Central Pathology Laboratory Review of HER2 and ER in Early Breast Cancer: An ALTTO Trial [BIG 2-06 / NCCTG N063D (Alliance)] Ring Study – revised 23dec13

Authors: 1Ann E. McCullough, 2Patrizia Dell'Orto, 3Monica M. Reinholz, 4Richard D. Gelber, 1Amylou C. Dueck, 2Leila Russo, 5Robert B. Jenkins, 2Stefania Andrighetto, 5Beiyun Chen, 6Christian Jackisch, 7Michael Untch, 8Edith A. Perez, 9Martine J. Piccart-Gebhart, and 2Giuseppe Viale on behalf of the ALTTO Study Team.

Supplementary Web Appendix

A. Brief summary of ASCO/CAP Guidelines for HER2 and ER testing.

B. Procedures used by Mayo and IEO central laboratories

C. Final distribution of ALTTO HER2 and ER status determinations from the Mayo and IEO central laboratories

D. Concordance in HER2 status between central laboratories in Phase 1 of the ring study

E. Summary of steps taken to reduce discordance rates in Germany

1 Supplementary Appendix A. Brief summary of ASCO/CAP Guidelines for HER2 and ER testing (refs. 3 and 4, respectively)

For HER2 testing, a positive HER2 result is IHC staining of 3+ (uniform, intense membrane staining of >30% of invasive tumor cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2. A negative HER2 result is an IHC staining of 0 or 1+, a FISH result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8. An equivocal HER2 IHC result (2+) is complete membrane staining with obvious circumferential distribution in at least 10% but fewer than 30% of tumor cells. The equivocal range for FISH assays is defined as HER2/CEP 17 ratios from 1.8 to 2.2.

A positive estrogen receptor immunohistochemical result is defined as visually apparent nuclear staining in 1% or more tumor nuclei; a negative result is less than 1% nuclear staining. There is no equivocal result category in estrogen receptor immunohistochemical testing. Supplementary Appendix B. Procedures used by Mayo and IEO central laboratories.

Mayo Central Pathology Review Methods – Ann E. McCullough, M.D./Wilma Lingle Ph.D. (responsible for immunohistochemistry methods) and Robert B. Jenkins, M.D., Ph.D. (responsible for FISH methods)

Immunohistochemistry - ER alpha:

Formalin fixed paraffin embedded tissue sections were deparaffinized and antigen retrieval was carried out using EDTA in a preheated 98 degree steamer for 30 minutes; The EDTA is 1 mM pH 8.0. After cooling slides were loaded onto the Dako Autostainer for the subsequent staining procedure. Slides were treated with 3% H2O2 (Dako S2001) to inactivate endogenous peroxidase. Mouse monoclonal anti-Estrogen Receptor alpha antibody (Dako 1D5) was applied using a 1/50 dilution for 30 minutes at room temperature. Visualization was carried out using DAKO's Dual + Envision link (K4061, DAKO North America, Inc., Carpinteria, CA) followed by incubation with diaminobenzidine. Sections were counterstained with hematoxylin.

Immunohistochemistry - PR:

Formalin fixed paraffin embedded tissue sections were deparaffinized and antigen retrieval was carried out using EDTA in a preheated 98 degree steamer for 30 minutes. After cooling slides were loaded onto the Dako Autostainer for the subsequent staining procedure. Slides were treated with 3% H2O2 (Dako S2001) to inactivate endogenous peroxidase. Mouse monoclonal anti-Progesterone Receptor antibody was applied using a 1/100 dilution for 30 minutes at room temperature. Visualization was carried out using DAKO's Dual + Envision link (K4061, DAKO North America, Inc., Carpinteria, CA) followed by incubation with diaminobenzidine. Sections were counterstained with hematoxylin.

Immunohistochemistry – Dako HER2 kit:

Formalin fixed paraffin embedded tissue sections were deparaffinized and antigen retrieval was carried out using citrate buffer (Dako K5207) in a preheated 99-100 degree water bath for 40 minutes. After a 20 minute cool down slides were loaded onto the Dako Autostainer for the subsequent staining procedure. Slides were treated with 3% H2O2 (Dako K5207) to inactivate endogenous peroxidase. Prediluted rabbit anti-HER2 antibody (Dako) was applied for 30 minutes at room temperature. Visualization was carried out using goat anti-rabbit Visualization

3 reagent (K5207, DAKO North America, Inc., Carpinteria, CA) followed by incubation with diaminobenzidine. Sections were counterstained with hematoxylin.

