The metastatic microenvironment: Melanoma-microglia cross-talk promotes the malignant phenotype of melanoma cells.

Melanoma has the highest propensity to metastasize to the brain compared to other cancers, as brain metastases are found frequently high in patients who have prolonged survival with visceral metastasis. Once disseminated in the brain, melanoma cells communicate with brain resident cells that include astrocytes and microglia. Microglia cells are the resident macrophages of the brain and are the main immunological cells in the CNS involved in neuroinflammation. Data on the interactions between brain metastatic melanoma cells and microglia and on the role of microglia-mediated neuroinflammation in facilitating melanoma brain metastasis are lacking. To elucidate the role of microglia in melanoma brain metastasis progression, we examined the bidirectional interactions between microglia and melanoma cells in the tumor microenvironment. We identified the molecular and functional modifications occurring in brain-metastasizing melanoma cells and microglia cells after the treatment of each cell type with supernatants of the counter cell type. Both cells induced alteration in gene expression programs, cell signaling, and cytokine secretion in the counter cell type. Moreover, melanoma cells exerted significant morphological changes on microglia cells, enhanced proliferation, induced matrix metalloproteinase-2 (MMP-2) activation, and cell migration. Microglia cells induced phenotypic changes in melanoma cells increasing their malignant phenotype: increased melanoma proliferation, MMP-2 activity, cell migration, brain endothelial penetration, and tumor cells ability to grow as spheroids in 3D cultures. Our work provides a novel insight into the bidirectional interactions between melanoma and micoglia cells, suggesting the contribution of microglia to melanoma brain metastasis formation.

Evidence for an instructive mechanism of de novo methylation in cancer cells.

DNA methylation has a role in the regulation of gene expression during normal mammalian development but can also mediate epigenetic silencing of CpG island genes in cancer and other diseases. Many individual genes (including tumor suppressors) have been shown to undergo de novo methylation in specific tumor types, but the biological logic inherent in this process is not understood. To decipher this mechanism, we have adopted a new approach for detecting CpG island DNA methylation that can be used together with microarray technology. Genome-wide analysis by this technique demonstrated that tumor-specific methylated genes belong to distinct functional categories, have common sequence motifs in their promoters and are found in clusters on chromosomes. In addition, many are already repressed in normal cells. These results are consistent with the hypothesis that cancer-related de novo methylation may come about through an instructive mechanism.

Mutation spectrum in HNPCC in the Israeli population.

Hereditary non-polyposis colon cancer is caused by mutations in DNA mismatch repair genes. The mutation spectrum in the Israeli population is poorly documented except for the c.1906G>C Ashkenazi founder mutation in the hMSH2 gene. To report our experience in HNPCC screening, the mutations detected and the clinical features among a cohort of Israeli patients. Diagnostic work-up was done in a multi-step process guided by clinical and ethnic information. Tumors of suspected patients were tested for microsatellite instability and immunohistochemistry. Based on tumor analyses, we proceeded to mutation screening by DHPLC followed by sequence analysis and multiplex ligase dependent probe amplification. Ashkenazi Jews were first tested for the c.1906G>C founder mutation. Of the 240 families, 24, including Arabs and Jews from different ethnic origins, were tested positive. All tumors that lost expression of mismatch repair proteins also showed microsatellite instability. There was evidence for involvement of hMSH2 (15) hMLH1 (6) and hMSH6 (3) genes. Mutations were identified in 17/24 (71%) patients: 6 Ashkenazi families harbored the c.1906G>C mutation. Eleven other mutations (2 nonsense, 3 splice site and 6 small deletions) were detected. Three of the mutations are novel. No gross deletions or insertions were detected. This is the first report that characterizes the profile of HNPCC in a cohort of patients in Israel. Tumor testing indicated that the 3 main MMR genes are involved, and that mutation spectrum is broad.

[A new oncogenetic service of counseling and diagnosing for hereditary non-polyposis colorectal cancer (HNPCC)].

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant cancer predisposition syndrome associated with a high risk for colorectal cancer (up to 80%), endometrial cancer (up to 60%), and increased risk for other malignancies, mostly ovarian and urinary system tumors. HNPCC is caused by a germline mutation in one of the mismatch repair (MMR) genes, mainly hMLH1, hMSH2 and hMSH6. The tumors present with microsatellite instability (MSI) associated with loss of heterozygosity of the affected gene, and with loss of expression of the gene product. Diagnosis of HNPCC involves tumor testing for MSI, immunohistochemistry staining and germ line mutation analysis of the suspected gene. Proper genetic counseling is based on the synthesis of the clinical, pathological and molecular data. Directed surveillance shows significant reduction in colon cancer incidence, cancer mortality and overall mortality among HNPCC patients. GOAL: To establish a multidisciplinary service for patients suspected of having HNPCC. METHODS: We have established a service which is based on tight collaboration between clinical departments and laboratories. The clinical work-up was conducted by a special oncogenetic clinic and the laboratory service consisted of tissue testing for MSI and immunohistochemistry, denaturing high performance liquid chromatography (DHPLC) for suspected genes, and mutation testing. RESULTS: The efficiency of detection of patients with HNPCC was high, completed in a multistep process. In the first year of our collaborative work, we have provided genetic counseling to over 100 families and performed suitable tests for 46 families. Among them we have identified more than 16 families with HNPCC; 4 showed absence of hMLH1, 1 showed absence of hMSH6, and 11 showed absence of hMSH2. All tumors that showed MSI also showed absence of either one of the three MMR proteins. We present the clinical, pathological and molecular features of our patients and discuss the implication of this data on future recommendations.