Kristen E. Muller, DO, Jonathan D. Marotti, MD, Vincent A. Memoli, MD, Wendy A. Wells, MD, and Laura J. Tafe, MD

AJCP / Original Article Impact of the 2013 ASCO/CAP HER2 Guideline Updates at an Academic Medical Center That Performs Primary HER2 FISH Testing Increase in Equivocal Results and Utility of Reflex Immunohistochemistry Kristen E. Muller, DO, Jonathan D. Marotti, MD, Vincent A. Memoli, MD, Wendy A. Wells, MD, and Laura J. Tafe, MD From Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, and Geisel School of Medicine at Dartmouth, Hanover, NH. Key Words: HER2 FISH; HER2 IHC; Breast cancer; ASCO/CAP HER2 guidelines Am J Clin Pathol August 2015;144:247-252 ABSTRACT Objectives: The 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline updates lowered the threshold for HER2 positivity and altered the equivocal category. The goal of this study was to evaluate the impact of these changes on the distribution of HER2 fluorescence in situ hybridization (FISH) status. The utility of reflex HER2 immunohistochemistry (IHC) for FISH equivocal cases was also examined. Methods: We retrospectively reviewed all invasive breast cancers analyzed for HER2 via dual-probe FISH (PathVysion; Abbott Laboratories. Abbott Park, IL) 12 months before and after the HER2 guidelines updates were implemented. Reflex HER2 IHC results were recorded for HER2 FISH equivocal cases. Results: There was a significant increase in the number of HER2 FISH equivocal results after the guideline updates (4.9% vs 1.4%, P =.0087) that was independent of specimen type (core vs surgical, P =.6). All 17 FISH equivocal cases after the updates had reflex HER2 IHC: two (12%) of 17 were positive, 12 (71%) of 17 remained equivocal, and three (18%) of 17 were negative. Conclusions: Implementation of the 2013 ASCO/CAP HER2 guideline updates resulted in an increase in HER2 FISH equivocal results, which can be attributed to HER2 copy number, regardless of the HER2/CEP17 ratio. Reflex IHC for FISH equivocal cases is of limited utility; however, IHC does assign HER2 positivity or negativity in a small percentage of cases. Human epidermal growth factor receptor 2 (HER2), also known as ERBB2 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2), is amplified or overexpressed in approximately 15% to 20% of invasive breast cancers. 1 Accurately identifying HER2 status is a critical prognostic and predictive factor in breast cancer. HER2 evaluation enables proper patient selection for targeted therapy with trastuzumab or other anti-her2 agents. 2-5 In 2007, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) established guidelines for laboratories in an attempt to optimize HER2 testing for patients with breast cancer. 3 While these guidelines greatly improved the standardization of HER2 testing, several technical and interpretation issues remained, including definition of tumor heterogeneity, interpretation of challenging cases with aneusomy, necessity of repeat testing, and standardization of preanalytic variables. More important, many were concerned that patients otherwise eligible for the initial trastuzumab adjuvant trials based on the original immunohistochemistry (IHC) positive criterion of 3+ in more than 10% of tumor cells or a fluorescence in situ hybridization (FISH) HER2/CEP17 ratio of 2 or more would be excluded from anti-her2 therapy with the revised criterion (IHC 3+ in >30% of cells or HER2/CEP17 ratio 2.2). These concerns were addressed in the recent 2013 ASCO/CAP HER2 testing guideline update. 4 The categories for HER2 positive, equivocal, and negative were altered for both IHC and FISH. Notably, the FISH threshold for positivity was lowered back to a HER2/CEP17 ratio of 2.0 from the prior 2.2, and the FISH equivocal category is now based on HER2 gene copy number per cell. 4 Am J Clin Pathol 2015;144:247-252 247

