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

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1 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 the Department of Pathology, University of Miami, Miller School of Medicine, Jackson Health System and Sylvester Cancer Center, Miami, FL. Key Words: Estrogen receptor; ER; IHC; ASCO/CAP guidelines; Formalin fixation; Rapid processing; Conventional processing DOI: /AJCPO7Z4SFIYDSXN ABSTRACT Objectives: Recent studies have questioned the supporting evidence for the American Society of Clinical Oncology/ College of American Pathologists (ASCO/CAP) guidelines of the 8-hour minimum fixation time required for estrogen receptor immunohistochemistry (ER-IHC) assays in breast cancer. Methods: We investigated whether brief formalin fixation together with rapid tissue processing affects the sensitivity of ER in core breast biopsies. Five core samples each from 22 mastectomy specimens were collected and fixed in 10% formalin for periods ranging from 30 minutes to 1 week. Core 5 was fixed and processed according to the ASCO/CAP guidelines. ER-IHC was performed following heat-induced antigen retrieval using antibody 1D5. The proportion and intensity of reaction was recorded using the Q score. Results: Five of 22 cancers were ER negative in all cores. In 17 ER-positive cases, no differences were found in the intensity of reaction between 30 minutes and 1 week of formalin fixation. Similarly, no difference was observed in the Q scores of rapidly and conventionally processed control tumor cores. Conclusions: Brief formalin fixation along with rapid processing has no negative effect on the sensitivity of ER-IHC in breast core biopsies. This combination significantly reduces the turnaround time for preparing breast needle biopsy specimens. The decision to initiate treatment for patients with breast cancer is based on the results of tissue biomarker studies including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Breast core needle biopsy specimens are now the primary source for the performance of these tests, particularly if neoadjuvant therapy is being considered. Preanalytic variables may affect the accuracy of ER, PR, and HER2 results. Immunohistochemical detection of ER requires adherence to standardization of preanalytic variables including the cold ischemic time, type of fixative, and duration of fixation. 1-4 The guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommend a minimum of 8 hours of formalin fixation for breast tissue specimens regardless of the sample type. 5 This fixation time, however, may be appropriate for large breast tissue specimens, such as lumpectomy and mastectomy, but the evidence that it should also be applied to core needle biopsy specimens is insufficient. In fact, since the publication of the ASCO/CAP guidelines, several studies have showed that fixation times that are considerably shorter than 8 hours have no negative impact on the sensitivity of ER immunohistochemistry (IHC) assay in breast biopsies. 6-8 Similarly, although the ASCO/CAP guidelines recommend conventional tissue processing for breast samples, others have questioned the necessity for the longer processing time. 8 In this study, we investigated the minimum length of formalin fixation time for core biopsy specimens of breast cancer without affecting the sensitivity of ER expression. Materials and Methods Thirty-one consecutive cases of total or partial mastectomy samples from patients with biopsy-proven invasive 522 Am J Clin Pathol 2014;141: DOI: /AJCPO7Z4SFIYDSXN

2 mammary carcinoma were selected for this study. All specimens were collected fresh from the operating rooms within 30 minutes of resection. The samples were immediately examined for adequacy of tumor volume for diagnostic purposes; nine cases that were judged to have inadequate tumor mass were excluded from the study. A cm block of tumor was removed from each of the remaining 22 cases. The fresh tissue blocks were further subdivided into five cm cores and immediately immersed in 120 ml of 10% neutral phosphate-buffered formalin. The lengths of fixation for the cores were as follows: core 1 was fixed for 30 minutes, core 2 for 60 minutes, core 3 for 24 hours, and core 4 for 1 week (168 hours). Core 5 was fixed in parallel with the main mastectomy specimen for a period that ranged between 12 and 48 hours. This core served as the control specimen because it was fixed and processed according to ASCO/CAP guidelines. Cores 1 through 4 were processed in a formalin-free, microwave-assisted tissue processing system for a total processing time of 80 