A novel all-in-one intraoperative genotyping system for <Emphasis Type="Italic">IDH1</Emphasis>-mutant glioma

IDH1 gene mutation has been demonstrated to be an oncogenic driver in a majority of lower-grade gliomas (LGGs). In contrast to other central nervous neoplasms and normal brain tissue without IDH1 mutation, almost 80% of LGGs exhibit IDH1 mutation. Therefore, expeditious detection of IDH1 mutation is useful, not only for intraoperative diagnosis of these gliomas but also for determination of the border between the tumor and normal brain tissue. In this study, we established a rapid genotyping assay with a simple DNA extraction method, involving only incubation of the tumor specimen with Tris–EDTA buffer, which can be easily performed in an operating room. In all 11 tested cases, we could identify the IDH1 status within 90–100 min intraoperatively. In a case of anaplastic astrocytoma, IDH-mutant, we could detect the tumor border by IDH1 profiling. In addition, with this assay, we could detect IDH1 mutation using cell-free tumor DNA derived from cerebrospinal fluid in a case of glioblastoma, IDH-mutant. Considering that clinical trials of mutated IDH1 inhibitors are ongoing, less-invasive intraoperative IDH1 gene profiling might be useful for decision making of the overall treatment strategy of LGGs. Our assay might be a useful tool for precision medicine and surgery of IDH1-mutant gliomas.     

<Emphasis Type="Italic">BRAF</Emphasis>/K-<Emphasis Type="Italic">ras</Emphasis> mutation,microsatellite instability,and promoter hypermethylation of <Emphasis Type="Italic">hMLH1</Emphasis>/<Emphasis Type="Italic">MGMT</Emphasis> in human gastric carcinomas

Background The BRAF and K-ras genes are the most frequently mutated oncogenes in various human malignancies. We examined BRAF and K-ras mutations in human gastric cancer, and investigated their relationship with microsatellite instability (MSI) and the hypermethylation of promoter regions in hMLH1 and O ⁶ -methylguanine DNA methyltransferase (MGMT).Methods Sixteen gastric cancer cell lines and 62 gastric cancer tissue samples were screened for BRAF and K-ras mutations by direct sequencing. We also performed a microsatellite assay and investigated methylation status in the promoter regions of hMLH1 and MGMT.Results mutation was not found in any of the cancer cell lines examined. One (1.6%) cancer tissue sample showed a point mutation in the BRAF gene (GT_G GA_G; V599E). K-ras mutation (GG_T GA_T, G12D) was detected in five (31%) gastric cancer cell lines and in 1 (1.6%) gastric cancer tissue sample. In the gastric cancer tissue samples examined, MSI was detected in 23 (37%) samples. Hypermethylated promoter regions in hMLH1 and MGMT, respectively, were detected in 6 (10%) and 13 (21%) gastric cancer tissue samples. Microsatellite stable (MSS) tumors showed frequent lymphatic invasion (P = 0.050).Conclusion Although BRAF mutation has been reported in a variety of other human cancers, it is a rare event in the carcinogenesis and progression/development of gastric cancer.     

Induction of apoptosis by <Emphasis Type="Italic">p53</Emphasis>, <Emphasis Type="Italic">bax</Emphasis>, <Emphasis Type="Italic">bcl-2</Emphasis>, and <Emphasis Type="Italic">p21</Emphasis> expressed in colorectal cancer

Background We investigated the influence of genes on the apoptosis of colorectal tumor cells, based on DNA and mRNA kinetics.Methods In 30 colorectal cancer patients, we examined the mRNA expression of p53, bax, bcl-2, and p21 ^WAF1 , and we also investigated the development of tumor cell apoptosis, using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method.Results TUNEL-positive cells showed a positive correlation with bax (P = 0.010) and a negative correlation with p21 (P = 0.04). We also investigated the relationship between p53 point mutation, p21 immunostaining degree, and apoptosis, based on DNA ladder expression. A remarkable correlation (P = 0.0090) was found between p21 and apoptosis.Conclusions The present study findings suggest that tumor cell apoptosis is (1) strongly inhibited by p21, (2) induced by bax, and (3) influenced by bcl-2, which, presumably, inhibits tumor cell apoptosis.     

