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Effect of Ischemic Time, Fixation Time, and Fixative Type on HER2/neu Immunohistochemical and Fluorescence In Situ Hybridization Results in Breast Cancer

Neda A. Moatamed MD, Gouri Nanjangud PhD, Richard Pucci PA, Alarice Lowe MD, I. Peter Shintaku PhD, Saeedeh Shapourifar-Tehrani MPH, Nagesh Rao PhD, David Y. Lu MD, Sophia K. Apple MD
DOI: http://dx.doi.org/10.1309/AJCP99WZGBPKCXOQ 754-761 First published online: 1 November 2011


Accurate determination of HER2/neu status in breast carcinoma is essential. Alteration of preanalytic variables is known to affect HER2/neu results. American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) issued guidelines to standardize fixation for increased HER2/neu accuracy. We studied the effects of changing preanalytic variables on HER2/neu immunohistochemical and fluorescence in situ hybridization (FISH) results in a known HER2/neu+ invasive carcinoma. The clinical specimen was processed according to ASCO/CAP guidelines, with remaining tumor stored fresh without any fixatives for 4 days at 4°C and cut into core biopsy–sized pieces. Each was fixed in 10% formalin, 15% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura molecular fixative (Sakura Tissue-Tek Xpress, Torrance, CA), or zinc formalin for 0 to 168 hours. Immunohistochemical studies and FISH were performed. Compared with the clinical specimen, the samples showed no tumor degradation or marked difference by immunohistochemical studies, except the 1-hour 10% formalin and Bouin samples, or FISH, except the Bouin-fixed samples. Our study demonstrates that HER2/neu results remain accurate beyond ASCO/CAP-recommended preanalytic variables, with the exception of Bouin solution for FISH analysis.

Key Words:
  • Breast carcinoma
  • HER2/neu
  • Immunohistochemistry
  • Fluorescence in situ hybridization
  • Preanalytic variables

Trastuzumab (Herceptin) therapy is indicated as adjuvant chemotherapy for human epidermal growth factor receptor type 2 (HER2/neu) overexpressed or amplified breast cancer with positive, and, more recently, with negative lymph nodes. Therefore, an accurate assessment of HER2/neu status is critical for the selection of patients for trastuzumab therapy. Initially, overexpression and/or amplification of HER2/neu were reported to occur in 25% to 30% of patients with breast cancer.1 More recently, HER2/neu+ rates have varied from 3% to 30% in the literature.25 Our institution had an average rate of HER2/neu amplification by fluorescence in situ hybridization (FISH) of 17% from the years 2002 to 2009.

Two main methods exist to detect HER2/neu status: immunohistochemical analysis and FISH assays. Other methods that may be used to detect HER2/neu status include chromogenic in situ hybridization (CISH), silver in situ hybridization, polymerase chain reaction, and, more recently, enzyme-linked immunosorbent assay to measure the amount of circulating HER2 protein in the blood. In the United States, only the immunohistochemical, FISH, and CISH assays have been approved by the US Food and Drug Administration (FDA) for testing for HER2/neu. Although these assays are FDA-approved, there is no true “gold standard” for HER2/neu testing in breast cancer. Some authors have suggested that FISH should be used as the primary assay for HER2/neu testing owing to less subjectivity in interpretation and less day-to-day variability in laboratory settings as compared with the immunohistochemical method. Moreover, initial clinical trials showed better clinical response rates to HER2/neu-amplified breast tumors determined by FISH than to HER2/neu+ tumors detected by immunohistochemical assays. However, there is no gold standard for evaluating the accuracy of an HER2/neu test; no assay exists that can identify with accuracy and precision all patients who are expected to benefit from trastuzumab therapy.6

Problems have also been encountered with regard to local vs centralized HER2/neu testing. Reddy et al7 have shown that HER2/neu testing is most accurate when performed at high-volume central (reference) laboratories. The difference between test results from such central laboratories and results from local, community-based laboratories can be significant. In one study, the discordance rate between low-volume local laboratory and high-volume central laboratory HER2/neu immunohistochemical and FISH results was documented to be as high as 26%.8 Another study showed a discordance rate of 18% between local and central laboratories for HER2/neu evaluated by immunohistochemical studies and FISH.9 Adding another dimension to central vs local testing is the comparison between central FISH results and outside laboratory immunohistochemical results. Using centralized FISH test results as the gold standard, Press et al10 demonstrated a 21.8% false-positive rate and an 8.9% false-negative rate for HER2/neu immunohistochemical studies performed in local laboratories.

