Clinical effectiveness of droplet digital polymerase chain reaction technology for human epidermal growth factor receptor 2/neu gene testing in breast cancer compared to conventional methods

A Decade of Human Epidermal Growth Factor Receptor 2 (HER2) Testing in Breast Cancer in Spain: A Comprehensive Real-World Data Analysis (2013-2023).

Introduction: A focused update of American Society of Clinical Oncology (ASCO) and College of American Pathologist's (CAP) guideline recommendations for HER2 testing in breast cancer (BrCa) was published in May, 2017 for public comment (http://www.cap.org/ShowProperty?nodePath=/UCMCon/Contribution Folders/WebContent/pdf/her2-breast-summary-draft-recommendations-2017.pdf). The focused update for in situ hybridization (ISH) addresses following uncommon scenarios: 1) HER2/CEP17 ratio ≥ 2.0, and HER2 /cell <4.0; 2) HER2/CEP17 ratio <2.0 and HER2 /cell ≥6.0; 3) HER2/CEP17 <2.0 and HER2 /cell ≥4.0 and <6.0. The first two groups are currently reported as positive, the third as equivocal. It is recommended that immunohistochemistry (IHC) performed by the same lab for all these groups. If the IHC testing is 3+ or 0/1+ the final diagnosis will be reported as positive or negative respectively. If the reflex IHC is 2+, a recount of the original ISH area of carcinoma with IHC 2+ staining is recommended. The purpose of this study is to review the results of targeted FISH testing following IHC to predict the effects of proposed guidelines in a high volume national reference lab. Materials and methods: HER2 FISH tests performed on BrCa between 4/2015-5/2017 are included. Our lab offers HER2 testing by IHC (HercepTest™ Dako) and dual probe FISH (Dako IQ). Equivocal (2+) cases showing ≥10% weak or moderate circumferential membrane staining or intense but <10% circumferential staining are circled by a pathologist and reflexed to HER2 FISH with preferential counting performed in the circled areas. Equivocal FISH cases are re-tested with the alternate RAI1 probe (Agilent Technologies). FISH scoring is done manually by 2 people following 2013 ASCO/CAP guidelines with at least 20 cells counted in amplified and non-amplified cases and 40 cells counted in equivocal cases. Results: 2460 HER2 FISH test requests were received during the study period. 7 cases failed at initial testing and 13 cases failed at repeat testing of equivocal cases with reflex probe. 389 (16.2%) cases were amplified, 1686 (68.7%) non amplified, and 369 (15.0%) were equivocal with FDA approved probe set. 116 (32.6%) of equivocal cases re-tested with alternate probe were amplified increasing overall amplification rate to 21%. The table below shows cases that fall under 3 uncommon categories that are proposed for changes in draft guidelines. HER2/CEP17 RatioAverage HER2/CellNumber of Cases with IHC (Total Number)IHC n(result)Results per 2013 ASCO/CAP before Reflex ProbeResults per 2013 ASCO/CAP after Reflex ProbeProposed Guidelines≥ 2< 48 (28)7 (2+)POSITIVE-NEGATIVE 1 (1+) < 2≥ 69 (31)9 (2+)POSITIVE-NEGATIVE < 2≥ 4-698 (372)96 (2+)EQUIVOCAL32/98 (32.7%) POSITIVE1/98 (1%) POSITIVE 1 (3+) 1 (0) Conclusions: In our lab where all FISH tests with prior IHC are counted on targeted areas, all of the cases within the first two groups will be reclassified as negative. The use of reflex probe in equivocal cases is not recommended in the draft recommendations further decreasing positivity rates. Approximately 5% decrease is expected in HER2 FISH positivity rates following proposed recommendations in reference laboratory setting. Citation Format: Johnson E, Gulbahce E. Draft recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer will decrease HER2 positivity rates [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-06.

