Inga Nagel

Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

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.

High-resolution genomic analysis suggests the absence of recurrent genomic alterations other than SMARCB1 aberrations in atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumor (AT/RT) is a rare malignant pediatric brain tumor characterized by genetic alterations affecting the SMARCB1 (hSNF5/INI1) locus in chromosome band 22q11.2. To identify potential additional genetic alterations, high‐resolution genome‐wide analysis was performed using a molecular inversion probe single‐nucleotide polymorphism (MIP SNP) assay (Affymetrix OncoScan formalin‐fixed paraffin‐embedded express) on DNA isolated from 18 formalin‐fixed paraffin‐embedded archival samples. Alterations affecting the SMARCB1 locus could be demonstrated by MIP SNP in 15 out of 16 evaluable cases (94%). These comprised five tumors with homozygous deletions, six tumors with heterozygous deletions, and four tumors with copy number neutral loss of heterozygosity (LOH) involving chromosome band 22q11.2. Remarkably, MIB SNP analysis did not yield any further recurrent chromosomal gains, losses, or copy neutral LOH. On MIP SNP screening for somatic mutations, the presence of a SMARCB1 mutation (c.472C>T p.R158X) was confirmed, but no recurrent mutations of other cancer relevant genes could be identified. Results of fluorescence in situ hybridization, multiplex ligation‐dependent probe amplification, and SMARCB1 sequencing were highly congruent with that of the MIP SNP assay. In conclusion, these data further suggest the absence of recurrent genomic alterations other than SMARCB1 in AT/RT.

Detection of genomic imbalances in microdissected Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma by array-based comparative genomic hybridization

Cytogenetic analysis of classical Hodgkin’s lymphoma is limited by the low content of the neoplastic Reed-Sternberg cells in the affected tissues. The present study demonstrates that array comparative genomic hybridization of microdissected Hodgkin-Reed-Sternberg cells is suitable for identifying and characterizing chromosomal imbalances. Se related perspective article on page 1292. Background Cytogenetic analysis of classical Hodgkin’s lymphoma is limited by the low content of the neoplastic Hodgkin-Reed-Sternberg cells in the affected tissues. However, available cytogenetic data point to an extreme karyotype complexity. To obtain insights into chromosomal imbalances in classical Hodgkin’s lymphoma, we applied array-based comparative genomic hybridization (array comparative genomic hybridization) using DNA from microdissected Hodgkin-Reed-Sternberg cells. Design and Methods To avoid biases introduced by DNA amplification for array comparative genomic hybridization, cHL cases rich in Hodgkin-Reed-Sternberg cells were selected. DNA obtained from approximately 100,000 microdissected Hodgkin-Reed-Sternberg cells of each of ten classical Hodgkin’s lymphoma cases was hybridized onto commercial 105 K oligonucleotide comparative genomic hybridization microarrays. Selected imbalances were confirmed by interphase cytogenetics and quantitative polymerase chain reaction analysis and further studied in an independent series of classical Hodgkin’s lymphoma. Results Gains identified in at least five cHL affected 2p12-16, 5q15-23, 6p22, 8q13, 8q24, 9p21-24, 9q34, 12q13-14, 17q12, 19p13, 19q13 and 20q11 whereas losses recurrent in at least five cases involved Xp21, 6q23-24 and 13q22. Copy number changes of selected genes and a small deletion (156 kb) of the CDKN2B (p15) gene were confirmed by interphase cytogenetics and polymerase chain reaction analysis, respectively. Several gained regions included genes constitutively expressed in cHL. Among these, gains of STAT6 (12q13), NOTCH1 (9q34) and JUNB (19p13) were present in additional cHL with the usual low Hodgkin-Reed-Sternberg cell content. Conclusions The present study demonstrates that array comparative genomic hybridization of microdissected Hodgkin-Reed-Sternberg cells is suitable for identifying and characterizing chromosomal imbalances. Regions affected by genomic changes in Hodgkin-Reed-Sternberg cells recurrently include genes constitutively expressed in cHL.

A recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma

The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.

DNA methylome analysis in Burkitt and follicular lymphomas identifies differentially methylated regions linked to somatic mutation and transcriptional control

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation–positive Burkitt lymphoma, nine BCL2 translocation–positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.

SMARCA4-mutated atypical teratoid/rhabdoid tumors are associated with inherited germline alterations and poor prognosis.

