Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer

Mutation analysis of the KRAS oncogene is now established as a predictive biomarker in colorectal cancer (CRC). Large prospective clinical trials have shown that only CRCs with wild-type KRAS respond to anti-epidermal growth factor receptor (EGFR) treatment. Therefore, mutation analysis is mandatory before treatment, and reliable benchmarks for the frequency and types of KRAS mutations have to be established for routinely testing large numbers of metastatic CRCs.     

SQSTM1 mutations in Han Chinese populations with sporadic amyotrophic lateral sclerosis

Mutations in the sequestosome 1 gene (SQSTM1) have recently been identified in patients with amyotrophic lateral sclerosis, accounting for 1.11%–4.92% of familial ALS and 2.42%–4.37% of sporadic amyotrophic lateral sclerosis (SALS). The mutation spectrum of SQSTM1 in Chinese patients with SALS remains unknown. Three hundred and six patients with SALS from the Department of Neurology, West China Hospital of Sichuan University were recruited for this study. From the same region, 350 healthy individuals were recruited as a control group. The encoding regions of SQSTM1 were screened by direct sequencing. Three novel nonsynonymous mutations— p. I99L, p. D337E, and p. L341V—were identified in 3 patients with SALS, none of which were found in healthy controls. The male patient carrying mutation p. I99L presented limb symptom at age of 34 and died in 34 months. Two late-onset patients carrying D337E and p. L341V mutations had bulbar and limb onset, respectively. Moreover, a c.1166-14_1166-11delTACT mutation in the intron 7 was found in a living male patient with limb onset at age of 62. None of the patients carrying SQSTM1 mutation showed clinical evidence of concomitant Paget disease of bone or mutation of the valosin-containing protein gene. The mutation frequency of SQSTM1 was 0.98% in Chinese patients with SALS, which was lower than those in other racial populations.     

Tumour-specific methylation of PTPRG intron 1 locus in sporadic and Lynch syndrome colorectal cancer

DNA methylation is a hallmark in a subset of right-sided colorectal cancers. Methylation-based screening may improve prevention and survival rate for this type of cancer, which is often clinically asymptomatic in the early stages. We aimed to discover prognostic or diagnostic biomarkers for colon cancer by comparing DNA methylation profiles of right-sided colon tumours and paired normal colon mucosa using an 8.5 k CpG island microarray. We identified a diagnostic CpG-rich region, located in the first intron of the protein-tyrosine phosphatase gamma gene (PTPRG) gene, with altered methylation already in the adenoma stage, that is, before the carcinoma transition. Validation of this region in an additional cohort of 103 sporadic colorectal tumours and 58 paired normal mucosa tissue samples showed 94% sensitivity and 96% specificity. Interestingly, comparable results were obtained when screening a cohort of Lynch syndrome-associated cancers. Functional studies showed that PTPRG intron 1 methylation did not directly affect PTPRG expression, however, the methylated region overlapped with a binding site of the insulator protein CTCF. Chromatin immunoprecipitation (ChIP) showed that methylation of the locus was associated with absence of CTCF binding. Methylation-associated changes in CTCF binding to PTPRG intron 1 could have implications on tumour gene expression by enhancer blocking, chromosome loop formation or abrogation of its insulator function. The high sensitivity and specificity for the PTPRG intron 1 methylation in both sporadic and hereditary colon cancers support biomarker potential for early detection of colon cancer.     

The prognostic value of DAPK1 hypermethylation in gliomas: A site-specific analysis

Background and aims

The gene of death associated protein kinase 1 (DAPK1) has been reported to be methylated in various cancers including gliomas. However, its prognostic value for gliomas is still controversy, and the methylation at specific CpG sites of DAPK1 has not been investigated. The aim of this study was to prognostically evaluate the methylation level of different CpG sites within DAPK1 promoter region in gliomas.

Surgically treated ovarian endometriosis association with BRCA1 and BRCA2 mutations

Endometriosis is associated with an increased risk of ovarian cancer. Few studies have also shown increased risk of breast cancer. BRCA1/2 mutations are linked to an increased risk of breast and ovarian cancers but their relation to endometriosis is unknown.     

