Hotspot mutations in cancer genes may be missed in routine diagnostics due to neighbouring sequence variants.

The detection of hotspot mutations in key cancer genes is now an essential part of the diagnostic work-up in molecular pathology. Nearly all assays for mutation detection involve an amplification step. A second single nucleotide variant (SNV) on the same allele adjacent to a mutational hotspot can interfere with primer binding, leading to unnoticed allele-specific amplification of the wild type allele and thereby false-negative mutation testing. We present two diagnostic cases with false negative sequence results for JAK2 and SRSF2. In both cases mutations would have escaped detection if only one strand of DNA had been analysed. Because many commercially available diagnostic kits rely on the analysis of only one DNA strand they are prone to fail in cases like these. Detailed protocols and quality control measures to prevent corresponding pitfalls are presented.

Deregulation of RB1 expression by loss of imprinting in human hepatocellular carcinoma.

The tumour suppressor gene RB1 is frequently silenced in many different types of human cancer, including hepatocellular carcinoma (HCC). However, mutations of the RB1 gene are relatively rare in HCC. A systematic screen for the identification of imprinted genes deregulated in human HCC revealed that RB1 shows imprint abnormalities in a high proportion of primary patient samples. Altogether, 40% of the HCC specimens (16/40) showed hyper- or hypomethylation at the CpG island in intron 2 of the RB1 gene. Re-analysis of publicly available genome-wide DNA methylation data confirmed these findings in two independent HCC cohorts. Loss of correct DNA methylation patterns at the RB1 locus leads to the aberrant expression of an alternative RB1-E2B transcript, as measured by quantitative real-time PCR. Demethylation at the intron 2 CpG island by DNMT1 knock-down or aza-deoxycytidine (DAC) treatment stimulated expression of the RB1-E2B transcript, accompanied by diminished RB1 main transcript expression. No aberrant DNA methylation was found at the RB1 locus in hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5) and their corresponding adjacent liver tissue specimens. Deregulated RB1 expression due to hyper- or hypomethylation in intron 2 of the RB1 gene is found in tumours without loss of heterozygosity and is associated with a decrease in overall survival (p = 0.032) if caused by hypermethylation of CpG85. This unequivocally demonstrates that loss of imprinting represents an important additional mechanism for RB1 pathway inactivation in human HCC, complementing well-described molecular defects.

Diagnostic utility of ESR1 mutation detection in liquid biopsy of metastatic breast cancer patients.

Molecular analysis of circulating cell-free DNA (cfDNA) extracted from peripheral blood plasma samples of metastatic breast cancer (BC) patients is of rising interest to find optimal therapeutic strategies. Detection of emerging resistance mutations against endocrine therapy is possible with this approach. Here we present the applicability of a laboratory-developed NGS assay in molecular pathology routine diagnostic, covering four genes with therapeutic (ESR1, PIK3CA, ERBB2) and prognostic (TP53) consequences in metastatic BC. We analyzed 162 liquid biopsy samples and 25 corresponding metastases from metastatic BC patients. In the liquid biopsies, we detected ESR1 mutations in 42 cases (25.9%) and ERBB2 mutations in six cases (3.7%), arguing for a change in therapy to fulvestrant, elacestrant, or neratinib. Furthermore, 17 cases had detectable TP53 mutations, associated with resistance against endocrine therapy. We conclude that liquid biopsy testing is a noninvasive, sensitive, and helpful method to optimize therapeutic decisions in metastatic BC.

Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker.