Muller et al / Equivocal FISH With 2013 HER2 Guidelines Table 1 2007 and 2013 ASCO/CAP Guidelines for HER2 FISH Interpretation a Result Multiple techniques are available to assess HER2 status, including evaluation of HER2 protein overexpression via IHC or gene amplification using in situ hybridization (ISH). To date, there is no single gold-standard method for determining HER2 status, and the ASCO/CAP do not recommend one assay over another. 4 In our laboratory, we use dual-probe FISH for HER2 gene amplification as the primary method on all newly diagnosed primary breast cancers and metastases, with reflex IHC testing for equivocal cases. Following the implementation of the 2013 guidelines, we anecdotally noticed an increase in the number of HER2 FISH equivocal cases. Therefore, our main objective was to investigate the impact of the 2013 guideline changes on the distribution of HER2 FISH results at a laboratory that performs primary HER2 FISH on all newly diagnosed breast cancers. Furthermore, we sought to determine if reflex HER2 IHC clarified HER2 status in cases that were equivocal by FISH. Materials and Methods Case Cohort FISH: HER2/CEP17 Ratio FISH: HER2 Copy Number 2007 2013 2007 b 2013 Positive >2.2 2 >6 6 Equivocal 1.8-2.2 <2 with HER2 copy # 4 and <6 4-6 4 and <6 Negative <1.8 <2 with HER2 copy # <4 <4 <4 ASCO, American Society of Clinical Oncology; CAP, College of American Pathologists; FISH, fluorescence in situ hybridization. a Summarized from Wolff et al. 3,4 b In the 2007 guidelines, the HER2 copy number was only considered when using a single HER2 probe assay. In comparison, the 2013 guidelines consider a HER2/ CEP17 ratio of 2 or more or a HER2 copy number of 6 or more per cell amplified for dual-probe assays. This study was approved by the Institutional Review Board at Dartmouth-Hitchcock Medical Center. We retrospectively identified all consecutive primary and metastatic invasive breast cancer cases with HER2 FISH testing during the 12 months before (October 1, 2012, to September 30, 2013; 2007 guidelines cohort ) and after (October 1, 2013, to September 30, 2014; 2013 guidelines cohort ) the HER2 ASCO/CAP guideline updates were implemented. All cases analyzed for HER2 via dual-probe FISH were included. Specimens consisted of breast core needle biopsy (CNB) samples, biopsy specimens from metastatic sites, and surgical excisions (partial and full mastectomy specimens); both in-house and outside consultation cases were evaluated. HER2 FISH results were extracted from the molecular pathology reports, and the distribution of HER2 FISH positive, equivocal, and negative cases was determined for each time period. The HER2 IHC results were recorded for HER2 FISH equivocal cases. FISH Formalin-fixed, paraffin-embedded breast cancer tissue specimens were tested for HER2 gene amplification using the PathVysion HER-2 DNA Probe Kit according to the manufacturer s protocol (PathVysion; Abbott Laboratories, Abbott Park, IL). Our laboratory switched from a manual to an automated slide-processing technique in October 2013 using the Thermobrite Elite system (Leica Biosystems, Buffalo Grove, IL), which has been described in detail previously. 6 Validation of the Thermobrite Elite system was based on the 2007 guidelines and showed 100% concordance with manual processing. HER2 FISH results for the present study were interpreted based on the guidelines that were in place at the time of testing and are summarized in Table 1. 3,4 Immunohistochemistry Immunohistochemical staining was performed on 4-µm thick formalin-fixed, paraffin-embedded tissue sections using standardized automated methodology (Leica Bond; Leica Microsystems, Bannockburn, IL). HER2 protein overexpression was evaluated using the c-erb-b2 antibody (either clone CB11 [Leica, 1:30, prior to September 2013] or clone 4B5 [Ventana Medical Systems, Oro Valley, AZ, after September 2013]). Standardized immunohistochemical protocols were followed with control slides as appropriate. HER2 IHC was interpreted based on the guidelines that were in place at the time of testing. Statistical Analysis Fisher exact test was used to analyze proportions. Unpaired t test was used to evaluate significance of means. A P value less than.05 was considered significant. Results 2007 Guidelines Cohort In the 12-month period preceding the ASCO/CAP HER2 guideline update, our laboratory processed 355 specimens for HER2 using FISH. The distribution of HER2 FISH results is summarized in Table 2. Mean ± SD patient age was 62.9 ± 13.2 years, and patients were predominantly female (99.4% female, 0.6% male). Overall, 200 (56%) samples were obtained from breast CNB specimens, 149 (42%) from surgical excisions, and six 248 Am J Clin Pathol 2015;144:247-252

