Novel somatic and germline mutations in cancer candidate genes in glioblastoma, melanoma, and pancreatic carcinoma.

A recent systematic sequence analysis of well-annotated human protein coding genes or consensus coding sequences led to the identification of 189 genes displaying somatic mutations in breast and colorectal cancers. Based on their mutation prevalence, a subset of these genes was identified as cancer candidate (CAN) genes as they could be potentially involved in cancer. We evaluated the mutational profiles of 19 CAN genes in the highly aggressive tumors: glioblastoma, melanoma, and pancreatic carcinoma. Among other changes, we found novel somatic mutations in EPHA3, MLL3, TECTA, FBXW7, and OBSCN, affecting amino acids not previously found to be mutated in human cancers. Interestingly, we also found a germline nucleotide variant of OBSCN that was previously reported as a somatic mutation. Our results identify specific genetic lesions in glioblastoma, melanoma, and pancreatic cancers and indicate that CAN genes and their mutational profiles are tumor specific. Some of the mutated genes, such as the tyrosine kinase EPHA3, are clearly amenable to pharmacologic intervention and could represent novel therapeutic targets for these incurable cancers. We also speculate that similar to other oncogenes and tumor suppressor genes, mutations affecting OBSCN could be involved in cancer predisposition.

BRAF alterations are associated with complex mutational profiles in malignant melanoma.

To evaluate the mutational profiles associated with BRAF mutations in human melanoma, we have studied BRAF, RAS, PTEN, TP53, CDKN2A and CDK4 genes and their expression in melanoma lesions. Owing to the lack of sufficient material from fresh specimens, we employed short-term cell lines obtained from melanoma biopsies. In all, 41 melanoma obtained from eight primary lesions, 20 nodal, 11 cutaneous and two visceral metastases from patients with sporadic (n=31), familial (n=4) and multiple melanoma (n=2) were analysed. The results revealed novel missense mutations in the BRAF, PTEN, CDKN2A and CDK4 genes. Overall, activating mutations of BRAF and loss of functional p16 and ARF were detected in the majority of melanomas (29/41, 36/41 and 29/41, respectively), while PTEN alterations/loss, NRAS and TP53 mutations occurred less frequently (6/41, 6/41 and 10/41, respectively). In the resulting 12 mutational profiles, p16/ARF loss associated with mutated BRAFV599E was the most represented (n=15). In addition, TP53 and PTEN mutations were always accompanied with BRAF alterations, while PTEN loss was found in association with CDKN2A or TP53 mutations in the absence of BRAF activation. The p16/ARFDelta+BRAF/RAS profile was significantly associated with a longer survival, while complex mutational profiles were detected in highly aggressive disease and poor survival. These data support the existence of several molecularly defined melanoma groups which likely reflect different clinical/biological behaviour, thus suggesting that a more extensive molecular classification of melanoma would significantly impact its clinical management.

Genetic progression of metastatic melanoma.

Melanoma progression is well defined in its clinical, histopathological and biological aspects, but the molecular mechanism involved and the genetic markers associated to metastatic dissemination are only beginning to be defined. The recent development of high-throughput technologies aimed at global molecular profiling of cancer is switching on the spotlight at previously unknown candidate genes involved in melanoma, such as WNT5A and BRAF. In fact, several tumor suppressors and oncogenes have been shown to be involved in melanoma pathogenesis, including CDKN2A, PTEN, TP53, RAS and MYC, though they have not been related to melanoma subtypes or validated as prognostic markers. Here, we have reviewed the published data relative to the major genes involved in melanoma pathogenesis, which may represent important markers for the identification of genetic profiles of melanoma subtypes.

Cutaneous melanoma in childhood and adolescence shows frequent loss of INK4A and gain of KIT.

Childhood cutaneous melanoma is a rare disease with increasing incidence. It is not clear whether it differs from adult melanoma in etiology and clinical evolution. To genetically characterize childhood melanoma, 21 pediatric patients were studied by germ-line analysis of CDKN2A, CDK4, and MC1R genes. In addition, alterations in CDKN2A, c-Kit, BRAF, and NRAS genes were evaluated at the somatic level by direct gene sequencing, fluorescence in situ hybridization analysis, and immunohistochemistry. As a control group of susceptible patients, we studied patients from 23 melanoma-prone families. At the germ-line level, CDKN2A and MC1R gene variants were detected in 2/21 and 12/21 pediatric patients and in 9/23 and 19/22 in familial patients. At the somatic level, most lesions (9/14) from pediatric patients showed CDKN2A locus homozygous deletions and a null p16 immunophenotype, whereas most lesions (5/8) from familial patients were disomic and immunoreactive. A c-Kit low-polysomy profile seems to parallel CDKN2A homozygous deletions in pediatric melanoma whereas the single activating mutation observed segregates with familial patients. Loss of KIT protein expression was frequent (7/14) in pediatric melanomas, where metastatic cases were prevalent. BRAF(V600E) mutation occurred at a similar rate (approximately 50%) in lesions from pediatric and familial patients, whereas no NRAS mutations were detected.