Introduction: Human epidermal growth factor receptor 2 (HER2) amplification is commonly detected in breast cancer tissue samples by immunohistochemistry (IHC) and/or fluorescent in situ hybridization (FISH) tests in clinical practice. It has been reported that cell-free DNA (cfDNA) may better capture the heterogeneity of acquired resistance than tumor biopsy. This study aims to develop a noninvasive digital polymerase chain reaction (PCR) assay for the detection of HER2 amplification in the plasma cfDNA of breast cancer patients. We further examine the concordance rate of HER2 amplification detected by blood-based digital PCR with tissue-based IHC/FISH tests. Materials and Methods: Plasma samples from 32 breast cancer patients were prospectively collected at Queen Elizabeth Hospital (Hong Kong SAR, China). According to previous IHC/FISH records, 15 patients were scored as HER2 amplified (IHC score 3 and/or FISH positive) and 17 patients as HER2 non-amplified. The detection of plasma cfDNA was performed by droplet digital PCR (ddPCR). The EFTUD2 gene was used as a reference and HER2:EFTUD2 ratio was assessed by ddPCR on the plasma DNA from cancer samples. Results: A median of 1.47 (range 0.92-3.83) was detected in the HER2 amplified patients and a median of 1.03 (range 0.76-1.23) was detected in the HER2 non-amplified patients by ddPCR. Our results showed that using 1.30 as the cutoff, ddPCR assay could well detect HER2 amplification. Receiver operating characteristic analysis was used to evaluate the diagnostic ability of this ddPCR assay and it returned an area under the curve of 0.898. A diagnostic test was used to evaluate the concordance of this ddPCR assay with the IHC/FISH tests and determine the sensitivity (73.33%), specificity (100%), accuracy (87.5%), positive predictive value (100%), and negative predictive value (81%) of the ddPCR assay. Conclusion: Accurate reporting of HER2 amplification status is a prerequisite for the appropriate choice of targeted therapy. We have obtained a high level of concordance in comparison to tissue-based IHC/FISH when cfDNA ddPCR assay was used to determine HER2 amplification in breast cancer patients. Most importantly, we have established a great accuracy of the ddPCR assay. Increasing studies have reported that HER2 levels may change during targeted therapy, but there are additional risks of patients by the invasive nature of IHC/FISH tests. Our results support the potential application of blood-based ddPCR assay to monitor the changes of HER2 amplification status in breast cancer patients during targeted therapy. Citation Format: William C. Cho, Eunice Y. Lau, Jeffrey C. Chan, Anna Y. Tai, Alex K. Leung, Anthony K. Leung, Michelle O. Szeto, Elizabeth Y. Chuk, Tony Y. Yuen, Molly W. Fung, Roger K. Ngan. Noninvasive detection of HER2 amplification in breast cancer with plasma DNA digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5155.

Objective: To investigate the correlation of immunohistochemistry (IHC) and in situ hybridization (ISH) for the assessment of human epidermal growth factor receptor 2 (HER-2) in breast cancer in Buddhasothorn Hospital. Materials and Methods: The present study design was a retrospective study. Breast cancer patients aged 18 years or older, diagnosed with stage I to IV according to the American Joint Committee on Cancer (AJCC) Eighth Edition between January 1, 2018 and September 30, 2023, in Buddhasothorn Hospital and had IHC and ISH for the assessment of HER-2 according to the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) 2018 were included. There were 131 breast cancer patients. Results: The present study included 131 breast cancer patients with average age of 54 years, tumor size greater than 20 mm but not exceeding 50 mm (T2) at 45.4% (59/131), and 1 to 3 lymph nodes involvement (N1) at 34.6% (45/131). Early breast cancer was presented in 83.2% (109/131) of the cases, with estrogen receptor positivity at 42% (55/131). HER-2 testing by IHC showed equivocal (2+) in 52% (68/131) and positive (3+) in 48% (63/131). The Ki-67 proliferation index was 34±21.6%. The result of HER-2 testing by IHC positive (3+) highly correlates with ISH at 96.83% (61/63), which was statistically significant (Spearman’s rho=0.54, p<0.001). The result of HER-2 testing by IHC positive (2+) correlated with ISH at 48.53% (33/68). These data demonstrated a correlation of HER-2 testing by IHC positive (3+) and ISH. Conclusion: The HER-2 test using the IHC method at Buddhasothorn Hospital in the IHC positive (3+) group had a statistically significantly high correlation with the ISH test. Therefore, there is no need for ISH testing. In the IHC equivocal (2+) group, the correlation with ISH results did not differ from other studies. This needs to be verified using ISH according to the standard.

