Mitochondrial DNA instability in malignant melanoma of the skin is mostly restricted to nodular and metastatic stages

Ultraviolet (UV) radiation is thought to be a major contributor to the development of sporadic malignant melanoma of the skin. It may induce alterations in genomic or mitochondrial DNA (mtDNA), especially C to T or CC to TT changes. Mutations or other alterations in mtDNA have been reported in a variety of human cancers and may be due to different mechanisms. In this study, we have attempted to elucidate whether aberrations in the mtDNA of melanoma are due to UV radiation or other factors by investigating two parts of the mitochondrial D-loop and two mitochondrial genes, as well as looking for the delta4977 mtDNA deletion and mtDNA duplications, in 61 primary malignant melanomas and neighbouring normal skin tissue (in 70% of primary tumours; otherwise, corresponding blood samples). Point mutations were a rare feature, occurring in only seven tumour samples and never as a C to T change, whereas mtDNA instability in the D-loop (mtMSI) was found in 13% of primary nodular tumours and 20% of metastases. A de novo delta4977 mtDNA deletion was demonstrated in 10% of melanomas; in 20% of patients, mtDNA duplications and/or the delta4977 mtDNA deletion was detectable. Our data indicate that mtDNA alterations in malignant melanoma are not induced by UV radiation. In addition, point mutations and mtMSI were mostly a feature of nodular and metastatic melanoma samples.     

Mutational analysis of ras gene family in lung cancer in Thai

H-, K- and N-ras gene mutations were analyzed in lung cancer from Thai patients. Thirteen out of 58 cases (22%) harbored the mutations. Ten cases showed K-ras gene mutations at codon 12, 1 case presented a mutation at codon 13 and another case exhibited a mutation at codon 63. Silent mutations of N-ras gene in codons 57 and 62 were seen in one patient, whilst no H-ras mutation was found in these patients. Bases change in K-ras gene were G right curved arrow T transversion (62%), G right curved arrow A transition (15%) and G right curved arrow C transition (15%), whereas T right curved arrow G transversion and A right curved arrow G transition were detected in N-ras mutant gene.     

ATM gene mutations in former uranium miners of SDAG Wismut: a pilot study

Ataxia-telangiectasia is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. Heterozygous carriers of an ataxia-telangiectasia gene mutation are predisposed to epithelial cancers. We initiated a study to elucidate the frequency and clinical relevance of ATM gene mutations in former uranium miners exposed to high levels of radiation from radon and its decay products. Former uranium miners with Schneeberg lung cancer (n=48), former uranium miners suffering from silicosis (n=60) and uranium miners without occupational lung disorders (n=102) were investigated for nine mutations in the ATM gene. One gastric and one prostate cancer occurred in the group of miners without occupational lung diseases. Mutation analyses for S707P, IVS10-6Tright curved arrow G, 2250Gright curved arrow A, E1978X, R2443X, 3801delG, S49C and D2625E-A2626P were performed using genomic DNA obtained from peripheral blood samples. Three ATM gene alterations (S707P, S49C or IVS10-6Tright curved arrow G) were observed. Of all cancer patients, 8.0% were heterozygous, but only 1.9% of the non-cancer controls were [OR=4.6; 95% confidence interval (CI), 0.8-26.8]. In this pilot study a major role of six ATM gene mutations could not be revealed for cancer predisposition in former uranium miners. The results leave the possibility of a moderate risk associated with more subtle ATM gene alterations.     

Analysis of skin cancer risk factors in immunosuppressed renal transplant patients shows high levels of UV-specific tandem CC to TT mutations of the p53 gene