The siRNA targeted to <Emphasis Type="Italic">mdr1b</Emphasis> and <Emphasis Type="Italic">mdr1a</Emphasis> mRNAs <Emphasis Type="Italic">in vivo</Emphasis>sensitizes murine lymphosarcoma to chemotherapy

Background  

One of the main obstacles for successful cancer polychemotherapy is multiple drug resistance phenotype (MDR) acquired by tumor cells. Currently, RNA interference represents a perspective strategy to overcome MDR via silencing the genes involved in development of this deleterious phenotype (genes of ABC transporters, antiapoptotic genes, etc.).     

Managing Patient with Mutations in <Emphasis Type="Italic">PALB2</Emphasis>, <Emphasis Type="Italic">CHEK2</Emphasis>, or <Emphasis Type="Italic">ATM</Emphasis>

Purpose of Review

The use of panel testing of multiple cancer-causing genes has allowed to find a subset of patients with harmful mutations in moderate penetrance genes. While extensive information is available regarding patients with BRCA1 and BRCA2 pathogenic variants, information regarding these less common genes and their management remains scarce. The aim of this review is to discuss penetrance, incidence, and management recommendations for PALB2, ATM, and CHEK2.

Recent Findings

NCCN guidelines now provide management recommendation for patients with pathogenic variants in these genes. In addition, more widespread testing has provided more information on penetrance and incidence. Although this is a huge step toward improving quality of care, prospective studies are still needed. We summarize the NCCN and other guidelines/suggestions for these genes and deliver our insight on the matter based on the best information we could find.

Summary

PALB2, ATM, and CHEK2 are less penetrant than BRCA1–2 and have a different spectrum, suggesting differing management. Data about incidence and penetrance along with management recommendations for these genes are provided.

     

Genetic variation in <Emphasis Type="Italic">IGF1</Emphasis>, <Emphasis Type="Italic">IGFBP3</Emphasis>, <Emphasis Type="Italic">IRS1</Emphasis>, <Emphasis Type="Italic">IRS2</Emphasis> and risk of breast cancer in women living in Southwestern United States

Background An insulin-related pathway to breast cancer has been hypothesized. Methods We examine the 19 CA repeat of the IGF1 gene, the -202 C > A IGFBP3, the G972R IRS, and the G1057D IRS2 polymorphisms among 1,175 non-Hispanic white (NHW) and 576 Hispanic newly diagnosed breast cancer cases and 1,330 NHW and 727 Hispanic controls living in Arizona, Colorado, New Mexico, and Utah. Results Among post-menopausal women not recently exposed to hormones, not having the 19 CA repeat of IGF1 gene was associated with breast cancer among NHW women [odds ratio (OR) 2.14, 95% confidence interval (CI) 1.21–3.79] and having an R allele of G972R IRS1 increased breast cancer risk among Hispanic women (OR 2.70, 95% CI 1.13–6.46). Among post-menopausal Hispanic women recently exposed to hormones the A allele of the -202 C > A IGFBP3 polymorphism increased risk of breast cancer (OR 1.57, 95% CI 1.06–2.33). The IGF1 19 CA repeat polymorphism interacted with hormone replacement therapy (HRT) among NHW post-menopausal women; women who had the 19/19 IGF1 genotype were at reduced risk of breast cancer (OR 0.64, 95% CI 0.47–0.88) if they did not use HRT. We also observed interaction between body mass index and IGF1 19 CA repeat (p=0.06) and between weight gain and the -202 C > A IGFBP3 polymorphism (p=0.05) in NHW post-menopausal women not recently exposed to hormones. Conclusions Our data suggest that associations between insulin-related genes and breast cancer risk among women living in the Southwestern United States may be dependent on estrogen exposure and may differ by ethnicity.     