AJCP / Original Article Table 2 HER2 FISH Result Distribution for 12 Months Before and After Guideline Updates HER2 FISH Result (2%) from metastatic sites (skin, lung, liver, pleural fluid, and bone). Most (87.6%) cases were negative for HER2 by FISH, while 10.7% were positive for HER2 amplification. Five (1.4%) specimens were HER2 equivocal by FISH. One case had insufficient material for testing. The equivocal cases consisted of two CNB specimens and three surgical excisions. Of the five equivocal specimens (from four patients), four underwent reflex HER2 IHC that resulted in one positive, two equivocal, and one negative result. One of the FISH equivocal cases (HER2/CEP17 ratio = 2.2) showed rare amplified tumor cells (HER2/CEP17 ratio ~7.0) in less than 1% of the total tumor cell population. The reflex IHC displayed rare tumor cells (1%-5%) with 3+ complete membranous staining. These findings were attributed to tumor heterogeneity. One FISH equivocal CNB specimen had a repeat FISH on the excision specimen, which was also equivocal. Four (80%) of five of the equivocal cases had HER2/CEP17 ratios between 2.0 and 2.2 and would have been classified as positive based on the 2013 guidelines. 2013 Guidelines Cohort 2007 Guidelines Cohort (n = 355) No. (%) 2013 Guidelines Cohort (n = 345) P Value Negative 311 (87.6) 291 (84.3).232 Equivocal 5 (1.4) 17 (4.9).0087 a Positive 38 (10.7) 31 (9.0).45 Insufficient 1 (0.3) 6 (1.7).066 FISH, fluorescence in situ hybridization. a Statistically significant (P <.05). In the 12-month period following the implementation of the ASCO/CAP HER2 guideline update, our laboratory processed 345 specimens for HER2 FISH. Mean ± SD patient age was 62.02 ± 12.9 years, and patients were predominantly female (98.6% female, 1.4% male). Overall, 233 (68%) samples were obtained from breast CNB specimens, 101 (29%) from surgical excision specimens, and 11 (3%) from metastatic sites (skin, bone, brain, pleural fluid, liver, and omentum). Significantly more tumors were tested on CNB specimens in the 2013 cohort compared with the 2007 cohort (P =.0024). Most (84.3%) cases were negative for HER2 by FISH, while 8.9% were positive for HER2 amplification. Interestingly, 4.9% (17 specimens from 15 patients) were HER2 equivocal by FISH, which was a significant increase from the prior year (P =.0087). No significant changes were observed in HER2 positive or negative groups (P =.45 and.23, respectively). Six cases had insufficient material for testing, the majority due to small tumor quantity and prior decalcification of metastatic bone biopsy specimens (not significantly changed in comparison to the 2007 cohort, P =.06). The equivocal cases consisted of 13 core biopsy specimens and 4 excisions. All 17 equivocal specimens underwent reflex HER2 IHC with two positive (10%-20% of cells with 3+ staining), 12 equivocal, and three negative results Image 1. None of the cases showed HER2 heterogeneity by FISH or IHC. Among the remaining 12 equivocal cases by both IHC and FISH, four were paired biopsy specimens and subsequent excisions from two patients, four cases did not have subsequent material available for repeat testing (two consults, one excision, and one metastatic site), and four were biopsy specimens that had repeat HER2 FISH testing performed on the subsequent surgical specimens. Of these four repeat cases, two were negative and two were positive. Thus, a total of six equivocal cases from the 2013 cohort had repeat testing with the following results: two negative, two equivocal, and two positive. Clinicopathologic Features of Equivocal Cases The clinical and histopathologic features of the equivocal cases are summarized in Table 3. Overall, a total of 22 cases were equivocal by HER2 FISH, with a significant increase in the number of HER2 FISH cases occurring after the 2013 guideline update. The 22 specimens were obtained from 19 patients, with a mean ± SD patient age of 66.6 ± 12.5 years (100% female). Specimens tested included 15 breast CNB specimens and seven surgical excisions. The percentage of equivocal cases was independent of specimen type (CNB vs surgical excision, P =.6). Reflex IHC was performed on 21 of 22 specimens. IHC assigned HER2 positivity in three cases and negativity in four cases; 14 remained equivocal. Most tumors were highgrade, estrogen receptor positive (ER+), invasive ductal carcinomas of no special type (13/19 [68%], 16/19 [84%], and 16/19 [84%], respectively). Of the seven equivocal cases (one from the 2007 cohort and six from the 2013 cohort) that underwent repeat HER2 FISH testing on subsequent specimens, two were positive, three were equivocal, and two were negative. The two positive repeat cases had HER2/CEP17 ratios of 2.2 and 2.0; these cases would have been equivocal with the 2007 guidelines. As expected, the average HER2/CEP17 ratio of equivocal cases was significantly different before and after the guideline updates (2.03 ± 0.16, and 1.67 ± 0.27, respectively; P =.01). The mean HER2 gene copy numbers were not significantly different between 2007 and 2013 equivocal cohorts (4.53 ± 0.44 and 4.62 ± 0.36, respectively; P =.6). Am J Clin Pathol 2015;144:247-252 249