Immunosuppressed renal transplant recipients (RTRs) are predisposed to non-melanoma skin cancers (NMSCs), predominantly squamous cell carcinomas (SCCs). We have analyzed skin lesions from RTRs with aggressive tumors for p53 gene modifications, the presence of Human Papillomas Virus (HPV) DNA in relation to the p53 codon 72 genotype and polymorphisms of the XPD repair gene. We detected 24 p53 mutations in 15/25 (60%) NMSCs, 1 deletion and 23 base substitutions, the majority (78%) being UV-specific C to T transitions at bipyrimidine sites. Importantly, 35% (6/17) are tandem mutations, including 4 UV signature CC to TT transitions possibly linked to modulated DNA repair caused by the immunosuppressive drug cyclosporin A (CsA). We found 8 p53 mutations in 7/17 (41%) precancerous actinic keratosis (AK), suggesting that p53 mutations are early events in RTR skin carcinogenesis. Immunohistochemical analysis shows a good correlation between p53 accumulation and mutations. HPV DNA was detected in 78% of skin lesions (60% Basal Cell Carcinomas, 82%AK and 79% SCCs). Thus, immunosuppression has increased the risk of infections by HPVs, predominantly epidermodysplasia verruciformis, speculated to play a role in skin cancer development. No association is found between HPV status and p53 mutation. Moreover, p53 codon 72 or frequencies of three XPD genotypes of RTRs are comparable with control populations. The p53 mutation spectrum, presenting a high level of CC to TT mutations, shows that the UV component of sunlight is the major risk factor and modulated DNA repair by immunosuppressive drug treatment may be significant in the skin carcinogenesis of RTRs.     

Mitochondrial DNA mutations in chemical carcinogen-induced rat bladder and human bladder cancer

Mitochondrial (mt) DNA mutations have been described recently in different tumors, whereas similar studies focusing on bladder cancer are scarce. In an effort to understand the significance of mtDNA mutations in bladder cancer, we investigated the mtDNA alterations in both clinical human bladder cancer and in a carcinogen-induced rat bladder cancer model. Human bladder cancer tissues were obtained by radical cystectomy and transurethral resection of bladder tumors. Rat bladder tumors were induced in SD rats by treatment with N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for 24-28 weeks. Genomic DNA was extracted from tumor specimens and microdissected normal bladder mucosae. Mitochondrial genes and D-loop region were amplified by PCR. The amplified PCR fragments were either cloned into plasmid vector or used for direct DNA sequencing. The results of DNA sequence revealed numerous point mutations in the non-coding D-loop region and different mtDNA genes in both human and rat bladder cancers. In addition, we also detected deletions of variable lengths in mononucleotide repeats in the D-loop region, ND2, ATPase 8 and COIII genes in human bladder cancer samples. Our results show that mtDNA exhibits a high rate of mutations in both human and rat bladder cancers. We also demonstrate that the repetitive sequences of mononucleotides within the mt genome are unstable and subjected to deletions. The high incidence of mtDNA mutations in bladder cancer suggests that mtDNA and mitochondria could play an important role in the process of carcinogenesis and also mtDNA could be valuable as a marker for early bladder cancer diagnosis.     

Mitochondrial microsatellite instability of colorectal cancer stroma

Mitochondrial microsatellite instability (mtMSI) and mutations of mitochondrial DNA has been reported in cancer epithelia of carcinomas. However, mtMSI in cancer stroma has not yet been identified in human cancers. In this study, we attempted to determine if mtMSI occurs in the cancer stroma of sporadic colorectal cancers, and if the stromal mtMSI has any correlations with stromal nuclear MSI (nMSI) and cancer epithelial mtMSI. Nine microsatellite sequences within the D-loop and 5 coding genes for mtMSI, and 9 microsatellites for nMSI were analyzed in the microdissected cancer epithelia and adjacent stromas of 48 sporadic colorectal cancers. Overall, 23 somatic mitochondrial DNA alterations were detected in 15 cancer epithelia (31.2%) and 5 stromas (10.4%). The mutations consisted of 19 D-loop mtMSI alterations, and 1 missense and 3 framshift mutations of repeat sequences within the coding genes. All of the 5 stromal genetic alterations showed D-loop mtMSI. In regards to other MSI status, the stromal mtMSI had no association with stromal nMSI or epithelial mtMSI, either. These findings indicate that in addition to the cancer epithelia the cancer stroma harbor mtMSI, and suggest a possible role of stromal mtMSI in the pathogenesis of colorectal cancers. Furthermore, the data suggest that stromal mtMSI may occur independently of stromal nMSI and epithelial mtMSI in sporadic colorectal cancers.     

