Methylation of the nonhomologous end joining repair pathway genes does not explain the increase of translocations with aging

Chromosome translocations are especially frequent in human lymphomas and leukemias but are insufficient to drive carcinogenesis. Indeed, several of the so-called tumor specific translocations have been detected in peripheral blood of healthy individuals, finding a higher frequency of some of them with aging. The inappropriate repair of DNA double strand breaks by the nonhomologous end joining (NHEJ) pathway is one of the reasons for a translocation to occur. Moreover, fidelity of this pathway has been shown to decline with age. Although the mechanism underlying this inefficacy is unknown, other repair pathways are inactivated by methylation with aging. In this study, we analyzed the implication of NHEJ genes methylation in the increase of translocations with the age. To this aim, we determined the relationship between translocations and aging in 565 Spanish healthy individuals and correlated these data with the methylation status of 11 NHEJ genes. We found higher frequency of BCL2-JH and BCR-ABL (major) translocations with aging. In addition, we detected that two NHEJ genes (LIG4 and XRCC6) presented age-dependent promoter methylation changes. However, we did not observe a correlation between the increase of translocations and methylation, indicating that other molecular mechanisms are involved in the loss of NHEJ fidelity with aging.Fil: Martin Guerrero, Idoia. Universidad del Pais Vasco; EspañaFil: de Prado, Elena. Universidad del Pais Vasco; EspañaFil: López López, Elixabet. Universidad del Pais Vasco; EspañaFil: Ardanaz, Maite. Hospital Txagorritxu; EspañaFil: Vitoria, Juan Carlos. Hospital de Cruces; EspañaFil: Parada, Luis Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: García Orad, Cristina. Hospital General Valencia; EspañaFil: García Orad, Africa. BioCruces Health Research Institute; España. Universidad del Pais Vasco; Españ

EMBR-25. Genome-wide genetic and epigenetic assessment of group 4 Medulloblastoma for improved, biomarker driven, prognostication and risk-stratification

Introduction: Medulloblastoma (MB) is the most common malignant brain tumour in children. The most frequent molecular subgroup, Group 4 (MBGrp4) accounts for ~35/40% of cases, however it has the least understood underlying biology. Clinical outcomes are heterogeneous in MBGrp4 and are not accounted for by established clinico-pathological risk factors. There is now a requirement for a comprehensive study of MBGrp4, considering established clinico-pathological features and novel molecular biomarkers to enhance risk-stratification and identify novel therapeutic targets. Methods: A clinically-annotated, retrospective MBGrp4 discovery cohort (n = 420) was generated from UK CCLG institutions, collaborating European centres and SIOP-UKCCSG-PNET3 and HIT-SIOP-PNET4 clinical trials. Contemporary, multi-omics profiling was performed. Focal and arm level copy number aberrations (CNAs) were determined from molecular inversion probe (MIP) or DNA methylation array which additionally provided next generation non-WNT/non-SHH (Grp3/Grp4) subtype classifications. Targeted next-generation DNA sequencing was performed to overlay the mutational landscape. Survival modelling was carried out with patients >3 years old who received craniospinal irradiation. Results: MBGrp4 subtypes were assigned to 88% of tumours with available data. Subtype VIII was strongly associated with i17q (p<0.0001). The favourable-risk cytogenetic signature (2 or 3 of; chromosome 7 gain, chromosome 8 loss and/or chromosome 11 loss) associated with both subtypes VI and VII (p<0.0001). MYCN amplifications were strongly associated with subtype V (p<0.0001) in addition to 16q loss (p<0.0001). The high-risk CNA group was enriched for mutations in genes involved in chromatin remodelling (p<0.0001). Risk factors were identified from multivariate survival modelling. Subtype and CNA groups contributed to improved risk-stratification models that outperformed current clinical schemes. Conclusion: Comprehensive genetic and epigenetic profiling in this large retrospective cohort has improved our understanding of the molecular and clinical heterogeneity within MBGrp4. Incorporation of molecular biomarkers improved risk-stratification for MBGrp4

Pipeline for Large-Scale Microdroplet Bisulfite PCR-Based Sequencing Allows the Tracking of Hepitype Evolution in Tumors

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into “hepitypes” and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer

MEDB-65. Molecular subclassification of a national cohort of pediatric medulloblastoma based on methylation profile