minutes (Xpress, Sakura Finetek, Torrance, CA). Core 5 was processed overnight (10 hours) in a conventional tissue processor (VIP, Sakura Finetek). The VIP instrument has two stations containing 10% neutral-buffered formalin that provides an additional 90 minutes of formalin exposure. The processed samples from both machines were embedded in paraffin, and the blocks were stored at room temperature until all 22 cases were processed and ready for microtomy. Three-micrometer sections from the paraffin blocks of each case were placed onto two glass slides in the following manner: slide A contained cores 1, 2, and 3, and slide B contained cores 4 and 5 Figure 1. Slides A and B from each case were stained with H&E and reviewed to ensure the presence of invasive cancer in each core. Additional 3-mm paraffin sections were prepared for ER-IHC. The stepwise procedure for ER-IHC is outlined in Table 1. Briefly, following heat-induced antigen retrieval, the slides were exposed to monoclonal anti-er antibody ID5 and the FLEX polymer detection system. DAB was used as chromogen in the presence of hydrogen peroxide. The proportion of positive nuclei and the intensity of reaction were semiquantitatively assessed using the Q score. 3 In brief, the score for intensity from 0 to 3 was added to the score for proportion of positive nuclei from 0 to 4. This resulted in Q scores with a range of 0 to 7, with a score of 0 being negative, scores of 1 to 5 moderately positive, and scores of 6 and 7 strongly positive. Two observers independently scored each case. Results Review of H&E-stained slides confirmed the presence of invasive carcinoma in all five cores of each case; they were all infiltrating ductal carcinomas of no special type. In all cases the histologic features of five cores on slides A and B, as well as their tinctorial properties, were comparable. Five of the 22 tumors were negative for ER in all five cores. In three of these cases, ER-positive non-neoplastic epithelial elements were present in at least one of the five cores, min min F F 24 hf 1 wk F 12 hf Table 1 Stepwise Procedure for ER Immunohistochemistry a Rapid tissue processor Slide A Cores 1-3 H&E and ER-IHC Overnight processor Slide B Cores 4-5 H&E and ER-IHC Figure 1 Schematic presentation of sample preparation, fixation, and processing of 22 breast cancer specimens. ER, estrogen receptor; F, formalin; IHC, immunohistochemistry. Paraffin sections cut at 3 mm Melt paraffin at 58 C, dewax in xylene Rehydrate tissue in decreasing grades of ethanol Target retrieval at ph 9 TBS rinse Peroxidase blocking ER-1D5 at 1:50 dilution; 30 minutes Linker solution; 15 minutes HRP polymer; 30 minutes Diaminobenzidine; 10 minutes Hematoxylin; 5 minutes Dehydrate in decreasing grades of ethanol, xylene, coverslip ER, estrogen receptor. a Steps 5 through 11 were carried out in Autostainer Link 48 (Dako, Carpinteria, CA). All reagents were from Dako. Am J Clin Pathol 2014;141: DOI: /AJCPO7Z4SFIYDSXN 523

3 Sujoy et al / ER-IHC and Brief Fixation in Breast Biopsies serving as the internal positive controls. Table 2 summarizes the mean Q scores of 17 ER-positive tumors. All tumors showed Q scores of 6 to 7 (strongly positive) regardless of the length of fixation Image 1. There were no significant differences in the intensity or the percentage of positive nuclei in cores 1, 2, 3, and 4 with fixation times of 30 minutes to 168 hours. Similarly, the Q scores of all cores processed by the microwave-assisted rapid processor were identical to the control cores (core 5) that were processed overnight in the conventional instrument. The semiquantitative Q scores that were determined independently by the two observers were concordant for all ER-positive cases. Table 2 The Mean Q Scores of ER-IHC in 17 ER-Positive Carcinomas in Relation to Length of Formalin Fixation and the Processing Method Core Fixation Time Processing Mean Q Score 1 30 min Rapid min Rapid h Rapid 6-7 a h Rapid h Conventional 7 ER, estrogen receptor; IHC, immunohistochemistry. a In 1 of 17 cases, the Q score of core 3 with 24-hour fixation was 6 (proportion of positive cells = 4; intensity of reaction = 2). Discussion Core needle biopsies are now the primary source of samples used for the histologic diagnosis, classification, and grading of breast cancer. Furthermore, these biopsies are the primary source for performance of biomolecular testing, such as ER, PR, and HER2, to guide the clinician in choosing the appropriate mode of treatment. Specifically, the ultimate goal of testing for ER is to identify patients who will or will not benefit from endocrine therapy. Tissue detection of ER with IHC, however, may be affected by a number of preanalytic factors including the length of warm and cold ischemic times, type of fixative, duration of fixation, and the processing method. In addition, a number of analytical variables inherent to the immunohistochemical technique are also involved: the type, affinity, and specificity of the primary antibody; the nature of the antigen retrieval step; the sensitivity of the detection technique; and the overall reproducibility of the results. 