Frequency and molecular analysis of hprt mutations induced by estradiol in Chinese hamster V79 cells

The natural hormone estradiol (E2) induces tumors in rodents and various types of DNA damage in vitro and in vivo, but has not been mutagenic in bacterial or mammalian assays. Recent reports of chromosomal and genetic lesions induced by E2 has led us to re-examine the mutation frequency and molecular alterations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in Chinese hamster V79 cells. E2 at both physiological and pharmacological concentrations (10-11, 10-10, and 10-7, 10-6 M) significantly increased the mutation frequency of the hprt gene by 2. 57-, 3.45-, 2.63-, and 8.78-fold, respectively, compared to the controls, while 10-13, 10-12, 10-9, or 10-8 M E2 induced little change (< or =0.93-fold). PCR and a molecular analysis of the hprt coding sequence identified genetic lesions in the cDNA and/or genomic DNA in 15 of the 21 picked E2-induced mutants (71%). Simple base substitutions, such as Tright curved arrow G or Tright curved arrow A transversions, were the most common mutations (8/21 or 38%) and frequently occurred at 122 bp or 407 bp of the hprt coding sequence. Deletion mutations were detected in 6 of the 21 clones (29%). An Aright curved arrow G and a Cright curved arrow T transition and a four-base insertion (TATT) were identified each in one mutant clone. A RT-PCR analysis demonstrated an abundant expression of the estrogen receptor-alpha (ERalpha). However, ICI 182,780, an antagonist of ERalpha, acted in an additive manner with E2 and increased the hprt mutation frequency. In conclusion, E2 induces a low frequency of mutations (deletions and point mutations) in V79 cells, which is consistent with the weak carcinogenic activity of this hormone. The mutagenic effects of E2 in V79 cells are not mediated by the ERalpha.     

The oncogenic B-raf V599E mutation occurs more frequently in melanomas at sun-protected body sites

Downstream of Ras, the serine/threonine kinase, B-raf, has recently been reported to be mutated, among other carcinomas, in a substantial subset of primary melanomas with a preponderance of the oncogenic V599E transition. As the risk of melanoma is enhanced by intermittent ultraviolet (UV) exposure but is less with chronic UV exposure, we here studied B-raf kinase domain (exon 15) mutations in primary cutaneous melanomas with respect to anatomical locations reflecting chronic versus intermittent UV exposure. Investigating a representative number of 101 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain by polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 32 cases (32%) which harbour somatic B-raf exon 15 mutations. As to the B-raf protein sequence, the V599E mutation was predicted in 66% of these positive melanomas, followed in frequency by the V599K transition (16%). Only two Cright curved arrow T transitions, considered to be induced by UV irradiation, occurred in two melanomas located on the head. Among 23 melanomas located at body sites with chronic UV exposure, only a single tumour harboured the B-raf V599E mutation (4%), which was a significantly lower frequency in comparison to melanomas from sun-protected body sites (26%; Fisher's exact test, p=0.038; odds ratio, 7.59). Our observation parallels the epidemiological data of intermittent sunlight exposure on unacclimatised skin increasing the risk of melanoma development.     

Germline mutations of BRCA1 in two Korean hereditary breast/ovarian cancer families

Testing for cancer susceptibility gene, in particular mutations in the BRCA1 gene in association with hereditary breast/ovarian cancer has been extensively studied. We investigated germline mutations in the BRCA1 gene from two Korean hereditary breast/ovarian cancer families using direct DNA sequencing. Blood samples of the thirteen family members were studied. We found three missense mutations; 3232 Aright curved arrow G, 2731 Cright curved arrow T, 3667 Aright curved arrow G. These mutations were involved in the altered coding of amino acids. According to the BIC database, clinical significance of these mutations is regarded as favor polymorphisms. Therefore, these genetic variations are not believed to be involved in the development of the disease, but may be associated with breast/ovarian cancers in another yet undefined way. For further clinical significance of these variations, additional study such as a case-controlled haplotyping study is needed.     

Search for mutations of the hRAD54 gene in sporadic meningiomas with deletion at 1p32.

