Epigenetic Mechanisms of Aging and Aging-Associated Diseases.

Aging is an inevitable outcome of life, characterized by a progressive decline in tissue and organ function. At a molecular level, it is marked by the gradual alterations of biomolecules. Indeed, important changes are observed on the DNA, as well as at a protein level, that are influenced by both genetic and environmental parameters. These molecular changes directly contribute to the development or progression of several human pathologies, including cancer, diabetes, osteoporosis, neurodegenerative disorders and others aging-related diseases. Additionally, they increase the risk of mortality. Therefore, deciphering the hallmarks of aging represents a possibility for identifying potential druggable targets to attenuate the aging process, and then the age-related comorbidities. Given the link between aging, genetic, and epigenetic alterations, and given the reversible nature of epigenetic mechanisms, the precisely understanding of these factors may provide a potential therapeutic approach for age-related decline and disease. In this review, we center on epigenetic regulatory mechanisms and their aging-associated changes, highlighting their inferences in age-associated diseases.

Italian Case Report with a Double Mutation in PSEN1 (K311R and E318G).

Alzheimer's disease (AD) is the most common cause of dementia worldwide. The clinical spectrum of suspected AD has been extended from mild cognitive impairment (MCI) to preclinical AD which includes people who have typical cognitive function but harbor the underlying biological features of AD. We report the first case of an Italian patient affected by MCI (MMSE 24\30), characterized by a double mutation p.Lys311Arg (K311R) and p.Glu318Gly (E318G) in Presenilin-1 but with the absence of abnormal accumulation of amyloid beta.

CT-Detected MTA Score Related to Disability and Behavior in Older People with Cognitive Impairment.

Our study aims to investigate the relationship between medial temporal lobe atrophy (MTA) score, assessed by computed tomography (CT) scans, and functional impairment, cognitive deficit, and psycho-behavioral disorder severity. Overall, 239 (M = 92, F = 147; mean age of 79.3 ± 6.8 years) patients were evaluated with cognitive, neuropsychiatric, affective, and functional assessment scales. MTA was evaluated from 0 (no atrophy) to 4 (severe atrophy). The homocysteine serum was set to two levels: between 0 and 10 µmol/L, and >10 µmol/L. The cholesterol and glycemia blood concentrations were measured. Hypertension and atrial fibrillation presence/absence were collected. A total of 14 patients were MTA 0, 44 patients were MTA 1, 63 patients were MTA 2, 79 patients were MTA 3, and 39 patients were MTA 4. Cognitive (p < 0.0001) and functional (p < 0.0001) parameters decreased according to the MTA severity. According to the diagnosis distribution, AD patient percentages increased by MTA severity (p < 0.0001). In addition, the homocysteine levels increased according to MTA severity (p < 0.0001). Depression (p < 0.0001) and anxiety (p = 0.001) increased according to MTA severity. This study encourages and supports the potential role of MTA score and CT scan in the field of neurodegenerative disorder research and diagnosis.

Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors.

BACKGROUND: The KEAP1/NRF2 pathway has been widely investigated in tumors since it was implicated in cancer cells survival and therapies resistance. In lung tumors the deregulation of this pathway is mainly related to point mutations of KEAP1 and NFE2L2 genes and KEAP1 promoter hypermethylation, but these two genes have been rarely investigated in low/intermediate grade neuroendocrine tumors of the lung. METHODS: The effects of KEAP1 silencing on NRF2 activity was investigated in H720 and H727 carcinoid cell lines and results were compared with those obtained by molecular profiling of KEAP1 and NFE2L2 in a collection of 47 lung carcinoids. The correlation between methylation and transcript levels was assessed by 5-aza-dC treatment. RESULTS: We demonstrated that in carcinoid cell lines, the KEAP1 silencing induces an upregulation of NRF2 and some of its targets and that there is a direct correlation between KEAP1 methylation and its mRNA levels. A KEAP1 hypermethylation and Loss of Heterozygosity at KEAP1 gene locus was also observed in nearly half of lung carcinoids. CONCLUSIONS: This is the first study that has described the effects of KEAP1 silencing on the regulation of NRF2 activity in lung carcinoids cells. The epigenetic deregulation of the KEAP1/NRF2 by a KEAP1 promoter hypermethylation system appears to be a frequent event in lung carcinoids.

