Hormonal and targeted human epidermal growth factor 2 (HER2) therapies are breakthroughs in the treatment of breast cancer.1–3 The status of biomarkers including estrogen receptor (ER), progesterone receptor (PR), and HER2 not only guides treatment options, but also provides prognostic information. Furthermore, expression levels of these biomarkers have also been indicated as surrogates for defining the molecular subtypes of breast cancer.4,5 The clinical importance of these biomarkers in the management of breast cancer has made their assessment mandatory for every newly diagnosed and recurrent tumor by American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP), and therefore, accurate assessment of ER/PR/HER2 is critical to ensure appropriate patient management.
Currently, the evaluation of the breast biomarkers is largely performed by immunohistochemical (IHC) analysis. IHC has replaced the radio-ligand binding assay in evaluating hormonal receptors in 1990s because it is radioactivity-free, less labor intensive, easier to interpret, applicable to small samples, and equivalent to superior in predicting response to hormonal therapy.6,7 After the adoption of automated staining platforms, the development of more sensitive antibody clones, and the usage of Food and Drug Administration (FDA)-cleared standard test kits, the accuracy and reproducibility of the biomarker assessment has improved significantly.6 However, due to some preanalytic, analytic, and postanalytic factors, the results of these biomarkers may still be affected. To assure the possible factors are well-controlled, a panel of experts published the “Recommendations for validating estrogen and progesterone receptor immunohistochemistry assays” in 2010.6 The ongoing assay assessment procedure was included in this recommendation, which required all the laboratories to monitor the overall positive and negative ER rates at least semiannually. In addition, it has been frequently reported that testing inaccuracy is identified as a major issue in the interpretation of IHC results of HER2.8–11 This led to monitoring of HER2 positivity rates via recommendations from both the ASCO/CAP and the UK-updated guidelines.12,13 In this study, we report our 10-year longitudinal experience on the positivity rates of biomarkers, resulting from the ongoing assay assessment procedures in a large centralized laboratory specialized in breast pathology. In addition, the results of participation in external quality-assurances programs with the CAP and NordiQC were also included in this study.
MATERIALS AND METHODS
Antibody clones for ER/PR/HER2 were changed from 6F11/1A6/CB11 to SP1/1E2/4B5 at our institution in April 2008. Therefore, cases with ER/PR/HER2 results from April 1, 2008 to December 31, 2017 were included in the current study. The cases included: (1) all cases with new diagnosis of invasive breast carcinoma, recurrent or metastatic breast cancer on core biopsies; and (2) all cases with repeat tests on the surgical resection specimen, including cases that were limited on biopsy, the presence of a different (high-grade component) in the excision specimen that was not sampled on core biopsy and any unexpected results on core biopsy material.
For all of the cases included in this study, the cold ischemic time for tissue fixation was <1 hour. The tissue specimens were fixed in 10% neutral buffered formalin between 6 and 72 hours and embedded in paraffin. Sections were cut at 4 µm and IHC staining was performed with the BenchMark autostainer (Ventana-Roche, Tucson, AZ), using the antibody clones SP1 for ER, 1E2 for PR, and 4B5 for HER2. All the procedures were performed in the IHC laboratory of Magee-Womens hospital of the University of Pittsburgh Medical Center and the results were interpreted by pathologists who specialize in breast pathology.
Tumors were defined as hormone receptor positive if ≥1% of tumor cell nuclei were stained or negative if <1% tumor cell nuclei were stained. HER2 by IHC was interpreted according to 2007 ASCO/CAP guidelines14 until October 2013, and thereafter using 2013 guidelines update.12 HER2 IHC 2+ cases were reflex tested by fluorescence in situ hybridization (FISH), using either the Vysis dual probe HER2 system (until February 2016) or the Dako IQ dual probe system (from February 2016 onwards). The FISH reporting system followed the ASCO/CAP guidelines. Annual HER2 IHC/FISH percent positive agreement (as defined by FDA) was performed yearly according to the ASCO/CAP guidelines.
The results of ER/PR/HER2 metrics were continuously monitored quarterly from April 1, 2008 to December 31, 2017 and the recorded data were reviewed by the medical director. The laboratory subscribed to the CAP hormone receptor and HER2 program during this time, along with the NordiQC program.
The laboratory received acceptable results from both CAP and NordiQC for both hormone receptors and HER2 during this monitoring period.
In the period between 2008 and 2017, a total of 9564 cases of invasive breast carcinomas with accompanying hormone receptors studies were identified (Table 1). Of these, 7976 cases (83.4%) were positive for ER and the ER-positive rates varied yearly between 81.4% and 86.8% with a SD of 1.8% during this period of time. Among them, ER+/PR+ was 71.0% (68.6% to 74.5%) and ER+/PR− was 12.3% (11.0% to 14.2%). There were 1588 cases (16.6%) with negative ER results. The rates of ER−/PR− and ER−/PR+ were 16.1% (13.5% to 17.7%), and 0.6% (0.2% to 1.0%), respectively.
A total of 8314 cases were submitted for HER2 IHC evaluation and the overall positivity rate was 13.7% with the lowest being in 2014 (10.2%) and highest in 2009 (17.9%) and a SD of 3.0%. Of these, 9.9% (n=823) were positive based on IHC 3+ scores and 3.8% (n=315) were positive based on FISH with original HER2 2+ results. The HER2 IHC–positive rates varied between 7.3% and 11.9% and FISH-positive rates were between 1.9% and 5.9%, respectively. The HER2 positivity rate by IHC and FISH between 2008 and 2017 is listed in Table 2. The overall frequency of HER2 negative, equivocal, and positive cases by IHC were 55.3%, 34.8%, and 9.9%, respectively, in the study period (Table 3). For all of the cases with HER2 2+ by IHC, average FISH amplification rate was 11.1% and it varied between 5.8% and 19.2% (Table 4). Annual quality-assurance check for HER2 IHC/FISH percent positive and percent negative agreement (as defined by FDA) was 96% to 100%.