Background: In 2013, the ASCO/CAP consensus panel published updated guidelines for HER2 testing in breast cancer that modified the definition of HER2 amplification by in situ hybridization (ISH), creating five new prognostic categories (group 1: classic amplified, group 2: monosomy, group 3: co-amplified (polysomy), group 4: equivocal, and group 5: classic non-amplified). Patients determined to be ISH amplified, were considered eligible for HER2-directed therapy. Concern over whether patients from non-classic groups 2-4 would benefit from treatment has led to the recent publication of the 2018 HER2 focused update. This update has modified the criteria for interpreting these ISH categories, recommending that the final diagnosis take into consideration a combination of HER2 immunohistochemistry (IHC) and ISH results. With increased emphasis on the HER2 protein assessment, it has prompted us to quantitatively examine HER2 protein expression in the ISH categories, using two different novel technologies. Materials & Methods: A cohort of 170 cases (URMC) and 102 cases (PSHMC) of invasive breast cancers, which had previously undergone HER2 IHC and ISH testing, were selected for this study. Cases were sorted and categorized into the HER2 ISH categories defined by ASCO/CAP. HER2 protein expression was quantitatively measured in the URMC and PSHMC cohorts using a novel immunodetection methodology (streptavidin-coated Phosphor-Integrated Dot (PID) fluorescent nanoparticles), and a novel dual-antibody, proximity-binding immunoassay (HERmark® Breast Cancer Assay, Monogram Biosciences, South San Francisco, California), respectively. HER2 protein expression was compared to the HER2 FISH and IHC results by ASCO/CAP category. Results: Cases in group 1 had a significantly (p < 0.01) higher average PID/cell and HERmark compared to cases in groups 2-5 (Table 1). Cases in groups 2-4 showed lower quantitative levels of HER2 protein expression, similar to the classic non-amplified cases (group 5). Group 1 was further divided into three subgroups (Table 2): Group A - ISH high-level amplified (ratio > 2, HER2 > 6, CEP17 < 2.7), Group B - amplified with elevated CEP17 (ratio > 2, CEP17 > 2.7), and Group C - low-level amplified (ratio > 2, HER2 > 4 and < 6). Group A and B had a significantly (p < 0.01) higher average PID/cell and HERmark compared to Group C. Group C was more comparable to cases in groups 2-5 (Table 1). Conclusion: Our results suggest that quantitative assessment of HER2 protein expression may help to further classify cases for HER2 status for targeted therapy, supporting the 2018 ASCO/CAP recommendation that non-classic ISH results might be resolved by evaluating protein expression. Follow up studies with a larger patient cohort and dual quantitative assessment are warranted. Average PID/cell and HERmark in ASCO category groupsASCO category groupN (URMC)PID/cell (URMC)*N (PSHMC)HERmark (PSHMC)*18888.07761.521011.20N/A32016.0213.84238.5315.95296.3208.3*averageTable 2:Average PID/cell and HERmark in subgroups of Group 1SubgroupN (URMC)PID/cell (URMC)*N (PSHMC)HERmark (PSHMC)*A24157.66465.7B34101.61044.1C3016.9329.8*average Citation Format: Buscaglia B, Turner B, Goda H, Huang W, Leitzel K, Natori T, Nakano Y, Okada H, Sperinde J, Ali S, Vasekar M, D'Aguiar M, McMahon L, Henry J, Lipton A, Hicks D. ASCO/CAP human epidermal growth factor receptor-2 (HER2) in situ hybridization (ISH) categories evaluated by quantitative HER2 protein diagnostic methodologies: A comparative analysis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-03-02.