Epigenetic inactivation by aberrant DNA methylation has been reported for many microRNA genes in various human malignancies. However, relatively little is known about microRNA gene methylation in hepatocellular carcinoma (HCC). Therefore, a systematic screen for identification of aberrantly hypermethylated microRNA genes in HCC was initiated. The methylation status of 39 intergenic CpG island associated microRNA genes was analyzed in HCC cell lines (n = 7), immortalized hepatocytes (n = 2) and normal liver samples (n = 5). Subsequently, 13 differentially methylated microRNA genes were analyzed in primary human HCC samples (n = 40), benign liver tumors (n = 15) and the adjacent liver tissues employing pyrosequencing. Expression of microRNA genes was measured using quantitative real-time polymerase chain reaction (RT-PCR). In addition, DNA methylation and expression of microRNA genes were measured after DNMT1 knockdown or DNMT inhibition. Aberrant hypermethylation and concomitant reduction in expression of intergenic microRNA genes is a frequent event in human HCC: hsa-mir-9-2 (23%), hsa-mir-9-3 (50 %), hsa-mir-124-1 (20%), hsa-mir-124-2 (13%), hsa-mir-124-3 (43%), hsa-mir-129-2 (58%), hsa-mir-596 (28%) and hsa-mir-1247 (38%). Altogether, it affects 90% of the HCC specimens under study. MicroRNA gene methylation is not found in hepatocellular adenoma (n = 10) and focal nodular hyperplasia (n = 5). DNMT1 knockdown or DNMT inhibition reduced microRNA gene methylation and stimulated expression. In primary human HCC specimens hypermethylation and expression of microRNA genes showed an inverse correlation. Concordant hypermethylation of three or more microRNA genes is a highly specific marker for the detection of HCC and for poor prognosis.

Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution.

BACKGROUND: New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed. RESULTS: To this end the methylation patterns of 12 loci (GSTπ1, p16INK4a, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31, and LINE-1) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r2 = 0.927). CONCLUSIONS: Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 as new targets of aberrant DNA methylation in human hepatocellular carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.

Identification of differentially expressed microRNAs in human male breast cancer.

BACKGROUND: The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. METHODS: The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. RESULTS: Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. CONCLUSIONS: Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

Epigenetic inactivation of tumour suppressor gene KLF11 in myelodysplastic syndromes*.

The identification of aberrantly hypermethylated genes may lead to the development of new diagnostic markers and the identification of novel targets of epigenetic therapy in myelodysplastic syndromes (MDS). We therefore investigated the methylation status of transcription factor genes KLF5, KLF11, and MAFB, shown to be aberrantly methylated in myelogeneous leukaemia cells, in a series of 115 MDS patient as well as in 25 control subjects. Using quantitative high-resolution pyrosequencing methodology, KLF11, MAFB, and KLF5 were shown for the first time to be hypermethylated in 17 (15%), 8 (7%), and 2 (1.7%) cases, respectively, but not in any of the patients with an isolated 5q-deletion. Patient samples harbouring KLF11 methylation displayed reduced KLF11 mRNA expression and KLF11 hypermethylation correlated with a high International Prognostic Scoring System score (P < 0.05). In conclusion, epigenetic inactivation and subsequent transcriptional repression of the KLF11 gene is quite frequent in MDS. Patients with an isolated 5q-deletion seem to harbour a distinct epigenetic profile.

Comprehensive genetic and functional characterization of IPH-926: a novel CDH1-null tumour cell line from human lobular breast cancer.

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E-cadherin. ILBCs also lack beta-catenin expression and show aberrant cytoplasmic localization of the E-cadherin binding protein p120-catenin. The lack of a well-characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH-926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH-926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH-926 expressed various epithelial cell markers but lacked expression of E-cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH-926 from this particular tumour. IPH-926 also lacked beta-catenin expression and showed aberrant cytoplasmic localization of p120-catenin. Array-CGH analysis of IPH-926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH-926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH-926 cells were anti-cancer drug-resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH-926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH-926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo.

Feasibility of Combined Detection of Gene Mutations and Fusion Transcripts in Bone Marrow Trephines from Leukemic Neoplasms.

Chromosomal translocations resulting in fusion genes represent important oncogenic drivers and potential therapeutic targets in rare leukemia subtypes. Formalin-fixed, paraffin-embedded trephines are frequently used in hematologic diagnostic tests and provide relevant access to leukemic cells for further studies, for example, phenotyping in bone marrow fibrosis. However, high-throughput molecular analysis of nucleic acids obtained from this material is challenging, especially the reliable detection of RNA transcripts. Sixty-three formalin-fixed, paraffin-embedded bone marrow trephines of patients with chronic eosinophilic leukemia, chronic myeloid leukemia, acute myeloid leukemia, and myeloproliferative neoplasms were analyzed for gene mutations and the presence of fusion transcripts with a commercial amplicon-based next-generation sequencing approach. Fusion transcripts relevant for diagnosis and therapy could be detected and validated (by RT-PCR) in 25 patients (39.7%). Retrospectively selected material, up to 10 years old, was used for this purpose, and only one sample failed in the RNA analysis (1.6%). This study concludes that amplicon-based fusion transcript detection in bone marrow trephines is feasible and that bone marrow trephines taken for histologic assessment can also be applied for high-throughput molecular analysis.