The hRAD54 gene is related to a family of genes involved in DNA recombination and repair and encodes a protein with DNA helicase activity. hRAD54 has been mapped to 1p32, a region frequently involved in deletions in a variety of tumor types, including atypical and anaplastic meningiomas. To determine whether alterations of hRAD54 are a common event in meningeal tumors, by means of polymerase chain reaction-single-stranded conformation analysis we examined 29 tumor samples characterized by 1p deletions for hRAD54 mutations. Although 18 tumors displayed allelic loss at the gene region (1p32) as determined by microsatellite marker analysis, the sole coding-sequence alteration detected corresponded to a T-->C transition, with no amino-acid change. The genotype distribution was 10.34% TT, 44.8% TC, and 44.8% CC, whereas in the normal controls it was 3.77% TT, 13.2% TC, and 83.01% CC, and most meningiomas with 1 p32 deletion retained allele C. Another polymorphism due to a T-->C change was evidenced at nt 3008, in the 3' untranslated region. This change was evidenced in all cases we sequenced. These results appear to exclude the involvement of the hRAD54 gene in the pathogenesis of the nontypical meningiomas, although a detrimental effect of the hRAD54 polymorphisms cannot be ruled out.     

乳腺癌线粒体基因组控制区突变的研究

目的:通过测定乳腺癌线粒体基因组控制区突变的频率和分布,探讨其在肿瘤发生中的作用。方法:提取30例乳腺癌患者的癌及癌旁正常组织细胞总DNA,PCR扩增线粒体DNA(mitochondrialDNA,mtDNA)基因组控制区D-loop区,单链构象多态性(single-strandconformationpolymorphism,SSCP)筛查突变标本,基因测序分析突变。结果:在30例乳腺癌组织中9例(30%)乳腺癌共发现14个新的突变位点,其中6例在多聚胞嘧啶区(D310区)的311~313位点出现了CC或CCC删除。结论:乳腺癌线粒体DNA显示了高频率的D-loop区的突变,D310区是点突变、插入/缺失突变的热点。D-loop区尤其是D310区的变化在肿瘤的形成中可能扮演了重要角色。     

Characterization of a highly aberrant plasma cell leukemia karyotype: a case report

We report on a 72-year-old patient with a clinically diagnosed plasmocytoma which developed to a plasma cell leukemia (PCL) with so far unrecorded complex translocations. As GTG-banding was not able to resolve all karyotypic changes, multiplex-fluorescence in situ hybridization (M-FISH) in combination with microdissection based comparative genomic hybridization (micro-CGH) and multicolor banding (MCB) have been done. Using these molecular cytogenetic approaches the karyotype of the PCL case can be described as: 51,XY,-1,-1,+3,+der(5)t(5;11;1)(5pter right curved arrow 5q13-q14::11q24 right curved arrow 11q25::1q12 right curved arrow 1qter),+7 or +der(7)t(7;1)(7qter right curved arrow 7p15::1p31.1 right curved arrow 1pter),+8,+der(9)t(1;9)(1qter right curved arrow 1q12::9q12 right curved arrow 9pter),der(11)t(1;11;1)(1pter right curved arrow 1p31.1::11p15.5 right curved arrow 11q25::1q12 right curved arrow 1qter),-13,der(14)t(X;14)(Xqter right curved arrow Xq21.3::14pter right curved arrow 14qter),+15,+18,der(19)t(9;19)(9qter right curved arrow 9q12::19q11 right curved arrow 19pter),+i(19)(q10). The case shows one of the most complex karyotypic rearrangements ever described in PCL and indicates two additional chromosomal regions which may contain genes of interest for the development of this hematological disorder: loss of 1p10-p31.1 material and gain of Xq21.3-qter.     