IntroductionOverexpression of the human epidermal growth factor receptor 2 (HER2) as a result of HER2 gene amplification is associated with a relatively poor prognosis in breast cancer and is predictive of HER2-targeting therapy response. False-positive rates of up to 20% for HER2 testing have been described. HER2-testing laboratories are therefore encouraged to participate in external quality control schemes in order to improve HER2-testing standardization.MethodsThis study investigated the feasibility of retesting large numbers of invasive breast cancers for HER2 status on tissue micro-array (TMA) as part of a quality control scheme. For this assessment different HER2 testing methods were used including HER2 detecting antibodies SP3, 4B5, Herceptest and mono color silver in situ hybridization (SISH) and dual color SISH. Final HER2 status for each tumor on the TMA was compared to the local testing result for the same tumor. Discordances between these two results were investigated further by staining whole tumor sections.ResultsFor this study, 1,210 invasive breast carcinomas of patients treated in six hospitals between 2006 and 2008 were evaluated. Results from the three immunohistochemistry (IHC) and two in situ hybridization (ISH) assays performed on the TMAs were compared. The final HER2 status on TMA was determined with SP3, 4B5 and mono color SISH. Concordance between local HER2 test results and TMA retesting was 98.0%. Discordant results between local and TMA retesting were found in 20 tumors (2.0%). False positive HER2 IHC results were identified in 13 (1.3%) tumors; false negative IHC results in seven (0.7%) tumors.ConclusionsRetesting large volumes of HER2 classified breast carcinomas was found to be feasible and can be reliably performed by staining TMAs with SP3, 4B5 and mono color SISH in combination with full-sized slides for discordant cases. The frequency of false-positive results was lower than previously reported in the literature. This method is now offered to other HER2-testing laboratories.

Background: In the 2013 ASCO/CAP HER2 update, new recommendations for HER2 diagnostic criteria in breast cancer organized in situ hybridization (ISH) results into five categories; group1 (amplified), group 2 (monosomy), group 3 (co-amplified), group 4 (equivocal) and group 5 (non-amplified). Patients falling into groups 2, 3 and 4 were potentially eligible for HER2 targeted therapy, however, there is uncertainty from limited prospective clinical trials that show patients in these uncommon groups would receive the same benefit as group 1. Concern over whether the interpretation criteria should be modified for these uncommon groups led to the recent publication of the 2018 HER2 focused update. This update has modified ISH criteria for groups 2, 3 and 4, recommending the final diagnosis take into consideration both immunohistochemistry (IHC) and ISH results. The publication of this new guideline has prompted us to investigate what impact this would have on our institution. Materials & Methods: A retrospective review of the URMC pathology database revealed 2,281 cases that had undergone HER2 FISH analysis since the 2013 update. IHC for initial HER2 screening was used, followed by reflex testing of all 2+ results and cases with histopathologic discordance. All 2,281 FISH cases were sorted into their 5 HER2 categories based on the 2013 guidelines. The final HER2 diagnosis for groups 2, 3 and 4 were then re-determined after applying the new criteria presented in the 2018 focused update. Results: The results from the 2,281 HER2 FISH cases are shown in Tables 1 and 2. The results for group 1 and group 5 cases remained the same. However, for ISH cases in groups 2, 3 and 4, there were alterations in the final HER2 results. All 25 monosomy cases, originally interpreted as HER2 positive by the 2013 guidelines, were now considered HER2 negative. All group 3 (co-amplified) cases remained positive (due to IHC 2+ results). The largest change was that the original 199 equivocal cases, based on the 2013 guidelines, became split into 198 HER2 negative (99.5%) and 1 HER2 positive (0.5%). Conclusion: Comparison of the HER2 FISH cases between 2013 and 2018 revealed differences in the final HER2 status. Using the 2018 guideline, 13.72% (313 cases) of the 2281 cases were HER2 positive in contrast to 14.77% (337 cases) from 2013. While that is only a difference of 1.05%, the reclassification of 198 of 199 equivocal cases to negative indicates that 9.82% of the final HER2 results for the entire patient cohort was affected. With 266,000 new cases of breast cancer annually, the final HER2 status of approximately 26,000 patients and their potential eligibility for targeted-therapy would change. Further study of the clinical significance of these changes is warranted. Final ISH HER2 Status (2013)AmplifiedMonosomyCo-AmplifiedEquivocalNon-Amplified272 cases (11.93%)25 cases (1.10%)40 cases (1.75%)199 cases (8.72%)1,745 cases (76.50%) Citation Format: Hicks DG, D'Aguiar M, Henry J, McMahon L, Buscaglia B, Turner B. Impact of the 2018 ASCO/CAP HER2 focused update on human epidermal growth factor receptor-2 (HER2) testing in breast cancer: A retrospective review of a single institutional cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-02-03.