Mutations associated with age-related clonal hematopoiesis in PMF patients with rapid progression to myelofibrosis.

Besides histopathological findings there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPN). Age-related clonal hematopoiesis (ARCH/CHIP) is a frequent finding in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared with prefibrotic PMF samples without development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, and DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations which are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of the tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when manifest already at first presentation.

LINE-1 hypomethylation in human hepatocellular carcinomas correlates with shorter overall survival and CIMP phenotype.

Reactivation of interspersed repetitive sequences due to loss of methylation is associated with genomic instability, one of the hallmarks of cancer cells. LINE-1 hypomethylation is a surrogate marker for global methylation loss and is potentially a new diagnostic and prognostic biomarker in tumors. However, the correlation of LINE-1 hypomethylation with clinicopathological parameters and the CpG island methylator phenotype (CIMP) in patients with liver tumors is not yet well defined, particularly in Caucasians who show quite low rates of HCV/HBV infection and a higher incidence of liver steatosis. Therefore, quantitative DNA methylation analysis of LINE-1, RASSF1A, and CCND2 using pyrosequencing was performed in human hepatocellular carcinomas (HCC, n = 40), hepatocellular adenoma (HCA, n = 10), focal nodular hyperplasia (FNH, n = 5), and corresponding peritumoral liver tissues as well as healthy liver tissues (n = 5) from Caucasian patients. Methylation results were correlated with histopathological findings and clinical data. We found loss of LINE-1 DNA methylation only in HCC. It correlated significantly with poor survival (log rank test, p = 0.007). An inverse correlation was found for LINE-1 and RASSF1A DNA methylation levels (r2 = -0.47, p = 0.002). LINE-1 hypomethylation correlated with concurrent RASSF1/CCND2 hypermethylation (Fisher's exact test, p = 0.02). Both LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation were not found in benign hepatocellular tumors (HCA and FNH). Our results show that LINE-1 hypomethylation and RASSF1A/CCND2 hypermethylation are epigenetic aberrations specific for the process of malignant liver transformation. In addition, LINE-1 hypomethylation might serve as a future predictive biomarker to identify HCC patients with unfavorable overall survival.

Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology.

BACKGROUND: During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology. RESULTS: A statistically highly significant correlation (Spearman r: 0.78 - 0.90, p < 0.0001) was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology. CONCLUSION: Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology.

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice: A Cross-Platform Comparison of Three Different Molecular Methods for Mutation Detection.

Circulating cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its metastases. Plasma cfDNA represents an alternative material for molecular monitoring of patients under therapy. Herein, we present a cross-platform comparison of three different molecular methods [digital PCR, next-generation sequencing (NGS), and quantitative PCR] to detect clinically relevant mutations in cfDNA. We validated our workflow with commercially available cfDNA reference material (5.0%, 1.0%, and 0.1% mutation frequency, respectively). Digital PCR and NGS detect reliably 0.1% allele frequency of the EGFR p.T790M mutation. Furthermore, we analyzed 55 cfDNA preparations from patients with lung cancer to compare reliability and sensitivity of the three methods under routine conditions and achieved 96.0% concordance of p.T790M results. A limit of detection for mutation calling using digital PCR (>0.1%) and NGS (>0.2%) was established. In total, 62.5% of known primary EGFR mutations were successfully detected in cfDNA. In 56.0% of the patients with detectable EGFR primary mutations, we identified a resistance conferring the EGFR p.T790M mutation.

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma.

AIM: To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC). METHODS: Knockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables. RESULTS: miRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes. CONCLUSION: Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.

