Andrea Haake

Distinct DNA methylation patterns in cirrhotic liver and hepatocellular carcinoma

Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array‐based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a “cirrhotic” methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.

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.

The mutational pattern of primary lymphoma of the central nervous system determined by whole-exome sequencing

To decipher the mutational pattern of primary CNS lymphoma (PCNSL), we performed whole-exome sequencing to a median coverage of 103 × followed by mutation verification in 9 PCNSL and validation using Sanger sequencing in 22 PCNSL. We identified a median of 202 (range: 139–251) potentially somatic single nucleotide variants (SNV) and 14 small indels (range: 7–22) with potentially protein-changing features per PCNSL. Mutations affected the B-cell receptor, toll-like receptor, and NF-κB and genes involved in chromatin structure and modifications, cell-cycle regulation, and immune recognition. A median of 22.2% (range: 20.0–24.7%) of somatic SNVs in 9 PCNSL overlaps with the RGYW motif targeted by somatic hypermutation (SHM); a median of 7.9% (range: 6.2–12.6%) affects its hotspot position suggesting a major impact of SHM on PCNSL pathogenesis. In addition to the well-known targets of aberrant SHM (aSHM) (PIM1), our data suggest new targets of aSHM (KLHL14, OSBPL10, and SUSD2). Among the four most frequently mutated genes was ODZ4 showing protein-changing mutations in 4/9 PCNSL. Together with mutations affecting CSMD2, CSMD3, and PTPRD, these findings may suggest that alterations in genes having a role in CNS development may facilitate diffuse large B-cell lymphoma manifestation in the CNS. This may point to intriguing mechanisms of CNS tropism in PCNSL.

Recurrent mutation of JAK3 in T-cell prolymphocytic leukemia.

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.

Genomic and epigenomic co-evolution in follicular lymphomas.

Follicular lymphoma (FL) with a t(14;18) is a B-cell neoplasm clinically characterized by multiple recurrencies. In order to investigate the clonal evolution of this lymphoma, we studied paired primary and relapse tumor samples from 33 patients with recurrent non-transformed t(14;18)-positive FL. We reconstructed phylogenetic trees of the evolution by taking advantage of the activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) active in the germinal center reaction using sequences of the clonal VHDHJH rearrangements of the immunoglobulin heavy chain (IGH) locus. Mutational analysis of the IGH locus showed evidence for ongoing somatic mutation and for counter-selection of mutations affecting the BCR conformation during tumor evolution. We further followed evolutionary divergence by targeted sequencing of gene loci affected by aberrant SHM as well as of known driver genes of lymphomagenesis, and by array-based genome-wide chromosomal imbalance and DNA methylation analysis. We observed a wide spectrum of evolutionary patterns ranging from almost no evolution to divergent evolution within recurrent non-transformed t(14;18) FL. Remarkably, we observed a correlation of the magnitude of evolutionary divergence across all genetic and epigenetic levels suggesting co-evolution. The distribution of coding mutations in driver genes and the correlation with SHM suggest CREBBP and AID to be potential modifiers of genetic and epigenetic co-evolution in FL.

A unique case of follicular lymphoma provides insights to the clonal evolution from follicular lymphoma in situ to manifest follicular lymphoma

To the editor: Follicular lymphoma in situ (FLIS) is characterized by the presence of a B-cell population with immunophenotypic and genotypic features of follicular lymphoma (FL) but exclusively localized to germinal centers (GCs) in morphologically reactive lymph nodes.[1][1] FLIS lesions are

Increasing genomic and epigenomic complexity in the clonal evolution from in situ to manifest t(14;18)-positive follicular lymphoma

Follicular lymphoma (FL) is characterized besides the t(14;18)(q32;q21), by recurrent chromosomal alterations and somatic mutations. In this study, we analyzed cases of FL in situ (FLIS) without manifest FL (mFL), partial involvement by FL (PFL) and paired cases of FLIS and mFL to detect possible early chromosomal imbalances, mutations, as well as DNA-methylation patterns of genomic regions of selected genes. We demonstrate that all paired FLIS and mFL cases were clonally related, based on IGH rearrangement patterns and BCL2 breakpoint sequences. FLIS and PFL had no or few secondary chromosomal imbalances detectable by array comparative genomic hybridization (FLIS 0.8 copy number alterations (CNA)/case; PFL 2.0 CNA/case; mFL 6.3 CNA/case) and a lower level of DNA methylation of genes recurrently de novo methylated in lymphomas, as compared with mFL. EZH2 Tyr641 mutations were detected in a subset of both FLIS (2/9) and PFL (1/3) cases. In conclusion, these findings provide evidence that FLIS represents a FL precursor lesion of long-lived clonal B cells carrying the t(14;18) with no or few secondary genetic changes. Our data suggest that there may be more than one distinct lesion driving the progression from FLIS to manifest lymphoma.

