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SummaryExtramedullary disease in patients with multiple myeloma is a rare event, occurring mostly in advanced disease or relapse. Outcome is poor and prognostic factors predicting the development of extramedullary disease have not been defined. We investigated cytogenetic alterations of myeloma cells in different extramedullary manifestations by adapting the fluorescence in situ hybridization (FISH) technique in combination with cytoplasmic immunoglobulin staining to study the cytogenetics of plasma cell tumours on paraffin embedded material. Thirty six patients were investigated: 19 with extramedullary disease, 11 with skeletal extramedullary disease and six with solitary extramedullary plasmacytoma. The first two groups showed the following results: del(17p13) 32% vs. 27%, del(13q14) 35% vs. 27%, MYC-overrepresentation 28% vs. 18% and t(4;14) 37% vs. 18%. We detected an overall higher incidence of del(17p13) in both groups compared to data from bone marrow samples of multiple myeloma reported to date (range 7-16%). The solitary extramedullary plasmacytomas presented overall less cytogenetic aberrations than the other groups. Most important, three patients with extramedullary disease and one with skeletal extramedullary disease presented different FISH findings in the extramedullary tumour compared to their bone marrow plasma cells. del(17p13), occurring additional in three of four cases, seems a strong marker for extramedullary progression of myeloma.

The recently discovered MLT/MALT1 gene is fused with the API2 gene in the t(11;18)(q21;q21), which characterizes about one-third of MALT lymphomas. In order to screen for variant translocations and amplifications of MLT/MALT1, we have developed a novel, undirected two-color interphase fluorescence in situ hybridization (FISH) assay with two PAC clones flanking MLT/MALT1. This assay was applied to 108 marginal zone B-cell lymphomas (MZBCLs), including 72 extranodal MALT lymphomas, 17 nodal, and 19 splenic MZBCL. In 19 MALT lymphomas (26%), but in none of the nodal or splenic MZBCL, separated hybridization signals of the MLT/MALT1 flanking probes, were found. Further FISH analyses showed that 12 of these 19 cases displayed the classical t(11;18) and the remaining seven cases revealed the novel t(14;18)(q32;q21), involving the MLT/MALT1 and IGH genes. The frequency at which these translocations occurred varied significantly with the primary location of disease. The t(11;18) was mainly detected in gastrointestinal MALT lymphomas, whereas the t(14;18) occurred in MALT lymphomas of the parotid gland and the conjunctiva. Amplification of MLT/MALT1 was not observed in any of the lymphomas analyzed. We conclude that the translocations t(11;18)(q21;q21) and t(14;18)(q21;q32) represent the main structural aberrations involving MLT/MALT1 in MALT lymphomas, whereas true amplifications of MLT/MALT1 occur rarely in MZBCL.

We analyzed the prognostic impact of the most frequent genetic abnormalities detected by fluorescence in situ hybridization in 101 patients with multiple myeloma, who underwent allogeneic hematopoietic stem cell transplantation (HSCT) after melphalan/fludarabine-based reduced conditioning. The incidences of abnormalities in the present analysis were as follows: del(13q14) (61%), t(11;14)(q13;q32) (14%), t(4;14)(p16.3;q32) (19%), MYC-gain gains (8q24) (21%), del(17p13) (16%) and t(14;16)(q32;q23) (5%). None of the patients had t(6;14)(p25;q32). The overall complete remission (CR) rate was 50% with no differences between the genetic abnormalities except for patients with del(17p13) who achieved less CR (7 vs 56%; P ¼ 0.001). Univariate analysis revealed a higher relapse rate in patients aged 450 years (P ¼ 0.002), patients with del(13q14) (P ¼ 0.006) and patients with del(17p13) (P ¼ 0.003). In multivariate analyses, only del(13q14) (HR: 2.34, P ¼ 0.03) and del(17p13) (HR: 2.24; P ¼ 0.04) significantly influenced the incidence of relapse, whereas for event-free survival, only age (HR 2.8; P ¼ 0.01) and del(17p13) (HR: 2.05; P ¼ 0.03) retained their negative prognostic value. These data show that del(17p13) is a negative prognostic factor for achieving CR as well as for event-free survival after HSCT. Translocation t(4;14) might be overcome by allogeneic HSCT, which will have implication for risk-adapted strategies.

Abstract The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein “preBLL”). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.

T-cell prolymphocytic leukemia (T-PLL) is an aggressive post-thymic T-cell malignancy characterized by the recurrent inv(14)(q11q32)/t(14;14)(q11;q32) or t(X;14)(q28;q11) leading to activation of either the TCL1 or MTCP1 gene, respectively. However, these primary genetic events are insufficient to drive leukemogenesis. Recently, activating mutations in JAK3 have been identified in other T-cell malignancies. Since JAK3 is essential for T-cell maturation, we analyzed a cohort of 32 T-PLL patients for mutational hot spots in the JAK3 gene using a step-wise screening approach. We identified 14 mutations in 11 of 32 patients (34%). The most frequently detected mutation in our cohort was M511I (seen in 57% of cases) previously described as an activating change in other T-cell malignancies. Three patients carried two mutations in JAK3. In two patients M511I and R657Q were simultaneously detected and in another patient V674F and V678L. In the latter case we could demonstrate that the mutations were on the same allele in cis. Protein modeling and homology analyses of mutations present in other members of the JAK family suggested that these mutations likely activate JAK3, possibly by disrupting the activation loop and the interface between N and C lobes, increasing the accessibility of the catalytic loop. In addition, four of the 21 patients lacking a JAK3 point mutation presented an aberrant karyotype involving the chromosomal band 19p13 harboring the JAK3 locus. The finding of recurrent activating JAK3 mutations in patients with T-PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis.

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.

BackgroundThe aim of this study was to determine the impact of a gain of the MALT1 gene on gene expression and clinical parameters in diffuse large B-cell lymphoma.