Quantitative measurement of HER2 expression to subclassify ERBB2 unamplified breast cancer.

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm2. Then by calculation of the limits of detection, quantification, and linearity of this assay we determined that low HER2 range expression in unamplified cell lines is between 2 and 20 attomol/mm2. Finally, application of this assay to a serial collection of 364 breast cancer cases from Yale shows 67% of the population has HER2 expression above the limit of quantification and below the levels seen in HER2 amplified breast cancer. In the future, this assay could be used to determine the levels of HER2 required for response to T-DXd or similar HER2 conjugated ADCs.

Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine.

Biospecimens acquired during routine medical practice are the primary sources of molecular information about patients and their diseases that underlies precision medicine and translational research. In cancer care, molecular analysis of biospecimens is especially common because it often determines treatment choices and may be used to monitor therapy in real time. However, patient specimens are collected, handled, and processed according to routine clinical procedures during which they are subjected to factors that may alter their molecular quality and composition. Such artefactual alteration may skew data from molecular analyses, render analysis data uninterpretable, or even preclude analysis altogether if the integrity of a specimen is severely compromised. As a result, patient care and safety may be affected, and medical research dependent on patient samples may be compromised. Despite these issues, there is currently no requirement to control or record preanalytical variables in clinical practice with the single exception of breast cancer tissue handled according to the guideline jointly developed by the American Society of Clinical Oncology and College of American Pathologists (CAP) and enforced through the CAP Laboratory Accreditation Program. Recognizing the importance of molecular data derived from patient specimens, the CAP Personalized Healthcare Committee established the Preanalytics for Precision Medicine Project Team to develop a basic set of evidence-based recommendations for key preanalytics for tissue and blood specimens. If used for biospecimens from patients, these preanalytical recommendations would ensure the fitness of those specimens for molecular analysis and help to assure the quality and reliability of the analysis data.

Programmed Death-Ligand 1 Immunohistochemistry Testing: A Review of Analytical Assays and Clinical Implementation in Non-Small-Cell Lung Cancer.

Purpose Three programmed death-1/programmed death-ligand 1 (PD-L1) inhibitors are currently approved for treatment of non-small-cell lung cancer (NSCLC). Treatment with pembrolizumab in NSCLC requires PD-L1 immunohistochemistry (IHC) testing. Nivolumab and atezolizumab are approved without PD-L1 testing, though US Food and Drug Administration-cleared complementary PD-L1 tests are available for both. PD-L1 IHC assays used to assess PD-L1 expression in patients treated with programmed death-1/PD-L1 inhibitors in clinical trials include PD-L1 IHC 28-8 pharmDx (28-8), PD-L1 IHC 22C3 pharmDx (22C3), Ventana PD-L1 SP142 (SP142), and Ventana PD-L1 SP263 (SP263). Differences in antibodies and IHC platforms have raised questions about comparability among these assays and their diagnostic use. This review provides practical information to help physicians and pathologists understand analytical features and comparability of various PD-L1 IHC assays and their diagnostic use. Methods We reviewed and summarized published or otherwise reported studies (January 2016 to January 2017) on clinical trial and laboratory-developed PD-L1 IHC assays (LDAs). Studies assessing the effect of diagnostic methods on PD-L1 expression levels were analyzed to address practical issues related to tissue samples used for testing. Results High concordance and interobserver reproducibility were observed with the 28-8, 22C3, and SP263 clinical trial assays for PD-L1 expression on tumor cell membranes, whereas lower PD-L1 expression was detected with SP142. Immune-cell PD-L1 expression was variable and interobserver concordance was poor. Inter- and intratumoral heterogeneity had variable effects on PD-L1 expression. Concordance among LDAs was variable. Conclusion High concordance among 28-8, 22C3, and SP263 when assessing PD-L1 expression on tumor cell membranes suggests possible interchangeability of their clinical use for NSCLC but not for assessment of PD-L1 expression on immune cells. Development of LDAs requires stringent standardization before their recommendation for routine clinical use.

The blood DNA virome in 8,000 humans.