FISH – HER2:

FISH analysis was performed on deparaffinized tissue sections (5-µm thickness) using the PathVysion HER2 DNA probe kit and the HER2/centromere 17(HER2/CEP17) probe mixture (Abbott Molecular, Abbott Park, Illinois) (Perez et al, 2010; Mansfield et al 2013). For each case, a parallel hematoxylin and eosin–stained slide was examined for regions of invasive carcinoma by a board-certified anatomic pathologist. The complete tissue section was scanned by two certified cytogenetic technologists to detect any subpopulation of amplified cells. Thirty representative nuclei from the invasive tumor were scored by each technologist (sixty nuclei total), with an overall evaluation performed by a board-certified pathologist (R.B.J.). When the red HER2 signals were clearly amplified, but highly clustered so as to be very difficult to enumerate, the nucleus was assigned >20 red signals and the green (CEP17) signals were counted. Samples exhibiting centromere 17 amplification (≥6 centromere 17 signals) were retested with a laboratory-developed probe set using probes directed toward HER2 and D17S122. D17S122 is rarely coamplified when HER2 and centromere 17 are coamplified (Mansfield et al. 2013). The Mayo Clinic criteria for HER2 anomalies follow the ASCO/CAP guidelines (Wolf et al. 2007; Vance et al. 2009) and are described in detail in Perez et al (2010).

Wolf AC, Hammond EH, Schwartz JN et al. American Society of Clinical Oncology/College of American Pathologists Guideline recommendation for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 2007. 131:18–43.

Vance GH, Barry TS, Bloom KJ, Fitzgibbons PL, Hicks DG, Jenkins RB, Persons DL, Tubbs RR, Hammond ME; College of American Pathologists.Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines. Arch Pathol Lab Med. 2009 Apr;133(4):611-2

Perez EA, Reinholz MM, Hillman DW, Tenner KS, Schroeder MJ, Davidson NE, Martino S, Sledge GW, Harris LN, Gralow JR, Dueck AC, Ketterling RP, Ingle JN, Lingle WL, Kaufman PA, Visscher DW, Jenkins RB. HER2 and chromosome 17 effect on patient outcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol 2010 Oct 1; 28(28):4307-15.

Mansfield AS, Sukov WR, Eckel-Passow JE, Sakai Y, Walsh FJ, Lonzo M, Wiktor AE, Dogan A, Jenkins RB. Comparison of Fluorescence In Situ Hybridization (FISH) and Dual-ISH (DISH) in the Determination of HER2 Status in Breast Cancer. Am J Clin Pathol. 2013 Feb;139(2):144-50. European Institute of Oncology Central Pathology Review Methods – Giuseppe Viale, M.D. (responsible pathologist)

Immunohistochemistry - ER alpha (ER/PR pharmDx TM Kit, Dako, Glostrup, Denmark):

Formalin fixed paraffin embedded tissue sections were deparaffinized and antigen retrieval was carried out using the Target Retrieval Solution 1x (K4071) at 125°C in a pressure cooker (Pascal Pressurized Heating Chamber, Dako) for 5 min, and then left to cool at 90°C for additional 30 min. After cooling slides were washed in tap water for 5 min and in the Wash Buffer (K4071) for 3 min before being loaded onto the Dako Autostainer for the subsequent staining procedure. Slides were treated with Peroxidase Blocking Reagent (Dako K4071) to inactivate endogenous peroxidase for 5 min. Prediluted mouse monoclonal anti-Estrogen Receptor alpha (cocktail of 1D5 and ER-2-123 monoclonal antibodies) was applied for 30 minutes at room temperature. The ER assay used at IEO was the only available FDA-approved assay at the time of the study. Visualization was carried out using the Visualization Reagent (Dako K4071) for 30 min, followed by incubation with DAB+Substrate Buffer+Chromogen (Dako K4071). Sections were then treated with 0.5% copper sulphate in 1M NaCl for 30 sec before being counterstained with hematoxylin.

Immunohistochemistry - PgR (ER/PR pharmDx TM Kit, Dako, Glostrup, Denmark):

Formalin fixed paraffin embedded tissue sections were deparaffinized and antigen retrieval was carried out using the Target Retrieval Solution 1x (K4071) at 125°C in a pressure cooker (Pascal Pressurized Heating Chamber, Dako) for 5 min, and then left to cool for additional 30 min. After cooling slides were washed in tap water for 5 min and in the Wash Buffer (K4071) for 3 min before being loaded onto the Dako Autostainer for the subsequent staining procedure. Slides were treated with Peroxidase Blocking Reagent (Dako K4071) to inactivate endogenous peroxidase for 5 min. Prediluted mouse monoclonal anti-Progesterone Receptor antibody (clone PgR 1294) was applied for 30 minutes at room temperature. Visualization was carried out using the Visualization Reagent (Dako K4071) for 30 min, followed by incubation with DAB+Substrate Buffer+Chromogen (Dako K4071). Sections were then treated with 0.5% copper sulphate in 1M NaCl for 30 sec before being counterstained with hematoxylin.

Immunohistochemistry -Dako HER2 kit:

Formalin fixed paraffin embedded tissue sections were deparaffinized and antigen retrieval was carried out using citrate buffer (Dako K5207) in a preheated 99-100 degree water bath for 40

5 minutes. After a 20 minute cool down slides were loaded onto the Dako Autostainer for the subsequent staining procedure. Slides were treated with 3% H2O2 (Dako K5207) to inactivate endogenous peroxidase. Prediluted rabbit anti-HER2 antibody (Dako) was applied for 30 minutes at room temperature. Visualization was carried out using goat anti-rabbit Visualization Reagent (K5207, Dako) followed by incubation with diaminobenzidine. Sections were then treated with 0.5% copper sulphate in 1M NaCl for 30 sec before being counterstained with hematoxylin.