Extraneuraxial hemangioblastoma: A clinicopathologic study of 10 cases with molecular analysis of the VHL gene.

Less than 250 extraneuraxial hemangioblastomas occurring in paraneuraxial or peripheral sites have been reported to date, sporadically or in the setting of von Hippel-Lindau disease. Seventeen such cases underwent molecular genetic analysis, using either the patient's peripheral blood in 9 cases or paraffin embedded tumor tissue in the rest. VHL gene mutations were documented in 3/9 cases in which DNA from peripheral blood lymphocytes was used, all with clinically manifest von Hippel-Lindau disease; instead, no VHL gene alterations were found in all of the 8 cases with sporadic extraneuraxial hemangioblastoma in which DNA from tumor tissue was analyzed. Our aim is to investigate the molecular genetic profile of the VHL gene in extraneuraxial hemangioblastoma using paraffin embedded tumor tissues. The clinical features, histopathology, and molecular investigations of 10 extraneuraxial hemangioblastomas (7 females, 3 males; median age: 47 years) are presented herein. The histopathologic diagnosis was supported by immunohistochemistry (10/10) and electron microscopy (4/10). Molecular genetic analysis was conducted (10/10) for VHL gene mutations, LOH, and gene promoter methylation. Two of the present cases were already published with only limited or no molecular investigations. Four tumors of the present series were paraneuraxial, and 6 peripheral (2 involved soft tissues, and 4 the kidney). One tumor was von Hippel-Lindau disease-associated, 1 was classified as "hemangioblastoma-only VHLD", 7 were sporadic, and one was unknown. All were histopathologically analogous to their counterpart located inside the central nervous system. Immunophenotypically, all tumors expressed vimentin, S-100, NSE, and alpha-inhibin (10/10). Ultrastructurally, unbound lipid droplets filled the cytoplasms of the stromal cells. Molecular analysis revealed 3 inactivating mutations (1 germline, two somatic) in the coding sequence of the VHL gene in 2 different extraneuraxial hemangioblastomas, and LOH in 4 (two as a double hit), all non-renal extraneuraxial hemangioblastomas. Methylation analysis failed to disclose promoter methylation in any case. In conclusion, we report eight new cases from the wide category of extraneuraxial hemangioblastomas (4 paraneuraxial, and 4 renal), one of which was von Hippel-Lindau disease-associated and 7 sporadic. VHL gene alterations were found not only in the von Hippel-Lindau disease-associated tumor, but - for the first time - also in 3 sporadic ones, two of which with novel mutations.

Large deletion at the CDC73 gene locus and search for predictive markers of the presence of a CDC73 genetic lesion.

The Hyperparathyroidism with Jaw-Tumours syndrome is caused by mutations of the CDC73 gene: it has been suggested that early onset of the disease and high Ca2+ levels may predict the presence of a CDC73 mutation. We searched for large deletions at the CDC73 locus in patients with: HPT-JT (nr 2), atypical adenoma (nr 7) or sporadic parathyroid carcinoma (nr 11) with a specific MLPA and qRT-PCR assays applied on DNA extracted from whole blood. A Medline search in database for all the papers reporting a CDC73 gene mutation, clinical/histological diagnosis, age at onset, Ca2+, PTH levels for familial/sporadic cases was conducted with the aim to possibly identify biochemical/clinical markers predictive, in first diagnosis, of the presence of a CDC73 gene mutation. A novel genomic deletion of the first 10 exons of the CDC73 gene was found in a 3-generation HPT-JT family, confirmed by SNP array analysis. A classification tree built on the published data, showed the highest probability of having a CDC73 mutation in subjects with age at the onset < 41.5 years (44/47 subjects, 93.6%, had the mutation). Whereas the lowest probability was found in subjects with age at the onset ≥ 41.5 years and Ca2+ levels <13.96 mg/dL (7/20 subjects, 35.0%, had the mutation, odds ratio = 27.1, p < 0.001). We report a novel large genomic CDC73 gene deletion identified in an Italian HPT-JT family. Age at onset < 41.5 ys and Ca2+ > 13.96 mg/dL are predictive for the presence of a CDC73 genetic lesion.

