Diagnostic Molecular Pathology最新文献

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are related members of the Herpesviridae family and are well-documented human pathogens causing a spectrum of diseases, from mucocutaneous disease to infections of the central nervous system. This study was carried out to evaluate and validate the performance of a multiplex real-time polymerase chain reaction (PCR) assay in detecting and differentiating HSV1, HSV2, and VZV from clinical samples. Consensus PCR primers for HSV were designed from the UL30 component of the DNA polymerase gene of HSV, with 2 separate hydrolysis probes designed to differentiate HSV1 and HSV2. Separate primers and a probe were also designed against the DNA polymerase gene of VZV. A total of 104 clinical samples were available for testing by real-time PCR, conventional PCR, and viral culture. The sensitivity and specificity of the real-time assay was calculated by comparing the multiplex PCR result with that of a combined standard of virus culture and conventional PCR. The sensitivity of the real-time assay was 100%, with specificity ranging from 98% to 100% depending on the target gene. Both PCR methods detected more positive samples for HSV or VZV, compared with conventional virus culture. This multiplex PCR assay provides accurate and rapid diagnostic capabilities for the diagnosis and differentiation of HSV1, HSV2, and VZV infections, with the presence of an internal control to monitor for inhibition of the PCR reaction.

EGFR-activating mutations predict responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients. Mutation screening is crucial to support therapeutic decisions and is commonly conducted using dideoxy sequencing, although its sensitivity is suboptimal in clinical settings. To evaluate the diagnostic performance of pyrosequencing and dideoxy sequencing, we examined EGFR mutation status in a retrospective cohort of 53 patients with NSCLCs clinically selected for TKI therapy and whose clinical outcome was available. Moreover, pyrosequencing quantitative results were compared with EGFR amplification data. EGFR mutations were investigated by pyrosequencing and by dideoxy sequencing. Detection rates of both methods were determined by titration assays using NCI-H1975 and HCC-827 cell lines. Increased EGFR copy number was assessed by fluorescence in situ hybridization (FISH). Pyrosequencing showed a higher detection rate than dideoxy sequencing. Tumor control rate of cases with mutant and wild-type EGFR was 86% and 29%, respectively. EGFR amplification was significantly associated with EGFR mutation and a positive correlation between high percentages of mutant alleles and clinical response to TKI was observed. We concluded that pyrosequencing is more sensitive than dideoxy sequencing in mutation screening for EGFR mutations. Detection rate of dideoxy sequencing was suboptimal when low frequencies of mutant alleles or low tumor cell contents were observed. Pyrosequencing enables quantification of mutant alleles that correlates well with increased EGFR copy number assessed by FISH. Pyrosequencing should be used in molecular diagnostic of NSCLC to appropriately select patients who are likely to benefit from TKI therapy.

Genetic analysis of fetal tissue provides valuable information regarding the underlying causes of miscarriage. However, current analysis techniques are limited and expensive. This trial compared a molecular multiplex, bead-based suspension array, KaryoLite Bacs on Beads, with conventional tissue culture and G-banded karyotype techniques. A 92% overall success rate was achieved. This method detected a cryptic deletion of a 7q subtelomeric region, a case of 25% mosaic trisomy 14, and 2 unbalanced subtelomeric rearrangements due to familial balanced translocations. Twenty (24%) of the 83 samples analyzed, that failed to yield a cytogenetic result due to culture failure, were successfully assayed using the suspension array. Genomic imbalances including trisomies and subtelomeric deletions were detected in 3 cases (15%) of previously failed cases. This method is limited by its inability to detect polyploidy, which is significant in first trimester loss. However, this can be readily overcome by prescreening using florescent in situ hybridization. Data indicates that KaryoLite BoBs molecular testing is superior to conventional cytogenetic evaluation in several key areas, including success rate (95% vs. 76%, for this study group), cost, turnaround time (2 vs. up to 28 d), and subjective result interpretation.

