Search

Objectives To determine cytokeratin 20 (CK 20) expression in tumor tissue, normal urothelial tissue, bladder washings, and urine from patients with urothelial carcinoma by real-time reverse transcriptase-polymerase chain reaction using the LightCycler Instrument. Methods Urine, bladder washings, tumor, and normal urothelial tissue were obtained from 33 patients with bladder carcinoma. RNA was subsequently extracted. Twenty tissue samples from healthy volunteers were used as controls. Real-time reverse transcriptase-polymerase chain reaction with the LightCycler was performed using fluorescence-labeled CK 20 primers as the target gene and the porphobilinogen deaminase housekeeping gene as the internal standard. Results CK 20 mRNA expression was detected in all samples investigated. CK 20 expression in the tissue and urine samples from patients with tumor was significantly increased compared with that in normal urothelial tissue (P = 0.028) or urine from the negative control group (P = 0.012). The average CK 20 expression was 70,140 arbitrary units (AU) in tumor tissue (30,533 AU in urine from patients with tumor) and 8460 AU in normal urothelial tissue (2234 AU in normal urine). A stage-related difference in CK 20 mRNA expression was observed in tumor tissue (P = 0.027) and for tumor grade G1-G2 versus G3 (P = 0.03). Conclusions CK 20 mRNA expression levels in tissue and urine were elevated in patients with urothelial carcinoma compared with the levels in healthy individuals. CK 20 expression was also detectable in normal urine or normal urothelial tissue, but at significantly lower levels than in tumor samples. Fluorescence-labeled quantitative polymerase chain reaction using the LightCycler helped to differentiate between low-level CK 20 expression in normal urothelial tissue and the levels in tumor tissue samples. CK 20 might be an excellent and sensitive marker for tumor monitoring and routine follow-up in patients with transitional cell carcinoma.

Cytokeratin 20 (CK20) is an epithelial protein expressed almost exclusively in the gastrointestinal (GI) tract and is widely used as immunohistochemical marker for routine diagnosis. In contrast, CK20 gene expression is not an established marker for the classification of tumours and the detection of disseminated cancer cells in colorectal cancer. Recently, real-time reverse transcriptase polymerase chain reaction (RT-PCR) has provided the means for reproducible and quantitative investigation of molecular markers. This report directly compares CK20 mRNA and protein expression in serial sections of archival, formalin-fixed, paraffin-embedded (FFPE) colorectal adenocarcinomas. CK20 expression was detected by immunohistochemistry (IHC) in 60/63 (95.2%) cases, by conventional RT-PCR in 58/60 (96.7%) and by quantitative RT-PCR using the LightCycler (LightCycler is a trademark of a Member of the Roche Group) System in 29/32 (90.6%) microdissected cases, one case yielding variable results. Despite the high detection rate of all three techniques, marked heterogeneity of CK20 expression was seen between different cases and also within individual cases. CK20 expression profiles were not related to particular histopathological features of the tumours. A good correlation (r = 0.8964) was found between CK20 mRNA and protein expression by comparing quantitative RT-PCR with IHC in 32 cases. This was also true for selected heterogeneous tumour cells within individual cases. Both RT-PCR and IHC are therefore valuable tools for CK20 detection in colorectal adenocarcinoma, with real-time RT-PCR providing supplementary quantitative information. This suggests a promising supportive role for quantitative RT-PCR in molecular pathology.

A prominent example of the potential application of pharmacogenomics and pharmacogenetics to oncology is the study of dihydropyrimidine dehydrogenase (DPD) in 5-fluorouracil (5-FU) metabolism. 5-FU is currently one of the most widely administered chemotherapeutic agents used for the treatment of epithelial cancers. DPD is the rate-limiting enzyme in the catabolism and clearance of 5-FU. The observation of a familial linkage of DPD deficiency from a patient exhibiting 5-FU toxicity suggested a possible molecular basis for variations in 5-FU metabolism. Molecular studies have suggested there is a relationship between allelic variants in the DPYD gene (the gene that encodes DPD) and a deficiency in DPD activity, providing a potential pharmacogenetic basis for 5-FU toxicity. In the last decade, studies have correlated tumoral DPD activity with 5-FU response, suggesting it may be a useful pharmacogenomic marker of patient response to 5-FU-based chemotherapy. This article reviews the basis and discusses the challenges of pharmacogenetic and pharmacogenomic testing of DPD for the determination of 5-FU efficacy and toxicity.

Background: Hepatocellular carcinoma (HCC) is characterized by hypoxia, hypoglycemia and lactic acidosis within the microenvironment due a highly glycolytic phenotype. However, routine culture conditions of HCC cells employ supra-physiological glucose, pH-buffered media, and normoxia. The aim of the study was to simulate the microenvironment features of HCC in vitro by culturing HCC cells in their native (NAT) conditions (hypoglycemia, hypoxia and lactic acidosis) and examine phenotypic, transcriptomic and pharmacological differences compared to cells cultured in standard (ST) culture conditions. Methods: Six HCC cell lines were cultured in NAT or ST conditions. Pharmacological response to cytotoxic drugs and targeted agents were evaluated using the MTS assay. Phenotypic differences were examined using standard techniques. Transcriptomic analysis was carried out using the Illumina HumanHT-12 expression kit. Results: HCC cells cultured in NAT conditions have higher doubling time than cells cultured in ST conditions. However, no remarkable differences in the cell cycle profiles were observed when cultured in either NAT or ST conditions. Protein analysis revealed an increase in phosphorylation of AKT and decrease in the levels of AMPK in all cells cultured in NAT but not in ST conditions. Increase in the protein levels of GLUT1, HK2, LDHA, and a decrease in PDHA was observed only in cells cultured in NAT conditions. Furthermore, the HCC cells in NAT conditions exhibited lower levels of reactive oxygen species and ATP consistent with the elevated glycolysis and inefficient oxidative phosphorylation phenotype of HCC. Thirty-one genes were found to be aberrantly expressed by gene expression analysis, most notably NDRG1, a hypoxia-associated gene was upregulated in NAT cells, validated by qPCR. Upregulated NDRG1 was maintained even up reversal of the NAT condition. The panel of thirty-one genes were found to be associated with poor prognosis exclusively in HCC based on data available via TCGA supporting the validity of NAT culture condition. Differences in the IC50 of doxorubicin, sorafenib, PI3K, c-MET and HDAC inhibitors but not AKT, MEK, mTOR, Wnt inhibitors were observed in cells cultured in NAT compared with ST conditions suggesting microenvironment modelling can influence pharmacological response of certain class of compounds. Additional genomic and metabolomics analysis are currently being undertaken to characterize the HCC cells in NAT conditions. Conclusion: In conclusion, HCC cells when cultured in NAT condition exhibit more of the tumor characteristics than cells in ST condition and this can be utilized as an informative platform for better understanding of disease biology and pharmacology. Citation Format: Mohd Feroz Mohd Omar, Benny Tang, Sheng Chun Chang, Sarah Hong Hui Low, Gim Hwa Tan, Richie Soong, Bhaskar Bhattacharya. A novel in vitro microenvironment modeling platform for HCC therapeutics and biomarker development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 165. doi:10.1158/1538-7445.AM2017-165