A case report of metastatic breast cancer successfully treated with lapatinib plus capecitabine therapy

Background: HER2-positive breast cancer is characterized by overexpression of HER2 protein and/or amplification of the HER2 gene, assessed by immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. According to the 2013 ASCO/CAP guidelines on HER2 testing in breast cancer, both protein and gene status can be classified into three categories: positive, equivocal and negative. Gene-protein assay (GPA) is a newly developed technique, in which IHC and dual color ISH (DISH) are simultaneously performed on a single slide. HER2 GPA slides allow bright-field analyses of both protein and gene expression status of each cell. Breast cancer cells can be classified into nine types based on the combination of protein and gene expression with GPA. Table 1. Three by three classification of cancer cells by the HER2 GPA analysis HER2 protein 3+ (positive)2+ (equivocal)0, 1+ (negative)HER2 gene copy number≥6 (positive)ABC 4, 5 (equivocal)DEF <4 (negative)GHI Purpose: We aimed to elucidate the relationship between the results of HER2 GPA analysis and the therapeutic effects of trastuzumab-based treatment. Methods: Fifty three (53) patients with HER2-positive breast cancer, who underwent neoadjuvant trastuzumab with chemotherapy, were analyzed for HER2 status and clinical outcome. First, HER2 protein and gene status in a pre-therapeutic biopsy material from each patient were separately assessed according to the ASCO/CAP guidelines using HER2 GPA slides. Second, all cancer cells in five representative microscopic images of GPA slides were assessed to determine protein expression and to count the gene copy number at individual cell levels. Finally, we investigated the relationship between the mixture composition of the nine cell types and pathological complete response (pCR) of trastuzumab-treated breast cancer. Results: The GPA results were concordant with the results of IHC in 98% of samples, and with DISH results in 100%. Two hundred eighty nine (289) cancer cells per patient were assessed on average, ranging from 137 to 490 cells. The mean proportion of type A cancer cells was 64%, ranging from 0% to 98%. Patients who had 50% or more of type A cancer cells achieved pCR in 72% of cases (28/39), while patients with less than 50% of type A cancer cells achieved pCR in 7% of cases (1/14) (P<0.0001). The mean proportion of type C cancer cells was 7%, ranging from 0% to 60%. Patients who had 10% or more of type C cancer cells achieved pCR in 10% of cases (1/10), while patients with less than 10% of type C cancer cells achieved pCR in 65% of cases (28/43) (P=0.0016). Conclusion: HER2 GPA analyses can more precisely predict therapeutic effect of trastuzumab-based treatment in primary HER2-positive breast cancer. Our study suggests that the HER2 GPA approach would further improve precision therapy in HER2-positive breast cancer. Citation Format: Horii R, Nitta H, Ito Y, Iwase T, Ohno S, Akiyama F. Simultaneous analyses of HER2 gene and protein status can more precisely predict pathological complete response (pCR) to neoadjuvant trastuzumab with chemotherapy in primary HER2-positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-08.

The human epidermal growth factor receptor 2 (HER2) is an important prognostic and predictive biomarker in the breast cancer. The American Society of Clinical Oncology/College of American Pathology (ASCO/CAP) has published HER2 testing guidelines in breast cancer. We herein reviewed the HER2 testing guidelines in breast cancer with a focus on the application of the current guidelines. The continual investigation of HER2 testing in breast cancer has resulted in updates in the HER2 testing guidelines. The current guidelines focus on the uncommon clinical scenarios and emphasize the coordination between immunohistochemistry and in situ hybridization results, in an effort to improve clarity and accuracy. The ASCO/CAP guidelines provide valuable recommendations to ensure the accurate evaluation of HER2 status in breast cancer patients through standardization. Additional studies, particularly those with long-term outcome data are still needed to validate the guideline recommendations, especially the uncommon cases.