Extraneuraxial Hemangioblastoma: Clinicopathologic Features and Review of the Literature.

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 VHL gene 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).

A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4-ALK fusion gene.

Inflammatory myofibroblastic tumors (IMT) in the head and neck region are rare neoplasms that generally mimic benign/low-grade neoplasms. Overexpression of anaplastic lymphoma kinase (ALK) has been reported in 50% of IMT cases, secondary to ALK activation by structural rearrangements in the ALK gene, which results in a fusion protein with echinoderm microtubule associated protein like 4 (EML4) in ~20% of cases. The present study describes a case of a 74-year-old woman with a malignant IMT in the right posterior hypopharynx harboring a previously unreported chromosomal rearrangement resulting in EML4 and ALK gene fusion. Strong ALK immunoreactivity was observed in neoplastic cells, while fluorescent in situ hybridization combined with fluorescent fragment analysis and direct sequencing identified the first case of the 3a/b variants of the EML4-ALK fusion gene in IMT. The results of the current study highlight the uncommon occurrence of ALK-positive IMT in the head/neck region and demonstrate the importance of integrating different molecular methodologies to identify unequivocal gene fusion characterization.

Keap1/Nrf2 pathway in kidney cancer: frequent methylation of KEAP1 gene promoter in clear renal cell carcinoma.

The Keap1/Nrf2 pathway is a master regulator of the cellular redox state through the induction of several antioxidant defence genes implicated in chemotherapeutic drugs resistance of tumor cells. An increasing body of evidence supports a key role for Keap1/Nrf2 pathway in kidney diseases and renal cell carcinoma (RCC), but data concerning the molecular basis and the clinical effect of its deregulation remain incomplete.Here we present a molecular profiling of the KEAP1 and NFE2L2 genes in five different Renal Cell Carcinoma histotypes by analysing 89 tumor/normal paired tissues (clear cell Renal Carcinoma, ccRCCs; Oncocytomas; Papillary Renal Cell Carcinoma Type 1, PRCC1; Papillary Renal Cell Carcinoma Type 2, PRCC2; and Chromophobe Cell Carcinoma).A tumor-specific DNA methylation of the KEAP1 gene promoter region was found as a specific feature of the ccRCC subtype (18/37, 48.6%) and a direct correlation with mRNA levels was confirmed by in vitro 5-azacytidine treatment. Analysis of an independent data set of 481 ccRCC and 265 PRCC tumors corroborates our results and multivariate analysis reveals a significant correlation among ccRCCs epigenetic KEAP1 silencing and staging, grading and overall survival.Our molecular results show for the the first time the epigenetic silencing of KEAP1 promoter as the leading mechanism for modulation of KEAP1 expression in ccRCCs and corroborate the driver role of Keap1/Nrf2 axis deregulation with potential new function as independent epigenetic prognostic marker in renal cell carcinoma.

The Post-Surgical Long-Term Behaviour of Lung Carcinoid Tumours.

Here, we report a retrospective evaluation of long-term behaviour of lung carcinoids after surgery. A total of 23 patients (17 with typical pulmonary carcinoids and 6 with atypical pulmonary carcinoids) were enrolled in our hospital from April 1994 to July 2009. All patients underwent intervention at the Unit of Surgery and then were followed at the Unit of Oncology. The standard protocol for patient monitoring consisted of follow-up at 3 months after surgery, 6 months after first control and annually for 5 years. The follow-up evaluations consisted in blood tests, imaging of chest and abdomen, bone scintigraphy, and brain computed tomography. In case of disease recurrence, patients underwent chemotherapy (etoposide, carboplatin) and radiotherapy. All patients were followed for a mean of follow-up period of 100 months, ranging between 20 and 203 months. In the group of typical carcinoid, the observed recurrence rate at 5 years was zero, at 10 years was 5.8 %, whereas the observed mortality rate at 5 and 10 years was zero. In the group of atypical carcinoid, both the recurrence rate and the mortality rate at 5 and 10 years were 16.6 %. A statistical significant difference (p = 0.002) in the recurrence rate between stage I and stage II was observed. The overall prognosis of pulmonary carcinoids was favourable, and the typical carcinoids presented a better prognosis than the atypical ones. The stage at time of diagnosis could be considered as a predictive prognostic factor.