MINCR is a MYC-induced lncRNA able to modulate MYC’s transcriptional network in Burkitt lymphoma cells

Significance Gains of the MYC gene are the most common imbalances in cancer and are associated with poor prognosis, particularly in B-cell lymphoma. Recent advances in DNA sequencing have revealed the existence of thousands of long noncoding RNAs (lncRNAs) with unknown functional relevance. We have here identified a MYC-regulated lncRNA that we named MYC-induced long noncoding RNA (MINCR) that has a strong correlation with MYC expression in cancer. We show that MINCR is functional and controls cell cycle progression by influencing the expression of MYC-regulated cell cycle genes. MINCR is, therefore, a novel player in MYC’s transcriptional network, with the potential to open new therapeutic windows in the fight against malignant lymphoma and, possibly, all cancers that rely on MYC expression. Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

The PCBP1 gene encoding poly(rc) binding protein i is recurrently mutated in Burkitt lymphoma

The genetic hallmark of Burkitt lymphoma is the translocation t(8;14)(q24;q32), or one of its light chain variants, resulting in IG‐MYC juxtaposition. However, these translocations alone are insufficient to drive lymphomagenesis, which requires additional genetic changes for malignant transformation. Recent studies of Burkitt lymphoma using next generation sequencing approaches have identified various recurrently mutated genes including ID3, TCF3, CCND3, and TP53. Here, by using similar approaches, we show that PCBP1 is a recurrently mutated gene in Burkitt lymphoma. By whole‐genome sequencing, we identified somatic mutations in PCBP1 in 3/17 (18%) Burkitt lymphomas. We confirmed the recurrence of PCBP1 mutations by Sanger sequencing in an independent validation cohort, finding mutations in 3/28 (11%) Burkitt lymphomas and in 6/16 (38%) Burkitt lymphoma cell lines. PCBP1 is an intron‐less gene encoding the 356 amino acid poly(rC) binding protein 1, which contains three K‐Homology (KH) domains and two nuclear localization signals. The mutations predominantly (10/12, 83%) affect the KH III domain, either by complete domain loss or amino acid changes. Thus, these changes are predicted to alter the various functions of PCBP1, including nuclear trafficking and pre‐mRNA splicing. Remarkably, all six primary Burkitt lymphomas with a PCBP1 mutation expressed MUM1/IRF4, which is otherwise detected in around 20–40% of Burkitt lymphomas. We conclude that PCBP1 mutations are recurrent in Burkitt lymphomas and might contribute, in cooperation with other mutations, to its pathogenesis.

A familial disorder of altered DNA-methylation

Background In a subset of imprinting disorders caused by epimutations, multiple imprinted loci are affected. Familial occurrence of multilocus imprinting disorders is rare. Purpose/objective We have investigated the clinical and molecular features of a familial DNA-methylation disorder. Methods Tissues of affected individuals and blood samples of family members were investigated by conventional and molecular karyotyping. Sanger sequencing and RT-PCR of imprinting-associated genes (NLRP2, NLRP7, ZFP57, KHDC3L, DNMT1o), exome sequencing and locus-specific, array-based and genome-wide technologies to determine DNA-methylation were performed. Results In three offspring of a healthy couple, we observed prenatal onset of severe growth retardation and dysmorphism associated with altered DNA-methylation at paternally and maternally imprinted loci. Array-based analyses in various tissues of the offspring identified the DNA-methylation of 2.1% of the genes in the genome to be recurrently altered. Despite significant enrichment of imprinted genes (OR 9.49), altered DNA-methylation predominately (90.2%) affected genes not known to be imprinted. Sequencing of genes known to cause comparable conditions and exome sequencing in affected individuals and their ancestors did not unambiguously point to a causative gene. Conclusions The family presented herein suggests the existence of a familial disorder of DNA-methylation affecting imprinted but also not imprinted gene loci potentially caused by a maternal effect mutation in a hitherto not identified gene.