Large genomic rearrangements of the <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> genes: review of the literature and report of a novel <Emphasis Type="Italic">BRCA1</Emphasis> mutation

Germline mutations in BRCA1 and BRCA2 increase the risk for developing breast and ovarian cancer. Previously, the techniques available allowed only for the identification of small genomic alterations, but the dawn of new technology now allows for the rapid detection of large genomic rearrangements (LGRs). LGRs in BRCA1 are responsible for between 0 and 27% of all BRCA1 disease-causing mutations identified in numerous populations. Such alterations are far less common in the BRCA2 gene. To determine the impact of BRCA1 and BRCA2 LGRs in South Africa, 52 hereditary breast and/or ovarian South African families (36 were Afrikaners) were screened for BRCA1 and BRCA2 LGRs using multiplex ligation-dependent probe amplification. These patients were previously shown to be BRCA1 and BRCA2 small mutation negative. One LGR was detected in BRCA1 in a South African family with Greek ancestry. This is a novel deletion of both exons 23 and 24 (NG_005905.2:g.169527_180579del). This first study of BRCA rearrangements in South Africa reveals that LGRs comprise ~3% of identified BRCA1 mutations, a low rate in comparison to other populations. In addition, we have reviewed all 98 previously characterized BRCA1/2 LGRs and re-named them according to the recommended HGVS nomenclature, using the recently released RefSeqGene records, NG_005905.2 and NG_012772.1 for BRCA1 and BRCA2. A standardized resource is now provided which will assist researchers in determining whether their LGRs are novel. Furthermore, we have clarified some of the previously misunderstood rules of nomenclature, which will make uniform reporting of BRCA1/2 easier in the future.     

<Emphasis Type="Italic">CYP1A1</Emphasis>, <Emphasis Type="Italic">GSTM1</Emphasis> and <Emphasis Type="Italic">GSTT1</Emphasis> polymorphisms,tobacco and alcohol status and risk of head and neck squamous cell carcinoma

We examined the influence of the CYP1A1 A4889G and T6235C, GSTM1 and GSTT1 polymorphisms, involved in carcinogen metabolism, on the head and neck (HN) squamous cell carcinoma (SCC) risk. DNA from 142 HNSCC patients and 142 controls was analysed by polymerase chain reaction (PCR)–restriction fragment length polymorphism or multiplex-PCR for the polymorphisms analyses. Excesses of the CYP1A1 4889AG+GG and 4889AG+GG plus GSTT1 null genotype were seen in patients with heavy tobacco habit compared with controls (41.9% versus 26.8%, P = 0.03; 26.2% versus 10.3%, P = 0.04, respectively). Carriers of the referred genotypes and heavy tobacco consumption were under a 2.0-fold and 2.8-fold increased risks for HNSCC than others, respectively. The CYP1A1 6235TC+CC plus GSTM1 and GSTT1 null genotypes were more common in pharyngeal SCC patients than in controls (5.3% versus 0.7%, P = 0.04). Carriers of the combined genotype had 16.0-fold increased risk for the disease than others. The frequency of one null genotype of the GSTM1 or GSTT1 gene was higher in patients with pharyngeal SCC and heavy smoking status than in controls (76.3% versus 57.7%, P = 0.04). Carriers of the referred genotype and heavy tobacco status had a 2.4-fold increased risk for pharyngeal SCC than others. In contrast, the CYP1A1 6235TC+CC genotype was more common in controls than in laryngeal SCC patients (35.9% versus 21.6%, P = 0.01). Carriers of the genotype had a 0.2-fold decreased risk for the disease than others. Our data present preliminary evidence that inherited combined CYP1A1 A4889G and T6235C abnormalities and GSTM1 and GSTT1 pathways are important determinants of HNSCC, particularly pharyngeal SCC in heavy smoking individuals from south-eastern Brazil.     