CD74-NRG1 fusions in lung adenocarcinoma.

UNLABELLED: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-ß3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE: CD74­NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.

Molecular dissection of the VHL gene in solitary capillary hemangioblastoma of the central nervous system.

Capillary hemangioblastomas (HGBs) of the CNS occur either sporadically or as part of the von Hippel-Lindau (VHL) syndrome. Molecular characterizations of the VHL gene in sporadic HGBs at the somatic level have been limited to date. We investigated the VHL gene in 57 patients most of whom (55 [96%] of 57) had a solitary CNS HGB at the time of surgery. Tissues from 23 HGBs of these patients (2 VHL related and 21 unrelated) were also investigated at genetic and epigenetic levels. Two of the 51 patients with apparently sporadic HGBs and no additional evidence of VHL (∼4%) were found to have a germline VHL gene mutation; both of these patients subsequently developed evidence of VHL syndrome. Somatic VHL gene mutations were found in 11 (52%) of the 21 non-VHL-related cases. A germline mutation was identified in 5 (84%) of 6 VHL-associated HGBs; double gene inactivation was observed in tumor tissue from VHL syndrome patients. Seven different previously unreported VHL gene alterations (6 somatic and 1 germline) were identified; double hits were identified in 7 (12%) of 57 cases. Our findings confirm the usefulness of VHL gene analysis at the germline level in patients who present with apparently solitary HGB. Moreover, the genetic and epigenetic VHL gene investigations performed support a key role for functional alterations of the VHL gene in sporadic neuraxial HGB.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

VHL gene alterations in Italian patients with isolated renal cell carcinomas.

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 VHL tumor suppressor gene. VHL germline mutations are associated with hereditary ccRCCs in VHL disease. However, somatic VHL gene defects may also occur in sporadic ccRCCs. In this study, we analyzed the frequency and the spectrum of VHL gene alterations in 35 Italian patients with sporadic renal cell carcinoma (RCC). Tumor-specific intragenic VHL pathogenic 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 VHL gene 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 VHL gene alterations also had loss of heterozygosity for at least 1 microsatellite marker. Our results confirm that somatic inactivation of the VHL gene may play a pivotal role in the tumorigenesis of sporadic ccRCCs in Italian patients and suggests that mutation analysis of the VHL gene may be helpful for discriminating sporadic, VHL-gene-related ccRCCs from those related to VHL disease.

Aberrant Keap1 methylation in breast cancer and association with clinicopathological features.

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 KEAP1 promoter 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 KEAP1 gene 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, Her2 expression and KEAP1 methylation on mortality was investigated by RECPAM multivariable statistical analysis, identifying four prognostic classes at different mortality risks. Triple-negative breast cancer patients with KEAP1 methylation 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 KEAP1 methylation 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 KEAP1 gene is involved in breast cancerogenesis. In addition, identifying patients with KEAP1 epigenetic abnormalities may contribute to disease progression prediction in breast cancer patients.

Aberrant genes promoter methylation in neural crest-derived tumors.

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.

Frequent epigenetics inactivation of KEAP1 gene in non-small cell lung cancer.

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 KEAP1 expression. 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 KEAP1 promoter 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 KEAP1 double 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 KEAP1 genetic and epigenetic abnormalities may contribute to disease progression prediction and response to therapy in lung cancer patients.

Gene expression of somatostatin receptor subtypes SSTR2a, SSTR3 and SSTR5 in peripheral blood of neuroendocrine lung cancer affected patients.

