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The singular BRCA1/2 mutational landscape of Asturias is updated 10 years after the first study. We analyzed BRCA1 and BRCA2 pathogenic variants in 1653 index cases. In total, 238 families were identified to carry a pathogenic variant, 163 families in BRCA1 and 75 families in BRCA2. This yielded a prevalence rate of 14.4%. Seven recurrent variants were found accounting for 55% of the cases. Among them, three are widely distributed (BRCA1 c.211A>G, c.470_471del and c.3331_3334del) and four had been reported as novel in Asturias: two in BRCA1 (c.1674del and c.2901_2902dup) and two in BRCA2 (c.2095C>T and c.4040_4035delinsC). A common haplotype was established for all recurrent variants indicating a shared ancestral origin. Three splicing analyses are shown: BRCA1:c.5152+3A>C and BRCA1:c.5333-3T>G that lead to skipping of exon 18, and 22 respectively, and BRCA1:c.5278-1G>T giving rise to two transcripts, one lacking exon 21 (p.Ille1760Glyfs*60) and one lacking the first 8 nucleotides of exon 21 (p.Phe1761Asnfs*14), supporting pathogenicity for these variants., (© 2024 The Author(s). Clinical Genetics published by John Wiley & Sons Ltd.)

Mantle cell lymphoma (MCL) is genetically characterized by the IG::CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG::CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH::CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2, TNFAIP3 and KMT2D. These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL., (© 2024. The Author(s).)

MALAT1 long non-coding RNA has oncogenic roles but has been poorly studied in indolent B-cell neoplasms. Here, MALAT1 expression was analyzed using RNA-seq, microarrays or qRT-PCR in primary samples from clinico-biological subtypes of chronic lymphocytic leukemia (CLL, n = 266), paired Richter transformation (RT, n = 6) and follicular lymphoma (FL, n = 61). In peripheral blood (PB) CLL samples, high MALAT1 expression was associated with a significantly shorter time to treatment independently from other known prognostic factors. Coding genes expressed in association with MALAT1 in CLL were predominantly related to oncogenic pathways stimulated in the lymph node (LN) microenvironment. In RT paired samples, MALAT1 levels were lower, concordant with their acquired increased independency of external signals. Moreover, MALAT1 levels in paired PB/LN CLLs were similar, suggesting that the prognostic value of MALAT1 expression in PB is mirroring expression differences already present in LN. Similarly, high MALAT1 expression in FL predicted for a shorter progression-free survival, in association with expression pathways promoting FL pathogenesis. In summary, MALAT1 expression is related to pathophysiology and more aggressive clinical behavior of indolent B-cell neoplasms. Particularly in CLL, its levels could be a surrogate marker of the microenvironment stimulation and may contribute to refine the clinical management of these patients., (© 2023. Springer Nature Limited.)

The cost reduction in sequencing and the extensive genomic characterization of a wide variety of cancers are expanding tumor sequencing to a wide number of research groups and the clinical practice. Although specific pipelines have been generated for the identification of somatic mutations, their results usually differ considerably, and a common approach is to use several callers to achieve a more reliable set of mutations. This procedure is computationally expensive and time-consuming, and it suffers from the same limitations in sensitivity and specificity as other approaches. Expert revision of mutant calls is therefore required to verify calls that might be used for clinical diagnosis. This step could take advantage of machine learning techniques, as they provide a useful approach to incorporate expert-reviewed information for the identification of somatic mutations. Here we present RFcaller, a pipeline based on machine learning algorithms, for the detection of somatic mutations in tumor-normal paired samples that does not require large computing resources. RFcaller shows high accuracy for the detection of substitutions and insertions/deletions from whole genome or exome data. It allows the detection of mutations in driver genes missed by other approaches, and has been validated by comparison to deep and Sanger sequencing., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFβ signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations., Significance: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis., (©2022 American Association for Cancer Research.)

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the 'CLL map,' we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS) high -B cell receptor (BCR) low -signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT., (© 2022. The Author(s).)