Mutation profile of the p53, fhit, p16INK4a/p19ARF and H-ras genes in Indian breast carcinomas

Breast cancer is the second most prevalent cancer affecting Indian women. Genetic alterations of oncogenes and tumor suppressor genes were attributed to the development of breast carcinomas. In the present study, human breast tumor DNAs from untreated, non-familial, Indian patients were analysed for the presence of mutations in p53, fhit, p16INK4a/p19ARF and H-ras genes. Polymerase chain reaction-single strand conformation polymorphism and sequencing analysis were used to detect point mutations. Exons 5-8 of p53, exons 1-2 of p16INK4a, exon 2 of p19ARF, exons 5-9 of fhit gene and exons 1-2 of H-ras genes were amplified and analysed individually using exon-flanking primers. Only 12% of the tumors had mutation in p53, 8% had mutation in fhit gene and none of the tumors showed evidence for mutation in p16INK4a/p19ARF and H-ras genes. Tumor B18 exhibited two novel mutations in the p53 gene, ATGright curved arrow GTG (Metright curved arrow Val) at codon 237 and AATright curved arrow GAT (Asnright curved arrow Asp) at codon 263. Both of these mutations are hitherto unreported in breast carcinomas. Tumor B20 had a non-sense mutation CGAright curved arrow TGA (Argright curved arrow Stop) at codon 306 of p53 gene. In fhit gene, tumor B1 exhibited TTCTright curved arrow TACT mutation at intron 8 and tumor B15 had a silent mutation GAGright curved arrow GAA (Gluright curved arrow Glu) at codon 123. Our results indicate that, among the genes analysed, the p53 gene was more frequently mutated than fhit, p16INK4a/p19ARF and H-ras genes in Indian mammary tumors. Transcribable point mutations of fhit gene were found to be extremely uncommon in these tumors. Mutations in the above genes are mutually exclusive and are infrequent in fhit, p16INK4a/p19ARF and H-ras genes suggesting that these genes may not play a major role in Indian breast carcinomas. However, the significant frequency of mutations in the p53 gene suggest that p53 could be one of the genes involved in the genesis of sporadic breast carcinomas in Indian women.     

Complex chromosomal rearrangements in a secondary acute myeloblastic leukemia after chemotherapy in TRAPS

We report on the cytogenetic findings from a patient with a de novo TNF-receptor-associated periodic syndrome (TRAPS), who showed first symptoms at the age of four months. Thus, he obtained a long-term therapy with cortisone, chlorambucile, methotrexate and cyclophosphamide. At the age of 14 he developed a secondary acute myeloblastic leukemia. Highly complex chromosomal rearrangements were detected after banding analysis. The exact definition of karyotype and the involved breakpoints could only be resolved after application of sophisticated multicolor-FISH techniques: 44,XY,-5,der(6)t(6;7)(6pter right curved arrow 6q12::7p22.2 right curved arrow 7pter or 7pter right curved arrow 7p22.2), dic(7;19)t(6;19;6;7;19;7;19)(19qter right curved arrow 19q12::7p13 right curved arrow 7p11.1::19q12 right curved arrow 19p12 or 19p12 right curved arrow 19q12::7p11.1 right curved arrow 7q21.3::6q12 right curved arrow 6q26::19p13.3 right curved arrow 19p12::6q26-6qter),dic(12;13)(13qter right curved arrow 13p11.2::12p13.1 right curved arrow 12qter),ace(12;13)(13pter right curved arrow 13p11.2::12p13.1 right curved arrow 12pter), -19. The simultaneous presence of two dicentric chromosomes has not been reported previously and is striking, as such chromosomes are suggested to be instable. However, such chromosomes are observed frequently after chemo- or radiotherapy and in secondary, i.e. therapy related AML (tAML). Thus, AML in this case may result from a long-term therapy of TRAPS with methotrexate, cyclophosphamide, chlorambucile and cortisone.     