The importance of determining the biomarker status of breast carcinoma for therapeutic and prognostic implications has been emphasized extensively. With the widespread use of the IHC method to assess these biomarkers, it is very important to monitor the performance of IHC to assure accurate results. Although a number of studies on the quality assessment of the biomarker results have been reported,11,15–17 these data largely represent multi-institutional surveys, which mostly reflect interlaboratory variation. In addition, the false negative rates for ER have been reported to be 15% to 40% even in the laboratories within United States, Europe, and Canada.18,19 The Cameron Commission, in particular, which investigated a 40% false negative ER rate over a 7-year time period, was a sobering reminder of the importance of continuous quality monitoring.20,21 In this study, we report 10-year longitudinal experience on monitoring the biomarker metrics of breast carcinoma in our laboratory, a large centralized institution specializing in breast pathology, which serves a patient population of 3.4 million with a median breast cancer age of 63 years.
The premise of this biomarker practice was to be able to monitor metrics to define any large drift in metrics early, to avoid adverse patient impact.11,20,21 One of the authors (D.J.D.) advised the Cameron Commission for adopting such a practice in Canada. This notion was also highlighted in the recommendations by ASCO/CAP as the ongoing assay assessment procedures.6,12,13 The findings in this study show the high fidelity of breast biomarkers results performed in this 10-year period when adhered to the ASCO/CAP guidelines with in vitro diagnostic class antibodies. During the 10-year period, no interventions were necessary to correct the course of the biomarkers.
In the current study, the overall ER and PR-positive rates of this large-scale assessment of 9564 cases are 83.4% and 71.6%, respectively, and the positive rates are consistent with the 2010 SEER result, which reported the overall HR+ rate of ∼83.0% in a large portion (∼28%) of the US population.22 However, our data shows slightly higher hormone receptor positivity rate than the available data reported before year 2008. A SEER study23 on 155,175 cases reported the overall ER positivity of 76.0% and PR positivity of 66.0% and another SEER study24 investigated the incidence of hormonal receptors on invasive breast cancer from 1992 to 1998 and found the overall ER-positive breast cancer was 75.4% to 77.5% and PR-positive cases were 65.0% to 67.7%. An Australian study17 reported that 74.9% cases were ER-positive and 61.4% cases were PR-positive after 8128 cases were evaluated. The nominal increase in the hormone receptors in our study likely reflects the older patient population with breast cancer in the geographic area of service in Western Pennsylvania. The use of the antibody clones may have a minor impact on our results as well. Cheang et al25 compared ER clone SP1, with ER 1D5 and showed SP1was 8% more sensitive than 1D5. Furthermore, a previous study from our institution reviewed 464 breast cancer cases evaluated by both IHC and oncotype DX assay for estrogen and PR (from the year 2008 and 2009) and found good correlation between IHC and Real-Time Polymerase Chain Reaction results. The concordance rates were 98.9% for ER and 94.2% for PR.26
Older published studies of HER2 testing showed substantial variability in HER2 positivity assessment, ranging 9% to 74% with an average of 22.2%,2 mainly due to the testing quality issue among local pathology laboratories and central testing centers.8–10,27 Paik et al8 found that 18% of the community-based assays, which were used to establish the eligibility of patients to participate in the B-31 study, could not be confirmed by HercepTest IHC or FISH by a central testing facility. The poor concordance between local and central testing for HER2 status was confirmed by additional studies.9,10,27 These earlier findings highlight the importance of participating in active quality-assurance programs, to improve the process of selecting patients likely to benefit from HER2-targeted therapy. The current investigation in a centralized, subspecialized laboratory reveals the overall HER2 positivity of 13.7%, and this is consistent with the ranges of recently published data with ranges of 13% to 20%. The studies reported recently showed the HER2 positivity rates have trended below 20% with most investigators currently reporting that the true positive rate is in the range of 13% to 20%.11,22,28 Interestingly, although the guideline updated in 2013 regarding the criteria defining HER2-positive status changed, the HER2 positivity rates by IHC or by FISH, as well as the FISH amplification rates were relatively stable in this study period (2008 to 2017, Tables 2–4). In our practice, the FISH test was performed as a reflex test on the IHC equivocal (2+) cases. No further testing was performed on IHC 0, 1+, and 3+ cases. To avoid the undertreatment or overtreatment of HER2-targeted therapy, we follow the ASCO/CAP guidelines, but generally use a low threshold for 2+ cases and high threshold for 3+ cases when interpreting the HER2 IHC results. This practice also contributes the slight higher percentage of HER2 2+ case (average, 34.8%) in the current study, as compared with the previous reported range of 12.5% to 23.2%.11,16,17,28,29 In addition, our observation also found approximately one third of HER2-positive cases, the hormonal receptors were negative (data not shown).
In summary, this report is the largest assessment from a single institution to report the experience of the ongoing assay assessment procedure on breast biomarkers. Our investigation of 9564 cases shows a high fidelity of breast biomarkers results, and a desired outcome when following the ASCO/CAP guidelines with in vitro diagnostic class antibodies. In addition, the participation in the ongoing assay assessment procedures as recommended by the expert panel can provide assurance of IHC biomarker reliability in the individual laboratory.
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