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.

HER2 staining intensity in HER2-positive disease: relationship with FISH amplification and clinical outcome in the HERA trial of adjuvant trastuzumab.

BACKGROUND: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. PATIENTS AND METHODS: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. RESULTS: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). CONCLUSIONS: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. HERA TRIAL NO: NCT00045032.

[Her2 testing in gastric cancer. What is different in comparison to breast cancer?]. / Her2-Diagnostik beim Magenkarzinom. Was ist anders im Vergleich zum Mammakarzinom?

Based on data from a large multicenter phase III trial (ToGA study) trastuzumab has very recently been approved by the EMEA for metastatic gastric cancer and adenocarcinoma of the gastro-esophageal junction. Only patients with tumors which over express Her2 as defined by IHC2+ and a confirmatory FISH+ result, or IHC 3+, determined by an accurate and validated assay are eligible for trastuzumab therapy. However, testing of Her2 status by immunohistochemistry (IHC) differs from breast cancer in core aspects: 1. IHC2+/3+ is scored even though membranous staining is incomplete if membrane staining is clearly detectable even at low magnification (2.5x/5x, 3+) or medium magnification (10x/20x, 2+). 2. Additionally, membrane staining at the appropriate intensity found in at least 10% of tumor cells is restricted to resection specimens. Evaluation of Her2 in situ hybridization (ISH) is similar to breast cancer with ratio values of > or =2.0 indicating Her2 gene amplification. Taking these modifications into account and defining the HER2 positive subgroup as IHC 3+ and IHC2+/FISH+, approximately 16% of gastric cancers are considered Her2 positive, affecting mainly tumor regions with intestinal (gland forming) type carcinoma. In contrast to breast cancer, up to one-third of gastric cancers show a heterogeneous Her2 status both at IHC and ISH levels which favors bright field ISH over FISH.

[ErbB2 diagnostics in breast cancer--an update]. / Aktuelles zur ErbB2-Diagnostik beim Mammakarzinom.

Determining ErbB2/Her-2/neu status has become an essential part of breast cancer diagnosis and a prerequisite before considering a patient's eligibility for treatment with trastuzumab. Currently the most common techniques to assess ErbB2 status in routine practice are the identification of receptor overexpression by means of immunohistochemistry (IHC) and the analysis of gene amplification by means of dual color fluorescence in situ hybridisation (FISH). According to recent recommendations ("ASCO/CAP Guidelines" and German S3 guidelines for breast cancer) the choice of primary test procedure--IHC or ISH - is left to the individual institution. Both techniques are of equal predictive value provided that strict quality precautions have been taken: internal test validation by comparing IHC and (F)ISH, carrying out controls, and annual participation in round-robin tests. Equivocal IHC (score 2+) has to be checked by ISH for amplification. Borderline ISH (ratio 1.8-2.2 or gene copy number 4.0-6.0) should be retested by counting additional cells or performing IHC. In approximately 5% of cases these criteria give conflicting results and the gene copy number alone generates over 90% of the equivocal ISH cases, mostly due to chromosome 17 polysomy. These cases need to be tested by IHC since over-expression is very exceptional and only these tumors have the potential to be trastuzumab responders.

Assessment of a HER2 scoring system for gastric cancer: results from a validation study.

AIMS: Human epidermal growth factor receptor 2 (HER2) overexpression/amplification is implicated in the development of various solid tumour types. Validated methods and scoring systems for evaluating HER2 status exist in breast cancer, but not in gastric cancer. The aim was to establish a HER2 scoring system for gastric cancer to identify suitable patients for enrollment in a trial of trastuzumab (Herceptin) in advanced metastatic gastric cancer. METHODS AND RESULTS: Formalin-fixed paraffin-embedded gastric cancer samples were tested for HER2 status using the fluorescence in situ hybridization (FISH) pharmDxt kit (Dako Denmark A/S). Immunohistochemistry (IHC) was performed using the HercepTest (Dako). Concordance between FISH and IHC was 93.5% in 168 evaluable samples. Eleven samples were scored as FISH+ but IHC- or equivocal. CONCLUSIONS: IHC/FISH discrepancies were attributed to basolateral membranous immunoreactivity of glandular cells resulting in incomplete membranous reactivity and/or a higher rate of tumour heterogeneity in gastric cancer compared with breast cancer. With modifications to the IHC scoring system, the HercepTest is considered valid for the identification of HER2+ gastric tumours for this clinical trial. Correlation of HER2 scores with clinical outcomes will be needed to determine which patients might benefit from trastuzumab therapy.

Transcriptional profiling of transurethral resection samples provides insight into molecular mechanisms of hormone refractory prostate cancer.

The molecular mechanisms for hormone-resistant prostate cancer progression still remain elusive, mainly due to the limited availability of corresponding tissue. As transurethral resection (TUR) is a common palliative therapy for patients with hormone refractory prostate cancer (HRPC) who have subvesical obstruction, we aimed to demonstrate that TUR samples can be used to identify significantly affected biological pathways during the switch to HRPC using oligonucleotide microarray analysis. Among the most significantly deregulated pathways in HRPC, we observed an induction of oxidative phosphorylation and a repression of cytoskeletal components.

Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin) phase II monotherapy study: assessment of test sensitivity and impact of chromosome 17 polysomy.