Novel BRCA1/2 mutations in Serbian breast and breast-ovarian cancer patients with hereditary predisposition

Mutations in breast cancer susceptibility (BRCA) genes lead to defects in DNA repair processes resulting in elevated genome instability and predisposing to breast and ovarian cancer. The study was designed to detect mutational spectra of BRCA1/2 genes in a Serbian population. Using automated DNA sequencing, we tested individuals for BRCA mutations, based on positive family history of either breast or ovarian cancer or both. Two novel mutations (c.4765_4784del in BRCA1 exon 15 and c.4367_4368dupTT in BRCA2 exon 11) were detected, in three probands from two different families. These mutations have not been reported previously in the BIC or LOVD databases. Protein products of these mutated alleles lack domains necessary for their DNA repair functions, an indicator that these are deleterious mutations. Neither mutation was found in any proband from 50 other families with hereditary predisposition, so the two mutations are likely family-specific rather than population-specific. Although BRCA1-associated tumors are typically negative for estrogen receptor (ER), progesterone receptor (PR), and ERBB2, the novel BRCA1 mutation identified in this study was detected in a proband with ER- and PR-positive breast cancer. Steroid receptor-positive BRCA-related breast cancer in this proband supports the idea of characteristic pathological features and older age of onset among BRCA1-mutated ER-positive breast cancers.     

Methyltransferase expression and tumor suppressor gene methylation in sporadic and familial colorectal cancer

Molecular mechanisms underlying coordinated hypermethylation of multiple CpG islands in cancer remain unclear and studies of methyltransferase enzymes have arrived at conflicting results. We focused on DNMT1 and DNMT3B, DNA methyltransferases responsible for (de novo) methylation, and EZH2, histone (H3K27) methyltransferase, and examined their roles in tumor suppressor gene (TSG) methylation patterns we have previously established in sporadic and familial cancers. Our investigation comprised 165 tumors, stratified by tissue of origin (117 colorectal and 48 endometrial carcinomas) and sporadic vs. familial disease (57 sporadic vs. 60 familial, mainly Lynch syndrome, colorectal carcinomas). By immunohistochemical evaluation, EZH2 protein expression was associated with a TSG methylator phenotype. DNMT1, DNMT3B, and EZH2 were expressed at significantly higher levels in tumor vs. normal tissues. DNMT1 and EZH2 expression were positively correlated and higher in microsatellite‐unstable vs. microsatellite‐stable tumors, whether sporadic or hereditary. Ki‐67 expression mirrored the same pattern. Promoter methylation of the methyltransferase genes themselves was addressed as a possible cause behind their altered expression. While DNMT1 or EZH2 did not show differential methylation between normal and tumor tissues, DNMT3B analysis corroborated the regulatory role of a distal promoter region. Our study shows that methyltransferase expression in cancer depends on the tissue of origin, microsatellite‐instability status, cellular proliferation, and—in the case of DNMT3B—promoter methylation of the respective gene. Translation of methyltransferase expression into DNA methylation appears complex as suggested by the fact that except for EZH2, no clear association between methyltransferase protein expression and TSG methylation was observed. © 2015 Wiley Periodicals, Inc.     

维吾尔族和汉族散发性乳腺癌患者BRCA1/2基因突变的检测

目的探讨新疆维吾尔族和汉族散发性乳腺癌患者乳腺癌易感基因1/2(BRCA1/2)突变情况及与临床病理参数的关系。方法采用PCR和DNA直接测序法,对新疆地区230例散发性乳腺癌患者(维吾尔族、汉族各115例)石蜡组织进行BRCA1基因第2、11(11A和11B)、20号外显子和BRCA2基因第11号部分外显子,共5对引物进行突变检测。结果 230例乳腺癌患者中,BRCA基因突变率为6.96%(16/230),其中1例BRCA1基因-5 382位点的突变及7例新发突变位点;维吾尔族和汉族患者中BRCA基因突变检出率分别为7.83%(9/115)和6.09%(7/115);BRCA基因突变组发病年龄均≤50岁;突变组16例患者中绝经前患者(13例)的突变率明显高于绝经后患者(3例)(P0.05)。结论 BRCA1基因突变可能与新疆地区散发性乳腺癌发生相关。     

