Maria Cristina Sini

Main roads to melanoma

The characterization of the molecular mechanisms involved in development and progression of melanoma could be helpful to identify the molecular profiles underlying aggressiveness, clinical behavior, and response to therapy as well as to better classify the subsets of melanoma patients with different prognosis and/or clinical outcome. Actually, some aspects regarding the main molecular changes responsible for the onset as well as the progression of melanoma toward a more aggressive phenotype have been described. Genes and molecules which control either cell proliferation, apoptosis, or cell senescence have been implicated. Here we provided an overview of the main molecular changes underlying the pathogenesis of melanoma. All evidence clearly indicates the existence of a complex molecular machinery that provides checks and balances in normal melanocytes. Progression from normal melanocytes to malignant metastatic cells in melanoma patients is the result of a combination of down- or up-regulation of various effectors acting on different molecular pathways.

Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases.

Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Molecular alterations at chromosome 9p21 in melanocytic naevi and melanoma

Background  The chromosome 9p21 and its CDKN locus, with the p16 tumour suppressor gene (CDKN2A), are recognized as the genomic regions involved in the pathogenesis of melanoma.

Issues affecting molecular staging in the management of patients with melanoma.

•  Introduction •  Role of the RT‐PCR in prognostic prediction ‐  Melanoma micrometastases in peripheral blood ‐  Melanoma micrometastases in lymph nodes •  Molecular complexity of melanomagenesis ‐  Molecular alterations and disease phenotype •  Conclusions

Chromosomal abnormalities and microsatellite instability in sporadic endometrial cancer

Defective DNA mismatch repair and nonfunctional mechanisms controlling the proper progression of the cell cycle have been proposed as being responsible for the genomic instability and accumulation of karyotypic alterations in endometrial cancer (EC). To assess whether numerical chromosomal anomalies (aneuploidy) and microsatellite instability (MSI) might be representative of distinctive tumour behaviour, paraffin-embedded tissue samples from 86 patients with sporadic EC were evaluated by both fluorescence in situ hybridisation (FISH) and microsatellite analysis, using free nuclei and genomic DNAs (respectively). Approximately one-third of the tumours analysed (24/74; 32%) exhibited MSI, whereas 38/86 (44%) of the EC samples displayed aneuploidy. The majority of the unstable cases (15/24; 63%) were from advanced-stage patients. Conversely, 23 (61%) out of the 38 tumours with aneuploidy were from early-stage patients. No apparent correlation was found between MSI and aneuploidy, whereas the immunohistochemical (IHC) analysis revealed that inactivation of the MLH1 mismatch repair gene may be involved in the majority of the MSI+ sporadic ECs. No genetic or cytogenetic alteration analysed here seems to add any significant predictive value to the stage of disease.

Role of key-regulator genes in melanoma susceptibility and pathogenesis among patients from South Italy

BackgroundSeveral genetic alterations have been demonstrated to contribute to the development and progression of melanoma. In this study, we further investigated the impact of key-regulator genes in susceptibility and pathogenesis of such a disease.MethodsA large series (N = 846) of sporadic and familial cases originating from South Italy was screened for germline mutations in p16CDKN2A, BRCA2, and MC1R genes by DHPLC analysis and automated DNA sequencing. Paired primary melanomas and lymph node metastases from same patients (N = 35) as well as melanoma cell lines (N = 18) were analyzed for somatic mutations in NRAS, BRAF, and p16CDKN2Agenes.ResultsFor melanoma susceptibility, investigations at germline level indicated that p16CDKN2Awas exclusively mutated in 16/545 (2.9%) non-Sardinian patients, whereas BRCA2 germline mutations were observed in 4/91 (4.4%) patients from North Sardinia only. Two MC1R germline variants, Arg151Cys and Asp294His, were significantly associated with melanoma in Sardinia. Regarding genetic events involved in melanoma pathogenesis at somatic level, mutually-exclusive mutations of NRAS and BRAF genes were observed at quite same rate (about two thirds) in cultured and in vivo melanomas (either primary or metastatic lesions). Conversely, p16CDKN2Agene alterations were observed at increased rates moving from primary to metastatic melanomas and melanoma cell lines. Activation of the ERK gene product was demonstrated to be consistently induced by a combination of molecular alterations (NRAS/BRAF mutations and p16CDKN2Asilencing).ConclusionOur findings further clarified that: a) mutation prevalence in melanoma susceptibility genes may vary within each specific geographical area; b) multiple molecular events are accumulating during melanomagenesis.

