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.

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.

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.

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.

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.

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.

HER-2/neu testing in breast cancer.

The testing of newly diagnosed breast cancer specimens for HER-2/neu status has achieved "standard of practice" status for the management of breast cancer in the United States. The discussion as to the best method to determine HER-2/neu status in these samples continues, with the fluorescence in situ hybridization method gaining popularity owing to the recent evidence that it, in comparison with immunohistochemical analysis, might more accurately predict clinical responses to trastuzumab-based therapies. With trastuzumab achieving excellent results in the treatment of HER-2/neu-positive advanced disease and under extensive evaluation in major clinical trials for its potential efficacy when used at earlier clinical stages, the potential role(s) for HER-2/neu testing as a predictor of response to other therapies being resolved by large prospective clinical outcome studies, and the more convenient gene-based chromogenic in situ hybridization technique "waiting in the wings," the saga of HER-2/neu testing in breast cancer will continue to unfold over the next several years.

Assessment of Her-2/Neu status by immunohistochemistry and fluorescence in situ hybridization in mammary Paget disease and underlying carcinoma.

HER-2/Neu overexpression is seen in 20% to 30% of invasive breast carcinomas and has been reported in as many as 80% of high-grade infiltrating carcinomas. Earlier studies have suggested that 100% of the tumor cells in mammary Paget disease show overexpression of HER-2 protein. We undertook this study to assess HER-2 status of mammary Paget disease and of the underlying breast carcinoma, when present, by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Formalin-fixed, paraffin-embedded tissue from 20 cases of mammary Paget disease were analyzed for HER-2 status by IHC and FISH. IHC for estrogen receptor (ER) was also performed. The patients ranged in age from 34 to 88 years, with a mean age of 62 years. Eighty percent of the cases showed strong overexpression (3+) of HER-2 protein by IHC, and all of these cases showed more than 5-fold amplification of the HER-2 gene by FISH. The remaining 4 cases, which were negative for HER-2/Neu by IHC, showed no amplification by FISH. All of the latter cases expressed ER, whereas no case that overexpressed HER-2 expressed ER. Sixteen cases had an underlying tumor, which was in situ in 6 cases. The underlying tumors were identical to the Paget disease with respect to their HER-2/Neu overexpression by both IHC and FISH. HER-2 overexpression was identified in 80% of our cases of Paget disease. There was 100% concordance between HER-2 protein overexpression by immunohistochemistry and gene amplification in both the Paget and the underlying tumor. Moreover, all of the cases negative for HER-2 overexpression expressed ER, whereas those positive for HER-2 did not.

Laser therapy for small breast cancers.

BACKGROUND: Widespread mammography has resulted in the increased detection of breast cancer <1.5 cm. It may be possible to treat these small tumors with in-situ laser ablation. Prior to ablation tumor size is determined by ultrasound and mammogram. Histologic diagnosis and determination of prognostic factors are obtained from image-guided needle core samples. Invasive and in-situ tumors may be percutaneously ablated by a stereotactically guided laser needle and subsequently evaluated by imaging methods and needle biopsy. METHODS: Fifty-four patients (50 invasive, 4 in-situ); 51 mass, 3 microcalcification; mean diameter 12 (5 to 23) mm were treated by a stereotactically guided 805 nm laser beam via a fiber in a 16G needle delivered to the cancer. One to 8 weeks later the coagulated lesions were surgically removed for pathologic evaluation. In 2 additional patients, the laser-treated tumors were not removed but were monitored by mammography, ultrasonography, and needle core biopsy. RESULTS: None of the patients sustained any adverse effect. The average treatment time was 30 minutes. Pathology analysis revealed a 2.5 to 3.5 hemorrhagic ring surrounding the necrotic tumor. Under steady conditions, in two groups of 14 patients, 93% and 100% of the tumors showed complete destruction, with no residual cancer report. In the 2 unresected cases kept under surveillance for 6 to 24 months, the laser-treated tumors first showed shrinkage, followed by a 2 to 3 cm oil cyst. Fibrosis was demonstrated on needle core biopsies. CONCLUSIONS: Laser energy delivered through a stereotactically guided needle appears to ablate mammographically detected breast cancer. A multicenter clinical trail is planned.

Fine-needle aspiration of clinically suspicious palpable breast masses with histopathologic correlation.

BACKGROUND: The purpose of this study was to compare the diagnostic accuracy of fine-needle aspiration (FNA) of clinically suspicious palpable breast masses in women younger and older than 40 years of age. METHODS: All women who had FNA biopsy with subsequent tissue biopsy were included. The cytologic diagnoses were classified into three groups: malignant, suspicious, or benign. Histopathologic correlation was based on either a needle core biopsy, an excisional biopsy, or a mastectomy specimen. RESULTS: A total of 1,158 fine-needle aspirations performed between 1982 and 2000, on women being evaluated for a clinically palpable breast mass were included in the study. The patients were divided into two groups: group I consisted of 231 patients aged 40 years and younger, and group II consisted of 927 patients aged 41 years and older. In group I there were 117 (51%) malignant FNA diagnoses, and only 1 (1%) false-positive case, subsequently diagnosed on histopathologic material as an atypical papillomatosis. There were 20 (9%) cases diagnosed as suspicious on FNA. On histopathology 10 were malignant, and 10 were benign. Of the 91 (39%) cases interpreted as benign, only 1 (1%) was a false negative. In group II, which comprised 927 patients, there were 693 (74%) malignant FNA diagnoses, and 3 (less than 1%) false-positive cases, which on follow-up histopathologic examination revealed 2 atypical ductal hyperplasias and 1 atypical papilloma. There were 90 (10%) cases diagnosed as suspicious on FNA. On histopathology, 68 were malignant and 22 were benign. Of the 131 (14%) lesions interpreted as benign, there were 18 false-negative cases (14%), which included 17 infiltrating carcinomas and 1 ductal carcinoma in-situ. Twelve (1%) of the cases were inadequate for the study. CONCLUSIONS: The sensitivity, specificity, and positive predictive values were remarkably high and comparable in both groups: group I had 99% sensitivity, 99% positive predictive value, 99% specificity, and 99% negative predictive value; and group II had 98% sensitivity, 97% specificity, 99% positive predictive value, and 86% negative predictive value. The overall rate of false-positive (less than 1%) and false-negative cases (9%) is comparable with published literature. Suspicious cases should be further evaluated, as our study revealed more than 50% to be malignant. The incidence of malignancy in patients presenting with a clinically palpable breast mass with follow-up biopsy was 51% in patients aged 40 years and younger and 74% in patients aged 41 years and older. Fine-needle aspiration is an excellent diagnostic tool in assessing clinically palpable breast masses.

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.

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.

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.