Stefan Gesk

Germline Nonsense Mutation and Somatic Inactivation of SMARCA4/BRG1 in a Family with Rhabdoid Tumor Predisposition Syndrome

Rhabdoid tumors of early infancy are highly aggressive with consequent poor prognosis. Most cases show inactivation of the SMARCB1 (also known as INI1 and hSNF5) tumor suppressor, a core member of the ATP-dependent SWI/SNF chromatin-remodeling complex. Familial cases, described as rhabdoid tumor predisposition syndrome (RTPS), have been linked to heterozygous SMARCB1 germline mutations. We identified inactivation of another member of the SWI/SNF chromatin-remodeling complex, its ATPase subunit SMARCA4 (also known as BRG1), due to a SMARCA4/BRG1 germline mutation and loss of heterozygosity by uniparental disomy in the tumor cells of two sisters with rhabdoid tumors lacking SMARCB1 mutations. SMARCA4 is thus a second member of the SWI/SNF complex involved in cancer predisposition. Its general involvement in other tumor entities remains to be established.

Presence of the P2RY8-CRLF2 rearrangement is associated with a poor prognosis in non–high-risk precursor B-cell acute lymphoblastic leukemia in children treated according to the ALL-BFM 2000 protocol

High-level expression of the cytokine receptor-like factor 2 gene, CRLF2, in precursor B-cell acute lymphoblastic leukemia (pB-ALL) was shown to be caused by a translocation involving the IGH@ locus or a deletion juxtaposing CRLF2 with the P2RY8 promoter. To assess its possible prognostic value, CRLF2 expression was analyzed in 555 childhood pB-ALL patients treated according to the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster 2000 (ALL-BFM 2000) protocol. Besides CRLF2 rearrangements, high-level CRLF2 expression was seen in cases with supernumerary copies of the CRLF2 locus. On the basis of the detection of CRLF2 rearrangements, a CRLF2 high-expression group (n = 49) was defined. This group had a 6-year relapse incidence of 31% plus or minus 8% compared with 11% plus or minus 1% in the CRLF2 low-expression group (P = .006). This difference was mainly attributable to an extremely high incidence of relapse (71% +/- 19%) in non-high-risk patients with P2RY8-CRLF2 rearrangement. The assessment of CRLF2 aberrations may therefore serve as new stratification tool in Berlin-Frankfurt-Münster-based protocols by identifying additional high-risk patients who may benefit from an intensified and/or targeted treatment.

MALT lymphoma with t(14;18)(q32;q21)/IGH-MALT1 is characterized by strong cytoplasmic MALT1 and BCL10 expression

Mucosa‐associated lymphoid tissue (MALT) lymphoma is specifically associated with t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21). t(11;18)(q21;q21) fuses the N‐terminus of the API2 gene to the C‐terminus of the MALT1 gene and generates a functional API2‐MALT1 product. t(1;14)(p22;q32) and t(14;18)(q32;q21) bring the BCL10 and MALT1 genes respectively to the IGH locus and deregulate their expression. The oncogenic activity of the three chromosomal translocations is linked by the physiological role of BCL10 and MALT1 in antigen receptor‐mediated NFκB activation. In this study, MALT1 and BCL10 expression was examined in normal lymphoid tissues and 423 cases of MALT lymphoma from eight sites, and their expression was correlated with the above translocations, which were detected by molecular and molecular cytogenetic methods. In normal B‐cell follicles, both MALT1 and BCL10 were expressed predominantly in the cytoplasm, high in centroblasts, moderate in centrocytes and weak/negative in mantle zone B‐cells. In MALT lymphoma, MALT1 and BCL10 expression varied among cases with different chromosomal translocations. In 9/9 MALT lymphomas with t(14;18)(q32;q21), tumour cells showed strong homogeneous cytoplasmic expression of both MALT1 and BCL10. In 12/12 cases with evidence of t(1;14)(p22;q32) or variants, tumour cells expressed MALT1 weakly in the cytoplasm but BCL10 strongly in the nuclei. In all 67 MALT lymphomas with t(11;18)(q21;q21), tumour cells expressed weak cytoplasmic MALT1 and moderate nuclear BCL10. In MALT lymphomas without the above translocations, both MALT1 and BCL10, in general, were expressed weakly in the cytoplasm. Real‐time quantitative RT‐PCR showed a good correlation between MALT1 and BCL10 mRNA expression and underlining genetic changes, with t(14;18)(q32;q21)‐ and t(1;14)(p22;q32)‐positive cases displaying the highest MALT1 and BCL10 mRNA expression respectively. These results show that MALT1 expression pattern is identical to that of BCL10 in normal lymphoid tissues but varies in MALT lymphomas, with high cytoplasmic expression of both MALT1 and BCL10 characterizing those with t(14;18)(q32;q21). Copyright

Nonsense Mutation and Inactivation of SMARCA4 (BRG1) in an Atypical Teratoid/Rhabdoid Tumor Showing Retained SMARCB1 (INI1) Expression

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive brain tumors of early childhood poorly responding to therapy. The majority of cases show inactivation of SMARCB1 (INI1, hSNF5, BAF47), a core member of the adenosine triphosphate (ATP)-dependent SWI/SNF chromatin-remodeling complex. We here report the case of a supratentorial AT/RT in a 9-month-old boy, which showed retained SMARCB1 staining on immunohistochemistry and lacked genetic alterations of SMARCB1. Instead, the tumor showed loss of protein expression of another SWI/SNF chromatin-remodeling complex member, the ATPase subunit SMARCA4 (BRG1) due to a homozygous SMARCA4 mutation [c.2032C>T (p.Q678X)]. Our findings highlight the role of SMARCA4 in the pathogenesis of SMARCB1-positive AT/RT and the usefulness of antibodies directed against SMARCA4 in this diagnostic setting.