CDKN2A germ-line mutations in individuals with multiple cutaneous melanomas

Germ-line CDKN2A mutations are present in some kindreds with hereditary cutaneous melanoma, and in Sweden a founder mutation with an extra arginine in codon 113 (113insR) has been identified. We screened 80 individuals with at least two primary cutaneous melanomas, who were identified mainly by a search of a regional cancer registry, for germ-line CDKN2A mutations. In nine patients, CDKN2A alterations that may contribute to melanoma predisposition were detected. In six individuals with a family history of melanoma, the 113insR founder mutation was present. One patient, who also had a family history of melanoma, had a 24-bp deletion that included codons 62-69. An in vitro binding assay established that the resulting mutant p16 protein was unable to bind cyclin-dependent kinase 4 and cyclin-dependent kinase 6. Two patients without a family history of melanoma had CDKN2A alterations: (a) one had a mutation in the 5' noncoding sequence (-14C/T); and (b) the other had an insertion of an extra T in codon 28, which results in a stop signal in codon 43. The median age at diagnosis of the first melanoma was significantly lower, the number of primary melanomas was significantly higher, and the presence of a family history of melanoma was significantly more common in patients with CDKN2A mutations than in those without germ-line mutations. The proportion of CDKN2A mutation carriers was significantly higher among patients treated for three or more primary melanomas compared with those with two tumors only. We conclude that mutation screening of individuals with multiple primary melanomas is a useful strategy to identify new melanoma kindreds with CDKN2A germ-line mutations.     

The INK4a-ARF locus: role in the genetic predisposition to familial melanoma and in skin carcinogenesis]

The INK4a-ARF locus, localized on 9p21, encodes two tumor suppressor proteins, p16INK4a and p14ARF, acting respectively through the CDK4-pRb and the p53 pathways. Familial melanoma (comprising between 8 and 12% of all melanoma cases) is a genodermatosis transmitted as an autosomal dominant trait, often associated with clinically atypical moles (AN). Germline mutations of p16INK4a are found in up to 20-30% of melanoma prone families. Mutated families often contain more than three family members affected and/or comprise at least one relative with multiple melanomas. Most of these mutations have been shown to affect p16INK4a protein function (i.e. CDK4 binding or pRB phosphorylation). Germline mutations of p16INK4a are also found in a lesser extend in sporadic multiple melanoma and in familial pancreatic cancer. The INK4a-ARF locus plays also an important role in skin carcinogenesis. P16INK4a UV induced mutations (CC:GG > TT:AA tandem transition or C:G > T:A transition at dipyrimidic site) are found in 12% of sporadic skin carcinomas, mainly in epidermoid tumors, and seem to occur independently of p53 mutations. Xeroderma pigmentosum (XP) is characterized by an inheritable DNA repair defect (involving the nucleotid excision repair (NER) system) predisposing to skin carcinomas. In skin tumors from (XP) patients, p16INK4a UV induced mutations occur more frequently, are often multiple, and significantly associated with the presence of p53 mutations. Such data, which could be related to the XP genetic instability and indicates a possible cooperative effect of inactivation of these pathways in the tumoral process of XP skin tumors.     

Influence of methylenetetrahydrofolate reductase gene polymorphisms C677T and A1298C on age-associated risk for colorectal cancer in a caucasian lynch syndrome population.

Lynch syndrome is caused by germ-line mutations in the DNA mismatch repair (MMR) genes; mutation carriers are predisposed to a variety of cancers, most commonly colorectal and endometrial. The median age of colorectal cancer onset is 45 years and the lifetime risk is approximately 80%, but the onset age varies substantially. It is likely that other low-penetrance genes and environmental factors act as modifiers of the risk associated with the highly penetrant MMR gene mutations. Methylenetetrahydrofolate reductase plays a key role in folate metabolism. We investigated the association of C677T and A1298C, two common polymorphisms in the methylenetetrahydrofolate reductase gene, with risk for early onset colorectal cancer in Lynch syndrome. Subjects were 185 non-Hispanic whites with confirmed DNA MMR mutations. Kaplan-Meier estimates for the age at colorectal cancer onset according to C677T genotypes were significantly different for the CT and TT genotypes compared with the wild-type CC (P = 0.014, log-rank test; P = 0.004, trend test). The median ages at onset were 43 years for the CC genotype and 39 years for the combined CC and CT genotypes and the CC+CT genotypes were associated with a reduced age-associated risk for developing colorectal cancer (hazard ratio, 0.55; 95% confidence interval, 0.36-0.85). No differences in ages at onset or risk were found for the A1298C genotypes. This is the first report to our knowledge to provide evidence that the C677T polymorphism modifies the age at onset of colorectal cancer in Caucasian Lynch syndrome subjects with the 677T allele having a protective effect.