Martin Hasselblatt · Inga Nagel · Florian Oyen · Kerstin Bartelheim · Robert B. Russell · Ulrich Schuller · Reimar Junckerstorff · Marc Rosenblum · Ali H. Alassiri · Sabrina Rossi · Irene Schmid · Nicholas G. Gottardo · Helen Toledano · Elisabetta Viscardi · Milagros Balbin · Leora Witkowski · Qianhao Lu · Matthew J. Betts · William D. Foulkes · Reiner Siebert · Michael C. Fruhwald · Reinhard Schneppenheim

Immunoglobulin heavy chain locus chromosomal translocations in B-cell precursor acute lymphoblastic leukemia: rare clinical curios or potent genetic drivers?

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus define common subgroups of B-cell lymphoma but are rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Recent fluorescent in situ hybridization and molecular cloning studies have identified several novel IGH translocations involving genes that play important roles in normal hemopoiesis, including the cytokine receptor genes CRLF2 and EPOR, all members of the CCAAT enhancer-binding protein gene family, as well as genes not normally expressed in hemopoietic cells including inhibitor of DNA binding 4. IGH translocation results in deregulated target gene expression because of juxtaposition with IGH transcriptional enhancers. However, many genes targeted by IGH translocations are also more commonly deregulated in BCP-ALL as a consequence of other genetic or epigenetic mechanisms. For example, interstitial genomic deletions also result in deregulated CRLF2 expression, whereas EPOR expression is deregulated as a consequence of the ETV6-RUNX1 fusion. The possible clinical importance of many of the various IGH translocations in BCP-ALL remains to be determined from prospective studies, but CRLF2 expression is associated with a poor prognosis. Despite their rarity, IGH chromosomal translocations in BCP-ALL therefore define not only new mechanisms of B-cell transformation but also clinically important subgroups of disease and suggest new targeted therapeutic approaches.

Frequent triple-hit expression of MYC, BCL2, and BCL6 in primary lymphoma of the central nervous system and absence of a favorable MYC low BCL2 low subgroup may underlie the inferior prognosis as compared to systemic diffuse large B cell lymphomas

a breakpoint affecting the MYC locus, a frequency similar to systemic DLBCL [3]. One of these cases carried a concomitant BCL6 break and was classified as genetic DH pCNsL. The IGH locus was excluded as MYC partner in all cases. IHC applying cut-off levels similar to Horn et al. [3] identified 82 % (41/50) of pCNsL as MYCBCL2 and 90 % (45/50) as MYCBCL2 or MYCBCL6 De. 70 % (35/50) were MYCBCL2BCL6 triple expressing (Te) pCNsL. The high fraction of MYC BCL2 De pCNsL significantly exceeds systemic DLBCL (depending on study 21–29 % [2–4], p < 0.001, Fisher’s exact test). None of the pCNsL lacked both MYC and BCL2 expression or was triple negative. Three of the four pCNsL with MYC break showed strong MYC expression (≥80 %). similarly high MYC protein levels like in MYC translocated cases (≥70 %) were noticed in the majority of tumors (all pCNsL: 35/50, 70 %; pCNsL without MYC break: 32/46, 70 %). Thus, there was a striking discrepancy between the high frequency of prominent MYC protein overexpression and “Double-hit” (DH) systemic diffuse large B cell lymphomas (DLBCL), defined by molecular cytogenetics as breakpoint affecting the MYC locus concomitantly with a breakpoint affecting the BCL2 and/or BCL6 locus as well as double-expressing (De) lymphomas defined by immunohistochemistry (IHC) as coexpression of MYC with BCL2 proteins, carry a poor prognosis [2–4]. primary lymphoma of the CNs (pCNsL) is a specific DLBCL with particular target organ manifestation and a significantly adverse outcome. To investigate potential mechanisms leading to these features, we studied a series of 50 pCNsL (immunocompetent patients) for MYC, BCL2, and BCL6 breaks by FIsH and MYC, BCL2, and BCL6 expression by IHC. Breakpoints affecting the BCL6 locus were detected in 26 % (12/47) of pCNsL (Table 1), in accordance with previous reports [6] and similar with systemic DLBCL [3]. Breakpoints affecting the BCL2 locus were absent (0/49, 0 %). Four (4/49, 8 %) pCNsL harbored

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.