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p

Gastric carcinogenesis is a complicated process that involves environmental and genetic factors like interleukin-4 (IL-4) and IL-8. Single nucleotide polymorphisms in their genes are associated with changed levels of gene expression. Here, we investigated the association between IL4-590 CT and IL8-251TA and gastric cancer (GC) risk in Sichuan of Southwestern China.We surveyed the research subjects using a self-designed questionnaire with questions on demographic factors and putative risk factors. Approximately 2-5ml of whole blood was collected after field survey to analyze IL4-590 CT and IL8-251TA genotypes using MALDI-TOF MS.Our study recruited 308 pairs of GC patients and controls, including 224 (72.7%) men and 84 (27.3%) women in each group. There were 99 cardia and 176 noncardia GC patients in the case group. The case and control groups had an average age of 57.7±10.6 (mean±SD) and 57.6±11.1 years. GC patients reported a significantly greater proportion of family history of cancer (29.9% vs 10.7%, p0.01) and drinking (54.6% vs 43.2%, p0.01) than did controls. Variant genotypes of IL-4-590 CT and IL-8-251 TA were not associated with overall GC risk (adjusted OR, 0.89; 95%CI, 0.61-1.28 for CT or CC vs TT; adjusted OR, 1.14; 95%CI, 0.86-1.79 for TA or AA vs TT). Stratification analysis of two SNPs for risk by subsites only found that variant IL-8-251 TA or AA genotype was associated with increased noncardia GC risk (adjusted OR, 2.58; 95%CI, 1.19-5.57). We did not observe interactions between the IL-8-251 TA genotype and smoking (adjusted OR, 0.38; 95%CI, 0.08-1.79) or drinking (adjusted OR, 0.36; 95%CI, 0.08-1.65) for risk of noncardia GC.Our data indicate no association between the two SNPs of IL-4-590 and IL-8-251 with overall GC risk, while the IL-8-251 TA or AA genotype conferred risk of cardia GC. Our findings contribute to the evidence body for risk of SNPs associated with the development of gastric cancer in this region.

South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language–speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP). The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP). SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal) identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP., Author Summary Indians of South Asia has long been a population of interest to a wide audience, due to its unique diversity. We have deep-sequenced 38 individuals of Indian descent residing in Singapore (SSIP) in an effort to illustrate their diversity from a whole-genome standpoint. Indeed, among Asians in our population panel, SSIP was most diverse, followed by the Malays in Singapore (SSMP). Their diversity is further observed in the population's chromosome Y haplogroup and mitochondria haplogroup profiles; individuals with European-dominant haplogroups had greater proportion of European admixture. Among variants (single nucleotide polymorphism and small insertions/deletions) discovered in SSIP, 21.69% were novel with respect to previous sequencing projects. In addition, some 14 loss-of-function variants (LOFs) were associated to cancer, Type II diabetes, and cholesterol levels. Finally, D statistic test with ancient hominids concurred that there was gene flow to East Asians compared to South Asians.

Background Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC. Methods The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations. Results A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy. Conclusions High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation beyond CpG sites and CpG islands. These technologies have opened new avenues to understand the interplay between epigenetic events, chromatin plasticity and gene regulation. However, the processing, managing and mining of this huge volume of data require specialized computational tools and statistical methods that are yet to be standardized. Here, we describe a complete bisulfite sequencing analysis workflow, including recently developed programs, highlighting each of the crucial analysis steps required, i.e. sequencing quality control, reads alignment, methylation scoring, methylation heterogeneity assessment, genomic features annotation, data visualization and determination of differentially methylated cytosines. Moreover, we discuss the limitations of these technologies and considerations to perform suitable analyses.

Serum carcinoembryonic antigen (CEA) is the only marker recommended for surveillance of colorectal cancer (CRC) recurrence; its sensitivity and specificity, however, are suboptimal. This study sought to evaluate the values of postoperative serum methylation levels of 7 genes for prognostication and especially for recurrence detection after curative resection.This prospective cohort study included 150 patients with stage I-III CRC from whom 3 consecutive blood sampling was taken 1 week before, and 6 months and 1 year after operation. Methylation levels of 7 genes were evaluated via quantitative methylation-specific polymerase chain reaction. Serum CEA was measured in parallel. Univariate and multivariate survival analyses were followed by construction of receiver operating characteristic curves for recurrence detection.After a median follow-up of 59 months, 43 patients (28.7%) developed recurrent lesions. High serum methylation levels of TAC1 in serum at 6-month follow-up (6M-FU), and SEPT9 at 1-year follow-up (1Y-FU) were independent predictors for tumor recurrence and unfavorable cancer-specific survival (CSS) (P .05 in all tests). Serum NELL1 methylation levels were significant alone for CSS at both 6M-FU and 1Y-FU, but not for disease-free survival. Dynamic changes of TAC1 and SEPT9 with methylation increment were also independently predictive for recurrence (P .05 in all tests). More importantly, TAC1 at 6M-FU and SEPT9 at 1Y-FU exhibited earlier detection of potential recurrences compared with concurrent serum CEA.Levels of TAC1 and SEPT9 methylation detected in postoperative sera of patients with CRC appear to be novel promising prognostic markers and may probably be considered for monitoring of CRC recurrence.

Summary Metastatic spine tumour surgery (MSTS) and metastatic musculoskeletal tumour surgery (MMTS) are associated with substantial blood loss. Allogeneic blood transfusion is the present method used to replenish this blood. Intraoperative cell salvage (IOCS) is a viable alternative, but is contraindicated in tumour surgery because of the risk of tumour dissemination. Use of IOCS–leucocyte depletion filter (LDF) allows removal of tumour cells from blood salvaged during oncological surgery. However, no reports exist on use of IOCS in MSTS or MMTS. We systematically reviewed studies on IOCS in oncological surgery to investigate whether sufficient evidence exists to support its use in MSTS or MMTS.