Current somatic mutation callers are biased against repetitive regions, preventing the identification of potential driver alterations in these loci. We developed a mutation caller for repetitive regions, and applied it to study repetitive non protein-coding genes in more than 2200 whole-genome cases. We identified a recurrent mutation at position c.28 in the gene encoding the snRNA U2. This mutation is present in B-cell derived tumors, as well as in prostate and pancreatic cancer, suggesting U2 c.28 constitutes a driver candidate associated with worse prognosis. We showed that the GRCh37 reference genome is incomplete, lacking the U2 cluster in chromosome 17, preventing the identification of mutations in this gene. Furthermore, the 5'-flanking region of WDR74, previously described as frequently mutated in cancer, constitutes a functional copy of U2. These data reinforce the relevance of non-coding mutations in cancer, and highlight current challenges of cancer genomic research in characterizing mutations affecting repetitive genes., (© 2022. The Author(s).)

Germline pathogenic variants in BRCA1 confer a high risk of developing breast and ovarian cancer. The BRCA1 exon 11 (formally exon 10) is one of the largest exons and codes for the nuclear localization signals of the corresponding gene product. This exon can be partially or entirely skipped during pre-mRNA splicing, leading to three major in-frame isoforms that are detectable in most cell types and tissue, and in normal and cancer settings. However, it is unclear whether the splicing imbalance of this exon is associated with cancer risk. Here we identify a common genetic variant in intron 10, rs5820483 (NC_000017.11:g.43095106_43095108dup), which is associated with exon 11 isoform expression and alternative splicing, and with the risk of breast cancer, but not ovarian cancer, in BRCA1 pathogenic variant carriers. The identification of this genetic effect was confirmed by analogous observations in mouse cells and tissue in which a loxP sequence was inserted in the syntenic intronic region. The prediction that the rs5820483 minor allele variant would create a binding site for the splicing silencer hnRNP A1 was confirmed by pull-down assays. Our data suggest that perturbation of BRCA1 exon 11 splicing modifies the breast cancer risk conferred by pathogenic variants of this gene., (© 2021 Wiley Periodicals LLC.)

B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling, conferring aggressive behavior. Epigenetic studies have defined 3 CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL), and memory-like (m-CLL) B cells with different biological features. i-CLL carries a borderline IGHV mutational load and significantly higher use of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes, we characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases: 30 (38%) of 79 i-CLLs, 5 (1.7%) of 291 m-CLLs, and 1 (0.5%) of 189 n-CLLs. All stereotype subset 2 cases carried IGLV3-21R110, whereas 62% of IGLV3-21R110 i-CLL cases had nonstereotyped BCR immunoglobulins. IGLV3-21R110 i-CLL had a significantly higher number of SF3B1 and ATM mutations and total number of driver alterations. However, the R110 mutation was the sole alteration in 1 i-CLL and was accompanied only by del(13q) in 3. Although IGHV mutational status varied, IGLV3-21R110 i-CLL transcriptomically resembled n-CLL/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. In contrast, i-CLL lacking IGLV3-21R110 mirrored m-CLL/mutated IGHV. Patients with IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to those of n-CLL/unmutated IGHV patients, whereas patients with non-IGLV3-21R110 i-CLL had a good prognosis similar to that of patients with m-CLL/mutated IGHV. IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of IGHV mutational status and epigenetic subtype., (© 2021 by The American Society of Hematology.)

Mutational signatures have emerged as powerful biomarkers in cancer patients, with prognostic and therapeutic implications. Wider clinical utility requires access to reproducible algorithms, which allow characterization of mutational signatures in a given tumor sample. Here, we show how mutational signature fitting can be applied to hematological cancer genomes to identify biologically and clinically important mutational processes, highlighting the importance of careful interpretation in light of biological knowledge. Our newly released R package mmsig comes with a dynamic error-suppression procedure that improves specificity in important clinical and biological settings. In particular, mmsig allows accurate detection of mutational signatures with low abundance, such as those introduced by APOBEC cytidine deaminases. This is particularly important in the most recent mutational signature reference (COSMIC v3.1) where each signature is more clearly defined. Our mutational signature fitting algorithm mmsig is a robust tool that can be implemented immediately in the clinic.

