The Effect of Delay in Fixation, Different Fixatives, and Duration of Fixation in Estrogen and Progesterone Receptor Results in Breast Carcinoma

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1 Anatomic Pathology / Fixation Effects on ER and PR in Breast Cancer The Effect of Delay in Fixation, Different Fixatives, and Duration of Fixation in Estrogen and Progesterone Receptor Results in Breast Carcinoma Sophia Apple, MD, MS, Richard Pucci, Alarice C. Lowe, MD, Itsushi Shintaku, PhD, Saeedeh Shapourifar-Tehrani, MPH, and Neda Moatamed, MD Key Words: Estrogen receptor; Progesterone receptor; Fixatives; Fixation; Time factors; Breast carcinoma CME/SAM DOI: /AJCPB1RIT5YXMRIS Upon completion of this activity you will be able to: list preanalytical factors that may alter the accuracy of breast biomarkers by immunohistochemical stains. compare with our findings the preanalytical factors affecting breast biomarker analysis by consensus recommendations and guidelines for American Society of Clinical Oncology/College of American Pathologists. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 645. Exam is located at Abstract Accurate determination of estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinoma is essential. Preanalytic variation may contribute to discordant results. Recently, American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) made recommendations to normalize fixation for breast biomarkers. To evaluate this, a 4-cm invasive lobular carcinoma was processed according to ASCO/CAP guidelines. The remainder was stored fresh at 4 C for 4 days and cut into biopsy-sized pieces. Each was fixed in 10% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura Molecular Fixative (Sakura Tissue-Tek Xpress, Torrance, CA), zinc formalin, or 15% formaldehyde for times ranging between 1 and 168 hours. Immunohistochemical studies for ER and PR were performed and interpreted. After 4 days at 4 C, all samples showed no degradation or ER/PR staining differences, except 2 Bouin-fixed samples, in comparison with the patient s sample processed according to ASCO/CAP guidelines. In our study, the preanalytic variables of fixative type, fixation time, and 4 days of ischemic time did not affect immunohistochemical accuracy for ER/PR. Hormone receptor status, such as estrogen receptor (ER) and progesterone receptor (PR), is the single most important therapeutic predictive factor in breast cancer. There is growing concern that the error rate of ER test results by immunohistochemical analysis is unacceptably high. Consensus recommendations and guidelines for breast biomarker analysis were developed by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) in The guidelines addressed several variables, including preanalytic, analytic, and postanalytic factors. Preanalytic factor guidelines addressed promptly placing the specimens in fixative to minimize prolonged ischemia time, time to gross sectioning and transfer of samples to cassettes, minimum and maximum fixation times, and type of fixative. Lack of standards and stringent regulatory oversight may be a major cause of interlaboratory variability in ER testing. 2-4 Prolonged tumor ischemia induced by delays in formalin fixation is known to cause decreased ER and PR expression. Khoury et al 5 reported on the effects of delayed formalin fixation and concluded that without fixation, ER expression began to decline after 2 hours and PR expression after 1 hour. At the 8-hour mark, ER expression was completely negative in their study, and, hence, they recommend that specimens be fixed as soon as possible, ideally within 1 hour of receipt, and that specimens not be stored overnight at 4 C. In fact, their finding was that overnight storage at 4 C yielded similar results to leaving specimens without fixation for 8 hours. 5 An ad hoc group was formed to provide and develop evidence-based guidelines for best practices in the assessment of ER status by immunohistochemical stains in January Based on their guidelines and those from the ASCO/ CAP, some of the preanalytic recommendations were as 592 Am J Clin Pathol 2011;135: DOI: /AJCPB1RIT5YXMRIS

