A novel entity of massive multifocal osteolyses in the elderly.

Osteolyses are common findings in elderly patients and most frequently represent malignant or locally aggressive bone tumors, infection, inflammatory and endocrine disorders, histiocytoses, and rare diseases such as Gorham-Stout syndrome. We here report on a novel entity of massive multifocal osteolyses in both shoulders, the right hip and left knee joint and the dens of an 83-year-old patient not relatable to any previously known etiopathology of bone disorders. The soft tissue mass is of myxoid stroma with an unspecific granulomatous inflammatory process, aggressively destroying extensive cortical and cancellous bone segments and encroaching on articulating bones in diarthrodial large joints. Radiological, nuclear medical, serological, histological, and immunohistochemical analyses were incapable of further classifying the disease pattern within the existing scheme of pathology. Quantitative polymerase chain reaction and next generation sequencing revealed that mutations are not suggestive of any known hereditary or acquired bone disease. Possible treatment options include radionuclide therapy for pain palliation and percutaneous radiation to arrest bone resorption while surgical treatment is inevitable for pathological fractures. This case study shall increase the awareness of the musculoskeletal community and motivate to collect further information on this rare but mutilating disorder.

Mixed Adenocarcinomatous and Neuroendocrine Tumor of the Urinary Bladder With Concomitant Carcinoma In Situ: A Case Report With a Comprehensive Immunohistochemical Analysis and Review of the Literature.

Mixed adenoneuroendocrine carcinomas are rare and usually occur in the gastrointestinal tract. Although there have been several investigations regarding their developmental mechanism, the molecular origin of these tumors remains unclear. In this article, we present an exceedingly rare case of a mixed tumor of the urinary bladder with an adenocarcinomatous and a neuroendocrine component and a concomitant urothelial carcinoma in situ (CIS). Due to this extraordinary combination of tumor components, our goal was to extensively examine the 3 tumor components with regard to a representable common origin. Therefore, a comprehensive immunohistochemical analysis and review of the literature was performed. Besides expected outcome, our examination also revealed surprising staining results. Urothelial CIS, like the adenocarcinomatous component, showed strong staining for CDX2. In addition, parts of the adenocarcinoma were positive for synaptophysin like the neuroendocrine tumor component. All 3 components showed a significant overexpression of p53 and a moderate to strong membranous and cytoplasmatic staining for ß-catenin. To our knowledge, we are the first to describe a case of a mixed tumor of the urinary bladder with an adenocarcinomatous and a neuroendocrine component and a concomitant CIS. The components share striking molecular features that argue for a common clonal origin and a development of the invasive tumor via the urothelial precursor lesion.

Clonal evolution and heterogeneity in metastatic head and neck cancer-An analysis of the Austrian Study Group of Medical Tumour Therapy study group.

BACKGROUND: Tumour heterogeneity and clonal evolution within a cancer patient are deemed responsible for relapse in malignancies and present challenges to the principles of targeted therapy, for which treatment modality is often decided based on the molecular pathology of the primary tumour. Nevertheless, the clonal architecture in distant relapse of head and neck cancer is fairly unknown. PATIENTS AND METHODS: For this project, we analysed a cohort of 386 patients within the Austrian Registry of head and neck cancer. We identified 26 patients with material from the primary tumour, the distant metastasis after curative first-line treatment and a germline sample for analysis of clonal evolution. After pathological analyses, these samples were analysed using a targeted massively parallel sequencing (MPS) panel of 257 genes known to be recurrently mutated in head and neck cancer plus a genome-wide SNP-set. RESULTS: Despite histological diagnosis of distant metastasis, no corresponding mutation in the supposed metastases was found in two of 23 (8.6%) evaluable patients suggesting a primary tumour of the lung instead of a distant metastasis of head and neck cancer. We observed a branched pattern of evolution in 31.6% of the analysed patients. This pattern was associated with a shorter time to distant metastasis, compared with a pattern of punctuated evolution. Structural genomic changes over time were also present in 7 of 12 (60%) evaluable patients with metachronous metastases. CONCLUSION: Targeted MPS demonstrated substantial heterogeneity at the time of diagnosis and a complex pattern of evolution during disease progression in head and neck cancer. Copy number analyses revealed additional changes that were not detected by mutational analyses. Mutational and structural changes contribute to tumour heterogeneity at diagnosis and progression.