Muller et al / Equivocal FISH With 2013 HER2 Guidelines A B Image 1 HER2 fluorescence in situ hybridization (FISH; dual-color PathVysion FISH assay) and immunohistochemical assessment of a representative equivocal breast carcinoma. A HER2 equivocal case from 2013 cohort demonstrates (A) HER2 (red signals) to CEP17 (green signals) ratio of 1.9 and HER2 copy number of 4.8 (DAPI counterstain; 1,000) and (B) reflex HER2 immunohistochemistry (IHC) showing incomplete, weak to moderate membrane staining in more than 10% of tumor cells, scored as HER2 2+ ( 40) (inset, IHC HER2 3+ control, 40). Ten cases, representing eight patients, remained HER2 equivocal without a change in HER2 status by IHC or repeat FISH. Anti-HER2 therapy was administered to 50% (four of eight) of the patients who remained HER2 equivocal. Three specimens were received in consult by our laboratory specifically for HER2 FISH testing. Treatment and follow-up for these three patients are not known. Three FISH equivocal cases from the 2013 guideline cohort were analyzed for HER2 total protein expression via the HERMark assay (Monogram Biosciences, San Francisco, CA) at the request of one oncologist; all three were reported as HER2 positive, and two patients are currently receiving anti-her2 therapy. Discussion The 2007 ASCO/CAP HER2 guidelines greatly standardized HER2 testing, which resulted in more reliable and accurate identification of HER2 amplification in breast cancer. The subsequent changes reflected in the 2013 guideline update were made, in part, to identify more patients who would potentially benefit from anti-her2 therapy.4 However, the actual impact of reverting to the HER2-positive threshold by FISH back to a HER2/CEP17 ratio of 2.0 from 2.2, as well as altering the equivocal criterion, has yet to be fully evaluated in the routine practice of a major academic medical center that performs primary HER2 FISH testing. 250 Am J Clin Pathol 2015;144:247-252 Almost all (4/5 [80%]) of our equivocal cases from before the guideline changes would have been classified as positive using the 2013 criterion based on HER2/CEP17 ratio alone. In contrast, nearly half (8/17 [47%]) of the equivocal cases after the guideline update had a HER2/ CEP17 ratio less than 1.8 and thus would have been classified as negative using the 2007 guidelines. These results imply that the guideline updates are effective in capturing additional patients eligible for anti-her2 therapy, as well as identifying patients with equivocal results who may potentially benefit from anti-her2 therapy. We identified a significant increase in the number of HER2 FISH equivocal cases when comparing the 12 months before and after the 2013 guideline updates were implemented. Significant changes were not identified in the distribution of HER2 positive, negative, or insufficient groups. To date, the largest examination of HER2 equivocal breast cancers was performed by Sapino et al,7 who evaluated 957 IHC HER2 equivocal cases by dual-color FISH and assessed the results according to a variety of criteria (US Food and Drug Administration [FDA]/European Medicines Agency, ASCO/CAP 2007, and ASCO/CAP 2013). Similar to our study, they reported an increase in equivocal cases when using the ASCO/CAP 2013 ISH algorithm (12.3%) compared with the ASCO/CAP 2007 ratio criterion (2.4%).7 Additional evaluation of the data by Sapino et al7 reveals that although only 2.4% of the 2007 cases were equivocal by ratio, 13.5% would have been equivocal by copy number alone using the 2013 guidelines or by the