The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

Validation of an electronic program for pathologist training in the interpretation of a complex companion diagnostic immunohistochemical assay.

Companion diagnostics assay interpretation can select patients with the greatest targeted therapy benefits. We present the results from a prospective study demonstrating that pathologists can effectively learn immunohistochemical assay-interpretation skills from digital image-based electronic training (e-training). In this study, e-training was used to train board-certified pathologists to evaluate non-small cell lung carcinoma for eligibility for treatment with onartuzumab, a MET-inhibiting agent. The training program mimicked the live training that was previously validated in clinical trials for onartuzumab. A digital interface was developed for pathologists to review high-resolution, static images of stained slides. Sixty-four pathologists practicing in the United States enrolled while blinded to the type of training. After training, both groups completed a mandatory final test using glass slides. The results indicated both training modalities to be effective. Overall, 80.6% of e-trainees and 72.7% of live trainees achieved passing scores (at least 85%) on the final test. All study participants reported that their training experience was "good" and that they had received sufficient information to determine the adequacy of case slide staining to score each case. This study established that an e-training program conducted under highly controlled conditions can provide pathologists with the skills necessary to interpret a complex assay and that these skills can be equivalent to those achieved with face-to-face training using conventional microscopy. Programs of this type are scalable for global distribution and offer pathologists the potential for readily accessible and robust training in new companion diagnostic assays linked to novel, targeted, adjuvant therapies for cancer patients.

The Cancer Genomics Resource List 2014.

CONTEXT: Genomic sequencing for cancer is offered by commercial for-profit laboratories, independent laboratory networks, and laboratories in academic medical centers and integrated health networks. The variability among the tests has created a complex, confusing environment. OBJECTIVE: To address the complexity, the Personalized Health Care (PHC) Committee of the College of American Pathologists proposed the development of a cancer genomics resource list (CGRL). The goal of this resource was to assist the laboratory pathology and clinical oncology communities. DESIGN: The PHC Committee established a working group in 2012 to address this goal. The group consisted of site-specific experts in cancer genetic sequencing. The group identified current next-generation sequencing (NGS)-based cancer tests and compiled them into a usable resource. The genes were annotated by the working group. The annotation process drew on published knowledge, including public databases and the medical literature. RESULTS: The compiled list includes NGS panels offered by 19 laboratories or vendors, accompanied by annotations. The list has 611 different genes for which NGS-based mutation testing is offered. Surprisingly, of these 611 genes, 0 genes were listed in every panel, 43 genes were listed in 4 panels, and 54 genes were listed in 3 panels. In addition, tests for 393 genes were offered by only 1 or 2 institutions. Table 1 provides an example of gene mutations offered for breast cancer genomic testing with the annotation as it appears in the CGRL 2014. CONCLUSIONS: The final product, referred to as the Cancer Genomics Resource List 2014, is available as supplemental digital content.

Assessing the discordance rate between local and central HER2 testing in women with locally determined HER2-negative breast cancer.

BACKGROUND: The importance of human epidermal growth factor receptor 2 (HER2) as a prognostic and predictive marker in invasive breast cancer is well established. Accurate assessment of HER2 status is essential to determine optimal treatment options. METHODS: Breast cancer tumor tissue samples from the VIRGO observational cohort tissue substudy that were locally HER2-negative were retested centrally with both US Food and Drug Administration (FDA)-approved immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays, using FDA-approved assay cutoffs; results were compared. RESULTS: Of the 552 unique patient samples centrally retested with local HER2-negative results recorded, tumor samples from 22 (4.0%) patients were determined to be HER2-positive (95% confidence interval [CI] = 2.5%-5.7%). Of these, 18 had been tested locally by only one testing methodology; 15 of 18 were HER2-positive after the central retesting, based on the testing methodology not performed locally. Compared with the 530 patients with centrally confirmed HER2-negative tumors, the 22 patients with centrally determined HER2-positive tumors were younger (median age 56.5 versus 60.0 years) and more likely to have ER/PR-negative tumors (27.3% versus 22.3%). These patients also had shorter median progression-free survival (6.4 months [95% CI = 3.8-15.9 months] versus 9.1 months [95% CI = 8.3-10.3 months]) and overall survival (25.9 months [95% CI = 13.8-not estimable] versus 27.9 months [95% CI = 25.0-32.9 months]). CONCLUSIONS: This study highlights the limitations of employing just one HER2 testing methodology in current clinical practice. It identifies a cohort of patients who did not receive potentially efficacious therapy because their tumor HER2-positivity was not determined by the test initially used. Because of inherent limitations in testing methodologies, it is inadvisable to rely on a single test to rule out potential benefit from HER2-targeted therapy.

Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. METHODS: The assay's limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. RESULTS: A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test performance was not compromised by endogenous substances, therapeutic drugs, necrosis up to 85%, and common micro-organisms. All of the assessed less common mutations except one (exon 19 deletion mutation 2236_2248 > AGAC) were detected at a similar DNA input level as that for the corresponding predominant mutation. CONCLUSION: The cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.

Mammostrat as an immunohistochemical multigene assay for prediction of early relapse risk in the tamoxifen versus exemestane adjuvant multicenter trial pathology study.

PURPOSE: Some postmenopausal patients with hormone-sensitive early breast cancer remain at high risk of relapse despite endocrine therapy and, in addition, might benefit from adjuvant chemotherapy. The challenge is to prospectively identify such patients. The Mammostrat test uses five immunohistochemical markers to stratify patients regarding recurrence risk and may inform treatment decisions. We tested the efficacy of this panel in the Tamoxifen versus Exemestane Adjuvant Multicenter (TEAM) trial. PATIENTS AND METHODS: Pathology blocks from 4,598 TEAM patients were collected, and tissue microarrays (TMAs) were constructed. The cohort was 47% node-positive, and 36% of patients in the cohort were treated with adjuvant chemotherapy. Triplicate 0.6-mm(2) TMA cores were stained, and positivity for p53, HTF9C, CEACAM5, NDRG1, and SLC7A5 was assessed. Cases were assigned a Mammostrat risk score, and distant relapse-free survival (DRFS) and disease-free survival (DFS) were analyzed. RESULTS: In multivariate regression analyses, which were corrected for conventional clinicopathologic markers, Mammostrat provided significant additional information on DRFS after endocrine therapy in estrogen receptor (ER) -positive node-negative patients (n = 1,226) who did not receive chemotherapy (P = .004). Additional analyses in all patients not exposed to chemotherapy, irrespective of nodal status (n = 2,559) and in the entire cohort (n = 3,837) showed Mammostrat scores provided additional information on DRFS in these groups (P = .001 and P < .001, respectively; multivariate analyses). No differences were seen between the two endocrine treatment regimens. CONCLUSION: The Mammostrat score predicts DRFS for patients treated with exemestane and patients treated with tamoxifen followed by exemestane irrespective of nodal status and chemotherapy. The ability of this test to provide additional outcome data after treatment provides additional evidence of its use in risk stratification of ER-positive postmenopausal patients with breast cancer.

Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma.

CONTEXT: A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF V600 Mutation Test) was recently approved by the US Food and Drug Administration to select patients with BRAF-mutant metastatic melanoma for treatment with the BRAF inhibitor vemurafenib. OBJECTIVES: (1) To compare the analytic performance of the cobas test to Sanger sequencing by using screening specimens from phase II and phase III trials of vemurafenib, and (2) to assess the reproducibility of the cobas test at different testing sites. DESIGN: Specimens from 477 patients were used to determine positive and negative percent agreements between the cobas test and Sanger sequencing for detecting V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel pyrosequencing method (454) to resolve discordances between polymerase chain reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 sites by using 3 reagent lots and an 8-member panel of melanoma samples. RESULTS: A valid cobas result was obtained for all eligible patients. Sanger sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 specimens, positive percent agreement was 96.4% (215 of 223) and negative percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 454. The cobas test detected 70% of V600K mutations. In the reproducibility study, a correct interpretation was made for 100% of wild-type specimens and specimens with greater than 5% mutant alleles; V600E mutations were detected in 90% of specimens with less than 5% mutant alleles. CONCLUSIONS: The cobas test (1) had a lower assay failure rate than that of Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most V600K mutations, and (4) was highly reproducible.

