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 clinicopathological 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.
A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase‐type cell surface receptor HER2 (c‐erbB2), and the corresponding chromosome (chromosome 17 for c‐erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave‐assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c‐erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c‐erbB2 locus. Furthermore, a combination of microwave‐assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c‐erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c‐erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus‐specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less‐adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype–phenotype analysis of individual cells.
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.
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.
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.A survival analysis was performed for GC patients. Since TNK2 amplification was identified as a prognostic marker in the analysis, we also examined the functional effect of TNK2 overexpression on gastric cells.A Kaplan-Meier analysis showed that the prognosis of patients with GC exhibiting TNK2 or AURKA amplification was significantly poorer than the prognosis of patients with GC without TNK2 or AURKA amplification. A further multivariate analysis revealed that TNK2 amplification was an independent predictor of a poor survival outcome among patients with GC (hazard ratio, 3.668; 95% confidence interval, 1.513-7.968; P = 0.0056). TNK2-overexpressing GC cells showed an increase in cell migration and non-anchored cell growth. Finally, microarray and pathway analyses revealed the aberrant regulation of some cancer-related pathways in TNK2-overexpressing GC cells.These results suggested that TNK2 amplification is an independent predictor of a poor prognosis in patients with GC and leads to an increase in the malignant potential of GC cells.
Background: Biological variations in and the heterogeneity of gastrointestinal stromal tumors (GISTs) are well known, but chromosomal numerical abnormality (CNA) has not been fully examined especially in this context. The aim of this study is to test CNA as a possible biological predictor of biological behavior of GISTs. Method: We applied microwave-assisted FISH protocol to pathological archives of GIST tumors displaying different clinical features to characterize the CNA profile of these tumors. A panel of 18 centromere enumeration probes (CEP) and 24 bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) probes containing genes like Aurora kinases (AURKs) and other candidate genes involved in human carcinogenesis were used. CNA profiles, histopathological risk categorization and Ki-67 labeling indexes of 23 primary and/or metastatic GIST tumors of 12 subjects (both primary and metastatic in 7 subjects) were compared between primary GIST with and without metastases, and between metastatic and primary portions in 7 individuals. Results: CNA in the primary sites was more extensive in the GISTs with recurrence and metastasis than in those without, especially as to the loss of chromosome 20 and genomic imbalance of AURKA-containing BAC probe on 20q in the cases with metastasis. The consistent loss of one allele of chromosome 14q was also noted. Interestingly, both primary and metastatic tumors in identical individuals had similar CNA profiles. Conclusion: The extent of CNA differed between GISTS with and without recurrence or metastasis; thus, FISH analysis of specimens from the primary sites may predict the biological behavior of this tumor.