<Emphasis Type="Italic">Abstracts</Emphasis>

Summary Epidermal growth factor receptor (EGFR) overexpression correlates with both loss of estrogen receptor (ER) and poor prognosis in breast cancer. Interestingly, in normal breast EGFR appears to be expressed more frequently than in malignant tissue, and there may be a different relationship between ER and EGFR. A variety of cellular regulators, such as EGF, TGF, phorbol esters, and steroid hormones, are capable of altering the level of EGFR expression in breast cells. However, much work remains to be done on the mechanistic details of EGFR regulation in this disease. The significance of EGFR as an oncogene in breast cancer is compounded by its potential interactions with other oncogenes such as c-erbB-2 and c-myc. Additionally, several recent studies have placed EGFR prominently in the signal transduction pathway, demonstrating that the EGFR-ligand system may play important roles throughout the course of malignant progression in breast cancer.     

Prognostic significance of co-overexpression of the <Emphasis Type="Italic">EGFR</Emphasis>/<Emphasis Type="Italic">IGFBP-2</Emphasis>/<Emphasis Type="Italic">HIF-2A</Emphasis> genes in astrocytomas

Overexpression of the EGFR, IGFBP-2 and HIF-2A genes has been observed in high-grade astrocytomas and these genes seem to be functionally related to one another. This study aimed to define the profile of their expressions, interactions and correlation with clinical features and prognostic significance in microdissected tumor samples from 84 patients with astrocytomas of different grades and from 6 white matter non-neoplasic brain tissue sample. EGFR, IGFBP-2 and HIF-2A gene expression levels were analyzed by quantitative real-time PCR and differed significantly between grades I–IV astrocytic tumors (P < 0.0001, P < 0.0001 and P: 0.0013, respectively) when analyzed by the Kruskal–Wallis test. Grade I astrocytomas presented gene expression levels similar to those encountered in samples of microdissected white matter of non-neoplastic brain tissue Overexpression of the EGFR, IGFBP-2 and HIF-2A genes was significantly associated with lower 2-year survival (P: 0.009, P: 0.0002 and P: 0.008, respectively). Co-overexpression of these genes was strongly associated with high-grade gliomas and lower survival in univariate (P < 0.0001) and multivariate (P: 0.009) analysis, suggesting that the co-expression of the EGFR/IGFBP-2/HIF-2A pathway genes may have a more important clinical and biological impact than the expression of each individual gene alone. These data support the existence of a common pathway involving these genes that could contribute to the design of new target treatments.     

Selection of patients with germline <Emphasis Type="Italic">MLH1</Emphasis> mutated Lynch syndrome by determination of <Emphasis Type="Italic">MLH1</Emphasis> methylation and <Emphasis Type="Italic">BRAF</Emphasis> mutation

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1, MSH2 and more rarely MSH6, PMS2, MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.     

Genetic variants of <Emphasis Type="Italic">CYP3A5</Emphasis>, <Emphasis Type="Italic">CYP2D6</Emphasis>, <Emphasis Type="Italic">SULT1A1</Emphasis>, <Emphasis Type="Italic">UGT2B15</Emphasis> and tamoxifen response in postmenopausal patients with breast cancer

Introduction  

Tamoxifen therapy reduces the risk of recurrence and prolongs the survival of oestrogen-receptor-positive patients with breast cancer. Even if most patients benefit from tamoxifen, many breast tumours either fail to respond or become resistant. Because tamoxifen is extensively metabolised by polymorphic enzymes, one proposed mechanism underlying the resistance is altered metabolism. In the present study we investigated the prognostic and/or predictive value of functional polymorphisms in cytochrome P450 3A5 CYP3A5 (*3), CYP2D6 (*4), sulphotransferase 1A1 (SULT1A1; *2) and UDP-glucuronosyltransferase 2B15 (UGT2B15; *2) in tamoxifen-treated patients with breast cancer.     

A comprehensive analysis of common genetic variation in <Emphasis Type="Italic">MUC1</Emphasis>, <Emphasis Type="Italic">MUC5AC</Emphasis>, <Emphasis Type="Italic">MUC6</Emphasis> genes and risk of stomach cancer

Objective  

MUC1, MUC5AC, and MUC6 are main constituents of the mucus barrier in the stomach, which protects the underlying epithelium from acid, proteases, mechanical trauma, and pathogenic microorganisms. Accumulating evidence implicates potential roles of MUC1, MUC5AC, and MUC6 genetic variation in the development of stomach cancer.     