1-4 It is therefore reasonable to expect that ER-IHC has such an important predictive value to be accurate and reproducible. To that end, in 2008, an expert panel was convened by ASCO and CAP to address the issue. The ASCO/CAP practice guidelines recommend a minimum of 8 hours of formalin fixation for breast tissue specimens regardless of whether they were core biopsies or larger lumpectomy/mastectomy samples. 5 It is possible that this recommendation for minimum required fixation time was based on the result of a single study published in However, a number of other studies have demonstrated that shorter fixation time in formalin does not have a negative effect on the sensitivity of ER-IHC. 6-8 In 1995, for example, Jensen and Ladekarl 6 systematically exposed 25 breast cancers to formalin for different periods and concluded that quantitative estimation of ER and the proliferative index of tumors are not significantly affected by fixation times of less than 4 hours to more than 48 hours. 6 A number of recent studies have also questioned the supporting evidence for ASCO/CAP recommendations. In 2010, Ibarra and colleagues 8 studied tissue samples from 10 invasive breast cancers and exposed them to 10% formalin for 1, 3, 6, 9, and 10 hours. They performed ER-IHC using three commonly used anti-er antibodies, SP1, 6F11, and ID5. They found no significant differences in the intensity of staining or the percentage of positive cells, regardless of length of fixation or the type of antibody. In a similar recent study, Apple et al 7 fixed breast core biopsy specimens in 10% and 15% buffered formalin and in four other tissue fixatives. The cores were exposed to each fixative for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, and 168 hours. The ER- and PR-IHC were performed after antigen retrieval. They concluded that the preanalytic variables, such as fixative type and fixation time, did not affect the accuracy of ER/PR-IHC. In the current study, we show that fixation times as short as 30 minutes and as long as 168 hours have no negative effect on the sensitivity of ER-IHC on core biopsy specimens of the breast. Furthermore, there is no significant difference between conventional tissue processing for 10 hours overnight and the microwave-assisted, 80-minute rapid processing. Together, the short fixation time and rapid processing reduced the turnaround time for breast biopsies from a minimum of 18 hours to less than 2 hours. It is noteworthy that the Q scores for all samples were uniformly 7 except for one core in a single case that was fixed for 24 hours (Table 2). This lends credence to prior observations that, with monoclonal anti-er antibody ID5, most breast cancers show a bimodal ER expression profile In other words, either ER-positive tumors show moderate to strong positivity in 80% to 90% of neoplastic cells or the tumors are completely negative for ER. It is now well established that the epitopes identified by commonly used antibodies to ER-a (1D5, 6F11, and SP1) are fixation sensitive. Delayed or inadequate fixation therefore may lead to false-negative results. 14,15 For breast core biopsy specimens, delayed fixation, or cold ischemic time, is ordinarily not an issue because core needle biopsy specimens are rapidly immersed in fixatives most of the time. Cold ischemic time, on the other hand, may affect the ER-IHC results of larger specimens, particularly if they are not appropriately trimmed. 524 Am J Clin Pathol 2014;141: DOI: /AJCPO7Z4SFIYDSXN

4 A B C E D Image 1 Estrogen receptor immunohistochemistry of core sections from a single case of infiltrating ductal carcinoma fixed in formalin for 30 minutes (A), 60 minutes (B), 24 hours (C), and 1 week (D); all were processed by the rapid processing system. E, The same tumor fixed in formalin for 48 hours and processed in the overnight processor in parallel with the main specimen. Am J Clin Pathol 2014;141: DOI: /AJCPO7Z4SFIYDSXN 525

