8 In this study, we investigated the minimum length of formalin fixa- tion time for core biopsy specimens of breast cancer without affecting the sensitivity of ER
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17 oct 2013 · Published studies have documented that a minimum of 6-8 hours formalin fixation is needed to obtain consistent IHC assay results for ER; fixation for less than this time has been shown to cause false negative ER staining
Brief Formalin Fixation and Rapid Tissue Processing Do Not Affect
8 In this study, we investigated the minimum length of formalin fixa- tion time for core biopsy specimens of breast cancer without affecting the sensitivity of ER
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522 Am J Clin Pathol 2014;141:522-526522
DOI: 10.1309/AJCP
O7Z4SFIYDSXN © American Society for Clinical PathologyAJCP / Original Article
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; Rapi d processing; Conventional processingDOI: 10.1309/AJCPO7Z4SFIYDSXN
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 from22 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. Immunohisto chemical detection of ER requires adherence to standardization of preanalytic variables including the cold ischemic time, type of fixative, and duration of fixation. 1-4The 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. 5This fixation time, however, may be appro
priate 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. 8In this
study, we investigated the minimum length of formalin fixa tion 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 Downloaded from https://academic.oup.com/ajcp/article/141/4/522/1761005 by guest on 30 May 2023
Am J Clin Pathol 2014;141:522-526 523523
DOI: 10.1309/AJCP
O7Z4SFIYDSXN 523© American Society for Clinical PathologyAJCP / Original Article mammary carcinoma were selected for this study. All speci mens were collected fresh from the operating rooms within 30 minutes of resection. The samples were immediately exam ined for adequacy of tumor volume for diagnostic purposes; nine cases that were judged to have inadequate tumor mass were excluded from the study. A 1.0 × 1.0 × 0.2-cm block of tumor was removed from each of the remaining 22 cases. The fresh tissue blocks were further subdivided into five 1.0 × 0.2 × 0.2-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 and48 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, Tor rance, 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-bufferedformalin 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- m paraffin sections were prepared for ER-IHC. The stepwise procedure for ER-IHC is outlined inTable 1
. Briefly, following heat-induced antigen retriev- al, 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 reac tion 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 of0 being negative, scores of 1 to 5 moderately positive, and
scores of 6 and 7 strongly positive. Two observers indepen- dently 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 epi thelial elements were present in at least one of the five cores,Table 1
Stepwise Procedure for ER Immunohistochemistry
aParaffin sections cut at 3
mMelt 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, coverslipER, estrogen receptor.
a Steps 5 through 11 were carried out in Autostainer Link 48 (Dako, Carp interia, CA).All reagents were from Dako.
30minF60minF
Rapid tissue processor
Slide A
Cores 1-3Slide BCores 4-5Overnight processor
H&E and ER-IHCH&E and ER-IHC24
hF1wkF12hFFigure 1
Schematic presentation of sample preparation,
fixation, and processing of 22 breast cancer specimens. ER,estrogen receptor; F, formalin; IHC, immunohistochemistry.Downloaded from https://academic.oup.com/ajcp/article/141/4/522/1761005 by guest on 30 May 2023
524 Am J Clin Pathol 2014;141:522-526524
DOI: 10.1309/AJCP
O7Z4SFIYDSXN © American Society for Clinical PathologySujoy et al / ER-IHC and Brief Fixation i
n Breast Biopsies serving as the internal positive controls. Table 2 summa- rizes the mea scores of 17 ER-positive tumors. All tumors showed Q scores of 6 to 7 (strongly positive) regardless of the length of fixationImage 1
. There were no significant dif- ferences in the intensity or the percentage of positive nuclei in cores 1, 2, 3, and 4 with fixation times of 30 minutes to168 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.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 inher ent 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 detec tion 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 fixa tion time was based on the result of a single study published in 2003.9 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 can cers to formalin for different periods and concluded that quan titative 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. 6A number of recent studies have
also questioned the supporting evidence for ASCO/CAP rec ommendations.In 2010, Ibarra and colleagues
8 studied tissuesamples 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, regard-less 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,