BACKGROUND: In patients with Lynch syndrome, germline mutations in DNA mismatch repair (MMR) genes cause a high risk of developing a broad spectrum of cancers. To date, the management of patients with Lynch syndrome has represented a major challenge because of large variations in age at cancer onset. Several factors, including genetic anticipation, have been proposed to explain this phenotypic heterogeneity, but the molecular mechanisms remain unknown. Telomere shortening is a common event in tumorigenesis and also has been observed in different familial cancers. In this study, the authors investigated the possibility of a relation between telomere length and cancer onset in patients with Lynch syndrome. METHODS: The mean telomere length was measured using quantitative polymerase chain reaction in peripheral blood samples from a control group of 50 individuals, from 31 unaffected mutation carriers, and from 43 affected patients, and the results were correlated with both gene mutation and cancer occurrence. In affected patients, telomere attrition was correlated with age at cancer onset. In all patients, a t test was used to assess the linearity of the regression. RESULTS: A significant correlation between telomere length and age was observed in both affected and unaffected mutation carriers (P =.0016 and P =.004, respectively) and in mutS hornolog 2 (MSH2) mutation carriers (P =.0002) but not in mutL homolog 1 (MLH1) mutation carriers. Telomere attrition was correlated significantly with age at onset in MSH2 carriers (P =.004), whereas an opposite trend toward longer telomeres in patients with delayed onset was observed in MLH1 carriers. CONCLUSIONS: The current data suggested that telomere dynamics differ between MLH1 and MSH2 mutation carriers. It is possible that subtle, gene-specific mechanisms can be linked to cancer onset and anticipation in patients with Lynch syndrome. Cancer 2011;117:432535. (C) 2011 American Cancer Society.

Lynch syndrome (LS) is the most common form of inherited predisposition to develop cancer mainly in the co- lon and endometrium but also in other organ sites. Germline mutations in DNA mismatch repair (MMR) gene cause the transmission of the syndrome in an autosomal dominant manner. The management of LS patients is complicated by the large variation in age at cancer diagnosis which requires these patients to be enrolled in surveillance protocol starting as early as in their second decade of life. Several environmental and genetic factors have been proposed to explain this phe- notypic heterogeneity, but the molecular mechanisms remain unknown. Although the presence of genetic anticipation in Lynch syndrome has been suspected since 15 years, only recently the phenomenon has been increasingly reported to be present in different cancer genetic syndromes including LS. While the biological basis of earlier cancer onset in succes- sive generations remains poorly known, recent findings point to telomere dynamics as a mechanism significantly contrib- uting to genetic anticipation in Lynch syndrome and in other familial cancers. In this review, we summarize the clinical and molecular features of Lynch syndrome, with a particular focus on the latest studies that have investigated the molecu- lar mechanisms of genetic anticipation.

The Peutz-Jeghers Syndrome (PJS) is an autosomal dominant polyposis disorder with increased risk of multiple cancers. STK11/LKB1 (hereafter named STK11) germline mutations account for the large majority of PJS cases whereas large deletions account for about 30% of the cases. We report here the first thorough molecular characterization of 15 large deletions identified in a cohort of 51 clinically well-characterized PJS patients. The deletions were identified by MLPA analysis and characterized by custom CGH-array and quantitative PCR to define their boundaries. The deletions, ranging from 2.9 to 180 kb, removed one or more loci contiguous to the STK11 gene in six patients, while partial STK11 gene deletions were present in the remaining nine cases. By means of DNA sequencing, we were able to precisely characterize the breakpoints in each case. Of the 30 breakpoints, 16 were located in Alu elements, revealing non-allelic homologous recombination (NAHR) as the putative mechanism for the deletions of the STK11 gene, which lays in a region with high Alu density. In the remaining cases, other mechanisms could be hypothesized, such as microhomology-mediated end-joining (MMEJ) or non-homologous end-joining (NHEJ). In conclusion we here demonstrated the non-random occurrence of large deletions associated with PJS. All our patients had a classical PJS phenotype, which shows that haploinsufficiency for SBNO2, C19orf26, ATP5D, MIDN, C19orf23, CIRBP, C19orf24,and EFNA2, does not apparently affect their clinical phenotype.

