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Chronic lymphocytic leukemia (CLL) is a common disease with highly variable clinical course. Several recurrent chromosomal alterations are associated with prognosis and may guide risk-adapted therapy. We have developed a targeted genome-wide array to provide a robust tool for ascertaining abnormalities in CLL and to overcome limitations of the 4-marker fluorescence in situ hybridization (FISH). DNA from 180 CLL patients were hybridized to the qChip®Hemo array with a high density of probes covering commonly altered loci in CLL (11q22-q23, 13q14, and 17p13), nine focal regions (2p15-p16.1, 2p24.3, 2q13, 2q36.3-q37.1, 3p21.31, 8q24.21, 9p21.3, 10q24.32, and 18q21.32-q21.33) and two larger regions (6q14.1-q22.31 and 7q31.33-q33). Overall, 86% of the cases presented copy number alterations (CNA) by array. There was a high concordance of array findings with FISH (84% sensitivity, 100% specificity); all discrepancies corresponded to subclonal alterations detected only by FISH. A chromothripsis-like pattern was detected in eight cases. Three showed concomitant shattered 5p with gain of TERT along with isochromosome 17q. Presence of 11q loss was associated with shorter time to first treatment (P = 0.003), whereas 17p loss, increased genomic complexity, and chromothripsis were associated with shorter overall survival (P < 0.001, P = 0.001, and P = 0.02, respectively). In conclusion, we have validated a targeted array for the diagnosis of CLL that accurately detects, in a single experiment, all relevant CNAs, genomic complexity, chromothripsis, copy number neutral loss of heterozygosity, and CNAs not covered by the FISH panel. This test may be used as a practical tool to stratify CLL patients for routine diagnostics or clinical trials., (© 2015 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.)

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., (Copyright © 2014 Wiley Periodicals, Inc.)

Translocations involving immunoglobulin (IG) loci are the hallmarks of several subtypes of B-cell lymphoma. Common to these translocations is that cellular proto-oncogenes come under the influence of IG regulatory elements leading to deregulated expression. In case of a breakpoint in the IGH switch region, oncogene activation can take place on both derivative chromosomes, which means that in principle one translocation can result in concurrent activation of two genes. By fluorescence in situ hybridization (FISH), we identified a case of leukemic B-cell lymphoma in a child with an IGH break and unknown partner. Subsequent long-distance inverse PCR revealed fusion of IGH Sl in 14q32 and the 50 region of CBFA2T3 in 16q24.3, suggesting presence of the t(14;16)(q32;q24.3). Candidate oncogenes targeted through this translocation are CBFA2T3 and ACSF3, which could be activated on der(16) and der(14), respectively. FISH screening of a population-based cohort of B-cell lymphomas from a prospective trial for the treatment of lymphoma in childhood (BFM-NHL) identified additionally a follicular lymphoma Grade 3/diffuse large B-cell lymphoma with IGH-CBFA2T3/ACSF3 juxtaposition. Both lymphomas shared expression of CD10 and CD20 in the absence of TdT, suggesting a germinal center (GC) B-cell origin. Our data indicate that the CBFA2T3/ACSF3 locus is a novel recurrent oncogenic target of IGH translocations, which might contribute to the pathogenesis of pediatric GC-derived B-cell lymphoma., (© 2011 Wiley Periodicals, Inc.)

Gains or amplifications of the REL locus are frequently seen in primary mediastinal B-cell lymphoma (PMBL). In classical Hodgkin's lymphoma, genomic overrepresentation of REL correlated with nuclear REL protein accumulation. To investigate the correlation between REL gene copies and its RNA and protein expression in PMBL, we analyzed genomic, transcriptional, and protein levels in 20 PMBLs and the PMBL derived cell lines MedB-1 and Karpas1106P. We found gains/amplifications in 75% of the PMBLs by fluorescence in situ hybridization (FISH) and genomic REL overrepresentation in the PMBL lines. Three of the five PMBLs with amplifications displayed elevated REL transcripts, while only 3/10 PMBLs with gains showed increased REL transcripts by real-time PCR. One PMBL without gains displayed increased REL transcription. REL protein expression exhibited a variable pattern across the PMBLs except for a single case that was completely negative by immunohistochemistry despite having gained REL. Although transcript levels were generally low and nuclear REL staining was weak in the lymphoma cell lines, these nevertheless exhibited high NF-kappaB activation. By fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms, genomic gains/amplifications of REL significantly correlated with nuclear REL expression (P < 0.05). In conclusion, the frequent genomic overrepresentation of REL in PMBL does not necessarily trigger an increased transcription/translation of REL. However, combined genomic and protein analysis revealed a significant association of gained REL and nuclear REL accumulation at the single cell level., ((c) 2007 Wiley-Liss, Inc.)

Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling-1 (SOCS-1). We have previously shown that SOCS-1 is biallelically mutated in the primary mediastinal B-cell lymphoma (PMBL) cell line MedB-1, resulting in impaired JAK2 degradation and sustained phospho-JAK2 action. SOCS-1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS-1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB-1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB-1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS-1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL., (Copyright (c) 2005 Wiley-Liss, Inc.)

Many B-cell malignancies bear chromosomal translocations juxtaposing immunoglobulin (IG) genes with oncogenes, resulting in deregulated expression of the latter. Translocations affecting the IG heavy chain (IGH) locus in chromosomal region 14q32 are most prevalent. However, variant translocations involving the IG kappa (IGK) locus in 2p12 or the IG lambda (IGL) locus in 22q11 occur recurrently in B-cell neoplasias. No routine methods for the detection of all breakpoints involving IG light chain loci independently of the translocation partner have been described. For this reason, we have designed 2 novel interphase fluorescence in situ hybridization (FISH) assays using differentially labeled probes flanking the IGK and IGL locus, respectively. Based on extensive control studies, the diagnostic thresholds for the detection of breakpoints were set at 0.3% for IGK and 1.4% for IGL. Fifteen cases of B-cell malignancies with cytogenetically detectable chromosomal abnormalities in 2p11-14 were investigated with the FISH assay for IGK. Breakpoints affecting the IGK locus were detected in 7 cases including all 4 variant Burkitt's translocations t(2;8)(p12;q24) and a variant BCL2-associated translocation t(2;18)(p12;q21). Other translocation partners were chromosome bands 7q21 and 16q24. Ten cases with abnormalities in 22q11-12 were investigated with the FISH assay for IGL. Breakpoints in the IGL locus were diagnosed in 7 cases including both variant Burkitt's translocations t(8;22)(q24;q11) and a t(3;22)(q27;q11) involving the BCL6 locus. Other translocation partners were 2p13-14, 4q13 and 16p12. Our results show that these FISH assays provide flexible, simple and reliable tools in the diagnosis and characterization of genetic changes in B-cell malignancies., (Copyright 2002 Wiley-Liss, Inc.)

Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (BCP-, TCP-ALL), in differentiated and undifferentiated acute myeloblastic leukemia (AML), and in chronic myeloid leukemia (CML) at progression to blast crisis. The nature of these translocations and their pathologic consequences remain unknown. To begin to define the gene(s) involved on chromosome 13, we have performed fluorescence in situ hybridization (FISH) using a panel of YACs from the region, on a series of 10 cases of acute leukemia with t(12;13)(p12;q14) and 1 case each with "variant" translocations including t(12;13)(q21;q14), t(10;13)(q24;q14) and t(9;13)(p21;q14). In 8/13 cases/cell lines, the 13q14 break fell within a single 1.4 Mb CEPH MegaYAC. This YAC fell immediately telomeric of the forkhead (FKHR) gene, which is disrupted in the t(2;13)(q35;q14) seen in pediatric alveolar rhabdomyosarcoma. Seven of the 8 cases with breaks in this YAC were AML. In 4/13 cases, the 13q14 break fell within a 1.7-Mb YAC located about 3 Mb telomeric of the retinoblastoma (RB1) gene: all 4 cases were ALL. One case of myelodysplastic syndrome exhibited a break within 13q12, adjacent to the BRCA2 gene. These data indicate the presence of myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia.

The human trithorax homolog gene (MLL) is directly involved in over 90% of cases of acute leukemia with abnormalities of 11q23. However, involvement of other genes at 11q23 both centromeric and telomeric of MLL has been identified in different subtypes of leukemia and lymphoma. We describe a case of acute myelomonocytic leukemia (AMML; FAB type M4) with t(10;11)(p13;q23) in which the breakpoint at 11q23 was centromeric to the MLL gene and distinct from the breakpoint seen in promyelocytic leukemias with t(11;17)(q23;q22), thus providing further evidence of heterogeneity of breakpoints in 11q23 in acute leukemia. Rearrangements of immunoglobulin (IG) and T-cell receptor (TCR) genes were also observed, with no immunophenotypic evidence for commitment to the lymphoid lineages, indicating that inappropriate activation of the recombinases may be a feature of this particular variant translocation.