AIMS: To investigate the correlation between centrally assessed human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) results and response to treatment of patients with metastatic breast cancer enrolled in a first-line, phase II, open-label, 3-weekly trastuzumab (Herceptin) monotherapy trial (WO16229). METHODS: Samples from participants in the WO16229 trial were collected and tumour HER2 status determined by IHC and FISH. HER2 test results were interpreted according to manufacturers' test kit protocols. Responders were defined as patients showing either partial or complete responses. RESULTS: Response data were available for 103/105 patients; centrally confirmed HER2 status was available for 95 patients. Intra-laboratory concordance for central IHC and FISH results was 93%. Complete responses were seen in two patients; their samples were IHC 3+ and FISH positive. Partial responses were seen in 17 patients; all were IHC 3+ and 14 were FISH positive. IHC and FISH showed 100% and 84.2% sensitivity, respectively, in determining response to trastuzumab. Polysomy was observed in 27% of patients; six responded to trastuzumab treatment. All six responders showed HER2 overexpression (IHC 3+) and HER2 gene amplification; two were FISH negative due to chromosome 17 polysomy. CONCLUSIONS: HER2 determination by IHC and FISH correlates with clinical response data in the WO16229 trial with high concordance of IHC and FISH results. Polysomy is the major cause of response in FISH-negative cases; polysomic cases should be retested by strictly standardised IHC.

[Her-2/neu analysis--new data?]. / Her-2/neu Analyse--Neue Daten?

Her-2 status determination is an essential prerequisite before considering patient eligibility for treatment with trastuzumab. Currently the most common techniques to assess Her-2 status in routine practice are immunohistochemistry (IHC) and dual color FISH for receptor expression and gene amplification analysis, respectively. Despite both methods are well-established in breast cancer there are a variety of yet unsolved questions: 1. Do we really need IHC since interlab variation is still quite high (up to 30%)? 2. Are FISH and CISH equivalent techniques? 3. Are there any precautions to be taken if Her-2 is tested in non-breast cancer samples? 4. What is the value of Her-2 status in blood serum (ELISA)? 5. Do we get better response prediction if new Her2 antibodies, other techniques such as quantitative (q) RT-PCR or multiparameter assays according to downstream signalling pathways are applied? 6. Is Her-2 status still predictive when other therapeutic antibodies than trastuzumab (e. g. pertuzumab) or kinase inhibitors (e. g. lapatinib) are used? These questions will be discussed under the review of the recent literature and under own experiences obtained either by centralized Her-2 assessment in a variety of breast and non-breast cancer therapy studies and within international ring studies between reference labs from Australia (M. Bilous), Canada (W. Hanna), France (F. Penault-Llorcoa), Great Britain (M. Dowsett), Japan (R. Y. Osamura), and Netherlands (M. v. d. Vijver) in which we participated.

The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites.

We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein. Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B. rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2beta and tau minigenes in cotransfection experiments. This effect can be reversed by rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purine-rich sequence. Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing.

An alternative-exon database and its statistical analysis.

We compiled a comprehensive database of alternative exons from the literature and analyzed them statistically. Most alternative exons are cassette exons and are expressed in more than two tissues. Of all exons whose expression was reported to be specific for a certain tissue, the majority were expressed in the brain. Whereas the length of constitutive exons follows a normal distribution, the distribution of alternative exons is skewed toward smaller ones. Furthermore, alternative-exon splice sites deviate more from the consensus: their 3' splice sites are characterized by a higher purine content in the polypyrimidine stretch, and their 5' splice sites deviate from the consensus sequence mostly at the +4 and +5 positions. Furthermore, for exons expressed in a single tissue, adenosine is more frequently used at the -3 position of the 3' splice site. In addition to the known AC-rich and purine-rich exonic sequence elements, sequence comparison using a Gibbs algorithm identified several motifs in exons surrounded by weak splice sites and in tissue-specific exons. Together, these data indicate a combinatorial effect of weak splice sites, atypical nucleotide usage at certain positions, and functional enhancers as an important contribution to alternative-exon regulation.

The in vivo minigene approach to analyze tissue-specific splicing.

The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements (cis-factors), their interacting protein components (trans-factors) or the cell type to alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections. Here, we give a detailed description of this technique that allows analysis of large gene fragments (up to 10-12 kb) under in vivo condition. Furthermore, we provide a summary of 44 genes currently investigated to demonstrate the general feasibility of this technique.

Alternative splicing determines the intracellular localization of the novel nuclear protein Nop30 and its interaction with the splicing factor SRp30c.

We report on the molecular cloning of a novel human cDNA by its interaction with the splicing factor SRp30c in a yeast two-hybrid screen. This cDNA is predominantly expressed in muscle and encodes a protein that is present in the nucleoplasm and concentrated in nucleoli. It was therefore termed Nop30 (nucleolar protein of 30 kDa). We have also identified a related cDNA with a different carboxyl terminus. Sequencing of the NOP gene demonstrated that both cDNAs are generated by alternative 5' splice site usage from a single gene that consists of four exons, spans at least 1800 nucleotides, and is located on chromosome 16q21-q23. The alternative 5' splice site usage introduces a frameshift creating two different carboxyl termini. The carboxyl terminus of Nop30 is rich in serines and arginines and has been found to target the protein into the nucleus, whereas its isoform is characterized by proline/glutamic acid dipeptides in its carboxyl terminus and is predominantly found in the cytosol. Interaction studies in yeast, in vitro protein interaction assays, and co-immunoprecipitations demonstrated that Nop30 multimerizes and binds to the RS domain of SRp30c but not to other splicing factors tested. Overexpression of Nop30 changes alternative exon usage in preprotachykinin and SRp20 reporter genes, suggesting that Nop30 influences alternative splice site selection in vivo.

Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable beta-galactosidase.

Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable beta-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3'- and 5'-end of the amyE gene (encoding alpha-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.