5 Sujoy et al / ER-IHC and Brief Fixation in Breast Biopsies Recent studies have showed that, for such specimens, delayed fixation times of up to 2 hours at room temperature and up to 4 hours in a refrigerator (4 C) have no negative impact on ER-IHC. 16,17 Furthermore, other studies have concluded that, for both core biopsy and larger breast specimens, prolonged formalin fixation beyond the recommended 48 to 72 hours has no effect on the sensitivity of ER-IHC because of the universal use of antigen retrieval methods. 18,19 Our results also confirm the observations by Ibarra and colleagues 8 that, compared with routine processing, rapid tissue processing protocols show no adverse effect in ER-IHC. Together, these observations support what seasoned immunohistochemists have pointed out: that scientific evidence for a number of consensus recommendations are lacking because they represent largely faith-based exercises. 20 In summary, short fixation time in formalin and rapid processing appear to have no negative impact on the sensitivity of ER-IHC in core biopsies of the breast. The combination of short fixation and processing times enable pathology laboratories to significantly decrease the turnaround time of breast biopsies while ensuring the preservation of biomolecules. This is an important issue for patients and clinicians and calls for a revisit of ASCO/CAP-recommended minimum fixation time and processing method. It is hoped that revised guidelines will also remove the concern about the documentation of the length of formalin fixation for core breast biopsies. Address reprint requests to Dr Nadji: Dept of Pathology, University of Miami, Jackson Health System, 1611 NW 12th Ave, Holtz-2147, Miami, FL 33136; mnadji@aol.com. References 1. Yaziji H, Taylor CR, Goldstein NS, et al. Consensus recommendation on estrogen receptor testing in breast cancer by immunohistochemistry. Appl Immunohistochem Mol Morphol. 2008;16: Nofech-Mozes S, Vella ET, Dhesy-Thind S, et al. Systematic review on hormone receptor testing in breast cancer. Appl Immunohistochem Mol Morphol. 2012;20: Barnes DM, Harris WH, Smith P, et al. Immunohistochemical determination of estrogen receptor: comparison of various methods of assessment of staining and correlation with clinical outcomes of breast cancer patients. Br J Cancer. 1996;74: Gown AM. Current issues in ER and HER2 testing by IHC in breast cancer. Mod Pathol. 2008;21:S8-S Hammond MEH, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Arch Pathol Lab Med. 2010; 134: Jensen V, Ladekarl M. Immunohistochemical quantitation of oestrogen receptors and proliferative activity in oestrogen receptor positive breast cancer. J Clin Pathol. 1995;48: Apple S, Pucci R, Lowe AC, et al. The effect of delay in fixation, different fixatives and duration of fixation in estrogen and progesterone receptor results in breast carcinoma. Am J Clin Pathol. 2011;135: Ibarra JA, Rogers LW, Kyshtoobayeva A, et al. Fixation time does not affect the expression of estrogen receptor. Am J Pathol. 2010;133: Goldstein NS, Ferkowicz M, Odish E, et al. Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol. 2003;120: 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: Collin LC, Botero ML, Schnitt SJ. Bimodal frequency distribution of estrogen receptor immunohistochemistry results in breast cancer. Am J Clin Pathol. 2005;123: Schnitt SJ. Estrogen receptor testing of breast cancer in current clinical practice: what s the question? J Clin Oncol. 2006;24: Nadji M. Quantitative immunohistochemistry of estrogen receptors in breast cancer: much ado about nothing. Appl Immunohistochem Mol Morphol. 2008;16: Neumeister VM, Anagnostou V, Siddiqui S, et al. Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissue. J Natl Cancer Inst. 2012;104: Bai Y, Tolles J, Cheng H, et al. Quantitative assessment shows loss of antigenic epitopes as a function of preanalytic variables. Lab Invest. 2011;91: Qui J, Kulkarni S, Chandrasekhar R, et al. Effect of delayed formalin fixation on estrogen and progesterone receptors in breast cancer. Am J Clin Pathol. 2010;134: Yildiz-Aktas I, Dabbs DJ, Bhargava R. The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor and HER2 expression in invasive breast carcinoma. Mod Pathol. 2012;25: Arber DA. Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast markers. Appl Immunohistochem Mol Morphol. 2002;10: Tong LC, Nelson N, Tsourigiannis J, et al. The effect of prolonged fixation on the immunohistochemical evaluation of estrogen receptor, progesterone receptor and HER2 expression in invasive breast cancer: a prospective study. Am J Surg Pathol. 2011;35: Gown AM. Tweaking and nudging toward improved IHC quality (letter). Appl Immunohistochem Mol Morphol. 2009;17: Am J Clin Pathol 2014;141: DOI: /AJCPO7Z4SFIYDSXN