Risk-factors for nodular hyperplasia of parathyroid glands in sHPT patients.

INTRODUCTION: Nodular hyperplasia of parathyroid glands (PG) is the most probable cause of medical treatment failure in secondary hyperparathyroidism (sHPT). This prospective cohort study is located at the interface of medical and surgical consideration of sHPT treatment options and identifies risk-factors for nodular hyperplasia of PG. MATERIAL AND METHODS: One-hundred-eight resected PG of 27 patients with a broad spectrum of sHPT severity were classified according to the degree of hyperplasia by histopathology. Twenty routinely gathered parameters from medical history, ultrasound findings of PG and laboratory results were analyzed for their influence on nodular hyperplasia of PG by risk-adjusted multivariable binary regression. A prognostic model for non-invasive assessment of PG was developed and used to weight the individual impact of identified risk-factors on the probability of nodular hyperplasia of single PG. RESULTS: Independent risk-factors for nodular hyperplasia of single PG were duration of dialysis in years, PG volume in mm3 determined by ultrasound and serum level of parathyroid hormone in pg/mL. Multivariable analyses computed a model with an Area Under the Receiver Operative Curve of 0.857 (95%-CI:0.773-0.941) when predicting nodular hyperplasia of PG. Theoretical assessment of risk-factor interaction revealed that the duration of dialysis had the strongest influence on the probability of nodular hyperplasia of single PG. CONCLUSIONS: The three identified risk-factors (duration of dialysis, PG volume determined by ultrasound and serum level of parathyroid hormone) can be easily gathered in daily routine and could be used to non-invasively assess the probability of nodular hyperplasia of PG. This assessment would benefit from periodically collected data sets of PG changes during the course of sHPT, so that the choice of medical or surgical sHPT treatment could be adjusted more to the naturally changing type of histological PG lesion on an individually adopted basis in the future.

Clonal evolution in relapsed and refractory diffuse large B-cell lymphoma is characterized by high dynamics of subclones.

Little information is available about the role of certain mutations for clonal evolution and the clinical outcome during relapse in diffuse large B-cell lymphoma (DLBCL). Therefore, we analyzed formalin-fixed-paraffin-embedded tumor samples from first diagnosis, relapsed or refractory disease from 28 patients using next-generation sequencing of the exons of 104 coding genes. Non-synonymous mutations were present in 74 of the 104 genes tested. Primary tumor samples showed a median of 8 non-synonymous mutations (range: 0-24) with the used gene set. Lower numbers of non-synonymous mutations in the primary tumor were associated with a better median OS compared with higher numbers (28 versus 15 months, p=0.031). We observed three patterns of clonal evolution during relapse of disease: large global change, subclonal selection and no or minimal change possibly suggesting preprogrammed resistance. We conclude that targeted re-sequencing is a feasible and informative approach to characterize the molecular pattern of relapse and it creates novel insights into the role of dynamics of individual genes.

The clinical significance of fibrinogen plasma levels in patients with diffuse large B cell lymphoma.

BACKGROUND: Fibrinogen plays a crucial role in the pathophysiology of tumour cell growth, invasion and metastasis. The aim of this study was to evaluate the prognostic significance of pretreatment plasma fibrinogen levels in patients with diffuse large B cell lymphoma (DLBCL) METHODS: Data from 372 patients with DLBCL, diagnosed and treated between 2004 and 2013 at two Austrian centres, were evaluated retrospectively. The prognostic influences of plasma fibrinogen levels and other factors, including age, tumour stage and the National Comprehensive Cancer Network-International Prognostic Index, on 5-year overall survival (OS) and 5-year disease-free survival (DFS) were studied using Kaplan-Meier curves as well as univariate and multivariate Cox regression models. RESULTS: Kaplan-Meier analysis revealed that a high fibrinogen plasma level is associated with decreased 5-year OS and 5-year DFS in patients with DLBCL (p<0.001, log-rank test). Furthermore, in multivariate analysis, elevated serum fibrinogen was found to be an independent marker of poor clinical outcome: 5-year OS (HR=1.69, 95% CI 1.06 to 2.72, p=0.029) and 5-year DFS (HR=1.68, 95% CI 1.08 to 2.61, p=0.021). CONCLUSIONS: In the current study, we demonstrate that high plasma fibrinogen levels at diagnosis predict poor outcome in patients with DLBCL. TRIAL REGISTRATION NUMBER: 25-434 ex 12713 and 415-EP/73/127-2012.

