Concetta Santonocito

Association between cutaneous melanoma, Breslow thickness and vitamin D receptor BsmI polymorphism

Summary  Background  Literature data report an association between some vitamin D receptor (VDR) polymorphisms and different kinds of tumours, including malignant melanoma (MM). Only three VDR polymorphisms (FokI, TaqI and A‐1012G) have been investigated in association with the presence of cutaneous MM or the development of metastases.

GSTM1-null polymorphism as possible risk marker for hypertension: results from the aging and longevity study in the Sirente Geographic Area (ilSIRENTE study)

BACKGROUND Involvement of various gene mutations in hypertension have been already reported, including GST (detoxification Phase II enzymes), with contrasting results. This study analyzes a possible association between GSTM1-null and GSTT1-null polymorphisms and the hypertension status in a very old population (>80 years). METHODS 354 old patients were collected (255 were hypertensive and 99 were not). RESULTS GSTM1-null individuals (n=156) were significantly associated with increased risk of hypertension [OR=2.25 (1.36-3.72); p=0.005]. This association was confirmed both in 115 male and 239 female subjects. In contrast, no significant association was observed [p=0.52, OR=1.24 (0.67-2.29)] in the 57 hypertensive GSTT1-null genotypes vs 198 wild-type individuals. The above mentioned significances were obtained by multivariate logistic regression analysis after adjusting for confounder variables. Alcohol consumption and/or smoke habits were not significantly different between the two groups, as well as gender, marital status, education, number of concomitant diseases, and presence of cardiovascular diseases. No synergistic association was observed for the combined null genotypes of GSTT1 and GSTM1. CONCLUSIONS The knowledge of GSTM1 variant status seems to be potentially useful to predict a possible hypertensive status after 80 years of age. This study underlines a possible importance of the GSTM1 enzyme for blood pressure regulation.

Clinical impact on ovarian cancer patients of massive parallel sequencing for BRCA mutation detection: the experience at Gemelli hospital and a literature review.

Objective: Massive parallel sequencing (MPS) is the new frontier for molecular diagnostics. Twenty-four papers regarding BRCA analysis were considered for reviewing all pipelines evaluated in this field. Methods: Proposed here is an integrated MPS workflow able to successfully identify BRCA1/2 mutational status on 212 Italian ovarian cancer patients. The review of literature data is reported. Result: The pipeline can be routinely used as robust molecular diagnostic strategy, being highly sensitive and specific. Conclusion: Literature data report that efforts are being made in order to fully translate MPS-based BRCA1/2 gene assay into routine clinical diagnostics. However, this study highlights the need of an integrated MPS BRCA1/2 molecular workflow fulfilling the standardized requirements needed in the routine clinical laboratory practice.

Advanced tools for BRCA1/2 mutational screening: comparison between two methods for large genomic rearrangements (LGRs) detection

Abstract Background: Currently, multiplex ligation-dependent probe amplification (MLPA) is the most commonly used technique for the detection of large genomic rearrangements (LGRs) in the BRCA1/2 genes. However, a very fast assay, the BRCA1/2 multiplex amplicon quantification (MAQ), has been recently developed by Multiplicom. Methods: As no data regarding the application of MAQ method to BRCA1/2 genes are available in literature, here we compared for the first time the performance of the MAQ assay with MLPA by using several positive BRCA1/2 LGRs DNA samples (previously tested by MLPA). Results: MAQ method was able to detect all BRCA1/2 LGRs and no false-positive or -negative results were obtained in independent repetitive experiments. Conclusions: We can affirm that MAQ, as well as MLPA method, results to be valid and reproducible tools for molecular diagnostics and we are confident that this assay can be used for BRCA1/2 mutational screening as a fast and safe alternative to MLPA, particularly in the first line of analysis.

Polymorphisms in base excision DNA repair genes and association with melanoma risk in a pilot study on Central-South Italian population

Base excision repair plays a key role in the removing of DNA damage from exposure to endogenous and exogenous carcinogens. The BER pathway removes alterations of a single oxidized, reduced or methylated base. Recently some studies have explored the association between risk for cutaneous melanoma and non-synonymous single-nucleotide polymorphisms (nsSNPs) in DNA-repair genes, although with contradictory results. We hypothesized that common nsSNPs of BER genes, specifically ADPRT rs1136410, XRCC1 rs25487, rs25489, rs1799782, APEX1 rs1130409, OGG1 rs1052133, LIG3 rs3136025 and MUTYH rs3219466, may contribute to risk of melanoma. The aim of this study is to investigate whether or not a correlation between these nsSNPs and melanoma risk and/or aggressiveness is present. 167 melanoma patients and 186 healthy control subjects were analysed. By multivariate statistical analysis no association was found between nsSNP and melanoma aggressiveness, while only the two XRCC1 (rs25487 and rs25489) nsSNPs showed a strong correlation (p<0.001) with melanoma risk. To our knowledge this is the first study reporting an association between BER nsSNPs and melanoma risk in Central-South Italian individuals. Our findings, if confirmed in larger population studies, will allow the inclusion of these XRCC1 nsSNPs in a screening panel for those individuals at higher risk for melanoma.

