# Best Practices for IHC Detection and Interpretation of ER, PR, and HER2 Protein Overexpression in Breast Cancer

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Aileen Martin
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#1034 - Best Practices for IHC Detection and Interpretation of ER,

Cartun, MS, PhD Andrew Ricci, Jr, MD Department of Pathology

1 # Best Practices for IHC Detection and Interpretation of ER, PR, and HER2 Protein Overexpression in Breast Cancer Richard W. Cartun, MS, PhD Andrew Ricci, Jr, MD Department of Pathology Hartford Hospital Hartford, CT (860) Richard.Cartun@hhchealth.org

2 Speaker Disclosure In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation.

3 Hartford Hospital Hartford, Connecticut

4 Patient treatment for breast cancer depends on: Histologic type and grade Tumor size Lymph node status Estrogen (ER) and progesterone receptor (PR) status HER-2/neu status (protein overexpression and/or gene amplification) Gene expression testing (Oncotype DX)

5 This image cannot currently be displayed.

6 Do we have a problem with IHC predictive marker testing? Problems with ER and PR testing conducted between 1997 and 2005 in the Newfoundland and Labrador (Canada) health care systems. Repeat IHC testing performed by central testing laboratories in the United States has shown poor concordance with results from community hospitals (especially with HER2 testing).

7 Is there a problem with IHC predictive marker testing? (cont.) Unsatisfactory results with proficiency testing programs in the United States. Genomic Health (Oncotype DX) now reports ER, PR, and HER2 scores because some oncologists don t trust results from their own pathology laboratories (or laboratories at other medical institutions).

8 Invasive Breast Carcinoma Estrogen Receptor Protein

9 Breast Cancer Consultation Case - HER2 Positive? Outside HER2 IHC Repeat HER2 IHC

10 Ductal carcinoma in situ 3+ HER2 protein overexpression

11 IHC Assay Total Test Concept Preanalytic Specimen type, acquisition, transport time Fixation (type and time) Tissue processing, type, and temperature Tissue sectioning Analytic Antigen retrieval Testing protocol, control selection Reagent validation Technical staff training/certification Laboratory certification Postanalytic Control evaluation Interpretation of results Reporting of results Pathologist, experience, and CME Taylor CR: Arch Pathol Lab Med 2000;124;

12 Should we be doing ER, PR, and HER2 IHC testing on diagnostic biopsy tissue or wait for the excisional specimen?

14 HER2 Heterogeneity HER2 IHC (-) clone HER2 IHC (+) clone

15 Khoury T, Sait S, Hwang H, et al.: Delay to formalin fixation effect on breast biomarkers. Mod Pathol 2009;22: We recommend not to delay formalin fixation for more than 1 hour and do not store specimens overnight at 4 degrees C.

16 Goldstein NS, Hewitt SM, Taylor CR, et al.: Recommendations for improved standardization of immunohistochemistry. Appl Immunohistochem Mol Morphol 2007;15: Tissue should be fixed in 10% neutral-buffered formalin.

18 Inadequate Fixation Yes, we can re-process tissue, but...

20 Wolff AC, Hammond ME, Schwartz JN, et al.: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor 2 testing in breast cancer. Arch Pathol Lab Med 2007;131:18-43 breast tissue should be fixed for a minimum of 6 hours and no longer than 48 hours

21 Arber D: Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast markers. Appl Immunohistochem Mol Morphol 2002;10: The IHC reactivity of some breast prognostic markers is reduced, but only after extensive fixation that may not be clinically relevant.

22 Tong LC, Nelson N, Tsourigiannis J, et al.: The effect of prolonged fixation on the IHC evaluation of ER, PR, and HER2 expression in invasive breast cancer: A prospective study. Am J Surg Pathol 2011;35: fixation for limited periods beyond 72 hours does not result in a reduction in assay sensitivity in the determination of ER, PR, or HER2 IHC status.

23 HER-2/neu protein overexpression 48 Hours fixation 16 Days fixation

24 Minimum Fixation Time for Needle Core Biopsy Specimens? (4.5 Hours) SMM ER

25 Maximum Fixation Time? (1.5 Years) HER2 IHC HER2 FISH

26 Hartford Hospital Fixation Policy Needle core biopsies must be fixed for a minimum of 4.5 hours. This includes 0.5 hours pre-processor fixation and 4.0 hours of fixation on the tissue processor. All other specimens must be fixed for a minimum of 6 hours. Excisional specimens arriving after 3:30 PM are held for overnight fixation (following slicing).