Independent Prognostic Value of Serum Markers in Diffuse Large B-Cell Lymphoma in the Era of the NCCN-IPI.

BACKGROUND: Several serum parameters have been evaluated for adding prognostic value to clinical scoring systems in diffuse large B-cell lymphoma (DLBCL), but none of the reports used multivariate testing of more than one parameter at a time. The goal of this study was to validate widely available serum parameters for their independent prognostic impact in the era of the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) score to determine which were the most useful. PATIENTS AND METHODS: This retrospective bicenter analysis includes 515 unselected patients with DLBCL who were treated with rituximab and anthracycline-based chemoimmunotherapy between 2004 and January 2014. RESULTS: Anemia, high C-reactive protein, and high bilirubin levels had an independent prognostic value for survival in multivariate analyses in addition to the NCCN-IPI, whereas neutrophil-to-lymphocyte ratio, high gamma-glutamyl transferase levels, and platelets-to-lymphocyte ratio did not. CONCLUSIONS: In our cohort, we describe the most promising markers to improve the NCCN-IPI. Anemia and high C-reactive protein levels retain their power in multivariate testing even in the era of the NCCN-IPI. The negative role of high bilirubin levels may be associated as a marker of liver function. Further studies are warranted to incorporate these markers into prognostic models and define their role opposite novel molecular markers.

Anti-Hu Antibody Associated Paraneoplastic Cerebellar Degeneration in Head and Neck Cancer.

BACKGROUND: Paraneoplastic syndromes are most frequently associated with small cell lung carcinoma, hematologic and gynecologic malignancies while reports in head and neck cancer are rare. CASE PRESENTATION: We present the case of a 60-year old female patient who developed paraneoplastic cerebellar degeneration upon locoregional recurrence of a poorly differentiated spindle cell carcinoma of the nasal cavity and paranasal sinus. The neurological symptoms, especially ataxia, stabilized after resection of tumor recurrence and concomitant chemoradiotherapy whereas anti-Hu-antibodies remained positive. Despite the unfavorable prognosis of paraneoplastic neurological disorders associated with onconeural antibodies, the patient achieved long-standing stabilization of neurological symptoms. CONCLUSION: We report the first patient with anti-Hu antibodies and paraneoplastic cerebellar degeneration associated with a spindle cell carcinoma of the head and neck. We recommend that evaluation of neurological symptoms in patients with this tumor entity should also include paraneoplastic syndromes as differential diagnoses and suggest early extensive screening for onconeural antibodies.

CYP39A1 polymorphism is associated with toxicity during intensive induction chemotherapy in patients with advanced head and neck cancer.

BACKGROUND: Induction chemotherapy incorporating docetaxel, cisplatin and 5- fluorouracil before radiotherapy may improve the outcome of patients with advanced head and neck cancer. Nevertheless, the addition of docetaxel increases hematological toxicity and infectious complications. Therefore, genetic markers predicting toxicity and efficacy of this treatment regimen may help to identify patients, who would have the most benefit from this intensive treatment. METHODS: A cohort of 78 patients with advanced head and neck cancer treated with induction chemotherapy was assessed for clinical outcome and toxicity during treatment with curative intention. Genetic polymorphisms primary associated with treatment efficacy (ERCC2-rs13181, rs1799793, ERCC1-rs3212986, rs11615, XRCC1-rs25487) or with docetaxel caused toxicity (CYP39A1-rs7761731, SLCO1B3-rs11045585) were evaluated in all patients. The results of these analyses were correlated with the clinical outcome of the patients (loco regional control, progression free survival, overall survival) and treatment related toxicity during induction chemotherapy. RESULTS: Median progression free survival and overall survival was 20 and 31 months in an intention to treat analysis, respectively. Overall response rate to induction chemotherapy was high with 78.1 % of all patients. None of the polymorphisms tested was associated with the clinical outcome of the patients. Genotype A of the CYP39A1 rs7761731 polymorphism was associated with a higher incidence of leucopenia and infections or death during induction chemotherapy. CONCLUSIONS: Intensive induction chemotherapy results in a high response rate in the majority of patients. None of the polymorphisms tested was associated with the clinical outcome of the patients. The CYP39A1 polymorphism rs7761731 may help to identify patients at high risk for treatment related toxicity.