INTRODUCTION: Pediatric Medulloblastoma (MB) accounts for approximately 20% of all childhood brain tumors. Molecular subgroups namely WNT, SHH, Group 3 and Group 4, exhibit divergent biology, and clinical outcomes. DNA methylation analysis is a robust option to classify pediatric MB into molecular subgroups, which allows the optimization of diagnosis and stratification of the treatment. We review the first experience of molecular subclassification carried out at the national level in our country. METHODS: Multi-center centralized prospective and retrospective study of frozen tumor samples at diagnosis from pediatric MB patients diagnosed in Spanish hospitals, from April 2021 to December 2021. A registry was created with histology review, immunohistochemical (IHC) subgrouping, and a molecular subgrouping based on the Minimal Methylation Classifier (MIMIC) from Schwalbe et al., 2017. The time from the sample centralized reception to the study result was also collected. RESULTS: 25 frozen MB tumor samples from patients at diagnosis were included. 6 were retrospective and 19 prospective. IHC classified 19 cases (76%) as non-WNT/non-SHH MBs, 3 (12%) as WNT-activated and 3 (12%) as SHH-activated. MIMIC classified, in the non-WNT/non-SHH, 6 tumors (24%) as Group 3 and 13 (52%) as Group 4. 2 cases (8%) were WNT-activated MBs and 3 (12%) were SHH-activated MBs. Only 1 case (4%) was unclassified by MIMIC (WNT using IHC). Comparing both methods (IHC and MIMIC), diagnosis agreed in 96% of cases. The response time ranged from 5 to 10 days. CONCLUSIONS: DNA methylation profiling has proven to be a robust and quick option to classify MB into subgroups and it correlates with the IHC diagnosis. This tool was successfully implemented in our national routine diagnosis, enabling a reliable and rapid molecular subgrouping classification

microRNA sequencing for biomarker detection in the diagnosis, classification and prognosis of Diffuse Large B Cell Lymphoma

Abstract Despite being considered a single disease, Diffuse Large B Cell Lymphoma (DLBCL) presents with variable backgrounds, which results in heterogeneous outcomes among patients, with 40% of them still having primary refractory disease or relapse. Thus, novel biomarkers are needed. In addition, multiple factors regarding its pathogenesis remain unclear. In this context, recent investigations point to the relevance of microRNAs (miRNAs) in cancer. However, regarding DLBCL, there is inconsistency in the data reported. Therefore, in this work, the main goals were to determine a miRNA set with utility as biomarkers for DLBCL diagnosis, classification, prognosis and treatment response, as well as to decipher the mechanism of action of deregulated miRNAs in the origin of the disease. We analyzed miRNA expression in a cohort of 78 DLBCL patients and 17 controls using small RNA sequencing and performed a miRNA-mRNA interaction network analysis. This way, we were able to define new miRNA expression signatures for diagnosis, classification, treatment response and prognosis, and we identified plausible mechanisms of action by which deregulated miRNAs could be involved in DLBCL pathogenesis. In summary, our study remarks that miRNAs could play an important role in DLBCL

Genetic Variants in MiRNA Processing Genes and Pre-MiRNAs Are Associated with the Risk of Chronic Lymphocytic Leukemia

<div><p>Genome wide association studies (GWAS) have identified several low-penetrance susceptibility alleles in chronic lymphocytic leukemia (CLL). Nevertheless, these studies scarcely study regions that are implicated in non-coding molecules such as microRNAs (miRNAs). Abnormalities in miRNAs, as altered expression patterns and mutations, have been described in CLL, suggesting their implication in the development of the disease. Genetic variations in miRNAs can affect levels of miRNA expression if present in pre-miRNAs and in miRNA biogenesis genes or alter miRNA function if present in both target mRNA and miRNA sequences. Therefore, the present study aimed to evaluate whether polymorphisms in pre-miRNAs, and/or miRNA processing genes contribute to predisposition for CLL. A total of 91 SNPs in 107 CLL patients and 350 cancer-free controls were successfully analyzed using TaqMan Open Array technology. We found nine statistically significant associations with CLL risk after FDR correction, seven in miRNA processing genes (rs3805500 and rs6877842 in <i>DROSHA</i>, rs1057035 in <i>DICER1</i>, rs17676986 in <i>SND1</i>, rs9611280 in <i>TNRC6B</i>, rs784567 in <i>TRBP</i> and rs11866002 in <i>CNOT1</i>) and two in pre-miRNAs (rs11614913 in miR196a2 and rs2114358 in miR1206). These findings suggest that polymorphisms in genes involved in miRNAs biogenesis pathway as well as in pre-miRNAs contribute to the risk of CLL. Large-scale studies are needed to validate the current findings.</p></div

Secondary structures of the miRNAs showing significant SNPs.

<p>(A) miR196a2 and (B) miR1206, predicted by the RNAfold tool.</p