Lynch syndrome (LS), or hereditary non-polyposis colorectal cancer (HNPCC), is an autosomal dominant condition responsible for early onset cancer mostly in the colonrectum and endometrium as well as in other organ sites. Lynch syndrome is caused by germline mutations in mismatch repair genes, prevalently in hMSH2, hMLH1, and less frequently in hMSH6 and hPMS2. Twenty-nine non-related index cases with colorectal cancer (CRC) were collected from a region in southeast Italy (Apulia). Among this set of patients, fifteen fulfilled the Amsterdam criteria II. The presence of tumor microsatellite instability (MSI) was assessed in all index cases and 19 (15 AC+/4 AC-) were classified as MSI-H. Mutation analysis performed on all patients, identified 15 pathogenic mutations in hMLH1 and 4 in hMSH2. 4/15 mutations in hMLH1 and 2/4 hMSH2 mutations have not been previously reported. Three previously reported mutations were further investigated for the possibility of a common founder effect. Genetic counseling was offered to all probands and extended to 183 relatives after molecular testing and 85 (46%) mutation carriers were identified. Eighty mutation carriers underwent an accurate clinical and instrumental surveillance protocol. Our results confirm that the identification of LS patients based exclusively on family history may miss patients carrying germline mutations in the MMR genes. Moreover, our results demonstrated that molecular screening and subsequent instrumental surveillance are very effective in identifying CRCs at earlier stages and reducing the number of deaths from secondary cancers in HNPCC patients.

Prostate cancer (PCa) is the second leading cause of cancer-related death in men; however, the molecular mechanisms leading to its development and progression are not yet fully elucidated. Of note, it has been recently shown that conditional stk11 knockout mice develop atypical hyperplasia and prostate intraepithelial neoplasia (PIN). We recently reported an inverse correlation between the activity of the STK11/AMPK pathway and the MAPK/p38 cascade in HIF1A-dependent malignancies. Furthermore, MAPK/p38 overactivation was detected in benign prostate hyperplasia, PIN and PCa in mice and humans. Here we report that STK11 expression is significantly decreased in PCa compared to normal tissues. Moreover, STK11 protein levels decreased throughout prostate carcinogenesis. To get insight into the role of STK11-MAPK/p38 activity balance in PCa, we treated PCa cell lines and primary biopsies with a well-established MAPK14-MAPK11 inhibitor (SB202190), which has been extensively used in vitro and in vivo. Our results indicate that inhibition of MAPK/p38 significantly affects PCa cell survival in a STK11-dependent manner. Indeed, we found that pharmacologic inactivation of MAPK/p38 does not affect viability of STK11-proficient PCa cells due to the triggering of the AMPK-dependent autophagic pathway, while it induces apoptosis in STK11-deficient cells irrespective of androgen receptor (AR) status. Of note, AMPK inactivation or autophagy inhibition in STK11-proficient cells sensitize SB202190-treated PCa cells to apoptosis. On the other end, reconstitution of functional STK11 in STK11-deficient PCa cells abrogates apoptosis. Collectively, our data show that STK11 is a key factor involved in the early phases of prostate carcinogenesis, and suggest that it might be used as a predictive marker of therapeutic response to MAPK/p38 inhibitors in PCa patients.

BACKGROUND PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. METHODS: We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. RESULTS AND CONCLUSION: Our data indicate that patients' cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.

Background: Peutz-Jeghers syndrome (PJS) is a rare dominantly inherited disease characterized by the association of gastrointestinal hamartomatous polyposis, mucocutaneous hyperpigmentation, and increased risk of cancer at different target organs. Its occurrence with differentiated thyroid cancer, particularly papillary thyroid carcinoma (PTC), even if rare, has been described. Summary: We here present a case of PTC observed in a PJS patient and a review of the literature aiming at discussing the utility of thyroid surveillance in the management of these patients. A 22-year-old woman presenting with hyperpigmented lesions of the lips and hamartomatous polyps in the stomach, duodenum, jejunum, and ileum, leading to the suspicion of PJS, was submitted to genetic analysis. Mutation scanning of the Liver Kinase B1 (LKB1) gene identified the presence of the truncating mutation E265X, thus confirming the clinical diagnosis. Beside the endoscopic, radiologic, and echographic evaluations required by the standard surveillance guidelines, the patient had a neck ultrasound (US), which showed a 5 x 4 x 6 mm hypoechoic nodule in the right thyroid lobe. The nodule contained microcalcifications and a perinodular vascular pattern. The cytological preparations derived from US-guided fine-needle aspiration biopsy of the nodule demonstrated the presence of PTC. The patient underwent a video-assisted total thyroidectomy and the histological examination revealed a follicular variant of papillary microcarcinoma. Radioactive iodine therapy was not performed because of the small size of the lesion. The patient was started on levothyroxine therapy to keep the serum thyrotropin levels suppressed. Both the sequencing and the multiplex ligation-dependent probe amplification analysis could not identify any LKB1 mutation in the tumor specimen, and the methylation-specific polymerase chain reaction assay excluded hypermethylation of the LKB1 promoter as the mechanism of inactivation for the remaining normal allele in the tumor. Conclusions: Although other mechanisms of LKB1 silencing may be responsible for its inactivation in the thyroid cancer, we cannot rule out that the occurrence of thyroid carcinoma could be a coincidental finding in this patient. However, the case here presented suggests that US of the thyroid could possibly become an integral part of the evaluation and the follow-up program adopted for PJS patients.