2 Anatomic Pathology / Original Article follows: (1) Breast specimens must be sectioned and placed in fixative within 1 hour of removal. (2) Breast core biopsy and excisional specimens require a minimum fixation of 8 hours and maximum fixation of 48 hours, with fixation not exceeding more than 72 hours. The total fixation time should be recorded in the report. (3) Only 10% buffered formalin at ph 7.0 to 7.4 should be used as the fixative. Our aim was to determine the effect of altering many preanalytic factors on ER and PR immunohistochemical results as follows: (1) The breast specimen was stored in a fresh state without any fixative in the refrigerator at 4 C for 4 days (96 hours) to assess the effect of prolonged ischemic time. This duration was chosen because many laboratories may not process tissue samples from late Friday surgery during the weekend, and the waiting time for a long holiday weekend was used. (2) Breast core size tissue was fixed for varying durations, ranging from 1 hour to 168 hours (1 week). (3) In addition to 10% neutral buffered formalin, a variety of fixatives, such as 15% buffered formaldehyde, Bouin solution, Sakura Molecular Fixative (Sakura Tissue-Tek Xpress, Torrance, CA), Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), and zinc-formalin were used. All other variables such as other preanalytic, analytic, and postanalytic factors remained constant. Materials and Methods A mastectomy specimen with a 4-cm known invasive lobular carcinoma (ER+, PR+, and HER-2/neu amplification negative by core needle biopsy studies before mastectomy) underwent routine clinical sampling and breast biomarker immunohistochemical stains according to the ASCO/CAP guidelines: 10-hour fixation in 10% neutral buffered formalin. (We waited 1 year to obtain a substantial size of tumor to do this study because most of our institutional cases are T1 to T2 tumor size.) The remaining tumor was stored fresh at 4 C for 4 days and subsequently cut into 92 core needle biopsy sized pieces ( cm long and 0.2 cm in diameter). The proportion of sample size and fixative volume was constant among all samples. Each piece was fixed in 20 ml of 10% neutral buffered formalin for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, or 168 hours or 20 ml of Pen-Fix, Bouin solution, Sakura Molecular Fixative, zinc formalin, or 15% buffered formaldehyde at similar intervals (only Pen-Fix for 168 hours). The following fixatives were used: 10% neutral buffered formalin, catalog number 575A-5 gl, Medical Chemical, Torrance, CA; 15% buffered formaldehyde, catalog number gl, Medical Chemical; Pen-Fix, catalog number 6101, Richard-Allan Scientific; Bouin solution, catalog number 456A-1 gl, Medical Chemical; Molecular Fixative, catalog number 7120, Sakura Tissue-Tek Xpress; and 10% neutral buffered zinc formalin, catalog number 59201ZF, Richard- Allan Scientific. Samples were processed on the Sakura Tissue-Tek VIP tissue processor (Sakura) with no additional fixation: stations 1 through 3 were skipped to control the exact fixation time for our study Table 1. The 1- to 12-hour fixed samples were processed during the first day. The 24-, 48-, 72-, and 168-hour samples were processed on 4 separate days. One H&E-stained slide was prepared from each block. Immunohistochemical Methods Immunohistochemical stains were performed with ER and PR antibody clones with appropriate positive and negative control samples. Slides were baked for 1 to 4 hours in a 60 C oven and then deparaffinized in xylene and brought to tap water through graded alcohols (100% 4 times; 95% 2 times). Slides were then treated with 0.5% hydrogen peroxide in absolute methyl alcohol for 10 minutes to quench endogenous peroxidase activity and washed with tap water and deionized water. Heat-induced antigen retrieval was then performed by placing slides in 0.01 mol/l citrate buffer, ph 6.0, preheated to 95 C in a vegetable steamer (Black & Decker, Baltimore, MD); heat-treatment lasted for 25 minutes, followed by a 15-minute, room-temperature, cool-down period. Following rinsing in phosphate-buffered saline, slides were immunostained on a DAKO Autostainer (DAKO, Carpinteria, CA), using a goat antimouse HRP polymer secondary antibody (MACH2 Mouse HRP-Polymer, Biocare Medical, Concord, CA) as the detection system. The staining procedure involved a 45-minute primary antibody incubation with mouse monoclonal antibodies against ER, clone 6F11 (dilution 1/50; Biocare Medical), and PR, clone 636 (dilution 1/300; DAKO), followed by a phosphate-buffered saline wash and a 30-minute incubation in MACH2 mouse Table 1 Tissue Processing * Station Reagent Time (min) 1 NBF, 10% 45 2 NBF, 10% 45 3 NBF, 10% 30 4 Alcohol, 95% 35 5 Alcohol, 100% 40 6 Alcohol, 100% 40 7 Alcohol, 100% 40 8 Xylene 45 9 Xylene Xylene Paraffin Paraffin Paraffin Paraffin 30 NBF, neutral buffered formalin. * Breast fatty tissue samples were processed on the Sakura Tissue-Tek VIP tissue processor (Sakura, Torrance, CA) with no additional fixation. Stations 1-3 were skipped to control the exact fixation time. The last step was in the vacuum for 42 minutes. Am J Clin Pathol 2011;135: DOI: /AJCPB1RIT5YXMRIS 593