FISH – HER2:

The Abbott HER2 FISH probe package insert was followed. In general, 20 cells were counted in case of homogeneously amplified tumors and concordance with IHC and local assessment, 40 to 60 cells (2 or 3 different readers) in case of non-homogeneous tumors or when there was any discordance with the immunohistochemical findings or the local assessment. Supplementary Appendix C. Final distribution of ALTTO HER2 and ER status determinations from the Mayo and IEO central laboratories

Table S1 Final concordance between local and central HER2 status determinations a, b Central Laboratory Local HER2 status Mayo IEO Local HER2 positive Total cases 1029 9095 Centrally eligible 971 7766 Centrally not eligible 58 (5.6%) 1329 (14.6%) Local HER2 equivocal Total cases 27 1193 Centrally eligible 14 730 Centrally not eligible 13 (48.1%) 463 (38.8%) HER2 human epidermal growth factor receptor 2, Mayo Mayo Clinic central laboratory, IEO European Institute of Oncology central laboratory a In addition to the cases reported above, 601 evaluable cases were screened in China; 71 (11.8%) were not eligible (HER2 negative). b A case was centrally eligible (HER+ positive) if either central IHC or FISH were positive; positivity of one test was sufficient. Both IHC and FISH were performed on all carcinomas eligible for enrollment in the trial.

7 Table S2 Final concordance between local and central ER status determinations Central Laboratory Local ER status Mayo IEO Local ER positive Total cases 701 5625 Positive ≥ 10% 582 5254 Positive ≥ 1% and < 10% 17 131 Negative 102 (14.6%) 240 (4.3%) Local ER negative Total cases 345 4640 Positive ≥ 10% 9 (2.6%) 727 (15.7%) Positive ≥ 1% and < 10% 3 (0.9%) 249 (5.4%) Negative 333 3664 ER estrogen receptor, Mayo Mayo Clinic central laboratory, IEO European Institute of Oncology central laboratory Supplementary Appendix D. Concordance in HER2 Status Between Central Laboratories in Phase 1 of the Ring Study – All cases were HER2-positive in local laboratories; 20 IEO HER2-negative cases (i.e., false-positive) were submitted to Mayo, and 5 Mayo HER2- negative cases were submitted to IEO (total of 25 cases exchanged)

Table S3 Concordance in HER2 status between central laboratories - HER2 IHC Mayo IEO Negative (0-1+) Equivocal (2+) Negative (0-1+) 18 6 Equivocal (2+) -- 1

Table S4 Concordance in HER2 status between central laboratories - HER2 FISH Mayo IEO Not Amplified (<1.8) Equivocal (1.8-2.2) Not Amplified (<1.8) 22 3 Equivocal (1.8-2.2) -- --

Table S5 Concordance in HER2 status between central laboratories - ALTTO eligibility Mayo IEO Not Eligible Eligible Not Eligible 25 -- Eligible -- --

9 Supplementary Appendix E. Summary of steps taken to reduce discordance rates in Germany

Based on the ALTTO ring study project and the information on a substantial discordance rate between the German centers and the central pathology review, an ad hoc meeting was organized. Participants were Prof. Hans Kreipe, Head of the Pathology Department of the Medical School of Hannover and Chairman of the German Society of Pathology, Prof. Rueschoff, Head Pathologist of the Targos Institute of Pathology in Kassel, formerly involved in the HERA trial, Prof. Giuseppe Viale from the EIO in Milan, as well as Prof. Michael Untch and Prof. Christian Jackisch, Principal Investigators of their institutions and members of the ALTTO Executive Committee. All of the discordant cases were reviewed. The consequences were as follows:

1. Implementation of nationwide ring studies involving the pathologists and PI of the Certified Breast Cancer Centers (Certified by the German Cancer Society and the German Society of Senology) as an obligatory quality control tool. The participation was rewarded with a certificate which was obligatory to present for the biannual recertification of the Certified Breast Cancer Centers (www.onkzert.de).

2. Activation of interdisciplinary teaching activities between pathologists and clinicians on the importance of the discordancy of ER, PR and Her2neu detection and their clinical treatment implications.

3. More important from the clinician point of view was to provide advice about how to proceed with discordant cases which were not accepted for the ALTTO trial in the local institutions. The ad hoc working group advised clinicians and PIs of the ALTTO trial to ask for a “second opinion” by one of the experienced pathologists participating in the ad hoc working group.

4. As a special initiative of the ad hoc working group, the pathologists themselves agreed to contact the top ten centers being listed as the” TOP TEN Centers of Discordance” to discuss the matters on site that contributed to the reported discordance rate. This initiative was highly appreciated and added to the improved quality of the testing results.