling components. This provides an overview of possible modes of activation of this pathway in a large cohort of T-PLL. In light of new advances in JAK/STAT inhibitor development, we also outline translational contexts for harnessing active JAK/STAT signaling, which has emerged as a 'secondary' hallmark of T-PLL.

Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin (GRN), is significantly higher expressed in aggressive CD38+ZAP-70+ as compared to indolent CD38−ZAP-70− chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics (11q-, 17p-) as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn (HR = 2.06, 95%-CI = 1.13–3.76, p = 0.018), unmutated IGHV status (HR = 5.63, 95%-CI = 3.05–10.38, p<0.001), high risk as defined by the study protocol (HR = 2.06, 95%-CI = 1.09–3.89, p = 0.026) but not poor risk cytogenetics were independent prognostic markers. In summary our results suggest that Pgrn is a novel, robust and independent prognostic marker in CLL that can be easily measured by ELISA.

The pathogenesis of T-cell large granular lymphocytic leukemia (T-LGL) is poorly understood, as STAT3 mutations are the only known frequent genetic lesions. Here, we identified non-synonymous alterations in the TNFAIP3 tumor suppressor gene in 3 of 39 T-LGL. In two cases these were somatic mutations, in one case the somatic origin was likely. A further case harbored a SNP that is a known risk allele for autoimmune diseases and B cell lymphomas. Thus, TNFAIP3 mutations represent recurrent genetic lesions in T-LGL that affect about 8% of cases, likely contributing to deregulated NF-jB activity in this leukemia.

Patients chronically infected with hepatitis C virus (HCV) frequently develop mixed cryoglobulinemia (MC), a monoclonal expansion of immunoglobulin M (IgM) autoreactive B cells, and also have an increased B-cell lymphoma risk. Whether HCV infection also impacts the B-cell compartment and the B-cell receptor repertoire in patients not affected by MC or lymphomas is poorly understood. Flow cytometric analysis of peripheral blood B cells of 30 MC-negative HCV-infected patients and 15 healthy controls revealed that frequencies of class-switched memory B cells were increased in the patients, whereas frequencies of transitional and naive B cells were decreased. For 22 HCV patients and 7 healthy controls, we performed high-throughput sequencing of immunoglobulin heavy chain VDJ rearrangements of naive, mature CD5, IgM memory, and class-switched memory B cells. An increased usage of several IGHV genes, including IGHV1-69 and IGHV4-59, which are closely linked to MC and HCV-associated lymphomas, was specifically seen among IgM memory B cells of the patients. Moreover, many, and partly very large, expanded clones were seen predominantly among IgM memory B cells of all HCV-infected patients analyzed. Thus, chronic HCV infection massively disturbs the B-cell compartment even in patients without clinically detectable B-cell lymphoproliferation and generates many large B-cell clones, especially among non-class-switched memory B cells. Because B-cell clones in MC and lymphomas derive from this B-cell subset, this establishes IgM memory B cells as a general target of lymphoproliferation in HCV patients, affecting apparently all patients.

The pathogenesis of ocular adnexal marginal zone lymphomas of mucosa-associated lymphatic tissue-type (OAML) is not fully understood. We performed whole genome sequencing (WGS) and/or whole exome sequencing (WES) for 13 cases of OAML and sequenced 38 genes selected from this analysis in a large cohort of 82 OAML. Besides confirmation of frequent mutations in the genes transducin beta like 1 X-linked receptor 1 (TBL1XR1) and cAMP response element binding protein (CREBBP), we newly identifed JAK3 as a frequently mutated gene in OAML (11% of cases). In our retrospective cohort, JAK3 mutant cases had a shorter progression-free survival compared with unmutated cases. Other newly identified genes recurrently mutated in 5–10% of cases included members of the collagen family (collagen type XII alpha 1/2 (COL12A1, COL1A2)) and DOCK8. Evaluation of the WGS data of six OAML did not reveal translocations or a current infection of the lymphoma cells by viruses. Evaluation of the WGS data for copy number aberrations confirmed frequent loss of TNFAIP3, and revealed recurrent gains of the NOTCH target HES4, and of members of the CEBP transcription factor family. Overall, we identified several novel genes recurrently affected by point mutations or copy number alterations, but our study also indicated that the landscape of frequently (>10% of cases) mutated protein-coding genes in OAML is now largely known.