Sensitive multiplex detection of KRAS codons 12 and 13 mutations in paraffin-embedded tissue specimens.

BACKGROUND: Colorectal cancer patients harbouring KRAS mutations in codon 12 or 13 do not benefit from current anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. Efficient and robust methods are therefore required for routine clinical testing of KRAS mutation status. AIMS: To evaluate a novel multiplex assay for the rapid detection of common KRAS mutations in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: Genomic DNA was amplified by multiplex PCR using primers targeting the KRAS codon 12/13 region and an internal control gene. PCR products were hybridised on a liquid bead array containing target-specific probes and detected by particle flow cytometry. RESULTS: Analytical performance assessed with plasmid DNA and genomic DNA extracted from cell lines or model FFPE cell line dilutions showed specific detection of seven distinct KRAS mutations with a limit of detection equivalent to 1% tumour. The assay was evaluated at two independent sites with a total of 140 clinical specimens. At site 1, about 45% of the specimens from a set of 86 archived FFPE blocks with unknown KRAS mutation status were found positive for a KRAS mutation. At site 2, each of the seven mutations was detected in at least five independent specimens from a selected set of 54 residual genomic DNAs previously tested with an ARMS/Scorpion laboratory-developed test. CONCLUSIONS: This novel single-well assay is a sensitive tool compatible with the clinical laboratory workflow for the rapid assessment of KRAS mutations in solid tumour specimens. Its performance and multiplex format warrant the development of broader panels including other relevant mutations in the EGFR pathway.

Fixation time does not affect the expression of estrogen receptor.

The purpose of this pilot study was to determine the impact of the length of fixation in 10% buffered formalin on the expression of estrogen receptor by immunohistochemical analysis. We studied tissue samples from 10 invasive breast cancer cases after fixation for 1, 3, 6, and 9 to 10 hours. The tissue was processed immediately after fixation, resembling routine practice. Then the 40 blocks were incubated with antiestrogen receptors SP1, 6F11, and 1D5. The stained slides were reviewed and scored. We found no significant difference in the intensity of the stain or the percentage of cells stained regardless of the time in fixation or the antibody used. Fixation times between 1 and 9 hours in 10% formalin do not seem to have an impact on the expression of estrogen receptor by immunohistochemical analysis, at least in these high-expressing tumors.

The role of KRAS mutation testing in the management of patients with metastatic colorectal cancer.

CONTEXT: KRAS mutations can be detected in approximately 30% to 40% of all patients with colorectal cancer. Several recent studies have shown that patients with KRAS mutations in codons 12 or 13 in metastatic tumors do not benefit from anti-epidermal growth factor receptor therapy with cetuximab or panitumumab. OBJECTIVE: To review the literature on the role of KRAS mutation testing for management of patients with metastatic colorectal cancer and to discuss testing strategies. DATA SOURCES: This review is based on published, peer-reviewed literature; available information from medical organizations (eg, National Comprehensive Cancer Network, American Society of Clinical Oncology, College of American Pathologists); and information from clinical laboratories conducting KRAS mutation analysis. CONCLUSIONS: Multiple methods for detecting KRAS mutations in colorectal tumors are available, and all methods in current clinical use appear to have adequate clinical sensitivity for predicting a lack of response to cetuximab and panitumumab. Pathologist expertise is essential to quality KRAS testing and to determining effective treatment for patients with metastatic colorectal cancer.

Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines.

CONTEXT: Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. OBJECTIVES: To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. DESIGN: We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. RESULTS: Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. CONCLUSION: Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.

Enhanced accuracy and reliability of HER-2/neu immunohistochemical scoring using digital microscopy.