To address the issue of HER2/neu testing accuracy, the American Society of Clinical Oncology (ASCO), the College of American Pathologists (CAP), and the National Comprehensive Cancer Network (NCCN) recently set forth new guidelines and recommendations. One of the recommendations was to standardize certain preanalytic factors, such as the time between specimen collection and placement of the specimen into fixative (ischemic time), the duration of fixation, and the fixative type. The ASCO/CAP advocated promptly placing breast specimens into fixative to minimize ischemic time to 1 hour or less, using minimum and maximum fixation times of 6 hours and 48 hours, respectively, and fixing the tissue with only 10% neutral buffered formalin (NBF).

With this background in mind, the aim of the current study was to determine the effect of altering the aforementioned preanalytic factors on HER2/neu immunohistochemical and FISH results by the following: (1) storage of the breast specimen 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. Such duration was chosen because many laboratories may not process tissues from late Friday surgeries during the weekend, and, occasionally, tissue may not be processed for an even longer period (eg, long holiday weekends); (2) fixation of breast core needle–sized tissues for varying durations, ranging from 0 to 168 hours (1 week); and (3) fixation of breast tissues in a variety of fixatives, such as 15% NBF, Bouin solution, Sakura molecular fixative (SMF; Sakura Tissue-Tek Xpress, Torrance, CA), Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), and zinc formalin, in addition to routine 10% NBF.

All remaining variables such as other preanalytic, analytic, and postanalytic factors were kept constant.

Materials and Methods

We had a modified radical mastectomy specimen with a 10-cm, known grade 3 invasive ductal carcinoma (estrogen receptor [ER]–, progesterone receptor [PR]–, and HER2/neu+ [immunohistochemical score 3+ and FISH amplification] determined by core needle biopsy studies before mastectomy) for routine clinical sampling and breast biomarker immunohistochemical staining according to the ASCO/CAP guidelines. The specimen was received directly from the operating room. We immediately inked the specimen, “bread-loafed” it, and identified the 10-cm tumor; all this was done within 1 hour. More than 80% of the tumor sample was removed and stored at 4°C without any fixative. The remainder of the specimen was used for clinical purposes and fixed in 10% NBF for 12 hours.

The patient did not receive neoadjuvant chemotherapy before mastectomy. (We waited 2 years to obtain a tumor with a size substantial enough to do this study without neoadjuvant chemotherapy before surgery; most of our institutional cases are pathologic T1 to T2 in tumor size.) The remaining tumor was stored fresh at 4°C for 4 days and subsequently cut into 97 core needle biopsy–sized pieces (length, 0.5–1.5 cm; diameter, 0.2 cm). The proportion of sample size and fixative volume was constant among all samples. Each piece was fixed in 20 mL of 10% NBF for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, or 168 hours or in 20 mL of Pen-Fix, Bouin solution, SMF, zinc formalin, or 15% NBF at similar intervals Table 1.