3 Apple et al / Fixation Effects on ER and PR in Breast Cancer HRP-Polymer. The negative control reagent was mouse IgG1 or diluent alone. Diaminobenzidine was used as the chromogen to effect visualization of the peroxidase enzyme. Slides were then counterstained with Harris hematoxylin. Interpretation Analysis The immunohistochemical stains were independently interpreted by 2 pathologists (S.A. and N.M.) who did not know the different fixatives and times; the pathologists documented the intensity and percentage of tumor ER and PR staining. An average score is listed as the final result Table 2. Results The patient s tumor showed more than 95% of tumor cells with an intensity of 3+ for ER and PR by immunohistochemical staining in the clinical sample, which had been processed under the strict guidelines of ASCO/CAP Image 1. All 92 blocks had invasive lobular tumor, except 1, which had lobular carcinoma in situ, on which results were interpreted. After 4 days at 4 C, the tumor showed no degradation and no differences in the quality of tissue on H&E stains or ER and PR immunohistochemical staining for all samples fixed in 10% neutral buffered formalin Image 2. In fact, there was no significant difference in ER and PR staining for 15% buffered formaldehyde, zinc formalin, Pen-Fix, Sakura Molecular Fixative, and most of the Bouin-fixed samples. Only 2 Bouinfixed samples (48 and 72 hours) had a significant decrease in estrogen staining, but they were still interpreted as positive. Bouin-fixed sections showed more variable staining with PR than with ER, but the overall percentage and intensity were positive (Table 2). Discussion ER and PR studies by immunohistochemical staining along with HER-2/neu are required to be performed for all breast cancers removed for therapeutic purposes. Accurate determination of ER and PR status in breast carcinoma is essential because their presence warrants hormonal therapy. More important, they show a direct correlation between the likelihood of clinical response to hormonal therapies and the level of ER-α expression. 7 However, accurate testing and reporting has been problematic, and there are several recent and ongoing efforts to improve it, such as the recently published ASCO/CAP guidelines for HER2 testing and imminent guidelines for ER and PR testing. Several factors may alter immunohistochemical results, including preanalytic and interpretation errors. The preanalytic factors include delay to fixation (known as ischemic time), prolonged or inadequate fixation, different types of fixatives used, and choice of ER or PR antibody. Interpretive and analytic factors include different thresholds for reporting positivity and the widespread use of diverse staining procedures of unequal quality, resulting in error rates as high as 20%. 3,8-11 ASCO/CAP guidelines warrant that the presence of certain preanalytic factors is grounds for rejection of the specimen for immunohistochemical evaluation of HER2 status. These include tissue fixed in fixatives other than 10% neutral buffered formalin, core needle biopsy samples fixed in formalin less than 1 hour, and any specimen fixed in formalin longer than 48 hours. The ASCO/CAP guidelines recommend that each laboratory should document the total number of hours that samples are fixed in 10% formalin in the actual report on each breast cancer case. 1,6 ER expression is present in 66% to 70% of breast cancers. 12 PR expression is reported along with ER expression and is known to be independently associated with disease-free and overall survival; patients with ER+ and PR+ tumors have a better prognosis than patients with ER+ but PR or ER and PR tumors. 13 ER-α regulates the expression of PR, so the presence of progesterone usually indicates that the ER-α pathway is intact and functional. 14 Once expressed, PR is activated by the hormone progesterone to help regulate several important normal cellular functions, including proliferation, which, of course, is detrimental in breast cancers. PR is expressed in the nuclei of 60% to 70% of breast cancers, and the expression varies on a continuum ranging from 0% to nearly 100% positive. There is a direct correlation between PR levels and response to hormonal therapies, such that tumors with even very low levels of PR+ cells ( 1%) have a significant chance of responding. 