The molecular characterisation of medulloblastoma, the most common paediatric brain tumour, is crucial for the correct management and treatment of this heterogenous disease. However, insufficient tissue sample, the presence of tumour heterogeneity, or disseminated disease can challenge its diagnosis and monitoring. Here, we report that the cerebrospinal fluid (CSF) circulating tumour DNA (ctDNA) recapitulates the genomic alterations of the tumour and facilitates subgrouping and risk stratification, providing valuable information about diagnosis and prognosis. CSF ctDNA also characterises the intra-tumour genomic heterogeneity identifying small subclones. ctDNA is abundant in the CSF but barely present in plasma and longitudinal analysis of CSF ctDNA allows the study of minimal residual disease, genomic evolution and the characterisation of tumours at recurrence. Ultimately, CSF ctDNA analysis could facilitate the clinical management of medulloblastoma patients and help the design of tailored therapeutic strategies, increasing treatment efficacy while reducing excessive treatment to prevent long-term secondary effects.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm initially driven by CCND1 rearrangement with 2 molecular subtypes, conventional MCL (cMCL) and leukemic non-nodal MCL (nnMCL), that differ in their clinicobiological behavior. To identify the genetic and epigenetic alterations determining this diversity, we used whole-genome (n = 61) and exome (n = 21) sequencing (74% cMCL, 26% nnMCL) combined with transcriptome and DNA methylation profiles in the context of 5 MCL reference epigenomes. We identified that open and active chromatin at the major translocation cluster locus might facilitate the t(11;14)(q13;32), which modifies the 3-dimensional structure of the involved regions. This translocation is mainly acquired in precursor B cells mediated by recombination-activating genes in both MCL subtypes, whereas in 8% of cases the translocation occurs in mature B cells mediated by activation-induced cytidine deaminase. We identified novel recurrent MCL drivers, including CDKN1B, SAMHD1, BCOR, SYNE1, HNRNPH1, SMARCB1, and DAZAP1. Complex structural alterations emerge as a relevant early oncogenic mechanism in MCL, targeting key driver genes. Breakage-fusion-bridge cycles and translocations activated oncogenes (BMI1, MIR17HG, TERT, MYC, and MYCN), generating gene amplifications and remodeling regulatory regions. cMCL carried significant higher numbers of structural variants, copy number alterations, and driver changes than nnMCL, with exclusive alterations of ATM in cMCL, whereas TP53 and TERT alterations were slightly enriched in nnMCL. Several drivers had prognostic impact, but only TP53 and MYC aberrations added value independently of genomic complexity. An increasing genomic complexity, together with the presence of breakage-fusion-bridge cycles and high DNA methylation changes related to the proliferative cell history, defines patients with different clinical evolution., (© 2020 by The American Society of Hematology.)

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.

The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2 nd -3 rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

Chronic lymphocytic leukemia is a common disease in Western countries and has heterogeneous clinical behavior. The relevance of the genetic basis of the disease has come to the forefront recently, with genome-wide studies that have provided a comprehensive view of structural variants, somatic mutations, and different layers of epigenetic changes. The mutational landscape is characterized by relatively common copy number alterations, a few mutated genes occurring in 10-15% of cases, and a large number of genes mutated in a small number of cases. The epigenomic profile has revealed a marked reprogramming of regulatory regions in tumor cells compared with normal B cells. All of these alterations are differentially distributed in clinical and biological subsets of the disease, indicating that they may underlie the heterogeneous evolution of the disease. These global studies are revealing the molecular complexity of chronic lymphocytic leukemia and provide new perspectives that have helped to understand its pathogenic mechanisms and improve the clinical management of patients.

Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered-despite intensive searching 1,2 . Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1) 3-6 . By contrast, cancer-related alterations in the noncoding component of the spliceosome-a series of small nuclear RNAs (snRNAs)-have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes 1,7,8 . Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5' splice site via base-pairing. This mutation changes the preferential A-U base-pairing between U1 snRNA and the 5' splice site to C-G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes-including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions.

In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes 1-3 . Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5' splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5' cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes (PTCH1) and activates oncogenes (GLI2 and CCND2), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer.