CCN3/nephroblastoma overexpressed matricellular protein regulates integrin expression, adhesion, and dissemination in melanoma.

CCN3/nephroblastoma overexpressed belongs to the CCN family of genes that encode secreted proteins associated with the extracellular matrix (ECM) and exert regulatory effects at the cellular level. Overexpression of CCN3 was shown in metastatic melanoma cells compared with cells of the primary tumor from the same patient. Analysis of short-term cultures from 50 primary and metastatic melanomas revealed a heterogeneous expression pattern of both the 46-kDa full-length cytoplasmic/secreted protein and the 32-kDa nuclear-truncated form. The different protein expression patterns were not associated with gene alterations or polymorphisms. Like the metastatic cells expressing high levels of the 46-kDa CCN3, cells transfected to overexpress CCN3 showed increased adhesion to ECM proteins, whereas inhibition of CCN3 expression by small interfering RNA decreased adhesion to laminin and vitronectin. CCN3 overexpression induced increased expression of laminin and vitronectin integrin receptors alpha 7 beta 1 and alpha v beta 5 by increasing their mRNA production. Moreover, CCN3 secreted by melanoma cells acted as an adhesion matrix protein for melanoma cells themselves. Analysis of CCN3 protein expression with respect to melanoma progression detected the protein in all visceral metastases tested and in most nodal metastases from relapsing patients but in only a few nodal metastases from nonrelapsing patients and cutaneous metastases. Consistently, xenotransplantation in immunodeficient mice showed a higher metastatic potential of melanoma cells overexpressing CCN3. Together, these data indicate a role for CCN3 in melanoma cell interaction with the ECM by regulating integrin expression, resulting in altered cell adhesion and leading melanoma progression to aggressive disease.

Detection of mutated BRAFV600E variant in circulating DNA of stage III-IV melanoma patients.

BRAFV600E is the most represented somatic point mutation in cutaneous melanoma, thus providing a unique molecular marker for this disease. The development of efficient methods for its detection in free circulating DNA of patients may lead to the improvement of diagnostic and prognostic tools. With this aim, we evaluated whether BRAFV600E represents a detectable marker in the plasma/serum from melanoma patients in a pilot study. Circulating cell-free DNA was extracted from the serum or plasma of 15 healthy donors and 41 melanoma patients at different clinical stages and obtained either presurgery or after surgery during follow-up. Quantitative analysis showed higher levels of circulating free DNA in patients compared to controls, with the highest levels detected in samples obtained presurgery and at stage IV. Four different PCR methods were compared for their capacity to amplify a few copies of BRAFV600E in wild-type DNA. BRAFV600E was detectable in circulating DNA of 12 patients and in none of the controls; only 1 PCR method reproducibly amplified BRAFV600E. Positive samples were obtained from 8/13 patients at stage IV and from 4/24 patients at stage III, but not in 4 patients at stage I-II; half of the positives were obtained presurgery and half at follow-up. Correspondence between circulating DNA and related tumors were examined for 20 patients, and a correlation was found for stage IV patients. In conclusion, this method can be utilized for monitoring the disease in stage IV melanoma patients but it appears unsatisfactory for the early detection of melanoma.

DHCR24 gene expression is upregulated in melanoma metastases and associated to resistance to oxidative stress-induced apoptosis.

The DHCR24 gene encoding for the 3beta-hydroxysterol delta24-reductase, an oxidoreductase involved in cholesterol biosynthesis, was isolated by subtractive hybridization as highly expressed in a short-term melanoma cell line derived from a cutaneous metastases (S/M2) compared to that obtained from the autologous primary tumor (S/P). DHCR24 (alias seladin-1, diminuto/dwarf1 homolog) has been reported to act as an antiapoptotic factor in neurons. Gene expression analysis by Northern blot confirmed that DHCR24 was 5-fold upregulated in S/M2 compared to S/P cells. High levels of DHCR24 gene expression were detected in 13/25 melanoma metastases and in 1/7 primary melanomas by real-time PCR, indicating that upregulation of this gene may occur in melanoma progression. In S/M2 cells, high DHCR24 gene expression associated with resistance to apoptosis triggered by oxidative stress induced by exposure to hydrogen peroxide. DHCR24 gene transfer was shown to protect melanoma cells from H2O2-induced cytotoxicity. Although higher cholesterol levels were shown in S/M2 cells compared to S/P cells, DHCR24 gene transfer did not increase cholesterol content. To evaluate whether DHCR24 acts as an antiapoptotic factor in melanoma metastases, the cytotoxic effect of chemotherapeutic agents was tested in DHCR24 transfectants and in the presence of a DHCR24 inhibitor, U18666A. High DHCR24 gene expression in transfectants did not result in a higher resistance to cytotoxic agents; treatment with U18666A was cytotoxic in S/P cells with a lower DHCR24 content and showed additive cytotoxic effect only when associated with H2O2 and not with cysplatin or etoposide, indicating that the DHCR24 protective effect is exerted through an oxidative stress-specific mechanism.