To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to > 10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing. This guideline was developed through a collaboration between the American Society of Clinical Oncology and the College of American Pathologists and has been published jointly by invitation and consent in both Journal of Clinical Oncology and the Archives of Pathology & Laboratory Medicine.

To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing.

Gene protein detection platform--a comparison of a new human epidermal growth factor receptor 2 assay with conventional immunohistochemistry and fluorescence in situ hybridization platforms.

In December 1998, the US Food and Drug Administration simultaneously approved the therapeutic antibody trastuzumab (Herceptin; Genentech, South San Francisco, CA) for the treatment of human epidermal growth factor receptor 2 (HER2) overexpressing invasive breast cancer and the immunohistochemistry (IHC)-based diagnostic test (HercepTest; Dako, Glosstrup, Denmark) designed to select patients for this novel targeted treatment. Today, more than 11 years later, anti-HER2–targeted therapy for breast cancer has become a cornerstone for treatment of HER2-positive disease; success has been achieved with use of both trastuzumab and the more recently approved tyrosine kinase small molecule inhibitor, lapatinib (Tykerb; GlaxoSmithKline, Philadelphia, PA), in adjuvant, neoadjuvant, and metastatic disease settings. Given this proven impact on survival for anti-HER2–targeted therapy, the importance of accurately identifying HER2-positive breast cancer has never been greater. The article in this issue of Journal of Clinical Oncology by Perez et al explores various interactions between HER2 status determined by IHC and by fluorescent in situ hybridization (FISH; PathVysion; Abbott Molecular, Abbott Park, IL), the estrogen receptor and progesterone receptor status determined by IHC, and the local laboratory versus central or reference laboratory HER2 testing results in the North Central Cancer Treatment Group N9831 trastuzumab adjuvant trial of early-stage breast cancer. The mining of the N9831 database is of keen interest tospecialists inbreastcancer,giventhelargepatientcohortandcontinued follow-up of the clinical outcome database. In the article, Perez et al present their data on the impact of independently assessing the HER2 gene copy number and the chromosome 17 centromere (CEP17) copy number for their impact on trastuzumab benefit and interactions with other clinical outcome variables. The study by Perez et al raises several important questions about the current state of HER2 testing in breast cancer, which, despite several major attempts at revision and standardization, continues to remain a serious concern for oncologists as they meet with their patients to select therapy for the disease.

Purpose To update key recommendations of the American Society of Clinical Oncology/College of American Pathologists human epidermal growth factor receptor 2 (HER2) testing in breast cancer guideline. Methods Based on the signals approach, an Expert Panel reviewed published literature and research survey results on the observed frequency of less common in situ hybridization (ISH) patterns to update the recommendations. Recommendations Two recommendations addressed via correspondence in 2015 are included. First, immunohistochemistry (IHC) 2+ is defined as invasive breast cancer with weak to moderate complete membrane staining observed in > 10% of tumor cells. Second, if the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test may (not "must") be ordered on the excision specimen based on specific clinical criteria. The HER2 testing algorithm for breast cancer is updated to address the recommended work-up for less common clinical scenarios (approximately 5% of cases) observed when using a dual-probe ISH assay. These scenarios are described as ISH group 2 ( HER2/chromosome enumeration probe 17 [CEP17] ratio ≥ 2.0; average HER2 copy number < 4.0 signals per cell), ISH group 3 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 6.0 signals per cell), and ISH group 4 ( HER2/CEP17 ratio < 2.0; average HER2 copy number ≥ 4.0 and < 6.0 signals per cell). The diagnostic approach includes more rigorous interpretation criteria for ISH and requires concomitant IHC review for dual-probe ISH groups 2 to 4 to arrive at the most accurate HER2 status designation (positive or negative) based on combined interpretation of the ISH and IHC assays. The Expert Panel recommends that laboratories using single-probe ISH assays include concomitant IHC review as part of the interpretation of all single-probe ISH assay results. Find additional information at www.asco.org/breast-cancer-guidelines .