Taxanes are the mainstay of treatment in triple-negative breast cancer (TNBC), with de novo and acquired resistance limiting patient's survival. To investigate the genetic basis of docetaxel resistance in TNBC, exome sequencing was performed on matched TNBC patient-derived xenografts (PDX) sensitive to docetaxel and their counterparts that developed resistance in vivo upon continuous drug exposure. Most mutations, small insertions/deletions, and copy number alterations detected in the initial TNBC human metastatic samples were maintained after serial passages in mice and emergence of resistance. We identified a chromosomal amplification of chr12p in a human BRCA1 -mutated metastatic sample and the derived chemoresistant PDX, but not in the matched docetaxel-sensitive PDX tumor. Chr12p amplification was validated in a second pair of docetaxel-sensitive/resistant BRCA1 -mutated PDXs and after short-term docetaxel treatment in several TNBC/ BRCA1 -mutated PDXs and cell lines, as well as during metastatic recurrence in a patient with BRCA1 -mutated breast cancer who had progressed on docetaxel treatment. Analysis of clinical data indicates an association between chr12p amplification and patients with TNBC/basal-like breast cancer, a BRCA1 mutational signature, and poor survival after chemotherapy. Detection of chr12p amplification in a cohort of TNBC PDX models was associated with an improved response to carboplatin. Our findings reveal tumor clonal dynamics during chemotherapy treatments and suggest that a preexisting population harboring chr12p amplification is associated with the emergence of docetaxel resistance and carboplatin responsiveness in TNBC/ BRCA1 -mutated tumors. SIGNIFICANCE: Chr12p copy number gains indicate rapid emergence of resistance to docetaxel and increased sensitivity to carboplatin, therefore sequential docetaxel/carboplatin treatment could improve survival in TNBC/ BRCA1 patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4258/F1.large.jpg., (©2019 American Association for Cancer Research.)

Genome-wide association studies have provided evidence for inherited genetic predisposition to chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms underlying CLL risk we analyze chromatin accessibility, active regulatory elements marked by H3K27ac, and DNA methylation at 42 risk loci in up to 486 primary CLLs. We identify that risk loci are significantly enriched for active chromatin in CLL with evidence of being CLL-specific or differentially regulated in normal B-cell development. We then use in situ promoter capture Hi-C, in conjunction with gene expression data to reveal likely target genes of the risk loci. Candidate target genes are enriched for pathways related to B-cell development such as MYC and BCL2 signalling. At 14 loci the analysis highlights 63 variants as the probable functional basis of CLL risk. By integrating genetic and epigenetic information our analysis reveals novel insights into the relationship between inherited predisposition and the regulatory chromatin landscape of CLL.

Analysis of mutational signatures is becoming routine in cancer genomics, with implications for pathogenesis, classification, prognosis, and even treatment decisions. However, the field lacks a consensus on analysis and result interpretation. Using whole-genome sequencing of multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia, we compare the performance of public signature analysis tools. We describe caveats and pitfalls of de novo signature extraction and fitting approaches, reporting on common inaccuracies: erroneous signature assignment, identification of localized hyper-mutational processes, overcalling of signatures. We provide reproducible solutions to solve these issues and use orthogonal approaches to validate our results. We show how a comprehensive mutational signature analysis may provide relevant biological insights, reporting evidence of c-AID activity among unmutated CLL cases or the absence of BRCA1/BRCA2-mediated homologous recombination deficiency in a MM cohort. Finally, we propose a general analysis framework to ensure production of accurate and reproducible mutational signature data.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF ( KITLG , KIT , PTPN11 , GNB1 , KRAS and NRAS ) were mutated in 12 patients (2.6%), and genes downstream of BRAF ( MAPK2K1 , MAPK2K2 , and MAPK1 ) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P =0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited., (Copyright© 2019 Ferrata Storti Foundation.)

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1 - MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1 - /D2 - MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1 - /SOX11 + MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1 - MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1 - MCL cases were indistinguishable from cyclin D1 + MCL. In conclusion, virtually all cyclin D1 - MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1 - /D2 - /D3 - MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1 - MCL., (© 2019 by The American Society of Hematology.)