We evaluated the HER-2/neu status of 129 invasive breast cancer specimens for gene amplification by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemical analysis. Each immunohistochemically stained slide was interpreted on a standard microscope independently by 10 pathologists. Separately, each pathologist reviewed the same slide set with the assistance of digital microscopy. A total of 1,258 manual immunohistochemical scores and 1,269 digital microscopy immunohistochemical scores were completed. When the same 10 pathologists scored the same immunohistochemical slides with the assistance of digital microscopy, each reviewer improved concordance with FISH, and overall concordance with immunohistochemical analysis improved significantly, to 93% (P < .001). The interrater kappa was used to compare interobserver agreement in HER-2 immunohistochemical scoring for manual and digital microscopy interpretation. Significant improvement in interobserver agreement (kappa = 0.51 vs 0.86; P < .001) was achieved when HER-2 immunohistochemical analysis was scored with the assistance of the digital microscope. The assistance of digital microscopy improves the accuracy and reliability of HER-2 immunohistochemical analysis. These data suggest that documented discrepancies between HER-2 immunohistochemical analysis and FISH reflect predominantly errors in manual immunohistochemical interpretation as opposed to immunohistochemical reagent limitations.

Laser therapy of breast cancer with 3-year follow-up.

Breast cancer is the commonest female malignancy in the United States and is increasingly being detected at a non-palpable stage by annual mammography. Minimally invasive methods of its local treatment have been recently introduced as alternative to its surgical removal. The case reported in this article illustrates the advantages of in-situ ablation with minimal discomfort to the patient and deformity of the breast. More importantly, it demonstrates the absence of any adverse effect on health and survival of the patient during this intermediate period of follow-up.

HER-2/neu protein overexpression and gene amplification in human transitional cell carcinoma of the bladder.

OBJECTIVES: To assess HER-2/neu gene status by fluorescence in situ hybridization and protein expression by immunohistochemistry in human bladder transitional cell carcinoma (TCC). In breast carcinoma, HER-2/neu gene amplification and receptor protein overexpression are tightly correlated and have prognostic and therapeutic implications. METHODS: We used 54 randomly selected TCC specimens obtained from 1998 to 2000. Each specimen was fixated in 10% neutral-buffered formalin and embedded in paraffin. Of the 54 specimens, 7 were grade 1 (13%), 26 were grade 2 (48%), and 21 were grade 3 (39%); 36 (67%) were superficial (Stage Ta or T1) and 18 (33%) were invasive (Stage T2 or T3). The specimens were analyzed for HER-2/neu protein overexpression by immunohistochemistry and for gene amplification using fluorescence in situ hybridization. RESULTS: Of the 54 specimens, 14 (26%) were positive for protein overexpression. One (14%) of the 7 grade 1 tumors was positive for protein overexpression, 3 (12%) of 26 grade 2 tumors were positive, and 10 (48%) of 21 grade 3 tumors were positive (P = 0.0195). Six (17%) of 36 Stage Ta or T1 specimens and 8 (44%) of 18 Stage T2 or T3 specimens were positive for protein overexpression (P = 0.01). None of the 54 TCC specimens showed amplification of the HER-2/neu gene using fluorescence in situ hybridization. CONCLUSIONS: HER-2/neu protein overexpression is present in human bladder TCC, with a statistically significant increase in overexpression in grade 3, and invasive specimens. Gene amplification does not appear to be the mechanism of protein overexpression. The prognostic significance of these findings and the application of HER-2/neu in treatment needs additional investigation.

Targeted therapy in breast cancer: the HER-2/neu gene and protein.

The HER-2/neu oncogene, a member of the epidermal growth factor receptor or erb gene family, encodes a transmembrane tyrosine kinase receptor that has been linked to prognosis and response to therapy with the anti-HER-2-humanized monoclonal antibody, trastuzumab (Herceptin, Genentech, South San Francisco, CA) in patients with advanced metastatic breast cancer. HER-2/neu status has also been tested for its ability to predict the response of breast cancer to other therapies including hormonal therapies, topoisomerase inhibitors, and anthracyclines. This review includes an analysis of 80 published studies encompassing more than 25,000 patients designed to consider the relative advantages and disadvantages of the various methods of measuring HER-2/neu in clinical breast cancer specimens. Southern blotting, PCR amplification detection, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is reviewed along with the current studies designed to test whether HER-2/neu status can predict the response to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review will also evaluate the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.