Promoter hypermethylation of the death-associated protein kinase gene in breast cancer is associated with the invasive lobular subtype.

Expression of death-associated protein (DAP) kinase, a proapoptotic serine/threonine protein kinase, is frequently lost in human tumors. In a study of 134 primary breast cancer specimens hypermethylation of the DAP kinase gene was found in 13% of cases. A highly significant difference (P < 0.001) of DAP kinase inactivation was observed between invasive lobular cancer (n = 19) and invasive ductal cancer (n = 85; 53% versus 9%, respectively). Hypermethylation correlated with loss of RNA expression, estrogen receptor positivity (P < 0.01), and the absence of p53 overexpression (P < 0.01). In contrast to invasive lobular cancer, the in situ-growing precursor lesion lacked epigenetic modification of the DAP kinase promotor by aberrant methylation indicating a potential role in tumor progression. Unlike the DAP kinase gene, hypermethylation of the cyclin D2 and RASSF1A genes did not correlate with a particular histological subtype or to invasiveness [corrected]. We conclude that different histological subtypes of breast cancer may not only differ concerning specific chromosomal abnormalities and DNA mutations but also with regard to epigenetic inactivation patterns.

Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms.

Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions.To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847-1,460,412) and a mean coverage of 2,702 (range 707-6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information.

Down-regulation of the IGF-2/H19 locus during normal and malignant hematopoiesis is independent of the imprinting pattern.

H19 and IGF-2 are two growth regulatory genes located on chromosome 11p15 implicated in tumorigenesis. Both genes are imprinted and regulated reciprocally under many circumstances. In order to elucidate the contribution of H19 and IGF-2 to leukemogenesis, the mRNA expression level of both genes were quantitated in bone marrow biopsies and peripheral blood samples from normal (n=98), chronic myelomonocytic leukemia (CMML, n=43), chronic myelogenous leukemia (CML, n=40) and, acute myelogenous leukemia (AML, n=32) cases. A concomitant reduction of H19 and IGF-2 expression was observed in all leukemic samples compared to the healthy controls. This down-regulation was not accompanied by changes in methylation of the differentially methylated region (DMR). Whereas the H19 gene showed strict monoallelic expression in all informative normal (n=31) and leukemic (n=54) samples, the imprinting pattern of the IGF2 gene was found to be heterogeneous. No correlations between imprinting status (mono- versus biallelic expression), quantitative mRNA expression levels and course of disease were found for the IGF-2 gene. The data suggest a disturbed regulation of the IGF-2/H19 locus in myeloid leukemias which is not caused by loss of imprinting.

Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival.

BACKGROUND: Aberrant DNA methylation at imprinted loci is an important molecular mechanism contributing to several developmental and pathological disorders including cancer. However, knowledge about imprinting defects due to DNA methylation changes is relatively limited in hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide. Therefore, comprehensive quantitative DNA methylation analysis at imprinted loci showing ~50 % methylation in healthy liver tissues was performed in primary HCC specimens and the peritumoural liver tissues. RESULTS: We found frequent and extensive DNA methylation aberrations at many imprinted loci in HCC. Unsupervised cluster analysis of DNA methylation patterns at imprinted loci revealed subgroups of HCCs with moderate and severe loss of methylation. Hypomethylation at imprinted loci correlated significantly with poor overall survival (log-rank test, p = 0.02). Demethylation at imprinted loci was accompanied by loss of methylation at LINE-1, a commonly used marker for global DNA methylation levels (p < 0.001). In addition, we found that loss of methylation at imprinted loci correlated with the presence of a CTNNB1 mutation (Fisher's exact test p = 0.03). Re-analysis of publically available genome-wide methylation data sets confirmed our findings. The analysis of benign liver tumours (hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH)), the corresponding adjacent liver tissues, and healthy liver tissues showed that aberrant DNA methylation at imprinted loci is specific for HCC. CONCLUSIONS: Our analyses demonstrate frequent and widespread DNA methylation aberrations at imprinted loci in human HCC and identified a hypomethylated subgroup of patients with shorter overall survival.