Kiyoko Nagura

PIK3CA mutation and amplification in human lung cancer

To explore the significance of phosphatidylinositol‐3‐kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild‐type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over‐expressed wild‐type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage‐independent growth activity and migration activity of immortalized airway epithelium 16HBE14o– cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild‐type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

hOGG1 exon7 polymorphism and gastric cancer in case–control studies of Japanese Brazilians and non-Japanese Brazilians

Polymorphism of hOGG1 may be capable of serving as a genetic marker for individual susceptibility to various cancers because of its role in the repair of oxyradical DNA damage. We examined the distribution of the hOGG1 Ser326Cys polymorphism and its presumed correlation with gastric cancer risk in two case-control studies of different ethnic groups in São Paulo, Brazil. Potentially eligible Japanese (JB) and non-Japanese Brazilian (NJB) case subjects were defined as patients with newly diagnosed malignant neoplasms of the stomach in 13 hospitals in São Paulo. Ninety-six JBs and 236 NJBs were adopted as subjects. Two controls were matched for each JB case, and one control for each NJB case. The subjects were interviewed using a questionnaire and their blood samples were collected. A significant difference in the distribution of this polymorphism between the two ethnic groups was observed (chi(2)=58.3, P<0.01). The mutant type (Ser/Cys or Cys/Cys) was predominant (approximately 65%) in the JBs, but was only present in approximately 40% of the NJBs. Logistic regression analysis showed no significant increased risk for either the Ser/Cys or Cys/Cys type in either group. The odds ratios of the Cys allele for gastric cancer were 1.01 (95% confidence interval (CI): 0.52-1.93) in the JBs and 0.85 (95% CI: 0.57-1.26) in the NJBs. In the NJBs, a significant increased risk of smoking was shown only in the Ser/Ser type, and no increased risk was shown in the genotypes with the Cys allele. However, no statistically significant interactions were observed with smoking or other possible confounding factors. No statistically significant difference in the distribution of the polymorphism was observed between the intestinal type and diffuse type of gastric cancer in either the JBs or the NJBs. The ethnic difference in hOGG1 Ser326Cys polymorphism was much greater than the case-control difference, and this polymorphism is unlikely to be associated with gastric cancer.

Induction of centrosome amplification and chromosome instability in p53-deficient lung cancer cells exposed to benzo[a]pyrene diol epoxide (B[a]PDE)

Benzo[a]pyrene diol epoxide (B[a]PDE), the ultimate carcinogenic metabolite of benzo[a] pyrene, has been implicated in the mutagenesis of the p53 gene involved in smoking‐associated lung cancer. To further understand the role of B[a]PDE in lung tumour progression, we investigated its effect on the numerical integrity of centrosomes and chromosome stability in lung cancer cells lacking p53. Exposure of p53‐deficient H1299 lung cancer cells to B[a]PDE resulted in S‐phase arrest, leading to abnormal centrosome amplification. Analysis of H1299 cells stably expressing fluorescence‐tagged centrin (a known centriolar marker) revealed that the centrosome amplification was primarily attributable to excessive centrosome duplication rather than to centriole splitting. Forced expression of POLK DNA polymerase, which has the ability to bypass B[a]PDE–guanine lesions in an error‐free manner, suppressed the B[a]PDE‐induced centrosome amplification. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2, 3, and 16 revealed that B[a]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. We extended these findings to primary lung carcinomas containing non‐functional p53, and found a strong association between centrosome amplification and a high level of B[a]PDE–DNA accumulation. Therefore B[a]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization as well as its mutagenic activity. Copyright

Detection of kinase amplifications in gastric cancer archives using fluorescence in situ hybridization

