High False- Negative Rate of HER2 Quantitative Reverse Transcription Polymerase Chain Reaction of the Oncotype DX Test: An Independent Quality Assurance Study

Magee-Womens Hospital of UPMC, 300 Halket St, Pittsburgh, PA, USA.
Journal of Clinical Oncology (Impact Factor: 18.43). 11/2011; 29(32):4279-85. DOI: 10.1200/JCO.2011.34.7963
Source: PubMed


HER2 (ERBB2) status is an important prognostic and predictive marker in breast carcinoma. In recent years, Genomic Health (GHI), purveyors of the Oncotype DX test, has been separately reporting HER2 by reverse transcription polymerase chain reaction (RT-PCR) to oncologists. Because of the lack of independent evaluation, this quality assurance study was undertaken to define the concordance rate between immunohistochemistry (IHC)/fluorescent in situ hybridization (FISH) and GHI RT-PCR HER2 assay.
All patients at three participating laboratories (Magee-Womens Hospital [Pittsburgh, PA], Cleveland Clinic [Cleveland, OH], and Riverside Methodist Hospital [Columbus, OH]) with available HER2 RT-PCR results from GHI were included in this study. All IHC-positive and equivocal patient cases were further evaluated and classified by FISH at respective laboratories.
Of the total 843 patient cases, 784 (93%) were classified as negative, 36 (4%) as positive, and 23 (3%) as equivocal at the three institutions using IHC/FISH. Of the 784 negative patient cases, 779 (99%) were also classified as negative by GHI RT-PCR assay. However, all 23 equivocal patient cases were reported as negative by GHI. Of the 36 positive cases, only 10 (28%; 95% CI, 14% to 45%) were reported as positive, 12 (33%) as equivocal, and 14 (39%) as negative.
There was an unacceptable false-negative rate for HER2 status with GHI HER2 assay in this independent study. This could create confusion in the decision-making process for targeted treatment and potentially lead to mismanagement of patients with breast cancer if only GHI HER2 information is used.

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    • "It has been suggested that HER2 status can be assessed with different approaches as a continuous variable and can be assessed on mRNA level [19]. HER2 gene amplification determined with FISH is strongly associated with elevated mRNA and protein levels and small studies have reported on the concordance of HER2 status by RT-PCR to that by IHC and FISH [20]–[22]; Two independent studies have reported on the concordance between HER2 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of the Oncotype Dx Test and the FDA-approved IHC/FISH assays and have yielded conflicting results [23], [24]. In the first study, a greater than 50% false-negative rate for Oncotype DX RT-PCR for HER2 assessment was reported. "
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    ABSTRACT: Background We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients. Methods A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan – Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates. Results HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson’s Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman’s Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31–0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22–0.70, p = 0.002). Conclusions The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.
    PLoS ONE 06/2014; 9(6):e99131. DOI:10.1371/journal.pone.0099131 · 3.23 Impact Factor
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    • "The 21 genes include five genes related to proliferation (KI-67, STK 15, SURVIVIN, CYCLIB B1, MYBL2), four genes related to invasiveness (STROMELYSIN 3, CATHEPSIN L2, HER2, GRB7), seven hormonal genes (ER, PR, BCL2, SCUBE2, GTSM1, CD68, BAG1) and five reference genes (β-ACTIN, GAPDH, RPLPO, GUS, TFRC). Unfortunately, the test based on real-time PCR (Oncotype DX) reports a higher percentage of false negatives with respect to the expression of HER2 [32]. "
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    ABSTRACT: Oncology indispensably leads us to personalized medicine, which allows an individual approach to be taken with each patient. Personalized oncology is based on pharmacogenomics and the effect of genetic differences in individuals (germline and somatic) on the way cancer patients respond to chemotherapeutics. Biomarkers detected using molecular biology tools allow the molecular characterization of cancer signatures and provide information relevant for personalized treatment. Biomarkers can be divided into two main subgroups: prognostic and predictive. The aim of the application of prognostic biomarkers, which provide information on the overall cancer outcome in patients, is to facilitate cancer diagnosis, usually with no need for putting invasive methods into use. Predictive biomarkers help to optimize therapy decisions, as they provide information on the likelihood of response to a given chemotherapeutic. Among the prognostic factors that identify patients with different outcome risks (e.g., recurrence of the disease), the following factors can be distinguished: somatic and germline mutations, changes in DNA methylation that lead to the enhancement or suppression of gene expression, the occurrence of elevated levels of microRNA (miRNA) capable of binding specific messenger RNA (mRNA) molecules, which affects gene expression, as well as the presence of circulating tumor cells (CTCs) in blood, which leads to a poor prognosis for the patient. Biomarkers for personalized oncology are used mainly in molecular diagnostics of chronic myeloid leukemia, colon, breast and lung cancer, and recently in melanoma. They are successfully used in the evaluation of the benefits that can be achieved through targeted therapy or in the evaluation of toxic effects of the chemotherapeutic used in the therapy.
    01/2014; 18(3). DOI:10.1007/s40291-013-0077-9
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    • "However, at least in part, the difficulties achieving a highly accurate test reflect chromosomal instability in breast cancer, and the difficulty in identifying a single region in the genome to act as a robust reference region. Analysis of HER2 mRNA over-expression has been reported to have high diagnostic accuracy [4], [8], although attempts to bring such HER2 RNA assessments to routine practice have met mixed results; analysis of HER2 status from the Oncotype DX® 21 gene recurrence score has been reported to have high diagnostic accuracy in some series [9], with discordance in other series [10]. "
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    ABSTRACT: Determination of the presence of HER2 amplification by quantitative PCR has been challenging, in part due to chromosomal instability and identification of a robust a reference region. We assessed the potential of digital PCR for highly accurate assessment of DNA concentration with EFTUD2 as chromosome 17 reference probe. We assessed a HER2:EFTDU2 ratio by digital PCR assay in the microdissected DNA from 18 HER2 amplified and 58 HER2 non-amplified cancers. The HER2:EFTUD2 ratio had high concordance with conventionally defined HER2 status with a sensitivity of 100% (18/18) and a specificity of 98% (57/58). The HER2:EFTUD2 digital PCR assay has potential to accurately assess HER2 amplification status.
    PLoS ONE 12/2013; 8(12):e83409. DOI:10.1371/journal.pone.0083409 · 3.23 Impact Factor
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