Andreas Gast

TERT Promoter Mutations in Familial and Sporadic Melanoma.

Promoter Mutations and Cancer Cancer genome sequencing projects have highlighted the pathogenic role of recurrent mutations within the protein-coding regions of genes. Now, two studies suggest that the scope of mutations in human tumors extends to gene regulatory regions. In a study of 70 melanomas, Huang et al. (p. 957, published online 24 January) found that 71% harbored one of two specific mutations in the promoter region of TERT, the gene coding for the catalytic subunit of telomerase, the enzyme that caps chromosome ends. Independently, Horn et al. (p. 959, published online 24 January) identified a disease-segregating germline mutation in the TERT promoter in a family predisposed to melanoma and found additional TERT promoter mutations in a high percentage of sporadic melanomas and melanoma cell lines. The mutations in both studies generated new binding sites for specific transcription factors and, in reporter assays, caused an increase in transcription. A large fraction of human melanomas harbor mutations in sequences that regulate the expression of telomerase. Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.

Variation in CDKN2A at 9p21.3 influences childhood acute lymphoblastic leukemia risk

Using data from a genome-wide association study of 907 individuals with childhood acute lymphoblastic leukemia (cases) and 2,398 controls and with validation in samples totaling 2,386 cases and 2,419 controls, we have shown that common variation at 9p21.3 (rs3731217, intron 1 of CDKN2A) influences acute lymphoblastic leukemia risk (odds ratio = 0.71, P = 3.01 × 10−11), irrespective of cell lineage.

Verification of the susceptibility loci on 7p12.2, 10q21.2, and 14q11.2 in precursor B-cell acute lymphoblastic leukemia of childhood

Recent genome-wide association data have implicated genetic variation at 7p12.2 (IKZF1), 10q21.2 (ARIDB5), and 14q11.2 (CEBPE) in the etiology of B-cell childhood acute lymphoblastic leukemia (ALL). To verify and further examine the relationship between these variants and ALL risk, we genotyped 1384 cases of precursor B-cell childhood ALL and 1877 controls from Germany and the United Kingdom. The combined data provided statistically significant support for an association between genotype at each of these loci and ALL risk; odds ratios (OR), 1.69 (P = 7.51 x10(-22)), 1.80 (P = 5.90 x 10(-28)), and 1.27 (P = 4.90 x 10(-6)), respectively. Furthermore, the risk of ALL increases with an increasing numbers of variant alleles for the 3 loci (OR(per-allele) = 1.53, 95% confidence interval, 1.44-1.62; P(trend) = 3.49 x 10(-42)), consistent with a polygenic model of disease susceptibility. These data provide unambiguous evidence for the role of these variants in defining ALL risk underscoring approximately 64% of cases.

B-RAF and N-RAS Mutations Are Preserved during Short Time In Vitro Propagation and Differentially Impact Prognosis

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients.

Somatic alterations in the melanoma genome: A high‐resolution array‐based comparative genomic hybridization study

We performed DNA microarray‐based comparative genomic hybridization to identify somatic alterations specific to melanoma genome in 60 human cell lines from metastasized melanoma and from 44 corresponding peripheral blood mononuclear cells. Our data showed gross but nonrandom somatic changes specific to the tumor genome. Although the CDKN2A (78%) and PTEN (70%) loci were the major targets of mono‐allelic and bi‐allelic deletions, amplifications affected loci with BRAF (53%) and NRAS (12%) as well as EGFR (52%), MITF (40%), NOTCH2 (35%), CCND1 (18%), MDM2 (18%), CCNE1 (10%), and CDK4 (8%). The amplified loci carried additional genes, many of which could potentially play a role in melanoma. Distinct patterns of copy number changes showed that alterations in CDKN2A tended to be more clustered in cell lines with mutations in the BRAF and NRAS genes; the PTEN locus was targeted mainly in conjunction with BRAF mutations. Amplification of CCND1, CDK4, and other loci was significantly increased in cell lines without BRAF‐NRAS mutations and so was the loss of chromosome arms 13q and 16q. Our data suggest involvement of distinct genetic pathways that are driven either through oncogenic BRAF and NRAS mutations complemented by aberrations in the CDKN2A and PTEN genes or involve amplification of oncogenic genomic loci and loss of 13q and 16q. It also emerges that each tumor besides being affected by major and most common somatic genetic alterations also acquires additional genetic alterations that could be crucial in determining response to small molecular inhibitors that are being currently pursued.

Folate metabolic gene polymorphisms and childhood acute lymphoblastic leukemia: a case–control study

We genotyped six folate metabolic pathway genes for 11 polymorphisms in 460 cases of childhood acute lymphoblastic leukemia (ALL) and 552 ethnically matched controls. None of the polymorphisms except the 66A>G (I22M) in the 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) gene showed any effect on disease risk. The carriers of the G-allele were associated with a marginal decreased risk of ALL (gender-adjusted global P=0.03; multiple-testing corrected P=0.25). Analysis of four polymorphisms in the MTRR gene showed statistically significant differences in haplotype distribution between cases and controls (global P<0.0001). The haplotypes GCAC (odds ratio (OR) 0.5, 95% confidence interval (CI) 0.4–0.6) and ATAC (OR 0.5, 95% CI 0.3–0.6) were associated with a reduced risk and the haplotypes ACAC (OR 2.3, 95% CI 1.8–2.9) and GTAC (OR 1.8, 95% CI 1.4–2.3) with an increased risk. The genotype-combination analyses indicated that the best model stratifies cases and controls based on the 66A>G and the 524C>T polymorphisms in the MTRR gene (global P=0.03). Our results suggest that, besides a weak association of childhood ALL with the 66A>G polymorphism, haplotypes within the MTRR gene may, in part, account for population-based differences in risk.