The causal association of NUDT1 (=MTH1) and OGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal contribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screening was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case-control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein stability), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended., (© 2018 Wiley Periodicals, Inc.)

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are 2 well-defined entities that diverge in their basic pathogenic mechanisms and clinical evolution but they share epidemiological characteristics, cells of origin, molecular alterations, and clinical features that differ from other lymphoid neoplasms. CLL and MCL are classically considered indolent and aggressive neoplasms, respectively. However, the clinical evolution of both tumors is very heterogeneous, with subsets of patients having stable disease for a long time whereas others require immediate intervention. Both CLL and MCL include 2 major molecular subtypes that seem to derive from antigen-experienced CD5 + B cells that retain a naive or memory-like epigenetic signature and carry a variable load of immunoglobulin heavy-chain variable region somatic mutations from truly unmutated to highly mutated, respectively. These 2 subtypes of tumors differ in their molecular pathways, genomic alterations, and clinical behavior, being more aggressive in naive-like than memory-like-derived tumors in both CLL and MCL. The pathogenesis of the 2 entities integrates the relevant influence of B-cell receptor signaling, tumor cell microenvironment interactions, genomic alterations, and epigenome modifications that configure the evolution of the tumors and offer new possibilities for therapeutic intervention. This review will focus on the similarities and differences of these 2 tumors based on recent studies that are enhancing the understanding of their pathogenesis and creating solid bases for new management strategies., (© 2018 by The American Society of Hematology.)

Understanding the mechanisms of cancer therapeutic resistance is fundamental to improving cancer care. There is clear benefit from chemotherapy in different breast cancer settings; however, knowledge of the mutations and genes that mediate resistance is incomplete. In this study, by modeling chemoresistance in patient-derived xenografts (PDXs), we show that adaptation to therapy is genetically complex and identify that loss of transcription factor 4 (TCF4; also known as ITF2) is associated with this process. A triple-negative BRCA1 -mutated PDX was used to study the genetics of chemoresistance. The PDX was treated in parallel with four chemotherapies for five iterative cycles. Exome sequencing identified few genes with de novo or enriched mutations in common among the different therapies, whereas many common depleted mutations/genes were observed. Analysis of somatic mutations from The Cancer Genome Atlas (TCGA) supported the prognostic relevance of the identified genes. A mutation in TCF4 was found de novo in all treatments, and analysis of drug sensitivity profiles across cancer cell lines supported the link to chemoresistance. Loss of TCF4 conferred chemoresistance in breast cancer cell models, possibly by altering cell cycle regulation. Targeted sequencing in chemoresistant tumors identified an intronic variant of TCF4 that may represent an expression quantitative trait locus associated with relapse outcome in TCGA. Immunohistochemical studies suggest a common loss of nuclear TCF4 expression post-chemotherapy. Together, these results from tumor xenograft modeling depict a link between altered TCF4 expression and breast cancer chemoresistance., Competing Interests: Competing interestsA. Villanueva and M.A.P. are recipients of unrestricted research grants from Roche Pharma and Astellas Pharma. A. Vidal and A. Villanueva are co-founders of Xenopat. No potential conflicts of interest were disclosed by the other authors., (© 2018. Published by The Company of Biologists Ltd.)

BRCA1 is a tumor suppressor that regulates DNA repair by homologous recombination. Germline mutations in BRCA1 are associated with increased risk of breast and ovarian cancer and BRCA1 deficient tumors are exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, uncovering additional components of this DNA repair pathway is of extreme importance for further understanding cancer development and therapeutic vulnerabilities. Here, we identify EDC4, a known component of processing-bodies and regulator of mRNA decapping, as a member of the BRCA1-BRIP1-TOPBP1 complex. EDC4 plays a key role in homologous recombination by stimulating end resection at double-strand breaks. EDC4 deficiency leads to genome instability and hypersensitivity to DNA interstrand cross-linking drugs and PARP inhibitors. Lack-of-function mutations in EDC4 were detected in BRCA1/2-mutation-negative breast cancer cases, suggesting a role in breast cancer susceptibility. Collectively, this study recognizes EDC4 with a dual role in decapping and DNA repair whose inactivation phenocopies BRCA1 deficiency.