27 Documentation of Fixation Time

28 Documentation of Formalin Contact Time

30 Best Practices for Breast Biospecimen Collection Minimize cold ischemic time to under one hour for excisional specimens. Do not allow the tissue to dry out. Fixation in 10% buffered formalin (1:10 ratio). Slice large specimens to facilitate fixation ASAP (hold for overnight fixation if needed). Submit gross sections no more than 2-3 mm in thickness. Document cold ischemic time and formalin contact time, and total time in formalin (if possible).

31 Needle Core Biopsy #2 Uniform Immunoreactivity

32 Needle Core Biopsy #1 Edge Effect Due To Drying

33 HER2 Protein Overexpression (tissue fixed in formalin 8 hours) Surface Center

35 Fixation Problem? Estrogen Receptor Protein

36 No, tissue sectioning problem. Estrogen Receptor Protein

37 Breast CA - Core Biopsy Estrogen Receptor Protein

38 Tissue Sectioning Recommendations: 4-5 Microns in thickness Sections should be placed in the center of the slide (not on the + or x ) Multiple sections for biopsy specimens Orientation (identical if possible) Make sure slides are dry before starting IHC testing Always verify patient identification and block designation No oven for unstained slides send-out

39 Slide Protocol for Diagnostic Biopsy Specimens Slide 1 - H&E Slide 2 - Unstain Slide 3 - Unstain Slide 4 - H&E Slide 5 - Unstain Slide 6 - Unstain Slide 7 - H&E

40 Should stored unstained slides be used for IHC testing? Estrogen Receptor Protein

41 Freshly Cut Unstained Slides Estrogen Receptor Protein

42 Xie R, Chung J-Y, Ylaya K, et al.: Factors influencing the degradation of archival formalin-fixed paraffinembedded tissue sections. J Histochem Cytochem 2011;59: the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss.

43 Shintaku IP and Said JW: Detection of ER with mabs in routinely processed formalin-fixed paraffin sections of breast CA; use of a DNase pretreatment to enhance sensitivity of the reaction. Am J Clin Pathol 1987;87: This method offers a reliable and reproducible alternative when tissue is not suitable or unavailable for DCC or frozen tissue analysis

44 ER Detection (clone H222) ER-ICA Breast CA

45 Non-neoplastic Breast Tissue H&E ER

46 Invasive Duct Carcinoma H&E ER

47 Immunohistochemical Testing at Hartford Hospital: All studies performed on Leica Biosystems Bond III automated IHC/ISH platform Bond Polymer Refine detection DAB chromogen Hematoxylin counterstain (off-line) ER (mouse mab clone 6F11) - Leica PR (mouse mab clone 16) - Leica HER2 (rabbit mab clone EP3) - Epitomics

48 ER - Clone 6F11

49 ER - Clone 6F11

50 PR - Clone 16

51 PR - Clone 16

52 HER2 - Clone EP3

53 HER2 - Clone EP3

54 Progesterone Receptor mabs Clone PgR636 Clone 16

55 HER2 clone EP3

56 Outside Hospital Consult ER clone 6F11

57 Personalized Antigen Retrieval There is no one retrieval method or time that is optimal for all antibodies and tissues. Control tissues may be over-fixed and, as a result, require more aggressive retrieval to provide optimal reactivity. Reduce time for small specimens. Reduce or increase time for specimens from other hospitals/laboratories.

58 Antibody Validation: Clinical response (unlikely to get this information from clinicians) Morphology and grade (low-grade tumors are ER+ and most ER- tumors are highgrade) Most HER2+ tumors are high-grade Compare results with other technologies (FISH for HER2 and Oncotype DX for ER, PR, and HER2) Interlaboratory comparisons and PT useful

59 Fitzgibbons PL, Murphy DA, Hammond MEH, et al.: Recommendations for validating ER and PR immunohistochemistry assays. Arch Pathol Lab Med 2010;134: will improve the accuracy of hormone-receptor testing, reduce interlaboratory variation, and minimize false-positive and false negative results.

60 ER, PR, and HER2 in Breast CA ER is expressed in 70% to 95% of invasive lobular carcinomas and in 70% to 80% of invasive ductal carcinomas PR is expressed in 60% to 70% of invasive breast carcinomas HER2 is overexpressed and/or amplified in 15% to 25% of invasive breast carcinomas Antibodies, detection systems, and interpretation guidelines with affect these numbers

61 Validating ER/PR IHC Results ER stronger than PR by IHC

62 Oncotype DX Test

63 Hammond MEH, Hayes DF, Dowsett M, et al.: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for estrogen and progesterone receptors in breast cancer. Arch Pathol Lab Med 2010;134:

64 Guideline Recommendations Positive for ER or PR if 1% of tumor cell nuclei are immunoreactive Specimen unsatisfactory if no tumor nuclei are immunoreactive and no internal positive control present Large, preferably multiple core biopsies of tumor are preferred Samples for ER and PR testing are fixed in NBF for 6 to 72 hours

65 Breast CA - ER Weak Positive Estrogen Receptor Protein (clone 6F11)

67 Best Practices for Interpretation: Print a copy of the pathology report (confirm case number/patient name and block designation; compare morphology). Evaluate all tissue fragments on biopsy specimens. Look for an internal positive control for ER/PR (benign breast tissue) if tumor cells are negative. Use LMW-CK (clone 5D3) to identify invasive tumor cells if not readily identified.