FUS, TARDBP, and SOD1 mutations in a Taiwanese cohort with familial ALS

The cause of familial amyotrophic lateral sclerosis (FALS) has been attributed to mutations in several genes. The authors analyzed these genes, including SOD1, FUS, VAPB, ANG, TDP-43, FIG4, and CHMP2B, in a cohort of 15 index patients of Han Chinese descent with adult-onset FALS. Seven different mutations in eight patients, including three in SOD1 (G85R, T137R, and G138E), two in exon 15 of FUS (H517D and R521H), and two in exon 6 of TARDBP (M337V and N378D) were identified. Among them, T137R SOD1, G138E SOD1, H517D FUS, and N378D TARDBP were novel. No mutation was found in VAPB, ANG, FIG4, or CHMP2B genes. Mutations in SOD1, FUS, and TARDBP account for 20%, 13.3%, and 20% of FALS, respectively. This study defined the distribution and frequency of mutations of FALS in a Taiwanese Han Chinese population, which not only broadens the spectrum of the mutations causing FALS, but also further highlights the importance of FUS and TARDBP in the pathogenesis of amyotrophic lateral sclerosis (ALS).     

Intratumoral heterogeneity of KRAS mutation is rare in non-small-cell lung cancer

Background

Several lines of evidence indicate that mutational activation of KRAS is an early event in the carcinogenesis of non-small cell lung cancer (NSCLC). Nonetheless, previous studies report high frequencies of divergent KRAS mutational status between primary NSCLC and corresponding metastases. This suggests heterogeneity of the primary tumor in respect to its KRAS status. We therefore aimed to examine the frequency and the extent of such intratumoral heterogeneity.

Methods

40 NSCLC were examined for intratumoral heterogeneity of KRAS mutation (20 adenocarcinomas, 10 squamous cell carcinomas and 10 large cell carcinomas). Three to eight different tumor areas were analyzed for KRAS mutation and up to four corresponding lymph node metastases were included for analysis in nineteen cases. A combination of different methods for screening of heterogeneity and its validation were used including direct sequencing, laser-capture microdissection for tumor cell enrichment and the very sensitive ARMS/S method.

Results

Mutations of KRAS were found in 13/30 adenocarcinomas and large cell carcinomas. No mutations were detected in 10 squamous cell carcinomas. Four cases showed heterogeneous KRAS results by direct sequencing. More sensitive methods for KRAS mutation analysis revealed false negative results due to admixture of non-neoplastic cells in all of these samples. Intratumoral heterogeneity of KRAS mutational status was therefore confirmed in none of the analyzed cases. In addition, identical KRAS mutations were present in the primary tumor and the corresponding lymph node metastases in 19 cases examined.

Conclusions

Intratumoral heterogeneity of KRAS mutational status is rare in NSCLC but highly sensitive tools are required to reliably identify these mutations. This finding is in line with the hypothesis that oncogenic activation of KRAS is an early event and a bona fide “driver mutation” in NSCLC. Furthermore, future therapies targeting KRAS will not be limited by intratumoral heterogeneity.     

No mutations in hnRNPA1 and hnRNPA2B1 in Dutch patients with amyotrophic lateral sclerosis,frontotemporal dementia,and inclusion body myopathy

Inclusion body myopathy (IBM) associated with Paget disease of the bone, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), sometimes called IBMPFD/ALS or multi system proteinopathy, is a rare, autosomal dominant disorder characterized by progressive degeneration of muscle, brain, motor neurons, and bone with prominent TDP-43 pathology. Recently, 2 novel genes for multi system proteinopathy were discovered; heterogenous nuclear ribonucleoprotein (hnRNP) A1 and A2B1. Subsequently, a mutation in hnRNPA1 was also identified in a pedigree with autosomal dominant familial ALS. The genetic evidence for ALS and other neurodegenerative diseases is still insufficient. We therefore sequenced the prion-like domain of these genes in 135 familial ALS, 1084 sporadic ALS, 68 familial FTD, 74 sporadic FTD, and 31 sporadic IBM patients in a Dutch population. We did not identify any mutations in these genes in our cohorts. Mutations in hnRNPA1 and hnRNPA2B1 prove to be a rare cause of ALS, FTD, and IBM in the Netherlands.