Background & Aims: Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC., Methods: We collected blood samples from 3 relatives with CRC in Spain (65, 62, and 40 years old at diagnosis) and performed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases (502 additional families). We carried out protein-dependent yeast growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants., Results: A total of 42 unique or rare (population minor allele frequency below 1%) nonsynonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11 of 503 CRC families, a significantly greater proportion than in the control population (34 of 4300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability, or expression and/or function., Conclusions: In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Proteases constitute up to 3% of all protein-coding genes in a vertebrate genome and participate in numerous physiological and pathological processes. The characterization of the degradome of one organism, the set of all genes encoding proteolytic enzymes, and the comparison to the degradome of other species have proved useful to identify genetic differences that are helpful to elucidate the molecular basis of diverse biological processes, the different susceptibility to disease, and the evolution of the structure and function of proteases. Here we describe the main procedures involved in the characterization of the degradome of an organism for which its genome sequence is available.

The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript. Using the CLIP-CHIP™ we mapped the expression profile of proteases and their inhibitors in 23 different human tissues and 6 ovarian cancer cell lines in 104 sample datasets. Hierarchical cluster analysis showed that expression profiles clustered according to their anatomic locations, cellular composition, physiologic functions, and the germ layer from which they are derived. The human ovarian cancer cell lines cluster according to malignant grade. 110 proteases and 42 inhibitors were tissue specific (1 to 3 tissues). Of these 110 proteases 69% (74) are mainly extracellular, 30% (34) intracellular and 1% intramembrane. Notably, 35% (197/565) of human proteases and 30% (47/156) of inhibitors were ubiquitously expressed in all 23 tissues; 27% (155) of proteases and 21% (32) of inhibitors were broadly expressed in 4-20 tissues. Our datasets provide a valuable resource for the community of baseline protease and inhibitor relative expression in normal human tissues and can be used for comparison with diseased tissue, e.g. ovarian cancer, to decipher pathogenesis, and to aid drug development. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

In the vast majority of pediatric patients with dilated cardiomyopathy, the specific etiology is unknown. Studies on families with dilated cardiomyopathy have exemplified the role of genetic factors in cardiomyopathy etiology. In this study, we applied whole-exome sequencing to members of a non-consanguineous family affected by a previously unreported congenital dilated cardiomyopathy syndrome necessitating early-onset heart transplant. Exome analysis identified compound heterozygous variants in the FLNC gene. Histological analysis of the cardiac muscle demonstrated marked sarcomeric and myofibrillar abnormalities, and immunohistochemical staining demonstrated the presence of Filamin C aggregates in cardiac myocytes. We conclude that biallelic variants in FLNC can cause congenital dilated cardiomyopathy. As the associated clinical features of affected patients are mild, and can be easily overlooked, testing for FLNC should be considered in children presenting with dilated cardiomyopathy., Competing Interests: The authors declare no conflict of interest. DMB and CB are compensated and serve as the chief medical officer and the laboratory director of Gene by Gene, respectively.