Translocations activating IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults

The prognosis of germinal center-derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma.

A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

Background Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.

Follicular lymphoma lacking the t(14;18)(q32;q21): identification of two disease subtypes.

Summary. The clinical and pathological features, including karyotype data and BCL2 protein expression pattern, of follicular lymphoma without a t(14;18)(q32;q21) have not been well defined. We have identified and conducted a detailed analysis of 50 cases with follicular lymphoma who lack the t(14;18). Fluorescent in situ hybridization (FISH) analysis was used to exclude cases with a cryptic IGH/BCL2 rearrangement. BCL2 protein expression level was assessed by immunohistochemistry. The karyotypes were assessed for recurrent sites of structural rearrangement, duplications and deletions on a band‐by‐band basis, and compared with a large cohort of cases with t(14;18). A distinct pattern of chromosomal alterations was identified in the cases without t(14;18). BCL2 protein overexpression was detected in 33% of 49 tested cases. In this minority, the karyotypes frequently showed increased copies of chromosome 18. The majority of cases (67%) did not show BCL2 overexpression and were characterized prominently by the presence of t(3;14)(q27;q32), implying a role for BCL6. Follicular lymphomas that lack a t(14;18) were segregated into two subgroups with distinct cytogenetic, phenotypic and possibly clinical features: one with BCL2 protein overexpression not related to an IGH/BCL2 rearrangement and a second without BCL2 overexpression. Objective identification of BCL2 expression level as well as BCL2 and BCL6 status by cytogenetic or FISH analysis has potential clinical utility and may yield insights into alternative genetic mechanisms associated with B‐cell lymphomas with a follicular growth pattern.

Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci.

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 Burkitts 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 Burkitts 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.

Clinicopathologic Correlations of Genomic Gains and Losses in Follicular Lymphoma

PURPOSE To evaluate the clinical relevance of genomic aberrations in follicular lymphomas (FLs). PATIENTS AND METHODS In this study, we analyzed 124 biopsy samples of patients with FL using comparative genomic hybridization. RESULTS In 87 cases (70%), genomic imbalances were detectable. The most frequent aberrations were gains of chromosome arms 7p (21 patients), 7q (21 patients), Xp (16 patients), 12q (15 patients), and 18q (14 patients) as well as losses on 6q (21 patients). Grades 2 and 3 according to the World Health Organization classification correlated with a more complex karyotype (P <.0001). In a subset of 82 patients, a comprehensive clinical data set was available. In a multivariate analysis including all clinical risk factors of the International Prognostic Index as well as genomic aberrations, the loss of material on chromosomal bands 6q25q27 was the strongest predictor of a shorter survival (P =.0001; hazard ratio, 6.5), followed by elevated serum lactate dehydrogenase level (P =.0009; hazard ratio, 4.9), the presence of more than one extranodal manifestation (P =.017; hazard ratio, 4.2), and age greater than 60 years (P =.022; hazard ratio, 2.6). CONCLUSION These data indicate that genomic aberrations may contribute significantly to risk assessment in patients with FL, the majority of whom are included in low-risk groups using established clinical prognostic scores.

A comprehensive genetic and histopathologic analysis identifies two subgroups of B-cell malignancies carrying a t(14;19)(q32;q13) or variant BCL3-translocation

The biologic and pathologic features of B-cell malignancies bearing a translocation t(14;19)(q32;q13) leading to a fusion of IGH and BCL3 are still poorly described. Herein we report the results of a comprehensive cytogenetic, fluorescence in situ hybridization (FISH), molecular and histopathological survey of a large series of B-cell malignancies with t(14;19) or variant translocations. A total of 56 B-cell malignancies with a FISH-proven BCL3 involvement were identified with the translocation partners being IGH (n=51), IGL (n=2), IGK (n=2) and a non-IG locus (n=1). Hierarchical clustering of chromosomal changes associated with the t(14;19) indicated the presence of two different groups of IG/BCL3-positive lymphatic neoplasias. The first group included 26 B-cell malignancies of various histologic subtypes containing a relatively high number of chromosomal changes and mostly mutated IgVH genes. This cluster displayed three cytogenetic branches, one with rearrangements in 7q, another with deletions in 17p and a third one with rearrangements in 1q and deletions in 6q and 13q. The second group included 19 cases, mostly diagnosed as B-cell chronic lymphocytic leukemia (B-CLL), and characterized by few additional chromosomal changes (e.g. trisomy 12) and unmutated IgVH genes. In conclusion, our study indicates that BCL3 translocations are not restricted to B-CLL but present in a heterogeneous group of B-cell malignancies.