15,16 The original methods of determining ER and PR status were biochemical, such as the dextran-coated charcoal method requiring prospective collection of fresh frozen tumor tissue without microscopic verification of tumor. The benefit of this test, however, was the quantitative determination of the ER content of the tumor, expressed as femtomoles of ER protein per milligram of cytosol protein. The dextran-coated charcoal method evaluated ER-α and ER-β. Currently, the principal method for assessment of hormonal receptors is by immunohistochemical stains using formalin-fixed, paraffin-embedded tissue. Immunohistochemical stains for ER detect only ER-α. It is critical that all immunohistochemical stains be done with positive and negative control samples. Normal breast tissue adjacent to tumor serves as an internal control sample. Prolonged or Inadequate Fixation Time It is known that formaldehyde produces reactive hydroxymethyl groups on amino acids that cross-link proteins and large molecules that are necessary for immunohistochemical 594 Am J Clin Pathol 2011;135: DOI: /AJCPB1RIT5YXMRIS

4 Anatomic Pathology / Original Article Table 2 Results for Estrogen and Progesterone Receptor Data * Estrogen Progesterone Estrogen Progesterone Receptor Receptor Receptor Receptor Hours/ Average Average Average Average Hours/ Average Average Average Average Sample No. Fixative Percentage Intensity Percentage Intensity Sample No. Fixative Percentage Intensity Percentage Intensity % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF % BF Bouin Molecular Pen-Fix Zinc % NBF Pen-Fix BF, buffered formaldehyde; NBF, neutral buffered formalin. * The results are the averages of the independent interpretations of 2 pathologists without knowledge of the fixatives and times. A positive estrogen receptor or progesterone receptor tumor shows 1% or more cells staining, and results are semiquantitated as shown. Am J Clin Pathol 2011;135: DOI: /AJCPB1RIT5YXMRIS 595

5 Apple et al / Fixation Effects on ER and PR in Breast Cancer A B C Image 1 Clinical sample processed after 10 hours of fixation in 10% neutral buffered formalin. A, H&E, 200. B, Immunohistochemical analysis for estrogen receptor ( 100). C, Immunohistochemical analysis for progesterone receptor ( 100). analysis. Prolonged tumor ischemia induced by delays in formalin fixation is known to cause decreased ER and PR expression. Khoury et al 5 reported on the effect of delay to formalin fixation and concluded that ER expression declined at the 2-hour mark and PR expression at the 1-hour mark. At the 8-hour mark, ER expression was completely negative in their study, and, hence, they recommend that specimens should be fixed as soon as possible, ideally within 1 hour, and that specimens should not be stored overnight at 4 C. In fact, their finding was that overnight storage at 4 C is similar to leaving the specimen without fixation for 8 hours. Goldstein et al 17 reported that at least 6 to 8 hours of formalin fixation time is required for breast biopsy specimens to exhibit reliable ER expression by immunohistochemical stains. The authors also noted that underfixation had more detrimental effects on immunohistochemical staining results than did overfixation. 17 Underfixed tissue can give unpredictable and unreliable results with standard antigen retrieval protocols. Overfixed tissue, with more than 72 hours of fixation, can be accepted for breast biomarker studies; however, the results should be validated by the laboratory director. We studied whether various preanalytic factors could alter ER and PR results from a mastectomy specimen with a 4-cm, known ER+, PR+, and HER-2/neu invasive lobular carcinoma, determined by previous core needle biopsy results. The patient s clinical tumor sample was grossed in a fresh state and fixed in 10% neutral buffered formalin for 10 hours before tissue processing, according to the ASCO/CAP guidelines. The remaining tumor tissue was stored fresh at 4 C for 4 days to test a long holiday weekend duration of ischemic time. The proportion of sample size and fixative volume was constant among all samples. A variety of fixatives, including 596 Am J Clin Pathol 2011;135: DOI: /AJCPB1RIT5YXMRIS