Chronic lymphocytic leukemia (CLL) is an adult B cell malignancy. Genome-wide association studies show that variation at 15q15.1 influences CLL risk. We deciphered the causal variant at 15q15.1 and the mechanism by which it influences tumorigenesis. We imputed all possible genotypes across the locus and then mapped highly associated SNPs to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding. SNP rs539846 C>A, the most highly associated variant (p = 1.42 × 10(-13), odds ratio = 1.35), localizes to a super-enhancer defined by extensive histone H3 lysine 27 acetylation in intron 3 of B cell lymphoma 2 (BCL2)-modifying factor (BMF). The rs539846-A risk allele alters a conserved RELA-binding motif, disrupts RELA binding, and is associated with decreased BMF expression in CLL. These findings are consistent with rs539846 influencing CLL susceptibility through differential RELA binding, with direct modulation of BMF expression impacting on anti-apoptotic BCL2, a hallmark of oncogenic dependency in CLL., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genomic studies have revealed the complex clonal heterogeneity of chronic lymphocytic leukemia (CLL). The acquisition and selection of genomic aberrations may be critical to understanding the progression of this disease. In this study, we have extensively characterized the mutational status of TP53, SF3B1, BIRC3, NOTCH1, and ATM in 406 untreated CLL cases by ultra-deep next-generation sequencing, which detected subclonal mutations down to 0.3% allele frequency. Clonal dynamics were examined in longitudinal samples of 48 CLL patients. We identified a high proportion of subclonal mutations, isolated or associated with clonal aberrations. TP53 mutations were present in 10.6% of patients (6.4% clonal, 4.2% subclonal), ATM mutations in 11.1% (7.8% clonal, 1.3% subclonal, 2% germ line mutations considered pathogenic), SF3B1 mutations in 12.6% (7.4% clonal, 5.2% subclonal), NOTCH1 mutations in 21.8% (14.2% clonal, 7.6% subclonal), and BIRC3 mutations in 4.2% (2% clonal, 2.2% subclonal). ATM mutations, clonal SF3B1, and both clonal and subclonal NOTCH1 mutations predicted for shorter time to first treatment irrespective of the immunoglobulin heavy-chain variable-region gene (IGHV) mutational status. Clonal and subclonal TP53 and clonal NOTCH1 mutations predicted for shorter overall survival together with the IGHV mutational status. Clonal evolution in longitudinal samples mainly occurred in cases with mutations in the initial samples and was observed not only after chemotherapy but also in untreated patients. These findings suggest that the characterization of the subclonal architecture and its dynamics in the evolution of the disease may be relevant for the management of CLL patients., (© 2016 by The American Society of Hematology.)

Purpose: Germ-line mutations in the exonuclease domains of POLE and POLD1 have been recently associated with polyposis and colorectal cancer (CRC) predisposition. Here, we aimed to gain a better understanding of the phenotypic characteristics of this syndrome to establish specific criteria for POLE and POLD1 mutation screening and to help define the clinical management of mutation carriers., Methods: The exonuclease domains of POLE and POLD1 were studied in 529 kindred, 441 with familial nonpolyposis CRC and 88 with polyposis, by using pooled DNA amplification and massively parallel sequencing., Results: Seven novel or rare genetic variants were identified. In addition to the POLE p.L424V recurrent mutation in a patient with polyposis, CRC and oligodendroglioma, six novel or rare POLD1 variants (four of them, p.D316H, p.D316G, p.R409W, and p.L474P, with strong evidence for pathogenicity) were identified in nonpolyposis CRC families. Phenotypic data from these and previously reported POLE/POLD1 carriers point to an associated phenotype characterized by attenuated or oligo-adenomatous colorectal polyposis, CRC, and probably brain tumors. In addition, POLD1 mutations predispose to endometrial and breast tumors., Conclusion: Our results widen the phenotypic spectrum of the POLE/POLD1-associated syndrome and identify novel pathogenic variants. We propose guidelines for genetic testing and surveillance recommendations.Genet Med 18 4, 325-332.

Germline mutations in UNC5C have been suggested to increase colorectal cancer (CRC) risk, thus causing hereditary CRC. However, the evidence gathered thus far is insufficient to include the study of the UNC5C gene in the routine genetic testing of familial CRC. Here we aim at providing a more conclusive answer about the contribution of germline UNC5C mutations to genetically unexplained hereditary CRC and/or polyposis cases. To achieve this goal we sequenced the coding region and exon-intron boundaries of UNC5C in 544 familial CRC or polyposis patients (529 families), using a technique that combines pooled DNA amplification and massively parallel sequencing. A total of eight novel or rare variants, all missense, were identified in eight families. Co-segregation data in the families and association results in case-control series are not consistent with a causal effect for 7 of the 8 identified variants, including c.1882&#95;1883delinsAA (p.A628K), previously described as a disease-causing mutation. One variant, c.2210G > A (p.S737N), remained unclassified. In conclusion, our results suggest that the contribution of germline mutations in UNC5C to hereditary colorectal cancer and to polyposis cases is negligible.