Previous studies on the prognostic significance of TP53 gene alterations in colorectal cancer (CRC) have led to conflicting results. The present study investigated the prognostic significance of TP53 gene mutation in a very large series of 995 Dukes' B and C CRC patients, the majority of whom did not receive chemotherapy. Mutations were found in 385 (39%) cases and were not associated with tumour stage, histological grade, patient age or sex. Significantly more mutations were found in tumours from the left-sided colon compared with those from the right side (43% versus 34%, P =0.006). TP53 gene mutation had no prognostic value in the overall series or in different site or stage subgroups. None of the different types of TP53 gene mutation showed prognostic value. A trend for association with worse survival was observed in the patient subgroup that received adjuvant chemotherapy (Hazard Ratio (HR) 1.4, 95% confidence interval (CI) 0.89–2.21, P =0.15). These results indicate that mutation of the TP53 gene is not a useful prognostic marker for CRC patients who do not receive adjuvant chemotherapy. Further study is required to determine whether different types of TP53 mutation might be of value in predicting the response of CRC patients to chemotherapy.

The replication error phenotype (RER+) represents an important new form of genetic alteration characterized by widespread instability in repetitive nucleotide sequences. The aim of this study was to compare the features of RER+ gastric tumours with those of RER+ colonic tumours. RER status was determined by analysis of size alterations in the BAT-26 mononucleotide repeat microsatellite. Twelve of 121 (10 per cent) gastric carcinomas from a low-incidence region were found to be RER+. BAT-26 instability was associated with tumours showing an absence of nodal invasion (p = 0·009) and with a trend for improved prognosis. These tumours were more frequent in older, female patients. Frameshift mutations in mononucleotide repeat sequences within the transforming growth factor-β receptor II (RII), insulin-like growth factor II receptor (IGFIIR), and BAX genes were observed in 83, 33, and 25 per cent, respectively, of RER+ tumours. Only 1/12 (8 per cent) RER+ tumours contained a p53 gene mutation compared with 29/109 (27 per cent) RER− tumours. RER+ gastric carcinomas therefore share several important features with RER+ colonic tumours, including less frequent nodal invasion, improved prognosis, a similar frequency of mutation in growth control genes containing repetitive nucleotide sequences, and a low frequency of mutation of the p53 tumour suppressor gene. Copyright © 1999 John Wiley & Sons, Ltd.

The presence of inactivating mutations in the transforming growth factor-β (TGF-β) type II receptor (RII) gene in colon cancer suggests that it may behave like a tumour suppressor gene. RII is mutated in the majority of colon tumours exhibiting widespread microsatellite instability, a characteristic generally referred to as the replication error phenotype (RER+). We investigated the association between RII mutations and various clinicopathological variables and genetic alterations in a large series of sporadic adenocarcinomas arising in the proximal colon. RII mutations were found in 17 per cent (36/210) of right-sided tumours and in 86 per cent (32/37) of those displaying RER+. They were associated with the absence of lymph node invasion (P=0·04), poor histological differentiation (P=0·006), and with a trend for improved patient survival. Tumours with an RII mutation also showed non-significant trends for a lower incidence of p53 protein overexpression and of p53, K-ras, and APC gene mutation compared with tumours with normal RII. These results indicate that right-sided colorectal tumours containing RII mutations resemble those with the RER+ phenotype in terms of their clinicopathological features and genetic alterations. © 1998 John Wiley & Sons, Ltd.

We examined the association between mutation of the p53 gene and survival in a large cohort of breast cancer patients. Using a rapid, non-isotopic single-strand conformation polymorphism (SSCP) method we screened for mutations in exons 4-10 of the p53 gene in 375 primary breast cancers from patients with a median follow-up of 57 months. Mutations were found in 19% of tumours. Statistically significant associations were found between p53 mutation and histological grade, hormone receptor status, ploidy and S-phase fraction. No association was found between p53 mutation and axillary lymph node involvement, histological type, tumour size, vascular invasion or patient age. In univariate survival analysis, p53 mutation was strongly associated with poor prognosis. This was maintained in the lymph node-negative and hormone receptor-positive patient subgroups. In multivariate analysis, p53 mutation was associated with poor survival independent of lymph node status, estrogen receptor status and S-phase fraction. Our results demonstrate the feasibility of using a rapid and simple polymerase chain reaction-SSCP screening procedure to detect p53 gene mutation in breast cancer for the provision of prognostic information.

We aim to evaluate the influence of covariates, including cytochrome P450 3A (CYP3A) genetic polymorphisms, on the pharmacokinetics of midazolam (MDZ) in Asian cancer patients, using a population pharmacokinetic approach. Pharmacokinetic data were obtained from 24 adult cancer patients who received an intravenous bolus dose of 1 mg MDZ as a CYP3A phenotyping probe, 1-day before starting FOLFIRI chemotherapy. Concentrations of MDZ and its major metabolites, 1'-hydroxymidazolam (1OHM) and 1'-hydroxymidazolam glucuronide (HMG) were measured using liquid chromatography/mass spectrometry. The population pharmacokinetic study was conducted using NONMEM. Demographics, clinical characteristics, and genetic polymorphisms were screened as covariates. A two-compartment model for MDZ and two sequential compartments representing 1OHM and HMG best described the data. The CYP3A5*3 and total bilirubin level significantly influenced MDZ clearance. The population typical MDZ clearance for CYP3A5*3 expressers was 22% lower than non-expressers. Baseline bodyweight was a statistically significant covariate for clearance and distribution volume of 1OHM. Creatinine clearance was positively correlated with HMG clearance. Our data indicate that CYP3A5*3, total bilirubin, bodyweight, and creatinine clearance are important predictors of MDZ and metabolite pharmacokinetics. Further studies in more patients are needed to explore the links between the identified covariates and the disposition of MDZ and its metabolites.

Inhibition of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a cancer treatment strategy that has entered into clinical trials. We performed a meta-analysis on the frequency of prominent genetic (PIK3CA mutation, PIK3CA amplification and PTEN deletion) and protein expression (high PI3K, PTEN loss and high pAkt) aberrations in the PI3K pathway in gastric cancer (GC) and colorectal cancer (CRC). We also performed laboratory analysis to investigate the co-occurrence of these aberrations. The meta-analysis indicated that East Asian and Caucasian GC patients differ significantly for the frequencies of PIK3CA Exon 9 and 20 mutations (7% vs. 15%, respectively), PTEN deletion (21% vs. 4%) and PTEN loss (47% vs. 78%), while CRC patients differed for PTEN loss (57% vs. 26%). High study heterogeneity (I2 > 80) was observed for all aberrations except PIK3CA mutations. Laboratory analysis of tumors from East Asian patients revealed significant differences between GC (n = 79) and CRC (n = 116) for the frequencies of PIK3CA amplification (46% vs. 4%) and PTEN loss (54% vs. 78%). The incidence of GC cases with 0, 1, 2 and 3 concurrent aberrations was 14%, 52%, 27% and 8%, respectively, while for CRC it was 10%, 60%, 25% and 4%, respectively. Our study consolidates knowledge on the frequency, co-occurrence and clinical relevance of PI3K pathway aberrations in GC and CRC. Up to 86% of GC and 90% of CRC have at least one aberration in the PI3K pathway, and there are significant differences in the frequencies of these aberrations according to cancer type and ethnicity.