6 Anatomic Pathology / Original Article A B C D E F Image 2 Immunohistochemical analysis for estrogen receptor (ER) and progesterone receptor (PR) after varying fixation times in 10% neutral buffered formalin. A, ER fixed for 1 hour ( 100). B, PR fixed for 1 hour ( 100). C, ER fixed for 72 hours ( 100). D, PR fixed for 72 hours ( 100). E, ER fixed for 168 hours (1 week; 100). F, PR fixed for 168 hours (1 week; 100). Am J Clin Pathol 2011;135: DOI: /AJCPB1RIT5YXMRIS 597

7 Apple et al / Fixation Effects on ER and PR in Breast Cancer Pen-Fix, Bouin solution, Sakura Molecular Fixative, zinc formalin, and 15% neutral formaldehyde, was also used to test for possible alterations in ER and PR results. Different fixation times were tested, from 1 to 168 hours. We found that after much delay in fixation time (ischemic time), the tumor showed no degradation and no differences in ER and PR staining for all samples fixed in 10% neutral buffered formalin. There was no significant difference in ER and PR staining for almost all fixatives. Only 2 Bouin-fixed samples (48 and 72 hours) had a significant decrease in ER staining, but they were still interpreted as positive. Bouin-fixed sections showed more variable staining with PR than with ER, but the overall percentage and intensity were positive. Fixation time did not alter the final results of ER and PR staining. We have not found that the preanalytic factors such as delay to fixation (ischemic time), different fixatives, including non 10% neutral buffered formalin, and different fixation times varying from 1 hour to 168 hours altered ER and PR immunohistochemical results significantly. We did not evaluate weakly ER+ and PR+ cases in this study. Perhaps if we had chosen cases that were weakly positive for ER and PR, the results may have been significantly altered with all the variables of preanalytic factors. Our findings apply only to the ER 6F11 and PR 636 antibody clones and the DAKO detection system used in our study. Additional studies need to be done to verify our study results before concluding that varied preanalytic factors do not significantly alter the results of ER and PR immunohistochemical studies. Conclusions In our study, the preanalytic variables of fixative type; fixation time, ranging from 1 hour to 1 week; and a delay in fixation of 4 days at 4 C did not affect the accuracy of ER and PR immunohistochemical results in core needle biopsy sized tumor sections. From Pathology and Laboratory Medicine, UCLA Center for the Health Sciences, Los Angeles, CA. Address correspondence to Dr Apple: Dept of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, UCLA Center for the Health Sciences, Le Conte Ave CHS, Los Angeles, CA References 1. 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. J Clin Oncol. 2007;25: Rhodes A, Jasani B, Balaton AJ, et al. Frequency of oestrogen and progesterone receptor positivity by immunohistochemical analysis in 7016 breast carcinomas: correlation with patient age, assay sensitivity, threshold value, and mammographic screening. J Clin Pathol. 2000;53: Rhodes A, Jasani B, Balaton AJ, et al. Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe: documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays. Am J Clin Pathol. 2001;115: Layfield LJ, Goldstein N, Perkinson KR, et al. Interlaboratory variation in results from immunohistochemical assessment of estrogen receptor status. Breast J. 2003;9: Khoury T, Sai S, Hwang H, et al. Delay to formalin fixation effect on breast biomarkers. Mod Pathol. 2009;22: Yaziji H, Taylor CT, Goldstein NS, et al. Consensus recommendations of estrogen receptor testing in breast cancer by immunohistochemistry. Appl Immunohistochem Mol Morphol. 2008;16: Harvey JM, Clark GM, Osborne CK, et al. Estrogen receptor status by immunohistochemistry is superior to the ligandbinding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999;17: Allred DC. Commentary: hormone receptor testing in breast cancer: a distress signal from Canada. Oncologist. 2008;13: Gown AM. Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol. 2008;21(suppl 2):S8-S Hede K. Breast cancer testing scandal shines spotlight on black box of clinical laboratory testing. J Natl Cancer Inst. 2008;100: , Mathews AW. Bad cancer tests drawing scrutiny. Wall Street Journal. January 4, 2008:B1-B Nadji M, Gomez-Fernandez C, Ganjei-Azar P, et al. Immunohistochemistry of estrogen and progesterone receptors reconsidered: experience with 5,993 breast cancers. Am J Clin Pathol. 2005;123: Bardou VJ, Arpino G, Elledge RM, et al. Progesterone receptor status. 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