AJCP / Original Article Table 3 Clinicopathologic Features of HER2 FISH Equivocal Cases Characteristic 2007 Guidelines Cohort 2013 Guidelines Cohort HER2 equivocal cases (patients), No. 5 (4) 17 (15) Age, mean ± SD, y 54.4 ± 15.6 70.2 ± 9.1 Female sex, No. (%) 4 (100) 15 (100) CNB specimens, No. 2 13 Surgical (excisions, metastases), No. 3 4 Histologic type, No. (%) IDC, NOS 4 (100) 12 (80) IDC focal special type features 3 (20) Grade, No. Low 1 Intermediate 1 3 High 3 10 Unknown 1 Hormone receptor status, No. ER+/PR+ 4 11 ER+/PR 1 ER /PR+ 1 ER /PR 2 HER2/CEP17 ratio, mean ± SD 2.03 ± 0.16 1.67 ± 0.27 HER2 copy number, mean ± SD 4.53 ± 0.44 4.62 ± 0.36 HER2 FISH repeated, No. of specimens 1 6 Positive 0 2 Equivocal 1 2 Negative 0 2 HER2 IHC, No. of specimens 4 17 Positive (3+) 1 2 Equivocal (2+) 2 12 Negative (0-1+) 1 3 Anti-HER2 treatment, No./total No. (%) 2/2 (100) 2/6 (33) CNB, core needle biopsy; ER+, estrogen receptor positive; ER, estrogen receptor negative; FISH, fluorescence in situ hybridization; IDC, invasive ductal carcinoma; IHC, immunohistochemistry; NOS, not otherwise specified; PR+, progesterone receptor positive; PR, progesterone receptor negative. 2007 criterion with a single-probe assay. In our study, all of the 2013 equivocal FISH cases had mean copy numbers per cell in the range of 4 to 6. Therefore, the increased frequency in equivocal cases is based on the HER2 absolute copy number regardless of HER2/CEP17 ratio. As a result, we expect a continued increase in the number of patients with equivocal HER2 FISH results using the 2013 criterion. The guideline updates acknowledge a lack of evidence on response to anti-her2 therapy in this subset of patients; optimal treatment regimens and clinical outcomes for patients with borderline increased HER2 copy number remain unclear. However, outcomes from the N9831 trial on the effects of adjuvant trastuzumab suggest anti-her2 therapy may be beneficial in patients with HER2 copy numbers greater than 4, irrespective of HER2 ratio. 8 Of note, most HER2 equivocal cases are ER+, and in the report by Sapino et al, 7 more than 50% also displayed a higher proliferation index based on Ki-67, findings that may guide non-her2 targeted therapy. The ASCO/CAP algorithm recommends reflex testing using a different modality for all HER2 equivocal cases. 3,4 Our laboratory is unique in that we perform upfront HER2 FISH testing with reflex IHC for equivocal cases. We moved to primary FISH testing more than 5 years ago largely due to an increasing demand by our clinicians to perform FISH testing regardless of IHC findings and the technical challenges of standardizing IHC. FISH is also more reproducible and resilient to preanalytic factors. 9 We now use IHC predominantly in a reflexive manner for equivocal FISH cases, for those cases with a limited amount of tumor present, or for a small group of referring hospitals that have requested HER2 IHC as the primary assay. As described in the Materials and Methods section, our switch in HER2 antibody clones coincided temporally with the guideline change, and therefore, we did not attempt to analyze the impact of the new guidelines on IHC testing results. In our 22 FISH equivocal cases, HER2 status was assigned using reflex IHC only in a minority of cases; the majority remained equivocal. Although there is a strong correlation between HER2 FISH and IHC testing, 10-12 the preferred method and testing algorithm remain controversial. Additional techniques for evaluation of HER2 status have been developed but have yet to be endorsed by the ASCO/CAP. These include DNA expression by microarray and messenger RNA expression by reverse transcriptase polymerase chain reaction. Measurement of HER2 total protein (HERMark; Monogram Biosciences) is commercially available and was requested on three of our equivocal cases. All three returned with a result of HER2 positive. Although analytically validated, 13 evidence for the routine clinical implementation of HERMark remains limited. Last, there has been an increasing demand to perform HER2 FISH or IHC on breast CNB specimens to guide and expedite treatment. Our laboratory has recently experienced this shift, which is reflected in the increased number of CNB specimens tested in the 2013 cohort compared with the 2007 cohort. Most prior studies have shown equivalent HER2 results between CNB and surgical specimens. 14 In our study, the increase in percentage of equivocal results was independent of specimen type. However, there are potential limitations to be aware of when performing HER2 FISH on CNB specimens. CNB specimens that yield small quantities of tumor may be insufficient for testing or may not reflect the full morphologic extent of tumor type and grade. Second, HER2 heterogeneity is reported to occur in 5% to 30% of breast cancers and could account for a change in HER2 status from CNB to excision. 15 We did observe a change in HER2 FISH status in six cases, which were initially HER2 equivocal via FISH on CNB specimens. HER2 heterogeneity via FISH and IHC was identified in one equivocal case from the 2007 cohort. Am J Clin Pathol 2015;144:247-252 251