Prostate screening uptake in Australian <Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> carriers

Men who carry mutations in BRCA1 or BRCA2 are at increased risk for prostate cancer. However the efficacy of prostate screening in this setting is uncertain and limited data exists on the uptake of prostate screening by mutation carriers. This study prospectively evaluated uptake of prostate cancer screening in a multi-institutional cohort of mutation carriers. Subjects were unaffected male BRCA1 and BRCA2 mutation carriers, aged 40–69 years, enrolled in the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab) and who had completed a mailed, self-report follow-up questionnaire 3 yearly after study entry. Of the 75 male carriers in this study, only 26 (35%) had elected to receive their mutation result. Overall, 51 (68%) did not recall having received a recommendation to have prostate screening because of their family history, but 41 (55%) had undergone a prostate specific antigen (PSA) test and 32 (43%) a digital rectal examination (DRE) in the previous 3 years. Those who were aware of their mutation result were more likely to have received a recommendation for prostate screening (43 vs. 6%, p = 0.0001), and to have had a PSA test (77 vs. 43%, p = 0.005) and a DRE (69 vs. 29%, p = 0.001) in the previous 3 years. The majority of unaffected males enrolled in kConFab with a BRCA1/2 mutation have not sought out their mutation result. However, of those aware of their positive mutation status, most have undergone at least one round of prostate screening in the previous 3 years.     

<Emphasis Type="Italic">PALB2</Emphasis> analysis in <Emphasis Type="Italic">BRCA2</Emphasis>-like families

BRCA2 and PALB2 function together in the Fanconi anemia (FA)–Breast Cancer (BRCA) pathway. Mono-allelic and bi-allelic BRCA2 and PALB2 mutation carriers share many clinical characteristics. Mono-allelic germline mutations of BRCA2 and PALB2 are risk alleles of female breast cancer and have also been reported in familial pancreatic cancer, and bi-allelic mutations cause a severe form of Fanconi anemia. In view of these similarities, we investigated whether the prevalence of PALB2 mutations was increased in breast cancer families with the occurrence of BRCA2 associated tumours other than female breast cancer. PALB2 mutation analysis was performed in 110 non-BRCA1/2 cancer patients: (a) 53 ovarian cancer patients from female breast-and/or ovarian cancer families; (b) 45 breast cancer patients with a first or second degree relative with pancreatic cancer; and (c) 12 male breast cancer patients from female breast cancer families. One truncating PALB2 mutation, c.509_510delGA, resulting in p.Arg170X, was found in a male breast cancer patient. We conclude that germline mutations of PALB2 do not significantly contribute to cancer risk in non-BRCA1/2 cancer families with at least one patient with ovarian cancer, male breast cancer, and/or pancreatic cancer.     

Mutations of the <Emphasis Type="Italic">SDHB</Emphasis> and <Emphasis Type="Italic">SDHD</Emphasis> genes

The succinate dehydrogenase (SDH) is a mitochondrial enzyme complex with an important role in oxydative phosphorylation and intracellular oxygene sensing and signaling. Mutations in the SDHB (1p35–36) and SDHD subunits (11q23) give rise to the paraganglioma syndromes (PGL), namely PGL 4 and PGL 1, and generate paraganglioma and pheochromocytoma. For both genes mutations have been described that result in a loss of function of the gene products. SDHBmutations were found in five of eight exons and in two introns, SDHD mutations in all four exons and one intron. Phenotypes and rate of malignancy of SDHB and SDHD seem to be different, with a higher frequency of head-and-neck tumors in SDHD and indications of a higher risk of malignancy in SDHB mutations. As routine diagnostic procedure all SDH mutation carriers should have urine catecholamine analysis as well as pelvic, abdominal, thoracic and skull/neck MRI.