Quality assessment of estrogen receptor and progesterone receptor testing in breast cancer using a tissue microarray-based approach.

Assessing hormone receptor status is an essential part of the breast cancer diagnosis, as this biomarker greatly predicts response to hormonal treatment strategies. As such, hormone receptor testing laboratories are strongly encouraged to participate in external quality control schemes to achieve optimization of their immunohistochemical assays. Nine Dutch pathology departments provided tissue blocks containing invasive breast cancers which were all previously tested for estrogen receptor and/or progesterone receptor expression during routine practice. From these tissue blocks, tissue microarrays were constructed and tested for hormone receptor expression. When a discordant result was found between the local and TMA result, the original testing slide was revised and staining was repeated on a whole-tissue block. Sensitivity and specificity of individual laboratories for testing estrogen receptor expression were high, with an overall sensitivity and specificity [corrected] of 99.7 and 95.4%, respectively. Overall sensitivity and specificity of progesterone receptor testing were 94.8 and 92.6%, respectively. Out of 96 discordant cases, 36 cases would have been concordant if the recommended cut-off value of 1% instead of 10% was followed. Overall sensitivity and specificity of estrogen and progesterone receptor testing were high among participating laboratories. Continued enrollment of laboratories into quality control schemes is essential for achieving and maintaining the highest standard of care for breast cancer patients.

Human epidermal growth factor receptor 2 overexpression and amplification in endoscopic biopsies and resection specimens in esophageal and junctional adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in a subset of esophageal adenocarcinomas. Frequently, biopsy material is used for evaluation of HER2 status. The aim of the study was to determine if HER2 expression in preoperative endoscopic biopsies is representative for the entire tumor. Preoperative endoscopic biopsies and matched resection specimens were collected from 75 patients who underwent esophagectomy for esophageal adenocarcinoma. Immunohistochemical staining (IHC) on HER2 and dual-color in situ hybridization (ISH) were performed. HER2 status was determined by following a clinical algorithm, first determining HER2 overexpression on immunohistochemistry and, when equivocal (2+), determining HER2 amplification on ISH. Seventy-one of 75 (95%) biopsies and 69/75 (92%) resection specimens could be analyzed due to technical failure. HER2 positivity was seen in 18/71 (25%) biopsies and in 15/69 (22%) resection specimens. Overall, HER2 status in the biopsy was concordant with HER2 status in the resection specimen in 94% of cases. Interobserver agreement on IHC scoring for all three observers was 83% in biopsies and 85% in resection specimens. HER2 positivity was detected in 22% of esophageal adenocarcinomas. Although interobserver agreement was moderate, HER2 status of a primary tumor can be reliably determined based on the endoscopically obtained pretreatment biopsy.

Establishment of cell lines from adenocarcinomas of the esophagus and gastric cardia growing in vivo and in vitro.

The aim of this study was to establish cell lines of adenocarcinomas of the gastro-esophageal junction(GEJ), which grow in vivo and in vitro. Primary esophageal and gastric cardia adenocarcinomas and corresponding lymph node metastases were xenografted subcutaneously to immunodeficient nude mice. In addition, tumor tissue was also used for in vitro culture. Xenografting of 70 primary adenocarcinomas and 17 metastases resulted in the initial growth of 22 and 6 tumors, respectively (total 32%). Upon retransplantation, six long-term xenografts [esophageal adenocarcinoma (OAC)P33X, OACP47X, OACP56X, OACP58X, OACP67X, OACP76X] from primary tumors and three (OACM2.1X, OACM30X, OACM53X) from metastases were obtained. In vitro culture attempts of 34 primary tumors and nine metastases resulted in the establishment of three (7%) permanent in vitro growing cell lines. From one patient, a cell line from the primary tumor (OACP4 C) and from a lymph node metastasis (OACM4.1 C) was established. The third cell line (OACM5.1 C) was also derived from a lymph node metastasis. The in vivo and in vitro cell lines were characterized using immunocytochemistry and microsatellite analysis to verify their epithelial and human tumor origin, respectively.

Molecular assessment of clonality leads to the identification of a new germ line TP53 mutation associated with malignant cystosarcoma phyllodes and soft tissue sarcoma.