CDKN2A and MC1R analysis in amelanotic and pigmented melanoma

Amelanotic melanoma (AM) is a rare subtype of melanoma with little or no clinically visible pigment; it is more difficult to diagnose than pigmented melanoma (PM), and has a worse prognosis. In the attempt to find a genetic explanation for the distinction between AM and PM, we conducted a case–case study, matching AM and PM patients, and testing them for germline mutations in high- (p16INK4A, p14ARF, CDK4) and low-penetrance (MC1R) melanoma susceptibility genes. Similar CDKN2A mutations were found in both sets of melanomas. A p14ARF splice germline mutation was detected for the first time in an Italian family with AM. This rare mutation, which has been described only once previously, may be involved in predisposition to the amelanotic phenotype in combination with germline MC1R variants and coordinate somatic expression of pigmentation genes and their regulators.

Discrepant alterations in main candidate genes among multiple primary melanomas

BackgroundAlterations in key-regulator genes of disease pathogenesis (BRAF, cKIT, CyclinD1) have been evaluated in patients with multiple primary melanoma (MPM).MethodsOne hundred twelve MPM patients (96 cases with two primary melanomas, 15 with three, and 1 with four) were included into the study. Paired synchronous/asynchronous MPM tissues (N = 229) were analyzed for BRAF mutations and cKIT/CyclynD1 gene amplifications.ResultsBRAF mutations were identified in 109/229 (48%) primary melanomas, whereas cKIT and CyclinD1 amplifications were observed in 10/216 (5%) and 29/214 (14%) tumor tissues, respectively. While frequency rates of BRAF mutations were quite identical across the different MPM lesions, a significant increase of cKIT (p < 0.001) and CyclinD1 (p = 0.002) amplification rates was observed between first and subsequent primary melanomas. Among the 107 patients with paired melanoma samples, 53 (49.5%) presented consistent alteration patterns between first and subsequent primary tumors. About one third (40/122; 32.8%) of subsequent melanomas presented a discrepant pattern of BRAF mutations as compared to incident primary tumors.ConclusionsThe low consistency in somatic mutation patterns among MPM lesions from same patients provides further evidence that melanomagenesis is heterogeneous and different cell types may be involved. This may have implications in clinical practice due to the difficulties in molecularly classifying patients with discrepant primary melanomas.

Long non-coding RNA CASC2 in human cancer

Long non-coding RNAs cover large part of the non-coding information of the human DNA, which represents more than 90% of the whole genome. They constitute a wide and complex group of molecules with more than 200 nucleotides, which generally lack an open reading frame, and are involved in various ways in the pathophysiology of cancer. Their roles in the regulation of gene expression, imprinting, transcription, and post-translational processing have been described in several types of cancer. CASC2 was discovered in 2004 in patients with endometrial carcinoma as a potential tumor suppressor. Since then, additional studies in other types of neoplasia have been carried out, and both mechanisms and interactions of CASC2 in cancer have been better elucidated. In this review, we summarize the current knowledge on the role of CASC2 in the genesis, progression, and clinical management of human cancer.

Unexpected Distribution of cKIT and BRAF Mutations among Southern Italian Patients with Sinonasal Melanoma

Background: Racial and geographic factors seem to affect the incidence of cutaneous and mucosal melanoma. Objective: To investigate the occurrence of BRAF and cKIT impairments in patients with sinonasal melanoma in Southern Italy. Methods: Eleven sinonasal melanomas were screened for BRAF mutations and cKIT alterations by immunohistochemistry (CD117), fluorescence in situ hybridization and sequencing analyses. Results: A high prevalence (4/11; 36%) of BRAF mutations and lack of cKIT mutations were observed. Amplification of cKIT was found in 18% of cases; cKIT expression was detectable in 18% non-overlapping cases. No correlation between CD117 and cKIT alterations was observed. One (6%) cKIT and two (12%) BRAF mutations were detected in an additional series of 17 acral/mucosal melanomas from the same geographic areas. Conclusion: Mutations of cKIT are infrequent in sinonasal melanoma in Southern Italy.