A modified scoring of the NCCN-IPI is more accurate in the elderly and is improved by albumin and ß2 -microglobulin.

The International Prognostic Index (IPI) has been used for decades in diffuse large B-cell lymphoma (DLBCL). A retrospective cancer registry analysis in North America showed significantly improved results when an enhanced IPI, the National Comprehensive Cancer Network (NCCN)-IPI was applied. This novel score puts more weight on age and high levels of lactate dehydrogenase (LDH). Nevertheless, it remains unclear if these results can be extrapolated to the general population. This retrospective bi-centre analysis included 499 unselected DLBCL patients who were treated with rituximab and anthracycline-based chemoimmunotherapy between 2004 and 2013. In our cohort, the NCCN-IPI was more accurate in identifying patients at low or high risk, despite older age, and more patients with increased LDH. Nevertheless, a modified scoring of the risk factors was required to more accurately identify elderly patients with a very favourable diagnosis, suggesting an impaired value of the original NCCN-IPI in the elderly. Serum ß2 -microglobulin and albumin were retained as independent prognostic factors for survival in a multivariate analysis. Our data confirm, for the first time, the superior prognostic power of the NCCN-IPI in an unselected, middle-European cohort. We furthermore propose a modified NCCN-IPI for more accurate prognostication in the elderly. Albumin and ß2 -microglobulin levels are likely to add significant information to the NCCN-IPI.

Presence of small parathyroid glands in renal transplant patients supports less-than-total parathyroidectomy to treat hypercalcemic hyperparathyroidism.

BACKGROUND: Parathyroid glands (PG) are rarely analyzed in renal transplant (RTX) patients. This study analyzes comparatively PG of RTX and end-stage renal disease (ESRD) patients. The clinical part of the study evaluates if total parathyroidectomy with autotransplantation (TPT+AT) treats appropriately hypercalcemic hyperparathyroidism in RTX patients. METHODS: TPT+AT was performed in 15 of 23 RTX and 21 of 27 ESRD patients. Remaining patients underwent less-than-total PT. Volume and stage of hyperplasia were determined from 86 PG of RTX and 109 PG of ESRD patients. Patients were categorized according to the presence of small PG (volume < 100 mm(3)). Calcium homeostasis and hyperparathyroidism were evaluated 2 years after PT in RTX patients. RESULTS: PG of RTX patients were significantly smaller, but similar hyperplastic in comparison to PG of ESRD patients. Small PG were more frequent in RTX than in ESRD patients (19% vs 6%) and mainly graded normal or diffuse hyperplastic (94%). Forty-seven percent of RTX, but only 14% of ESRD, patients receiving a total PT possessed ≥1 small PG (P < .05). Overall, PT treated successfully hypercalcemic hyperparathyroidism. However, TPT+AT caused permanent hypocalcemia in 50% of RTX patients without small PG and even in 83% of RTX patients with small PG. All RTX patients receiving less-than-total PT were normocalcemic at 2-year follow-up. Logistic regression revealed a 10.7 times greater risk of permanent hypocalcemia in RTX patients with small PG receiving TPT+AT compared with RTX patients without small PG receiving TPT+AT or RTX patients undergoing less-than-total PT. CONCLUSION: Surgeons performing PT should be aware of the high frequency of small and less diseased PG in RTX patients. In this context, TPT+AT might overtreat hypercalcemic hyperparathyroidism in RTX patients, especially when small PG are present.

SFRP1 CpG island methylation locus is associated with renal cell cancer susceptibility and disease recurrence.