The 17 Y-STR loci included in the AmpFLSTR Yfiler PCR Amplification Kit were analyzed in 98 unrelated healthy males from Apulia (Southern Italy). A total of 97 different haplotypes were identified, of which 96 haplotypes were unique and 1 occurred twice. Allele frequencies for each Y-STR locus in pooled sample and estimated value of gene diversity (GD) were evaluated. The lowest value of GD was observed for DYS392 (0.126) and the highest one (0.936) for DYS385. The HD (haplotype diversity) for the studied Y-STR set showed a value of 0.9994, with an HMP (haplotype match probability) value of 0.0006, while the overall DC was 98.98%. Microvariant alleles were found for the DYS458 and DYS385 markers and sequenced. Furthermore, Φ st-based genetic distance computation and pair-wise analysis of molecular variance (AMOVA) test were carried out. When comparing our population with the Apulia sample previously investigated, the AMOVA analysis detected no evidence for significant differentiation. The comparison with all Italian populations submitted to the YHRD website showed no relevant differences with all Southern Italian populations (San Giorgio La Molara, Belvedere, Trapani and Catania) and significant genetic deviation with all Northern Italian populations (Udine, Biella, La Spezia, Modena, Ravenna, Marche and North Sardinia). Moreover, the other populations and meta-populations belonging to the whole Mediterranean area (Croatia, Macedonia, Albania, Greece, Turkey, Israel, Libya, Tunisia, Algeria, Morocco and Spain) were different from our Apulia sample. The data were submitted to YHRD.

Molecular testing for KRAS and BRAF mutations in tumor tissue is a fundamental tool to identify patients with metastatic colorectal cancer (CRC) who are eligible for anti-EGFR monoclonal antibody therapy. We here report a molecular analysis by high-resolution melting analysis and direct sequencing of KRAS, BRAF and PIK3CA hot spot mutations in 209 Italian CRC patients. One hundred and ten patients (51%) were identified who were potentially nonresponders to anti-EGFR therapy: 90/209 patients (43%) harboring KRAS mutations, 13/117 (11.1%) with the V600E BRAF mutation, and 7/209 (3.3%) with mutations in PIK3CA exon 20. The prevalence of BRAF and PIK3CA mutations was significantly higher in patients older than 65 years (p=0.014 and p=0.018), while patients with triple-negative tumors were significantly younger than mutation carriers (p=0.000011). Patients with gene mutations also showed a trend towards preferential tumor location in the colon (p=0.026). Moreover, although involving a relatively small number of samples, we report the presence of a discordant mutational profile between primary tumors and secondary lesions (3/9 patients), suggesting that it is worthwhile to test other available tissues in order to better define the efficacy of targeted therapy. Further correlations of specific clinical features with tumor mutational profile could be helpful to predict the response of CRC patients to monoclonal antibody therapy.

In this study the impact of hUPF1 and hUPF2 knockdown on alternative splicing (AS) isoforms of different genes encoding subunits of respiratory chain complex I and complex IV is described. As expected, loss of both hUPF1 and hUPF2 led to impairment of nonsense-mediated mRNA decay (NMD) and accumulation of PTC-containing NMD substrates generated by both complex I and complex IV genes. The levels of some complex I splice variants, which did not contain PTC as well as the level of some complex I canonical transcripts were, however, affected only by hUPF1 knockdown. This finding confirms that NMD plays a role in the maintenance of the transcriptome integrity and reveals a specific impact of hUPF1 on the regulation of complex I genes.