Cystosarcoma phyllodes (CSP) is a rare breast neoplasm composed of stromal and epithelial elements. It usually runs a benign course but it may metastasize. In a 31-year-old patient with recurring CSP, a mesenchymal tumor in the leg developed. The question arose whether the latter tumor could be a metastasis from the CSP, which would have major treatment consequences. The problem was addressed using molecular methods, i.e., comparison of the pattern of polymorphic repeat markers on chromosome 17p as well as single strand conformation polymorphism analysis and sequencing of exons 5 to 8 of the TP53 gene in both tumor and normal tissue. An identical pattern of loss of heterozygosity in both breast tumors was demonstrated, but a different pattern was shown in the tumor in the leg. This led to the conclusion that the latter tumor had to be a new primary tumor. A mutation in codon 162 of the TP53 gene was found in the tumor tissue as well as in the normal tissue of this patient. This germ line mutation leads to the replacement of isoleucine by asparagine and most likely has functional consequences. In all four examined tumors of this patient, the normal TP53 allele was lost. This is strong evidence that this germ line TP53 mutation causes the genesis of these two rare primary mesenchymal tumors in this young patient. The current study exemplifies the power of molecular diagnostic methods in investigating the specific clinical problem of clonal relation between two separate tumors. The germ line mutation found in codon 162 of the TP53 gene and the association with cystosarcoma phyllodes have not been described previously.

Loss of heterozygosity of chromosome 8 microsatellite loci implicates a candidate tumor suppressor gene between the loci D8S87 and D8S133 in human prostate cancer.

To search for specific chromosome 8 aberrations in human prostate cancer, DNA was isolated from 44 human prostate tumor samples. Twenty six tumor samples were obtained from locally progressive tumors by transurethral resection, 12 were from radical prostatectomy specimens, and 6 were from lymph node metastases. Tumor DNAs were screened for allelic losses using 16 highly polymorphic microsatellite loci (14 covering the p arm, 2 on the q arm). In general, the detected deletions were large. In 59% of the tumor DNAs, allelic loss of 3 or more 8p loci was observed. Loss of 8p loci occurred in between 36 and 69% of the informative cases; for the two 8q markers, the percentages of loss were 11 and 25%, respectively, indicating preferential loss of (part of) 8p. In one tumor, two separate 8p deletions were found. The percentage of loss of heterozygosity was considerably higher in transurethral resection (65%) and lymph node metastases (83%) than in radical prostatectomy specimens (33%), suggesting that 8p deletion is a relatively late step in tumor progression. The maximal overlapping deleted region in all tumor DNAs is between the distal locus D8S133 and the proximal locus D8S87, indicating the localization of a candidate tumor suppressor gene within this region.

A practical approach to the detection of androgen receptor gene mutations and pedigree analysis in families with x-linked androgen insensitivity.

Androgen insensitivity syndrome (AIS) is an X-linked disorder in which defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. This survey reports the analysis of 11 AIS subjects. The androgen receptor gene of these subjects was analyzed using polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis and sequencing or sequencing of PCR-amplified androgen receptor gene fragments alone. In total, 10 single base changes and one partial gene deletion were detected. Seven single base changes resulted in an amino acid change, one resulted in the introduction of a premature stop codon, one event represented a single base insertion resulting in a frame-shift, and one single base change affected a donor splice site. The androgen receptor protein in genital skin fibroblasts from several patients was studied with respect to molecular mass after immunoprecipitation and SDS-PAGE. Two patients expressed a truncated receptor protein in agreement with the established genomic mutation. Pedigree analysis was performed to identify possible carriers for the syndrome in families of AIS patients using single-strand conformation polymorphism and restriction site analysis of PCR products. In one case, the polymorphic (CAG)n(CAA) repeat in exon 1 encoding a polyglutamine stretch was used to identify the mutant allele in a family with X-linked partial androgen insensitivity before the identification of the actual genomic mutation. PCR-single-strand conformation polymorphism analysis proved to be a fast and reliable technique to screen for androgen receptor gene mutations and to study the androgen receptor gene of family members of AIS-affected individuals.