Background & Aims Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil–based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T 17 intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin. We investigated whether HSP110 T 17 could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. Methods We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T 17 affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II–III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T 17 . Results HSP110 and HSP110DE9 interacted in a 1:1 ratio. Tumor cells with large deletions in T 17 had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T 17 were mostly biallelic in primary tumor samples with MSI. Patients with stage II–III cancer who received chemotherapy and had large HSP110 T 17 deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012–0.8; P = .03). We found a significant interaction between chemotherapy and T 17 deletion ( P = .009). Conclusions About 25% of patients with stages II–III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T 17 intron repeat of HSP110 in tumor DNA.

Immunohistochemical (IHC) detection of p53 protein was compared with the presence of p53 gene mutation in many colorectal ( n = 100), breast ( n = 92), endometrial ( n = 122), and gastric ( n = 116) carcinomas. Two commercially available antibodies, D07 and CMl, were used for IHC analysis of paraffin-embedded tissue sections. Screening for gene mutations in frozen and paraffin-embedded tumor samples was carried out using polymerase chain reaction—single-strand conformation polymorphism (PCR-SSCP). The frequency of nuclear staining with D07 or CMl for each tumor type, respectively, was colorectal (36%, 23%); breast (15%, 19%); endometrial (21%, 33%); and gastric (23%,-). Overall correlation between the two antibodies for nuclear staining was 90% for the 314 tumors analyzed. Cytoplasmic staining was observed with D07 in 7% of breast and 5% of gastric carcinomas and with CMl in 17% of breast and 54% of endometrial carcinomas. p53 gene mutation was found in 39% of colorectal, 28% of breast, 13% of endometrial, and 25% of gastric cancers. The concordance between p53 nuclear overexpression and gene mutation (both positive or both negative) was 68% for colorectal, 79% for breast, 76% for endometrial, and 73% for gastric carcinomas. This study provides further evidence that IHC detection of p53 protein accumulation does not always indicate the presence of a gene mutation and vice versa. Discordant results were observed in approximately 20% to 30% of the tumors studied, highlighting the need for careful characterization of both p53 gene and protein alterations when assessing the relationship between p53 status and tumor behavior.

The important role of the p53 gene in tumour progression and cellular response to DNA damage has prompted investigation of the clinical significance of alterations to this gene. We examined both p53 overexpression and mutation of the gene in endometrial carcinoma in order to evaluate the prognostic significance of these changes. Of 122 endometrial carcinomas, 33 (27%) showed overexpression of p53 in the nucleus and 66 (54%) in the cytoplasm. Mutation in the p53 gene was found in 16 (13%) cases but showed no significant association with patient survival. Nuclear p53 overexpression was associated with poor survival (48% vs 80% alive in negative tumours 5 years post operatively, P < 0.001). In contrast, cytoplasmic p53 overexpression was associated with better survival (85% vs 55%, P < 0.001). When patients were separated into prognostic subgroups according to established clinical markers, these associations remained significant within most subgroups examined. In multivariate analysis adjusted for surgical stage, histological grade and type and vascular invasion, both nuclear p53 overexpression [hazard ratio 4.9 (95% CI 1.3-17.6). P = 0.016] and cytoplasmic overexpression [0.25 (0.06-0.98), P = 0.047] were independent prognostic factors. Immunohistochemical assessment of p53 overexpression in the nucleus and cytoplasm could provide useful prognostic information for the management of patients with endometrial cancer. Images Figure 1 Figure 2

Hepatocellular carcinoma (HCC) is an endemic disease globally, with a 5-year survival rate of 15%. Current treatment of late-stage HCC involves the use of chemotherapy and sorafenib, a multi-kinase inhibitor. Metabolic studies have indicated HCC has a high glycolysis rate, and is surrounded by a hypoglycemic microenvironment. Given the need for therapeutic options for HCC and its glycolytic nature, this study explored the activity of novel GLUT1 inhibitors in varied glucose concentrations. To assess target inhibition, [3H] deoxy-D-glucose (2DG) uptake assays were performed in HEK293 cells stably over-expressing GLUT1-4 individually. Six HCC cell lines were cultured in media with standard high glucose (HG:25mM) and low glucose/lactic acidosis (LGLA: 5mM glucose/20mM lactic acid) to mimic the hypoglycemic microenvironment. IC50s were assessed using the SRB assay, and other phenotypes using standard methods. Four novel GLUT1 inhibitors (IOM1-4) from the same chemical series were identified from diversity screening. The compounds exhibited differential potencies to GLUT proteins at the sub-μM level. The inhibitors reduced the uptake of 2DG, the extrusion of lactate, and increased apoptosis. In HCC cells, IC50s were lowest with IOM1 and 2 and highest with IOM4 (Table 1). HEP3B cells displayed exceptional sensitivity to the inhibitors. The ranking of potency according to the IC50s correlated with the 2DG uptake studies. There was no correlation between GLUT protein levels and IC50s. Significant differences in sensitivity to inhibitors were observed in cells cultured in HG and LGLA conditions. Cells cultured in LGLA had consistently lower glucose uptake compared those in HG conditions. In LGLA conditions, cells with low levels of reactive oxygen species (ROS) were more sensitive to GLUT1 inhibitors compared to cells with high ROS levels. In conclusion, GLUT1 inhibition can be influenced by microenvironment glucose levels and could be a strategy for HCC treatment. IC50 values (uM) for respective inhibitors in respective HCC cell linesCELL LINESInhibitorCultureC3AHEP3BHUH7PLCSKHEP1SNU449IOM1HG>5.00.8±0.040.3±0.06>5.01.4±0.451.7±0.38IOM1LGLA>5.00.2±0.134.0±1.20>5.00.1±0.021.6±0.28IOM2HG4.7±0.445.00.1±0.080.2±0.093.2±1.290.1±0.021.3±0.42IOM3HG>5.00.2±0.040.9±0.222.3±0.251.1±0.641.6±0.40IOM3LGLA>5.05.00.4±0.092.3±1.13IOM4HG>5.02.8±0.354.3±1.494.6±1.404.7±0.85>5.0IOM4LGLA>5.01.4±0.493.8±1.10>5.03.1±0.56>5.0 Citation Format: Bhaskar Bhattacharya, Sanamerjit S. Mann, Min Ji Han, Sarah HH Low, Gim Hwa Tan, Barry E. McGuinness, Sarah C. Trewick, Phillip M. Cowley, Alan Wise, Richie Soong. Identification and activity of novel GLUT1 inhibitors in hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1283.

Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase have been described in a variety of human malignancies including non–small-cell lung cancer (NSCLC), cholangiocarcinoma and glioblastoma multiforme. Recently, clinicopathologic characteristics of c-ros oncogene 1, receptor tyrosine kinase (ROS1)-rearranged NSCLC patients have been described. Furthermore, anaplastic lymphoma kinase inhibitor, novel class of drugs targeting this tyrosine kinase receptor is currently under clinical trial in this molecular subset of NSCLC patients. This review will focus on the current knowledge of ROS1 rearrangements in NSCLC, methods to detect ROS1 rearrangement, and targeting ROS1-rearranged NSCLC patients with anaplastic lymphoma kinase inhibitors.

Histone arginine methylation has emerged as an important histone modification involved in gene regulation. Protein arginine methyltransferase (PRMT) 4 and 5 have been shown to play essential roles in early embryonic development and in embryonic stem (ES) cells. Recently, it has been reported that PRMT6-mediated di-methylation of histone H3 at arginine 2 (H3R2me2) can antagonize tri-methylation of histone H3 at lysine 4 (H3K4me3), which marks active genes. However, whether PRMT6 and PRMT6-mediated H3R2me2 play crucial roles in early embryonic development and ES cell identity remain unclear. Here, we have investigated their roles using gain and loss of function studies with mouse ES cells as a model system. We report that Prmt6 and histone H3R2 methylation levels increased when ES cells are induced to differentiate. Consistently, we find that differentiation of ES cells upon upregulation of Prmt6 is associated with decreased expression of pluripotency genes and increased expression of differentiation markers. We also observe that elevation of Prmt6 increases the methylation level of histone H3R2 and decreases H3K4me, Chd1, and Wdr5 levels at the promoter regions of Oct4 and Nanog. Surprisingly, knockdown of Prmt6 also leads to downregulation of pluripotency genes and induction of expression of differentiation markers suggesting that Prmt6 is important for ES cell pluripotency and self-renewal. Our results indicate that a critical level of Prmt6 and histone H3R2me must be maintained in mouse ES cells to sustain their pluripotency.

Since their first discovery in chronic myeloid leukaemia, fusion genes have been shown to play a central role in haematologic cancers. However, aberrations arising from this type of somatic mutations have been neglected in common solid tumours, largely because of limitations of current cytogenetic techniques. The recent discovery of recurrent gene fusions in prostate and lung cancer has led to a renewed interest in the identification of novel fusion genes in solid tumours. In this review, we discuss the technical challenges of studying gene fusions in solid tumours, appraise the application of newer molecular techniques and highlight emerging technologies, with the focus on next-generation sequencing and chromogenic in situ hybridisation, that have greatly enhanced the speed and reliability of gene fusion discovery in malignant solid tumours. Key Concepts: A fusion gene is a gene hybrid formed from linking two physically separated genes. A fusion gene can occur as a result of chromosomal rearrangements such as translocations, insertions, deletions and inversions, which lead to novel protein fusion products. The detection of fusion genes is being recognised as a promising clinical tool for the diagnosis, staging classification, prognosis and treatment of cancer. Gene fusions have been an underappreciated class of mutation in solid tumours. The identification of gene fusions in solid tumours is technically challenging due to the complex and often chaotic karyotypic profiles of solid cancers. Recent advances in genomics obtained from next generation sequencing, coupled with the emerging field of bioinformatics and ever-advancing molecular techniques, have uncovered many novel oncogenic fusion genes in solid tumours that were unable to be detected by traditional cytogenetic methods. Fluorescence In-Situ Hybridisation (FISH) is recognised as the most effective diagnostic methods for the detection of gene fusions. Transcriptome-sequencing (also known as RNA-sequencing), a recently introduced high-throughput method of characterising ribonucleic acid (RNA) transcribed from the genome is rapidly emerging as the key method for the discovery of gene fusions. Numerous challenges and unanswered issues remained with the discovery of novel gene fusions and their development as effective targeted cancer therapies or as clinical diagnostic and/or prognostic tools. Keywords: fusion genes; genomic rearrangement; next-generation sequencing; cancer genomics; chromogenic in-situ hybridisation; fluorescence in-situ hybridisation

Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups — Epi-A, Epi-B, Mes, Stem-A and Stem-B — exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients.

Aldo-ketoreductases have been implicated in the metabolism of doxorubicin. We sought to assess the influence of AKR1C3 genetic variants on doxorubicin metabolism.We sequenced AKR1C3 exon 5 and genotyped seven functional single nucleotide polymorphisms in CBR3, ABCB1 and SLC22A16 involved in doxorubicin pharmacology in 151 Asian breast cancer patients treated with doxorubicin-containing chemotherapy, and correlated these genotypes with doxorubicin pharmacokinetics and pharmacodynamics.Two previously reported AKR1C3 intronic variants, IVS4-212 CG and IVS4+218 GA, were detected. The AKR1C3 IVS4-212 GG genotype was associated with significantly lower cycle 1 day 15 leucocyte (mean leucocytes 2.49 ± 1.57 × 10(9) vs. 3.85 ± 3.42 × 10(9) l(-1) , P = 0.007) and neutrophil counts (mean neutrophils 0.70 ± 1.01 × 10(9) vs. 1.56 ± 2.80 × 10(9) l(-1) , P = 0.008) and significant improvement of progression-free survival [PFS, mean PFS 49.0 (95% confidence interval 42.2-55.8) vs. 31.0 (95% confidence interval 20.7-41.2) months, P = 0.017] and overall survival [OS; mean OS 64.4 (95% confidence interval 58.3-70.5) vs. 46.3 (95% confidence interval 35.1-57.5) months, P = 0.006] compared with those carrying at least one C allele. There was no significant association between AKR1C3 IVS4-212 CG and doxorubicin pharmacokinetics. Of the other seven single nucleotide polymorphisms genotyped, CBR3 G11A correlated with doxorubicinol area under the concentration-time curve and OS, ABCB1 G2677T/A correlated with doxorubicin clearance and platelet toxicity, while ABCB1 IVS26+59 TG correlated with OS. The AKR1C3 IVS4-212 CG genotype remained significantly correlated with both PFS and OS on multivariate analysis with clinical prognosticators.The AKR1C3 IVS4-212 GG genotype was associated with greater haematological toxicity and longer progression-free survival and overall survival after doxorubicin-based therapy, suggesting potential interaction of this variant with doxorubicin metabolism.

