A study of HER2 gene amplification and protein expression in gastric cancer

Background Gastric cancer is a leading cause of cancer-related mortality, and current treatment outcomes for advanced disease remain poor. HER2 has been identified as a potential candidate for targeted therapy in gastric cancers displaying HER2 gene amplification and protein overexpression.

Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer

Tissue microarrays allow high throughput molecular profiling of diagnostic or predictive markers in cancer specimens and rapid validation of novel potential candidates identified from genomic and proteomic analyses in a large number of tumor samples. To validate the use of tissue microarray technology for all the main biomarkers routinely used to decide breast cancer prognostication and postsurgical adjuvant therapy, we constructed a tissue microarray from 97 breast tumors, with a single 0.6 mm core per specimen. Inummostaining; of tissue microarray sections and conventional full sections of each tumor were performed using well-characterized prognostic markers (estrogen receptor ER, progesterone receptor PR and c-erbB2). The full section versus tissue microarray concordance for these stains was 97% for ER, 98% for PR, and 97% for c-erbB2, respectively, with a strong statistical association (kappa value more than 0.90). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification from the single-core tissue microarray was technically successful in about 90% (87/97) of the cases, with a concordance of 95% compared with parallel analyses with the full sections. The correlation with other pathological parameters was not significantly different between full-section and array-based results. It is concluded that the constructed tissue microarray with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinicopathological parameters, with no significant associated sampling bias.

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene

Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation.

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

Dual-colour HER2/chromosome 17 chromogenic in situ hybridisation enables accurate assessment of HER2 genomic status in ovarian tumours

Ovarian cancer is a leading cause of gynaecological cancer-related morbidity and mortality. There has been increasing interest in the potential utility of anti-human epidermal growth factor receptor 2 (anti-HER2) agents in the treatment of this disease, with the attendant need to identify suitable predictive biomarkers of response to treatment.

Dual-colour HER2/chromosome 17 chromogenic in situ hybridisation assay enables accurate assessment of HER2 genomic status in gastric cancer and has potential utility in HER2 testing of biopsy samples

Determination of HER2 protein expression by immunohistochemistry (IHC) and genomic status by fluorescent in situ hybridisation (FISH) are important in identifying a subset of high HER2-expressing gastric cancers that might respond to trastuzumab. Although FISH is considered the standard for determination of HER2 genomic status, brightfield ISH is being increasingly recognised as a viable alternative. Also, the impact of HER2 protein expression/genomic heterogeneity on the accuracy of HER2 testing has not been well studied in the context of gastric biopsy samples.

Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression

The oncogenic role of WNT is well characterized. Wntless (WLS) (also known as GPR177, or Evi), a key modulator of WNT protein secretion, was recently found to be highly overexpressed in malignant astrocytomas. We hypothesized that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signaling such as ovarian, gastric, and breast cancers. Immunohistochemical analysis using a TMA platform revealed WLS overexpression in a subset of ovarian, gastric, and breast tumors; this overexpression was associated with poorer clinical outcomes in gastric cancer (P=0.025). In addition, a strong correlation was observed between WLS expression and human epidermal growth factor receptor 2 (HER2) overexpression. Indeed, 100% of HER2-positive intestinal gastric carcinomas, 100% of HER2-positive serous ovarian carcinomas, and 64% of HER2-positive breast carcinomas coexpressed WLS protein. Although HER2 protein expression or gene amplification is an established predictive biomarker for trastuzumab response in breast and gastric cancers, a significant proportion of HER2-positive tumors display resistance to trastuzumab, which may be in part explainable by a possible mechanistic link between WLS and HER2.

Molecular pathology of prostate cancer:the key to identifying new biomarkers of disease

Microarray technology has recently accelerated the study of the molecular events involved in prostate cancer, offering the prospect of more precise prognosis and new therapeutic strategies. This review summarises current knowledge of the molecular pathology of prostate cancer. The expression and function of numerous genes have been shown to be altered in prostate cancer. Many of these genes are involved in cell cycle regulation, steroid hormone metabolism or regulation of gene expression. The mechanisms by which androgen independence arises are discussed, including cross-activation, gene amplification and point mutations of the androgen receptor. Analysis of changes in the levels of expression of large numbers of genes during prostate cancer progression have provided a better understanding of the basis of the disease, yielding new molecular markers, such as hepsin, with potential use in diagnosis and prognosis.

Autosomal dominant retinitis pigmentosa:linkage to rhodopsin and evidence for genetic heterogeneity

Retinitis pigmentosa (RP) is the most prevalent human retinopathy of genetic origin. Chromosomal locations for X-linked RP and autosomal dominant RP genes have recently been established. Multipoint analyses with ADRP and seven markers on the long arm of chromosome 3 demonstrate that the gene for rhodopsin, the pigment of the rod photoreceptors, cosegregates with the disease locus with a maximum lod score of approximately 19, implicating rhodopsin as a causative gene. Recent studies have indicated the presence of a point mutation at codon 23 in exon 1 of rhodopsin which results in the substitution of histidine for the highly conserved amino acid proline, suggesting that this mutation is a cause of rhodopsin-linked ADRP. This mutation is not present in the Irish pedigree in which ADRP has been mapped close to rhodopsin. Another mutation in the rhodopsin gene or in a gene closely linked to rhodopsin may be involved. Moreover, the gene in a second ADRP pedigree, with Type II late onset ADRP, does not segregate with chromosome 3q markers, indicating that nonallelic as well as perhaps allelic genetic heterogeneity exists in the autosomal dominant form of this disease.

Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation.

The c-kit proto-oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK-cells, and to early T-cell precursors and it is not expressed by normal B-cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkin's disease and anaplastic large-cell lymphoma. In the present paper we describe a case of a B-cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B-cell population. Further studies are necessary to explore both the functional role of c-kit expression in the neoplastic B-cells from this patient and its potential utility for the diagnosis and follow-up of patients with B-cell non-Hodgkin's lymphoma.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays:towards new standards for gene expression measurements

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

Identification of gene networks associated with acute myeloid leukemia by comparative molecular methylation and expression profiling

Around 80% of acute myeloid leukemia (AML) patients achieve a complete remission, however many will relapse and ultimately die of their disease. The association between karyotype and prognosis has been studied extensively and identified patient cohorts as having favourable [e.g. t(8; 21), inv (16)/t(16; 16), t(15; 17)], intermediate [e.g. cytogenetically normal (NK-AML)] or adverse risk [e.g. complex karyotypes]. Previous studies have shown that gene expression profiling signatures can classify the sub-types of AML, although few reports have shown a similar feature by using methylation markers. The global methylation patterns in 19 diagnostic AML samples were investigated using the Methylated CpG Island Amplification Microarray (MCAM) method and CpG island microarrays containing 12,000 CpG sites. The first analysis, comparing favourable and intermediate cytogenetic risk groups, revealed significantly differentially methylated CpG sites (594 CpG islands) between the two subgroups. Mutations in the NPM1 gene occur at a high frequency (40%) within the NK-AML subgroup and are associated with a more favourable prognosis in these patients. A second analysis comparing the NPM1 mutant and wild-type research study subjects again identified distinct methylation profiles between these two subgroups. Network and pathway analysis revealed possible molecular mechanisms associated with the different risk and/or mutation sub-groups. This may result in a better classification of the risk groups, improved monitoring targets, or the identification of novel molecular therapies.

Development and clinical validation of a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.