Loss of the secreted Fzd-related protein 1 (SFRP1) and concurrent alteration of the SFRP1/WNT pathway are frequently observed in human cancers such as in renal cell cancer (RCC). Whether methylation of a SFRP1 CpG island locus in normal human solid tissues is associated with increased tissue specific cancer risk has not been determined to date. Here we measure the cancer risk attributable to SFRP1 DNA methylation in renal tissue. Pyrosequencing of bisulfite treated DNA was used for a case-control study including 120 normal-appearing renal tissues of autopsy specimens and 72 normal-appearing tissues obtained from tumor adjacent areas, and a cross sectional study of 96 RCCs. Association of methylation with demographic risk factor age, clinicopathological parameters and course of patients was investigated. We show significant hypermethylation of a SFRP1 CpG island locus in normal-appearing renal tissues from RCC patients compared with normal-appearing autopsy kidney tissues. Inter quartile analysis revealed a 6-, 13- and 11-fold increased cancer risk for the second, third and fourth quartiles of methylation in the age matched subgroup of tissues (p = 0.001, p = 1.3E-6, p = 6.9E-6). Methylation in autopsy tissues increased with age and methylation in tumors was an independent predictor of recurrence free survival. SFRP1 DNA methylation, accumulates with age in normal-appearing kidney tissues and is associated with increased renal cancer risk, suggesting this CGI sub region as an epigenetic susceptibility locus for RCC. Our data underline the need to further analyze the tissue specific risks conferred by methylated loci for the development of human cancers.

GATA5 CpG island methylation in renal cell cancer: a potential biomarker for metastasis and disease progression.

UNLABELLED: GATA5 CpG island (CGI) methylation and transcriptional inactivation is involved in colorectal and gastric cancer. Whether DNA methylation of GATA5 affects clinical pathology is still unclear. In the present study, we analysed, for the first time, CGI methylation in RCC and its association with clinicopathological parameters and progression-free survival of patients. We show for the first time GATA5 CGI hypermethylation in RCC. Moreover, we found out that increased methylation is statistically associated with status of metastasis, progressive disease and shortened progression-free survival. The present study underline the necessity for further functional investigations as well as prospective survival analyses to clarify whether GATA5 promoter methylation can provide independent information for future clinical management of patients with RCC. OBJECTIVE: To investigate whether GATA5 CpG island (CGI) methylation occurs in renal cell carcinoma (RCC) and is associated with clinical, histopathological characteristics or progression-free survival of patients. PATIENTS AND METHODS: Methylation was quantified in 117 RCC samples and 89 paired adjacent normal tissues using quantitative combined bisulphite restriction analysis (COBRA). COBRA was evaluated in advance by pyrosequencing analyses of control RCC cell lines (coefficient of correlation, R = 0.95). Statistical analyses were carried out using the paired t-test for matched tumour tissue (TU) and adjacent normal tissue (adN) samples, logistic regression for comparisons of independent sample groups and Cox regression for analysis of progression-free survival. RESULTS: In the present study, we found a significant higher mean relative methylation in TU (20.4%) than in adN (7.9%, P < 0.001) in paired samples of all RCCs. Increased GATA5 methylation in tumours was associated with metastasis (P = 0.005) and decreased progression-free survival (P = 0.005, HR = 4.59) in the clear-cell RCC (ccRCC) group. CGI methylation in advanced ccRCCs (pT ≥3 and/or N1, M1 or G2-3/G3) exceeds those detected in localized tumours (pT ≤2, N0, M0, G1/G1-2) (27.8% vs 11.0%, P < 0.001). CONCLUSIONS: The association of GATA5 hypermethylation with metastasis and progression-free survival of patients indicates that epigenetic alterations of GATA5 participate in renal cell carcinogenesis. Moreover, GATA5 CGI methylation could serve as a biomarker for tumour progression, although prospective and functional investigations are necessary to clarify whether independent information for future clinical management of patients with RCC can be obtained.

Caveolin 1 protein expression in renal cell carcinoma predicts survival.

BACKGROUND: Caveolae play a significant role in disease phenotypes such as cancer, diabetes, bladder dysfunction, and muscular dystrophy. The aim of this study was to elucidate the caveolin-1 (CAV1) protein expression in renal cell cancer (RCC) and to determine its potential prognostic relevance. METHODS: 289 clear cell RCC tissue specimens were collected from patients undergoing surgery for renal tumors. Both cytoplasmic and membranous CAV1 expression were determined by immunohistochemistry and correlated with clinical variables. Survival analysis was carried out for 169 evaluable patients with a median follow up of 80.5 months (interquartile range (IQR), 24.5-131.7 months). RESULTS: A high CAV1 expression in the tumor cell cytoplasm was significantly associated with male sex (p = 0.04), a positive nodal status (p = 0.04), and poor tumor differentiation (p = 0.04). In contrast, a higher than average (i.e. > median) CAV1 expression in tumor cell membranes was only linked to male sex (p = 0.03). Kaplan-Meier analysis disclosed significant differences in 5-year overall (51.4 vs. 75.2%, p = 0.001) and tumor specific survival (55.3 vs. 80.1%, p = 0.001) for patients with higher and lower than average cytoplasmic CAV1 expression levels, respectively. Applying multivariable Cox regression analysis a high CAV1 protein expression level in the tumor cell cytoplasm could be identified as an independent poor prognostic marker of both overall (p = 0.02) and tumor specific survival (p = 0.03) in clear cell RCC patients. CONCLUSION: Over expression of caveolin-1 in the tumour cell cytoplasm predicts a poor prognosis of patients with clear cell RCC. CAV1 is likely to be a useful prognostic marker and may play an important role in tumour progression. Therefore, our data encourage further investigations to enlighten the role of CAV1 and its function as diagnostic and prognostic marker in serum and/or urine of RCC patients.

