HER2 testing in paired biopsy and excision specimens of gastric cancer: the reliability of the scoring system and the clinicopathological factors relevant to discordance.

BACKGROUND: Inclusion of trastuzumab in chemotherapy regimens is advantageous for patients with advanced or metastatic gastric cancer who overexpress HER2. Therefore, accurate assessment of HER2 status in tumor tissue is critical when weighing treatment options. METHODS: We examined HER2 expression in 180 paired endoscopic biopsy and surgical excision specimens of gastric cancers via immunohistochemistry (IHC). Equivocal IHC results (IHC 2+) were resolved by HER2 fluorescence in situ hybridization (FISH). The relationships of several clinical and pathological features with discordant HER2 results in paired specimens were determined. RESULTS: Fourteen biopsy specimens and surgical specimens (7.8%) were HER2-positive. Discordant HER2 IHC scores were observed in 90 paired specimens (50%) and 8 paired specimens (4.4%) had discordant results. The kappa coefficients for an HER2 diagnostic algorithm were 0.264, 0.339, and 0.690 for IHC scores, IHC categories, and final results, respectively (p < 0.001). Discordant HER2 results were significantly associated with discordant tumor differentiation in the paired biopsy and excision specimens (p = 0.01). Intratumoral heterogeneity did not predict HER2 discordance. There was no association between HER2 discordance and the number of biopsy tissue fragments (p = 0.764). CONCLUSIONS: Hofmann's HER2 scoring system is a fairly reliable tool for evaluating HER2 status in biopsy and excision specimens. Discordant HER2 results in paired specimens were observed in a small percentage of gastric cancers. Testing all available specimens should be considered in order to eliminate discrepancies, especially when discordant tumor differentiation is observed.

Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination.

The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription.

Detection of hepatitis delta virus recombinants in cultured cells co-transfected with cloned genotypes I and IIb DNA sequences.

It was reported previously that hepatitis delta virus (HDV), the only animal virus in which replication is performed by cellular RNA polymerase(s), undergoes RNA recombination. However, the previous RNA transfection system was somewhat limited in terms of practical application. Cultured cells were transfected with plasmids expressing replication-competent genotypes I and IIb HDV genomic RNAs to develop a better system for studying the fundamental aspects of HDV RNA recombination and HDV-related RNA species were examined using restriction fragment length polymorphisms and sequence analysis of cloned RT-PCR products. This novel experimental system generated efficiently recombinants between the two parental HDV sequences, but not between replication-defective HDV constructs. The genome organization of the HDV recombinants produced in this system resembled that observed previously in cultured cells co-transfected with genome I and IIb RNAs. These data indicate that replication-dependent HDV RNA recombination can be catalyzed by host RNA polymerases in cultured cells co-transfected with two cloned HDV sequences. This new DNA-based system is simpler than the previous RNA-based method of study, and generates a higher recombination frequency, facilitating study of HDV RNA recombination.