68 Best Practices for Interpretation: Use myoepithelial markers (SMM, Calponin, or p63) to confirm invasive tumor. Score % of positive invasive tumor cells and their intensity (weak, moderate, or strong); mark score on slide (record). Recommend repeat studies on excisional tumor when the biopsy specimen shows little tumor; ER/PR are negative and there is no internal control; or biopsy specimen shows a Triple-Negative immunoprofile.

73 Internal Positive Control ER (clone 6F11)

74 Internal Positive Control HER2 clone EP3

75 Allred DC, Harvey JM, Berardo M: Prognostic and predictive factors in breast cancer by immunhistochemical analysis. Mod Pathol 1998;11: Allred Scoring System

76 Allred Scoring System

77 Total Score (TS): Negative (0 or 2) Positive (3 and higher) Determined by cutpoint analysis of disease-free survival (DFS) in a study involving more than 1,900 patients separated into low and high risk groups (Clark GM, et al., Proc Am Soc Clin Oncol 1997;16:129A)

78 ER - 10x = 8/8

80 PR - 10x = 6-8/8

82 Fitzgibbons PL, Murphy DA, Hammond EH, et al: Recommendations for validating estrogen and progesterone receptor immunohistochemical assays. Arch Pathol Lab Med 2010;134: all laboratories with validated ER and PgR IHC assays must periodically reassess the assays to ensure that their analytic sensitivity has not drifted.

83 Published Benchmarks (ER/PR): For women over 65 years of age, the % of negative cases should not exceed 20% For low-grade invasive carcinomas, the proportion of negative cases should not exceed 5% If the proportion of negative cases exceeds these rates, investigation is warranted.

84 For those of you performing IHC detection of HER2 protein overexpression, what is your HER2 positive rate?

85 Ross J: Saving lives with accurate HER2 testing. Am J Clin Pathol 2010;134: A number of experts in the field have now agreed that a laboratory performing HER2 testing in the US patient population should have a HER2+ rate of approximately 16% with a range of 12% to 20%.

86 Lal P, Tan LK, Chen B: Correlation of HER2 status with ER and PR and histologic features in 3,655 invasive breast carcinomas. AM J Clin Pathol 2005;123: Studied the inverse relationship between HER2 and ER and PR, and correlated HER2 status with histologic features in 3,655 unselected invasive breast carcinomas.

87 Findings: Overall ER expression rate was 74% Overall PR expression rate was 49% Overall HER2 positive rate was 16% HER2 was positive in 11% of grade 2 and 28% of grade 3 ductal carcinomas and negative in all grade 1 ductal carcinomas Only 3 of 357 (0.8%) lobular carcinomas were positive for HER2 (pleomorphic)

88 CLP/Hartford Hospital Predictive IHC Testing 2011-Present Case number Block designation Formalin contact time Patient age ER PR HER2 FISH Oncotype DX Previous specimen Comments Repeat on excision Immunoprofile confirmed S/P neoadjuvant chemotherapy Treated with Herceptin

89 Pleural Fluid Metastatic Breast CA H&E Direct Smear Cell Block ER-6F11

91 Cell Block Preparation Collect specimen in RPMI or saline Centrifuge specimen Pour-off supernatant Add 4-5 drops of plasma to sediment; mix Add 2-3 drops of thrombin (5,000 IU); mix Mixture should clot within one minute Add formalin (divide if necessary) Wrap in filter paper and process

92 HER2 Detection in FNA Cytology Cytology FNA vs. Formalin-Fixed Tissue?

93 Decalcified Bone Marrow Core Biopsy ER clone 6F11

94 The successful IHC detection and interpreation of ER, PR, and HER2 in breast cancer occurs when there is a Partnership between breast surgeons, radiologists, oncologists, and, most importantly, the pathology staff.

Brief Formalin Fixation and Rapid Tissue Processing Do Not Affect the Sensitivity of ER Immunohistochemistry of Breast Core Biopsies

Brief Formalin Fixation and Rapid Tissue Processing Do Not Affect the Sensitivity of ER Immunohistochemistry of Breast Core Biopsies Victoria Sujoy, MD, Mehrdad Nadji, MD, and Azorides R. Morales, MD From