A new standardized absolute quantitative RT-PCR method for detection of tyrosinase mRNAs in melanoma patients: Technical and operative instructions

BACKGROUND To develop a new absolute quantitative real-time PCR method for blood mRNA tyrosinase assay and to compare this new method with standard RT-PCR nested. METHODS Ten blood of melanoma patients (stages I-III), 5 tissue samples, 2 surgical fresh metastatic skin and 3 lymph nodes paraffin-embedded slices were analysed, and 10 negative controls were used. Ten millilitres of blood was analysed for each individual. Three different protocols for RNA extraction and two reverse transcription methods were used. Specific human tyrosinase cDNA fragment was cloned into pcDNA3+ vector and then titrated for the standard curve construction (from 10(6) to 10(1)copies/microl). Recovery assays for RNA and cells were also performed. RESULTS Our method was able to detect less than 5 cells/10(8) WBC and about 100 fg of tyrosinase RNA. Very low CVs (<1.5%) were obtained on all samples run in triplicate. Sensitivity and specificity were of 100%. The amount of starting volume of blood was crucial for the determination of copy number since large volumes are necessary for patients monitoring. CONCLUSIONS Our absolute qRT-PCR assay could be proposed as a new standardized molecular method for the management of melanoma patients, particularly for the follow up of the highest AJCC stages.

FOXM1 expression is significantly associated with chemotherapy resistance and adverse prognosis in non-serous epithelial ovarian cancer patients

BackgroundEpithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro.MethodsGene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT–qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients’ prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR.ResultsA significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response.ConclusionsFOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology.

Common genetic variants of MUTYH are not associated with cutaneous malignant melanoma: application of molecular screening by means of high-resolution melting technique in a pilot case-control study

MUTYH glycosylase recognizes the 8-oxoG:A mismatch and is able to excise the adenine base using proofreading mechanisms. Some papers have reported a strong association between cancer development or aggressiveness and MUTYH gene mutations. The aim of this study was to find a possible association between the most frequent MUTYH mutations and melanoma in the context of a case-control pilot study. One hundred ninety-five melanoma patients and 195 healthy controls were matched for sex and age. Clinical and laboratory data were collected in a specific database and all individuals were analyzed for MUTYH mutations by high-resolution melting and direct sequencing techniques. Men and women had significantly different distributions of tumor sites and phototypes. No significant associations were observed between the Y165C, G382D and V479F MUTYH mutations and risk of melanoma development or aggressiveness. Our preliminary findings therefore do not confirm a role for MUTYH gene mutations in the melanoma risk. Further studies are necessary for the assessment of MUTYH not only in melanoma but also other cancer types with the same embryonic origin, in the context of larger arrays studies of genes involved in DNA stability or integrity.

Insulin-like growth factor I (CA) repeats are associated with higher melanoma's Breslow index but not associated with the presence of the melanoma. A pilot study

BACKGROUND IGF-I-(CA) repeats have been previously analysed in few types of cancer and the results, although discordant in different studies, showed possible associations between cancer and IGF-I(CA)(19) repeats. Aim of this pilot study was to detect a possible association between some of the IGF-I(CA) repeats and the presence of malignant melanoma and its Breslow index. METHODS Two hundred patients affected with cutaneous malignant melanoma and 100 control healthy subjects were analysed for IGF-I(CA) repeats by fragment analysis sequencing and, partially, confirmed by direct sequencing. RESULTS A significant association of IGF-I(CA)(19) repeats was observed with melanoma higher Breslow indices (P<0.001), while no association between melanoma patients and the different genotypes of IGF-I(CA) was found. The above mentioned association was confirmed after Bonferronis correction for multiple comparisons and also by logistic regression analysis adjusted for age, sex and BMI variables. A slight, significant difference (P=0.03) was observed for serum IGF-I values in IGF-I(CA)(19)-positive or IGF-I(CA)(19)-negative subjects. DISCUSSION The association observed for IGF-I(CA)(19) and malignant melanoma is in keeping with similar results obtained in prostate or breast cancers, suggesting that this type of repeat may be directly or indirectly important in controlling cancer induction and its severity.

IL-8 and eNOS polymorphisms predict bevacizumab-based first line treatment outcomes in RAS mutant metastatic colorectal cancer patients

Background Predictive biomarkers of efficacy and toxicity of bevacizumab have not yet been validated. This study assessed the influence of IL-8, eNOS and VEGF-A polymorphisms in RAS mutated metastatic colorectal cancer patients receiving bevacizumab-based chemotherapy. Methods 120 patients treated with first-line combination FOLFOX6 plus bevacizumab were included. A historical cohort of 112 RAS mutated colorectal cancer patients treated with FOLFOX6 alone served as control group. The following SNPs were analyzed: IL-8 c.-251T>A; eNOS c.-786T>C and c.-894G>T; VEGF-A c.936C>T, c.958T>C, c.1154A>G and c.2578C>A. Correlation of SNPs, baseline IL-8 serum levels and bevacizumab-efficacy was done. Results In the bevacizumab group, carriers of the IL-8 alleles c.-251TA+AA showed a shorter PFS (P=0.002) and OS (P=0.03) compared to TT alleles. Patients with pre-treatment IL-8 < 18.25 pg/ml showed significantly longer median PFS and OS (PFS: 10.9 vs 7.6 months, P=0.005; OS: 30.7 vs 18.2 months, P<0.001) compared to patients with IL-8 higher levels (>18,25 pg/ml). IL-8 c.-251TA+AA carriers had significantly higher IL-8 levels (P<0.0001). Multivariate analysis confirmed association of IL-8 polymorphism with PFS, and of IL-8 baseline levels with both PFS and OS. IL-8 SNP did not affect the outcome in the control group. The eNOS polymorphism c.-894G>T was found associated with higher severe toxicity (P=0.0002) in patients carrying the c.-894TT genotype. Conclusions Although our data need prospective validation, IL-8 and eNOS SNPs may be have a role as predictive biomarkers for bevacizumab efficacy and toxicity.