Association of Inherited Variation in Toll-Like Receptor Genes with Malignant Melanoma Susceptibility and Survival

The family of Toll-like receptors (TLRs) is critical in linking innate and acquired immunity. Polymorphisms in the genes encoding TLRs have been associated with autoimmune diseases and cancer. We investigated the genetic variation of TLR genes and its potential impact on melanoma susceptibility and patient survival. The study included 763 cutaneous melanoma cases recruited in Germany and 736 matched controls that were genotyped for 47 single nucleotide polymorphisms (SNPs) in 8 TLR genes. The relationship between genotype, disease status and survival was investigated taking into account patient and tumor characteristics, and melanoma treatment. Analysis of 7 SNPs in TLR2, 7 SNPs in TLR3 and 8 SNPs in TLR4 showed statistically significant differences in distribution of inferred haplotypes between cases and controls. No individual polymorphism was associated with disease susceptibility except for the observed tendency for TLR2-rs3804099 (odds ratio OR  = 1.15, 95% CI 0.99–1.34, p = 0.07) and TLR4-rs2149356 (OR = 0.85, 95% CI 0.73–1.00, p = 0.06). Both polymorphisms were part of the haplotypes associated with risk modulation. An improved overall survival (Hazard ratio HR 0.53, 95% CI 0.32–0.88) and survival following metastasis (HR 0.55, 95% CI 0.34–0.91) were observed in carriers of the variant allele (D299G) of TLR4-rs4986790. In addition various TLR2, TLR4 and TLR5 haplotypes were associated with increased overall survival. Our results point to a novel association between TLR gene variants and haplotypes with melanoma survival. Our data suggest a role for the D299G polymorphism in the TLR4 gene in overall survival and a potential link with systemic treatment at stage IV of the disease. The polymorphic amino acid residue, located in the ectodomain of TLR4, can have functional consequences.

Polymorphisms in the CD28/CTLA4/ICOS genes: role in malignant melanoma susceptibility and prognosis?

The appearance of vitiligo and spontaneous regression of the primary lesion in melanoma patients illustrate a relationship between tumor immunity and autoimmunity. T lymphocytes play a major role both in tumor immunity and autoimmunity. CD28, Cytotoxic T lymphocyte antigen 4 (CTLA4) and inducible costimulator (ICOS) molecules are important secondary signal molecules in the T lymphocyte activation. Single nucleotide polymorphisms (SNPs) in the CD28/CTLA4/ICOS gene region were reported to be associated with several autoimmune diseases including, type-1 diabetes, SLE, autoimmune thyroid diseases and celiac disease. In this study, we investigated the association of SNPs in the CD28, CTLA4 and ICOS genes with the risk of melanoma. We also assessed the prognostic effect of the different polymorphisms in melanoma patients. Twenty-four tagging SNPs across the three genes and four additional SNPs were genotyped in a cohort of 763 German melanoma patients and 734 healthy German controls. Influence on prognosis was determined in 587 melanoma cases belonging to stage I or II of the disease. In general, no differences in genotype or allele frequencies were detected between melanoma patients and controls. However, the variant alleles for two polymorphisms in the CD28 gene were differentially distributed in cases and controls. Similarly no association of any polymorphism with prognosis, except for the rs3181098 polymorphism in the CD28 gene, was observed. In addition, individuals with AA genotype for rs11571323 polymorphism in the ICOS gene showed reduced overall survival. However, keeping in view the correction for multiple hypothesis testing our results suggest that the polymorphisms in the CD28, CTLA4 and ICOS genes at least do not modulate risk of melanoma and nor do those influence the disease prognosis in the investigated population.

Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma

Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.

Single-nucleotide polymorphisms in DNA-repair genes and cutaneous melanoma.

Single-nucleotide polymorphisms in different DNA-repair genes are reported to modulate risk of various cancers including melanoma. We genotyped DNA from 1186 melanoma patients and 1280 healthy controls for 13 different polymorphisms in eight DNA-repair genes. Data analyses showed that none of the polymorphisms except T241M XRCC3 was associated with an increased risk for cutaneous melanoma. Carriers of the variant alleles were associated with a decreased risk (OR 0.83; 95% CI, 0.79-0.98). Three additional polymorphisms together with T241M XRCC3 that tagged the entire gene region and the neighbouring genes KLC1, ZFYVE21 and PPP1R13B were not associated with the disease risk; neither were the inferred haplotypes. Imputation showed association of comparable magnitude with 11 non-genotyped neighbouring polymorphisms. Finally, the combination of results for all polymorphisms genotyped in the present study with published data suggests that none of the investigated polymorphisms was associated with melanoma susceptibility. We conclude that 13 non-synonymous polymorphisms in eight DNA-repair genes that are frequently investigated with respect to modulation of cancer risk in populations are not associated with susceptibility to cutaneous melanoma.