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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    • "Whether mRNA PgR assessment is indeed more strongly associated with clinical outcome compared with IHC PgR assessment as suggested is a hypothesis to be tested when MINDACT outcome data will become available[15]. The positive agreement for HER-2 mRNA analysis in the current series is only 72 %, and in line with previously reported figures for mRNA analysis[16,17]. The agreement for ER and PgR between central pathology and TargetPrint results increases significantly using 10 % invasive tumor cells as an IHC cut-off instead of 1 %, reaching a concordance rate of 98 % (j = 0.91) for ER and of 90 % for PgR (j = 0.76). "
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    ABSTRACT: Accurate identification of breast cancer patients most likely to benefit from adjuvant systemic therapies is crucial. Better understanding of differences between methods can lead to an improved ER, PgR, and HER-2 assessment. The purpose of this preplanned translational research is to investigate the correlation of central IHC/FISH assessments with microarray mRNA readouts of ER, PgR, and HER-2 status in the MINDACT trial and to determine if any discordance could be attributed to intratumoral heterogeneity or the DCIS and normal tissue components in the specimens. MINDACT is an international, prospective, randomized, phase III trial investigating the clinical utility of MammaPrint in selecting patients with early breast cancer for adjuvant chemotherapy (n = 6694 patients). Gene-expression data were obtained by TargetPrint; IHC and/or FISH were assessed centrally (n = 5788; 86 %). Macroscopic and microscopic evaluation of centrally submitted FFPE blocks identified 1427 cases for which the very same sample was submitted for gene-expression analysis. TargetPrint ER had a positive agreement of 98 %, and a negative agreement of 95 % with central pathology. Corresponding figures for PgR were 85 and 94 % and for HER-2 72 and 99 %. Agreement of mRNA versus central protein was not different when the same or a different portion of the tumor tissue was analyzed or when DCIS and/or normal tissue was included in the sample subjected to mRNA assays. This is the first large analysis to assess the discordance rate between protein and mRNA analysis of breast cancer markers, and to look into intratumoral heterogeneity, DCIS, or normal tissue components as a potential cause of discordance. The observed difference between mRNA and protein assessment for PgR and HER-2 needs further research; the present analysis does not support intratumoral heterogeneity or the DCIS and normal tissue components being likely causes of the discordance.
    Full-text · Article · Jan 2016 · Breast Cancer Research and Treatment
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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.
    Full-text · Article · Jun 2014 · PLoS ONE
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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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    Full-text · Article · Jan 2014
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