Epigenetics refers to the heritable yet reversible chemical modifications to the deoxyribonucleic acid (DNA) sequence and histone protein such as DNA methylation and histone modification resulting in modulation of gene expression without direct alteration to the DNA sequence itself. It also includes post-transcriptional regulation of protein coding ribonucleic acids (RNAs) by microRNAs (miRNAs), which subsequently affect protein translation. Although the development of microarray technologies has transformed the interrogation of epigenetics from targeted regions to whole epigenome scale, next generation sequencing has brought another wave of technological revolution that allows a more thorough investigation of the epigenome at a single nucleotide resolution. Since the arrival of next generation sequencing technologies, several studies have applied next generation sequencing-based methods to dissect cancer epigenome, such as whole methylome sequencing of one colon cancer and adjacent normal as well as small RNA sequencing in several cancer tissues for discovery of novel miRNAs. In addition to individual studies, next-generation sequencing technologies have enabled international consortia such as NIH Roadmap Epigenomics Programme and the International Human Epigenome Consortium to perform in-depth investigation of epigenetics in a wide range of tissues. These international consortia ensures the standardisation of experimental methods across different groups to generate high quality data that can be compared and integrated into larger epigenomic datasets. The completion of these projects will undoubtedly contribute to significant advances in the knowledge and understanding of the roles of epigenetics underlying human diseases. Key Concepts: Epigenetics has advanced considerably after the arrival of whole genome interrogation tools such as microarray and next generation sequencing technologies. Epigenetic aberration plays an equally important role as with genetic aberration in contributing to diseases such as cancers. Next generation sequencing-based methods permit a thorough investigation of DNA methylation, histone modifications and miRNAs throughout the whole genome at a single nucleotide resolution. Sequencing of the entire methylome in embryonic stem cells has unravelled non-CG methylation patterns, which would not have been discovered using microarrays focus primarily on CpG sites. Sequencing of the first cancer methylome in colon cancer has also documented substantial differences in DNA methylation patterns between cancer and normal tissue. ChIP-seq has been used to compare histone modification patterns in human breast cancer and normal mammary epithelial cell lines revealing that H3K4me3 and H3K9/14ac were both highly enriched in promoter regions, whereas H3K4me1 was more broadly distributed. The production of a large number of sequence reads by NGS technologies has also allowed deep sequencing of noncoding RNAs (including miRNAs) or a more comprehensive analysis of RNA species. The arrival of NGS technologies has brought a paradigm shift in the approaches used for epigenomics studies, as well as improving our understanding of biology and diseases. As TGS technologies mature, the investigation of cancer epigenomics will likely be provided with a greater depth and breadth of analysis through single DNA molecule sequencing. Although NGS has brought new opportunities to the study of epigenetics, challenges ranging from technical difficulties to bioinformatics analysis must be noted. Keywords: next generation sequencing technologies; epigenetics; DNA methylation; histone modification; microRNA; cancer epigenome; whole genome bisulfite sequencing; chromatin immunoprecipitation sequencing; small RNA sequencing; microarray

It is increasingly recognized that differences in overall survival and toxicity exist between Asian and caucasian patients with small-cell and non-small-cell lung cancer, with a longer survival, higher response rates and greater toxicity to chemotherapy and targeted therapy reported in Asian patients. Two global studies are used to illustrate how the proportions of Asian patients can influence survival outcome. Ethnicity is an important and complex characteristic that should considered in the design and conduct of a global clinical study, as the safety, tolerability and response may vary between Asian and caucasian patients. Whether ethnic differences in lung cancer survival are attributed to genetic differences among races or are simply a surrogate marker of differences in access to healthcare because of socioeconomic differences is unclear. Carefully designed prospective studies investigating ethnic-specific determinants of sensitivity and toxicity to systemic therapy are warranted.

Phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors are an emerging class of anti-cancer agents. Here, we tested the hypothesis that the dual PI3K/mTOR inhibitor, PI103, could synergize with the chemotherapeutic agent, 5-fluorouracil (5-FU) by inhibiting E2F1, thymidylate synthase (TS) and enhancing DNA damage. Drug combination effects were assessed in gastric cancer cells using the median-effect equation. The specific effects of inhibition of E2F1 and PIK3CA were examined by siRNA, and mTOR by rapamycin exposure. Protein expression and apoptosis pre- and post-treatment was measured using standard methods. PI103 and 5-FU was synergistic in 3/5 gastric cancer cell lines tested. Synergy was associated with PI3KCA mutation, reduced TS and E2F1 protein levels, increased H2AX phosphorylation and apoptosis. E2F1 siRNA enhanced sensitivity to 5-FU only in cells displaying synergy. Excess thymidine exposure converted synergism to antagonism in all cells. Inhibition of PI3K and mTOR alone enhanced 5-FU cytotoxicity in only 2/3 cell lines that displayed synergy each. In AGS cells, PI3K inhibition alone enhanced 5-FU sensitivity as much as dual PI3K/mTOR inhibition. In HGC27 cells, dual inhibition increased 5-FU sensitivity more than single PI3K or mTOR inhibition. Combined PI103 and 5-FU treatment reduced in vivo tumor growth more than treatment with single agents. PI3K/mTOR inhibitors can enhance 5-FU cytotoxicity in vitro and in vivo, especially in PIK3CA mutant tumor cells. Dual, rather than single, PI3K/mTOR inhibitors may combine better with 5-FU due to cellular heterogeneity in sensitivity to PI3K and mTOR inhibition.