To test the feasibility of using bacterial artificial chromosomes (BAC) containing kinases for pathological diagnosis using fluorescence in situ hybridization (FISH), 10 BAC probes containing a gene amplified in 5% or more of a pilot cohort were selected from a previous survey using arbitrarily selected BAC clones harboring 100 kinases. In this report, we describe the prevalence and association with the clinico‐pathological profile of these selected 10 BAC probes in 365 gastric cancer tissues. FISH analyses using these 10 BAC probes containing loci encoding EGFR, ERBB2(HER2), EPHB3, PIK3CA, MET, PTK7, ACK1, STK15, SRC, and HCK showed detectable amplifications in paraffin‐embedded tissue in 2.83% to 13.6% of the gastric cancer tissues. Considerable numbers of the cases showed the co‐amplification of two or more of the probes that were tested. BAC probes located within a genome neighborhood, such as PIK3CA, EPHB3, and ACK1 at 3q26‐29 or HCK, SRC, and STK15 at 20q11‐13.1, were often co‐amplified in the same cases, but non‐random co‐amplifications of genes at distant genomic loci were also observed. These findings provide basic information regarding the creation of a strategy for personalizing gastric cancer therapy, especially when using multiple kinase inhibitors.

Fluorescence in situ hybridization analysis with a tissue microarray: 'FISH and chips' analysis of pathology archives

Practicing pathologists expect major somatic genetic changes in cancers, because the morphological deviations in the cancers they diagnose are so great that the somatic genetic changes to direct these phenotypes of tumors are supposed to be correspondingly tremendous. Several lines of evidence, especially lines generated by high‐throughput genomic sequencing and genome‐wide analyses of cancer DNAs are verifying their preoccupations. This article reviews a comprehensive morphological approach to pathology archives that consists of fluorescence in situ hybridization with bacterial artificial chromosome (BAC) probes and screening with tissue microarrays to detect structural changes in chromosomes (copy number alterations and rearrangements) in specimens of human solid tumors. The potential of this approach in the attempt to provide individually tailored medical practice, especially in terms of cancer therapy, is discussed.

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin‐fixed, paraffin‐embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.

Suppression of hydroxyurea-induced centrosome amplification by NORE1A and down-regulation of NORE1A mRNA expression in non-small cell lung carcinoma

The candidate tumor suppressor NORE1A is a nucleocytoplasmic shuttling protein, and although a fraction of the NORE1A in cells is localized to their centrosomes, the role of centrosomal NORE1A has not been elucidated. In this study we investigated the role of NORE1A in the numerical integrity of centrosomes and chromosome stability in lung cancer cells. Exposure of p53-deficient H1299 lung cancer cell line to hydroxyurea (HU) resulted in abnormal centrosome amplification (to 3 or more centrosomes per cell) as determined by immunofluorescence analysis with anti-γ-tubulin antibody, and forced expression of wild-type NORE1A partially suppressed the centrosome amplification. The nuclear export signal (NES) mutant (L377A/L384A) of NORE1A did not localize to centrosomes and did not suppress the centrosome amplification induced by HU. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2 and 16 showed that wild-type NORE1A, but not NES-mutant NORE1A, suppressed chromosome instability in HU-exposed H1299 cells that was likely to have resulted from centrosome amplification. We next examined the status of NORE1A mRNA expression in non-small cell lung carcinoma (NSCLC) and detected down-regulation of NORE1A mRNA expression in 25 (49%) of 51 primary NSCLCs by quantitative real-time-polymerase chain reaction analysis. These results suggest that NORE1A has activity that suppresses the centrosome amplification induced by HU and that NORE1A mRNA down-regulation is one of the common gene abnormalities in NSCLCs, both of which imply a key preventive role of NORE1A against the carcinogenesis of NSCLC.

TNK2 gene amplification is a novel predictor of a poor prognosis in patients with gastric cancer

We previously examined the amplification status of 10 kinase genes (PIK3CA, EPHB3, TNK2, PTK7, EGFR, MET, ERBB2, HCK, SRC, and AURKA) in gastric cancer (GC). This study aimed to determine the prognostic significance of these gene amplifications in GC.

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

BackgroundGenomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.Methods and ResultsA genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.ConclusionThese results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

Inverse relationship between the length of the EGFR CA repeat polymorphism in lung carcinoma and protein expression of EGFR in the carcinoma

There is a CA dinucleotide repeat polymorphism in the first intron of the epidermal growth factor receptor (EGFR) gene. Our aim was to examine the relationship between the CA repeat length in lung carcinoma and EGFR mutation, EGFR gene copy number, and EGFR protein expression level in the carcinoma.