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Extraneuraxial hemangioblastoma occurs in nervous paraneuraxial structures, somatic tissues, and visceral organs, as part of von Hippel-Lindau disease (VHLD) or in sporadic cases. The VHLgene plausibly plays a key role in the initiation and tumorigenesis of both central nervous system and extraneuraxial hemangioblastoma, therefore, the underlying molecular and genetic mechanisms of the tumor growth are initially reviewed. The clinical criteria for the diagnosis of VHLD are summarized, with emphasis on the distinction of sporadic hemangioblastoma from the form fruste of VHLD (eg, hemangioblastoma-only VHLD). The world literature on the topic of extraneuraxial hemangioblastomas has been comprehensively reviewed with ∼200 cases reported to date: up to 140 paraneuraxial, mostly of proximal spinal nerve roots, and 65 peripheral, 15 of soft tissue, 6 peripheral nerve, 5 bone, and 39 of internal viscera, including 26 renal and 13 nonrenal. A handful of possible yet uncertain cases from older literature are not included in this review. The clinicopathologic features of extraneuraxial hemangioblastoma are selectively presented by anatomic site of origin, and the differential diagnosis is emphasized in these subsets. Reference is made also to 10 of the authors’ personal cases of extraneuraxial hemangioblastomas, which include 4 paraneuraxial and 6 peripheral (2 soft tissue hemangioblastoma and 4 renal).

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney and belongs to the few human tumors known to develop from mutations of the VHLtumor suppressor gene. VHLgermline mutations are associated with hereditary ccRCCs in VHL disease. However, somatic VHLgene defects may also occur in sporadic ccRCCs. In this study, we analyzed the frequency and the spectrum of VHLgene alterations in 35 Italian patients with sporadic renal cell carcinoma (RCC). Tumor-specific intragenic VHLpathogenic mutations were detected in 38% (11/29) of the ccRCC patients and 33% (2/6) of the patients with other types of RCC. One novel 18-bp in-tandem duplication and 4 previously unreported nucleotide changes in the VHLgene were described. Microsatellite analysis showed loss of heterozygosity for at least 1 informative marker in 43% (9/21) of the ccRCCs and 50% (3/6) of the non-ccRCCs; 5 of the 13 tumors (38%) harboring VHLgene alterations also had loss of heterozygosity for at least 1 microsatellite marker. Our results confirm that somatic inactivation of the VHLgene may play a pivotal role in the tumorigenesis of sporadic ccRCCs in Italian patients and suggests that mutation analysis of the VHLgene may be helpful for discriminating sporadic, VHL-gene-related ccRCCs from those related to VHL disease.

Keap1 (Kelch-like ECH-associated protein 1) is an adaptor protein that mediates the ubiquitination/degradation of genes regulating cell survival and apoptosis under oxidative stress conditions. We determined methylation status of the KEAP1promoter in 102 primary breast cancers, 14 pre-invasive lesions, 38 paired normal breast tissues and 6 normal breast from reductive mammoplasty by quantitative methylation specific PCR (QMSP). Aberrant promoter methylation was detected in 52 out of the 102 primary breast cancer cases (51%) and 10 out of 14 pre-invasive lesions (71%). No mutations of the KEAP1gene were identified in the 20 breast cancer cases analyzed by fluorescence based direct sequencing. Methylation was more frequent in the subgroup of patients identified as ER positive-HER2 negative tumors (66.7%) as compared with triple-negative breast cancers (35%) (p = 0.05, Chi-square test). The impact of the interactions between Er, PgR, Her2expression and KEAP1methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1methylation had higher mortality risk than patients without triple-negative breast cancer (HR = 14.73, 95%CI: 3.65–59.37). Both univariable and multivariable COX regressions analyses showed that KEAP1methylation was associated with a better progression free survival in patients treated with epirubicin/cyclophosfamide and docetaxel as sequential chemotherapy (HR = 0.082; 95%CI: 0.007–0.934). These results indicate that aberrant promoter methylation of the KEAP1gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.

Disturbances in the epigenetic landscape by aberrant methylation of CpG islands can lead to inactivation of cancer-related genes in solid tumors. We analyzed the promoter methylation status of 6 genes previously reported as cancer-specific methylated (MCAM, SSBP2, NISCH, B4GALT1, KIF1A and RASSF1A) in 38 neural crest-derived tumors by quantitative methylation-specific real-time PCR (QMSP). The results demonstrated that the determination of the methylation status of RASSF1A is able to distinguish between normal and tumor samples in cutaneous melanomas, lung carcinoids and small bowel carcinoids. MCAM methylation levels were significantly higher in lung carcinoids tumors (p=0.001), suggesting that this alteration may represent a molecular biomarker in this tumor type.

The KEAP1/Nrf2 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumor cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1expression. We performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 47 non-small cell lung cancer tissues and normal specimens. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time. Methylation at the KEAP1promoter region was detected in 22 out of the 47 NSCLCs (47%) and in none of the normal tissues analyzed. Somatic mutations were detected in 7 out of the 47 tumors (15%) and loss of heterozygosity (LOH) in 10 out of the 47 (21%) of the cases. Overall, we found at least one molecular alteration in 57% of the cases. Approximately one third of the tumors had two alterations and this feature was associated with higher risk of disease progression in univariate COX regression analysis (HR = 3.62; 95% CI 1.24–10.65, p = 0.02). This result was confirmed by Kaplan-Meier analysis, which demonstrated an association between worst outcome and KEAP1double alterations (p = 0.01, Log rank test). Our results further suggest that deregulation of the NRF2/KEAP1 system could play a pivotal role in the cancerogenesis of NSCLC. In addition identifying patients with KEAP1genetic and epigenetic abnormalities may contribute to disease progression prediction and response to therapy in lung cancer patients.

In light with the view that KEAP1loss of function may impact tumour behavior and modify response to chemotherapeutical agents, we sought to determine whether KEAP1gene is epigenetically regulated in malignant gliomas. We developed a Quantitative Methylation Specific PCR (QMSP) assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by Receiving Operating Characteristics (ROC) curve analysis. The AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman’s Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1mRNA transcript in tumour tissue (Spearman’s Rho -0.656 P=0.0001) and in a cell line before and after treatment with 5-azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMTand KEAP1methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMTand KEAP1promoters was associated with the lowest risk to progress, patients showing only MGMTmethylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results further suggest that KEAP1expression is epigenetically regulated. In addition we demonstrated that KEAP1is frequently methylated in malignant gliomas and a predictor of patient’s outcome.