Cancer genome sequencing studies have identified tens of thousands of somatic mutations in various human cancers to date. This data has started to generate new insights into mutation patterns and their differences between various cancers. Further, the mutation patterns have also helped to elucidate mechanism involved in generating mutations in cancer genome such as deoxyribonucleic acid (DNA)-repair processes and mutagen exposures. With the introduction of next-generation sequencing technologies, cancer genome sequencing has evolved from a targeted sequencing approach to whole-exome sequencing and whole-genome sequencing (WGS) approaches. However, each sequencing approach has its strengths and limitations. It is widely anticipated that WGS would eventually replace targeted and exome sequencing. WGS offers a unique advantage to study structural variants or rearrangements and fusion genes in a single experiment, in addition to point mutations. However, currently the WGS is still prohibitively expensive for a large number of samples. Despite these technological advances, several challenges still remain, such as discerning driver mutations from benign mutations and collection of high-quality primary tumour tissues to minimise tissue heterogeneity. Ultimately, a comprehensive delineation of the somatic mutations in the cancer genome would require WGS of a large number of samples from various cancer types and subtypes. Congruent to this goal, the International Cancer Genome Consortium was initiated and upon completion of the project, its data is expected to further enhance our knowledge and understanding of the biological mechanisms underlying cancer development. Key Concepts: Several sequencing approaches are available to decipher the somatic mutation profile of cancer genome, including targeted sequencing, whole-exome sequencing (WES) and whole-genome sequencing (WGS). Targeted sequencing is a hypothesis-driven approach where candidate genes were often selected based on knowledge of previously reported mutated genes or their related functions in cancer development, such as the kinome or phosphatome. The ability to study different cancers and their subtypes in a comparatively higher throughput to exome and WGS is an important advantage of the targeted sequencing approach. The introduction of multiple commercial exome enrichment kits has circumvented the technical challenges in isolating the entire exome for sequencing. Although WES interrogates only approximately 1–2% of the entire human genome, the several hundred somatic mutations identified in WES studies have already prohibited all of them to be examined by follow-up studies in larger sample series. The advances of NGS technologies and rapidly declining sequencing cost have enabled the completion of a number of WGS studies for various cancers. The patterns of somatic mutations provided by WGS are useful in elucidating the mechanisms of generating the mutations in cancer genome such as DNA-repair processes and differences in mutagen exposure. A further advantage over the other two sequencing approaches of WGS is its ability to identify structural rearrangements. Although WES and WGS approaches have now been shown to be technically feasible, several challenges still remain. Delineating patterns of somatic mutations in the cancer genome require a comprehensive interrogation of cancer genomes (entire genome, exome or a large number of candidate genes) in series of up to hundreds of samples. Keywords: somatic mutation; targeted sequencing; whole-exome sequencing; whole-genome sequencing; next-generation sequencing; cancer genome; single nucleotide variation; copy number variation; structural rearrangement; fusion gene

Sclerosis (tissue hardening) development is a common occurrence in slow growing or benign osteolytic lesions. However, there is lack of knowledge on the mechanical and material property changes associated with sclerotic bone response. The immune system is postulated to play a relevant role in evoking sclerotic bone responses. In this study, localized sclerotic response in an immunocompetent model of Walker 256 breast carcinoma in SD rats showed an apparent increase in new reactive bone formation. Sclerotic rat femurs had significant increases in bone mineral density (BMD), bone mineral content (BMC), bone volume fraction (BV/TV), bone surface density (BS/TV), trabecular number (Tb.N) and a significant decrease in trabecular separation (Tb.Sp) and structural model index (SMI) as compared to control rat femurs. Significantly reduced creep responses (increased η) were observed for both trabecular and cortical bone in sclerotic bones while no significant difference was observed in elastic modulus (E) and hardness (H) values. Therefore, we conclude that viscoelastic creep property using nanoindentation would serve as a more sensitive indicator of localized bone modeling than elastic properties. Moreover, reduced viscoelasticity can contribute towards increased microcrack propagation and therefore reduced toughness. Since significant positive correlations between elastic properties (E) and (H) with viscosity (η) were also observed, our results indicate that sclerotic response of bone metastasis would cause reduced toughness (increased η) with stiffening of material (increased E and H).

Substantial progress has been made in human genetics and genomics research over the past ten years since the publication of the draft sequence of the human genome in 2001. Findings emanating directly from the Human Genome Project, together with those from follow-on studies, have had an enormous impact on our understanding of the architecture and function of the human genome. Major developments have been made in cataloguing genetic variation, the International HapMap Project, and with respect to advances in genotyping technologies. These developments are vital for the emergence of genome-wide association studies in the investigation of complex diseases and traits. In parallel, the advent of high-throughput sequencing technologies has ushered in the 'personal genome sequencing' era for both normal and cancer genomes, and made possible large-scale genome sequencing studies such as the 1000 Genomes Project and the International Cancer Genome Consortium. The high-throughput sequencing and sequence-capture technologies are also providing new opportunities to study Mendelian disorders through exome sequencing and whole-genome sequencing. This paper reviews these major developments in human genetics and genomics over the past decade.

The advances in next generation sequencing (NGS) technologies have had a significant impact on epigenomic research. The arrival of NGS technologies has enabled a more powerful sequencing based method--that is, ChIP-Seq--to interrogate whole genome histone modifications, improving on the conventional microarray based method (ChIP-chip). Similarly, the first human DNA methylome was mapped using NGS technologies. More importantly, studies of DNA methylation and histone modification using NGS technologies have yielded new discoveries and improved our knowledge of human biology and diseases. The concept that cytosine methylation was restricted to CpG dinucleotides has only been recently challenged by new data generated from sequencing the DNA methylome. Approximately 25% of all cytosine methylation identified in stem cells was in a non-CG context. The non-CG methylation was more enriched in gene bodies and depleted in protein binding sites and enhancers. The recent developments of third generation sequencing technologies have shown promising results of directly sequencing methylated nucleotides and having the ability to differentiate between 5-methylcytosine and 5-hydroxymethylcytosine. The importance of 5-hydroxymethylcytosine remains largely unknown, but it has been found in various tissues. 5-hydroxymethylcytosine was particularly enriched at promoters and in intragenic regions (gene bodies) but was largely absent from non-gene regions in DNA from human brain frontal lobe tissue. The presence of 5-hydroxymethylcytosine in gene bodies was more positively correlated with gene expression levels. The importance of studying 5-methylcytosine and 5-hydroxymethylcytosine separately for their biological roles will become clearer when more efficient methods to distinguish them are available.

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimising administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterised for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumours from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumours, CTPS2 was the gene with the highest probability of differential expression (p=0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analogue, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p=0.072), while those with high expression did (p=0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

S-1 is an oral fluoropyrimidine anti-neoplastic agent that is converted by CYP2A6 to 5-fluorouracil (5FU). We prospectively studied the pharmacokinetics and pharmacodynamics of S-1 in two groups of East Asian and Caucasian patients with solid malignancy refractory to standard chemotherapy, or for which 5FU was indicated, to elucidate differences in relation to CYP2A6 genotype and phenotype. S-1 was given orally at 30 mg/m(2) b.i.d. for 14 days every 21 days. Dose normalized AUC(0-48 h) for tegafur (P = 0.05) and gimeracil (P = 0.036) were higher in East Asians; conversely, AUC(0-48 h) of fluoro-β-alanine was higher in Caucasians (P = 0.044). Exposure to 5FU was similar in both groups (P = 0.967). Mean cotinine:nicotine ratio was 54% higher in the Caucasian group (P = 0.03), and correlated with oral clearance of tegafur (r = 0.59; P = 0.002). Grade 3/4 gastrointestinal toxicities were more common in Caucasians than Asians (21%vs 0%). Treatment with S-1 yields no significant difference in 5FU exposure between Caucasians and East Asians.