Cancer molecular investigation revealed a huge molecular heterogeneity between different types of cancers as well as among cancer patients affected by the same cancer type. This implies the necessity of a personalized approach for cancer diagnosis and therapy, on the basis of the development of standardized protocols to facilitate the application of molecular techniques in the clinical decision-making process. Ion channels encoding genes are acquiring increasing relevance in oncological translational studies, representing new candidates for molecular diagnostic and therapeutic purposes. Hence, the development of molecular protocols for the quantification of ion channels encoding genes in tumor specimens may have relevance for diagnostic and prognostic investigation. Two main hindrances must be overcome for these purposes: the use of formalin-fixed and paraffin-embedded samples for gene expression analysis and the physiological expression of ion channels in excitable cells, potentially present in the tumor sample. We here propose a method for hERG1 gene quantification in colorectal cancer samples in both cryopreserved and formalin-fixed and paraffin-embedded samples. An analytical method was developed to estimate hERG1 gene expression exclusively in epithelial cancer cells. Indeed, we found that the hERG1 gene was expressed at significant levels by myofibroblasts present in the tumor stroma. This method was based on the normalization on a smooth muscle-myofibroblast-specific gene, MYH11, with no need of microdissection. By applying this method, hERG1 expression turned out to correlate with VEGF-A expression, confirming previous immunohistochemical data.

Established clinicopathologic characteristics of non-small cell lung cancer patients define a subgroup responding better to EGFR-TK inhibitors: adenocarcinoma histology, ethnicity, sex, smoking status, presence of activating EGFR mutation, and/or K-RAS wild type. However, EGFR mutation does not automatically lead to increased activity of the protein influenced by several factors. As adenocarcinoma can be further divided into histologic subclasses, we compared adenocarcinomas without lepidic growth pattern (NLAC) to those characterized by pure or predominant lepidic growth (LAC) for EGFR protein expression and autophosphorylation activity (Y1173), as determined by immunohistochemistry. This pretarget therapy cohort comprised a total of 110 surgically operated patients of stage I non-small cell lung cancer: 49 NLAC and 61 LAC variants. The LAC group had a significantly better prognosis and the incidence of phospho-EGFR-positive tumors was significantly higher compared with NLAC. Patient sex did not influence EGFR activity, but the incidence of pEGFR-positive tumors was significantly lower among smoker patients. There was no statistically significant difference in EGFR or KRAS mutation frequencies between the 2 groups. In NLAC, pEGFR-positive tumors occurred exclusively among EGFR-mutant/K-RAS wild-type tumors. On the contrary, in LAC tumors, pEGFR-positive tumors were similarly frequent in the EGFR or K-RAS mutant groups indicating an interesting feedback activation of EGFR signaling in K-RAS mutant tumors. Our data also indicate that EGFR mutation leads to EGFR autophosphorylation only in a small fraction of adenocarcinoma patients, which might have clinical significance.

Hepatocellular carcinoma (HCC) is one of the rare tumors with well-defined risk factors. The multifactorial etiology of HCC can be explained by its complex molecular pathogenesis. In the current study, the methylation status of 7 genes involved in DNA repair mechanisms, namely MLH1, PMS2, MSH6, MSH2, MGMT, MSH3, and MLH3, was investigated in tumor samples from HCC patients, using the methylation-specific-multiplex ligated probe amplification method and the results were correlated with available clinical findings. The most common etiological factor in these cases was the presence of hepatitis B alone (47.2%). Among the 56 cases that were studied, promoter methylation was detected in at least one of the genes in 27 (48.2%) cases, only in 1 gene in 13 (23.2%) cases, and in >1 gene in 14 (25%) cases. Of the 7 genes investigated, methylation was most frequently observed in MSH3, in 14 (25%) cases. Methylation of at least 1 gene was significantly more frequent in patients with single tumors than multifocal tumors. There were significant differences regarding hepatitis B status, Child Class, tumor number, grade, and TNM stage in cases where PMS2 methylation was detected. Our results suggest that methylation of genes involved in mismatch repair may be responsible in the pathogenesis of HCC, and evaluating changes in multiple genes in these pathways simultaneously would be more informative. Despite being a robust and relatively inexpensive method, the methylation-specific-multiplex ligated probe amplification assay could be more extensively applied with improvements in the currently intricate data analysis component.