BACKGROUND: Somatostatin (SS) acts as a universal endocrine off-switch, and also inhibits the growth of neuroendocrine tumours through its specific receptors (SSTRs). Somatostatin receptors are G-protein-coupled receptors, which are encoded by five separate genes (SSTR1-5). Short peptide analogues demonstrate specific binding only for the subgroup consisting of SSTR2a, SSTR3 and SSTR5. Moreover, previous studies reported that expression of mRNA for SSTR2a correlated with therapeutic outcome in patients with carcinoid tumours treated with somatostatin analogs. PURPOSE: To develop and apply a Real Time Quantitative PCR technique (RT-qPCR) to compare and contrast the mRNA levels of SSTR2a, SSTR3 and SSTR5 in Neuroendocrine Lung Cancer affected patients. METHODS: Peripheral blood samples from 21 neuroendocrine lung cancer affected patients (14 SCLC, 6 LC and 1 LCNEC) subjected to scintigraphy with (111)In-DTPA-D-Phe(1)-octreotide (OctreoScan) and 24 healthy blood donors were investigated by RT-qPCR. mRNA levels for SSTR2a, SSTR3 and SSTR5 were measured in peripheral blood samples with a relative quantification method using plasmid dilutions as calibration curves and GAPDH as reference gene. RESULTS: A statistically significant increase in target genes/GAPDH copy number ratio was found for SSTR2a (median 38; IQR 22-141) and SSTR5 (median 51; IQR 19-499) in neuroendocrine lung cancer affected patients as compared with samples from healthy blood donors (P ≤ 0.0003 and P ≤ 0.0005). Since low levels of expression were detected in the control group for all three genes, optimal cut-off values were assessed using ROC curve analyses and were equal to 9.05 for SSTR2a and 16.97 for SSTR5. These cut off values resulted in a sensitivity of 86% (95%IC 65-95) for both markers and a specificity of 83% (95%IC 64-93%) and 79% (95%IC 60-91%) for SSTR2a and SSTR5 respectively. Comparison between OctreoScan results and RT-qPCR analysis demonstrated agreement in 76% of the cases. CONCLUSIONS: Our results suggest that SSTR2a and SSTR5 mRNAs are detectable in peripheral blood of neuroendocrine lung cancer affected patients using real-time quantitative PCR, with a good agreement with OctreoScan. The high sensitivity of this non-invasive molecular technique suggests that this method could represent a useful tool in the clinical management of neuroendocrine lung cancers.

Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome.

In light with the view that KEAP1 loss of function may impact tumour behavior and modify response to chemotherapeutical agents, we sought to determine whether KEAP1 gene 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 KEAP1 mRNA 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 MGMT and KEAP1 methylation 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 MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results further suggest that KEAP1 expression is epigenetically regulated. In addition we demonstrated that KEAP1 is frequently methylated in malignant gliomas and a predictor of patient's outcome.

Molecular analysis of the HuD gene in neuroendocrine lung cancers.

n-ELAV (neuronal-Embryonic Lethal, Abnormal Vision)-like genes belong to a family codifying for onconeural RNA-binding proteins, also called Hu antigens. Anti-Hu-antibodies (anti-Hu-Ab) are typically associated with paraneoplastic encephalomyelitis/sensory neuropathy (PEM/PSN), and low titres of anti-Hu-Ab were found in neural/neuroendocrine neoplasms, especially small cell lung cancer (SCLC). To date, few studies have been published focused on the genetic causes of their involvement in the pathogenesis of neuroendocrine tumors (NE). Here we analyzed 20 primary human neuroendocrine lung tumor tissues for somatic mutations in the HuD gene. Two inactivating mutations (a frameshift and a stop codon mutation) and 11 nucleotide changes were detected in the coding sequence of HuD gene in 7 different lung tumors. Our results on SCLC and carcinoid tissues support the hypothesis that alterations of nELAV genes could be involved in the onset and/or progression of a subset of neuroendocrine lung tumors.