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

Background Most previous studies of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) have been conducted on a relatively small numbers of CpG sites. In the present study we performed comprehensive DNA methylation profiling of CRC with the aim of characterizing CIMP subgroups. Methods DNA methylation at 1,505 CpG sites in 807 cancer-related genes was evaluated using the Illumina GoldenGate® methylation array in 28 normal colonic mucosa and 91 consecutive CRC samples. Methylation data was analyzed using unsupervised hierarchical clustering. CIMP subgroups were compared for various clinicopathological and molecular features including patient age, tumor site, microsatellite instability (MSI), methylation at a consensus panel of CpG islands and mutations in BRAF and KRAS. Results A total of 202 CpG sites were differentially methylated between tumor and normal tissue. Unsupervised hierarchical clustering of methylation data from these sites revealed the existence of three CRC subgroups referred to as CIMP-low (CIMP-L, 21% of cases), CIMP-mid (CIMP-M, 14%) and CIMP-high (CIMP-H, 65%). In comparison to CIMP-L tumors, CIMP-H tumors were more often located in the proximal colon and showed more frequent mutation of KRAS and BRAF (P < 0.001). Conclusions Comprehensive DNA methylation profiling identified three CRC subgroups with distinctive clinicopathological and molecular features. This study suggests that both KRAS and BRAF mutations are involved with the CIMP-H pathway of CRC rather than with distinct CIMP subgroups.

Ong C W, Kim L G, Kong H H, Low L Y, Wang T T, Supriya S, Kathiresan M, Soong R & Salto-Tellez M (2010) Histopathology56, 523–529 Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage Aims: Interpretation of immunohistochemistry is primarily done through human visual scoring while computer-assisted scoring is relatively uncommon. This study aimed to examine (i) the level of agreement between human visual and computer-assisted pathological scoring of immunoreactivity expression in colorectal cancers, (ii) whether computer-assisted scoring affects the prognostic significance of biomarkers, and (iii) whether computer-assisted pathological scoring provides any time-saving or reproducibility advantages. Methods and results: Tissue microarray blocks were constructed from the primary colorectal adenocarcinoma specimens of 486 patients. Scoring of the six markers [cytokeratin (CK) 7, CK20, cyclooxygenase-2, Ki67, p27 and p53] was done independently by a qualified pathologist, a trained scientist and the Ariol SL-50 (Applied Imaging). Univariate analysis showed that human visual and computer-assisted scoring were strongly correlated (all κ values >0.8). Both human visual and computer-assisted pathological scoring identified the same set of biomarkers with significant association with survival. Computer-assisted pathological scoring was shown to be a time-effective means of scoring larger numbers of slides (for high-throughput studies). Conclusions: Our results suggest that computer-assisted pathological scoring can be a viable alternative to pathologist scoring in a manner that is more practical and time-effective, but, interestingly, providing no analytical advantage.

The cancer stem cell hypothesis may explain why conventional chemotherapies are unable to fully eradicate cancers. In this study, we examined both the prognostic and predictive significance of putative cancer stem cell markers in colorectal cancer. In this study, immunohistochemistry for three candidate cancer stem cell markers (CD133, Oct-4 and Sox-2) and for six other postulated prognostic markers (CK7, CK20, Cox-2, Ki-67, p27 and p53) were performed using tissue microarrays containing 501 primary colorectal cancer cases. Receiver-operating characteristic analysis was used to determine cut-off scores for positive protein expression. Multivariate analysis revealed that positive expression for CD133 and Oct-4 was associated with significantly worse survival in patients treated by surgery alone (P=0.023 and P

Aim Accumulating evidence indicates that RUNX3 is an important tumour suppressor that is inactivated in many cancer types. This study aimed to assess the role of microRNA (miRNA) in the regulation of RUNX3 . Methods Four bioinformatic algorithms were used to predict miRNA binding to RUNX3 . The correlation between candidate miRNAs and RUNX3 expression in cell lines was determined by real-time reverse transcriptase quantitative PCR (RT-qPCR) and Western blot. Candidate miRNAs were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability, apoptosis and Bim expression. miRNA and RUNX3 expression, RUNX3 methylation and RUNX3 protein levels were assessed in gastric tissue by RT-qPCR, Methylight analysis and immunohistochemistry, respectively. Results Bioinformatics, gene and protein expression analysis in eight gastric cell lines identified miR-130b as the top candidate miRNA for RUNX3 binding. Overexpression of miR-130b increased cell viability, reduced cell death and decreased expression of Bim in TGF-β mediated apoptosis, subsequent to the downregulation of RUNX3 protein expression. In 15 gastric tumours, miR-130b expression was significantly higher compared to matched normal tissue, and was inversely associated with RUNX3 hypermethylation. Conclusion Attenuation of RUNX3 protein levels by miRNA may reduce the growth suppressive potential of RUNX3 and contribute to tumourigenesis.

Evidence that activating mutations of the KRAS oncogene abolish the response to anti-epidermal growth factor receptor therapy has revolutionized the treatment of advanced colorectal cancer. This has resulted in the urgent demand for KRAS mutation testing in the clinical setting to aid choice of therapy. The aim of this study was to evaluate six different KRAS mutation detection methodologies on two series of primary colorectal cancer samples. Two series of 80 frozen and 74 formalin-fixed paraffin-embedded tissue samples were sourced and DNA was extracted at a central site before distribution to seven different testing sites. KRAS mutations in codons 12 and 13 were assessed by using single strand conformation polymorphism analysis, pyrosequencing, high resolution melting analysis, dideoxy sequencing, or the commercially available TIB Molbiol (Berlin, Germany) or DxS Diagnostic Innovations (Manchester, UK) kits. In frozen tissue samples, concordance in KRAS status (defined as consensus in at least five assays) was observed in 66/80 (83%) cases. In paraffin tissue, concordance was 46/74 (63%) if all assays were considered or 71/74 (96%) using the five best performing assays. These results demonstrate that a variety of detection methodologies are suitable and provide comparable results for KRAS mutation analysis of clinical samples.