Muller et al / Equivocal FISH With 2013 HER2 Guidelines In summary, we evaluated the impact of the 2013 guideline updates on the distribution of HER2 classification as determined by primary FISH testing. The alteration of the equivocal category led to an increase in equivocal cases, many of which would have been classified as negative using the 2007 criteria. The decreased threshold for HER2 FISH positivity from 2.2 to 2.0 brings the ASCO/CAP guidelines in alignment with the FDA-approved threshold and is more inclusive of cases that were previously classified as equivocal. Last, the utility of IHC to further evaluate HER2 FISH equivocal cases is of limited value but does assign HER2 positivity in a minority of cases. The increase in HER2 equivocal cases, due largely to HER2 copy number, leaves a unique category of carcinomas that are predominantly ER+ and warrants additional investigation to better define treatment options and outcomes in this subset of patients. Corresponding author: Laura J. Tafe, MD, Dept of Pathology, Dartmouth-Hitchcock Medical Center, One Medical Center Dr, Lebanon, NH 03756; Laura.J.Tafe@Hitchcock.org. The data here were presented at the 104th United States and Canadian Academy of Pathology Annual Meeting; March 21-27, 2015; Boston, MA. Acknowledgments: The authors thank the staff of the Dartmouth-Hitchcock Medical Center s Department of Pathology Molecular Pathology Laboratory for technical support and Larry Dumont, PhD, for statistical advice. References 1. Burstein HJ. The distinctive nature of HER-2-positive breast cancer. N Engl J Med. 2005;353:1652-1654. 2. Hicks DG, Kulkarni S. HER2+ breast cancer: review of biologic relevance and optimal use of diagnostic tools. Am J Clin Pathol. 2008;129:263-273. 3. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131:18-43. 4. Wolff AC, Hammond ME, Hicks DG. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138:241-256. 5. Tafe LJ, Tsongalis GJ. The human epidermal growth factor receptor 2 (HER2). Clin Chem Lab Med. 2011;50:23-30. 6. Tafe LJ, Allen SF, Steinmetz HB, et al. Automated processing of fluorescence in-situ hybridization slides for HER2 testing in breast and gastro-esophageal carcinomas. Exp Mol Pathol. 2014;97:116-119. 7. Sapino A, Maletta F, Verdun Di Cantogno L, et al. Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method. Oncologist. 2014;19:1-9. 8. Perez EA, Reinholz MM, Hillman DW, et al. HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol. 2010;28:4307-4315. 9. Sauter G, Lee J, Bartlett JM, et al. Guidelines for human epidermal growth factor receptor 2 testing: biologic and methodologic considerations. J Clin Oncol. 2009;27:1323-1333. 10. Owens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer. 2004;5:63-69. 11. Middleton LP, Price KM, Puig P, et al. Implementation of American Society of Clinical Oncology/College of American Pathologists HER2 guideline recommendations in a tertiary care facility increases HER2 immunohistochemistry and fluorescence in situ hybridization concordance and decreases the number of inconclusive cases. Arch Pathol Lab Med. 2009;133:775-780. 12. Pauletti G, Dandekar S, Rong H, et al. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol. 2000;18:3651-3664. 13. Larson JS, Goodman LJ, Tan Y, et al. Analytical validation of a highly quantitative, sensitive, accurate, and reproducible assay (HERmark ) for the measurement of HER2 total protein and HER2 homodimers in FFPE breast cancer tumor specimens. Patholog Res Int. 2010;2010:814176. 14. Lee AH, Key HP, Bell JA, et al. Concordance of HER2 status assessed on needle core biopsy and surgical specimens of invasive carcinoma of the breast. Histopathology. 2012;60:880-884. 15. Vance GH, Barry TS, Bloom KJ, et al. Genetic heterogeneity in HER2 testing in breast carcinoma: panel summary and guidelines. Arch Pathol Lab Med. 2009;133:611-612. 252 Am J Clin Pathol 2015;144:247-252