Caveolin 1 mRNA is overexpressed in malignant renal tissue and might serve as a novel diagnostic marker for renal cancer.

BACKGROUND & AIM: Caveolae play a significant role in disease phenotypes, such as cancer, diabetes, bladder dysfunction and muscular dystrophy. The aim of this study was to elucidate the expression of caveolin (CAV)1 in the development of renal cell cancer (RCC) and to determine a possible prognostic relevance for optimal clinical management. MATERIAL & METHODS: 109 RCC and 81 corresponding normal tissue specimens from the same kidney were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Quantification of CAV1 mRNA expression was performed using real-time reverse transcription PCR with three endogenous controls for renal proximal tubular epithelial cells and the ΔΔCt method for calculation of relative quantities. Expression levels were correlated to clinical variables. RESULTS: Tissue-specific mean CAV1 expression was significantly increased in RCC compared with normal renal tissue (p = 0.0003; paired Wilcoxon rank sum test). CAV1 expression was increased 1.9-fold in clear cell RCC compared with papillary RCC (p = 1.48 × 10(-7); unpaired Wilcoxon rank sum test). Patients with advanced disease had higher CAV1 expression when compared with organ-confined disease (p = 0.019; unpaired Wilcoxon rank sum test). Moreover, mean tissue-specific CAV1 expression was increased in patients with distant metastasis at the time of diagnosis compared with patients without metastasis (p = 0.0058; unpaired Wilcoxon rank sum test). CONCLUSION: To our knowledge, this is the first study to show that CAV1 mRNA expression, using quantitative real-time PCR, is significantly higher in RCC compared with normal renal tissue and increases with tumor stage. CAV1 mRNA expression might serve as a candidate biomarker for objective prognosis indicating RCC aggressiveness. Our data encourage further investigations to determine the role of CAV1 in RCC.

Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver.

BACKGROUND: Gene silencing due to aberrant DNA methylation is a frequent event in hepatocellular carcinoma (HCC) and also in hepatocellular adenoma (HCA). However, very little is known about epigenetic defects in fibrolamellar carcinoma (FLC), a rare variant of hepatocellular carcinoma that displays distinct clinical and morphological features. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the methylation status of the APC, CDH1, cyclinD2, GSTπ1, hsa-mir-9-1, hsa-mir-9-2, and RASSF1A gene in a series of 15 FLC and paired normal liver tissue specimens by quantitative high-resolution pyrosequencing. Results were compared with common HCC arising in non-cirrhotic liver (n = 10). Frequent aberrant hypermethylation was found for the cyclinD2 (19%) and the RASSF1A (38%) gene as well as for the microRNA genes mir-9-1 (13%) and mir-9-2 (33%). In contrast to common HCC the APC and CDH1 (E-cadherin) genes were found devoid of any DNA methylation in FLC, whereas the GSTπ1 gene showed comparable DNA methylation in tumor and surrounding tissue at a moderate level. Changes in global DNA methylation level were measured by analyzing methylation status of the highly repetitive LINE-1 sequences. No evidence of global hypomethylation could be found in FLCs, whereas HCCs without cirrhosis showed a significant reduction in global methylation level as described previously. CONCLUSIONS: FLCs display frequent and distinct gene-specific hypermethylation in the absence of significant global hypomethylation indicating that these two epigenetic aberrations are induced by different pathways and that full-blown malignancy can develop in the absence of global loss of DNA methylation. Only quantitative DNA methylation detection methodology was able to identify these differences.