The fixatives were as follows: 10% NBF (catalog No. 575A-5 gl) and 15% NBF (catalog number 5806-5 gl), Medical Chemical, Torrance, CA; Pen-Fix (catalog No. 6101); Bouin solution (catalog No. 456A-1 gl), Medical Chemical; SMF (catalog No. 7120), Sakura Tissue-Tek Xpress; and 10% neutral buffered zinc formalin (catalog No. 59201ZF, Richard-Allan Scientific). Samples were processed on the Sakura Tissue-Tek VIP tissue processor (Sakura) with no additional fixation: stations 1–3 were skipped to control the exact fixation time for our study Table 2. Samples fixed for 0 to 12 hours were processed in the first day. Samples fixed for 24, 48, 72, and 168 hours were processed on 4 separate days. One H&E-stained slide was prepared from each block. The overall procedure undertaken was similar to that of another study performed by our group on the assessment of ER and PR results.11

View this table:
Table 1

Immunohistochemical Methods

The FDA-approved HercepTest was performed. This test uses the DAKO A0485 polyclonal antibody kit (DAKO, Carpinteria, CA).

Immunohistochemical Scoring

In accord with ASCO/CAP and NCCN guidelines, we placed HER2/neu immunohistochemical test results into 3 categories: negative for HER2 protein overexpression (scores 0 [no staining observed in invasive tumor cells] and 1+ [weak, incomplete membrane staining in any proportion of invasive tumor cells or weak, complete membrane staining in <10% of cells]), indeterminate (2+ [complete membrane staining that is nonuniform or weak but with obvious circumferential distribution in at least 10% of cells or intense complete membrane staining in ≤30% of tumor cells]), and overexpression (3+ [uniform, intense membrane staining of >30% of invasive tumor cells]).3,12,13 All samples were scored by 2 pathologists blindly (N.A.M. and S.K.A.). For each sample, an average score rounded to the nearest whole number was calculated, eg, when an average score of 2.5+ was obtained, it was rounded to 3+.

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Table 2
Image 1

Sample showing invasive ductal carcinoma (H&E, ×400).

FISH Methods

Slides containing 4-μm sections were coded 1 through 97 and submitted for FISH analysis. For each slide, based on the corresponding H&E-stained slide, the invasive tumor area(s) was(were) circled with a Secureline marker. No areas containing ductal carcinoma in situ or normal tissue were evaluated by HER2/neu FISH. Slides were baked overnight at 60°C and pretreated using the VP2000 tissue processor according to the manufacturer’s protocol (Abbott Molecular, Abbott Park, IL). Amplification of the HER2/neu gene was detected by using the PathVysion Kit; the instructions in the package insert were followed for the hybridization, posthybridization washing, and analysis steps (Abbott Molecular). HER2/neu amplification was defined as an HER2/CEP17 ratio of greater than 2.2, no amplification was defined as an HER2/CEP17 ratio of 1.8 or less, and equivocal was defined as an HER2/CEP17 ratio of greater than 1.8 to 2.2.


The patient’s tumor Image 1 showed an HER2/neu immunohistochemical score of 3+ and demonstrated an HER2/CEP17 ratio of 7.8 by FISH analysis under routine clinical sampling and processing, according to the ASCO/CAP guidelines. Of 97 blocks, 8 showed no tumor cells to test for HER2/neu. Blocks without tumor were as follows: 15% NBF at 1 and 4 hours, Bouin solution at 4 and 168 hours, SMF at 4 and 8 hours, Pen-Fix at 11 hours, and zinc formalin at 3 hours. After 4 days at 4°C in the fresh state, the tumor showed no degradation, and there were no differences in the quality of tissue sections on H&E-stained slides or immunohistochemical slides.

Effect of Fixative Type and Fixation Time on HER2/neu Immunohistochemical Results

Of 89 blocks that contained tumor, 87 showed 2+ or 3+ HER2/neu immunohistochemical staining, and 2 blocks (10% NBF and Bouin solution at 1 hour of fixation) showed 1+ staining. The unfixed sample showed 3+ staining Image 2. The most consistent score was 3+ from Pen-Fix fixation at all times. Fixation with 10% NBF resulted in a 1+ score at 1 hour, variable 2+ to 3+ staining from 2 to 48 hours, and 3+ staining at 72 or more hours Image 3, which is beyond the fixation duration espoused in the ASCO/CAP guidelines. Other fixatives such as 15% NBF, Bouin solution, SMF, Pen-Fix, and zinc formalin did not affect the overall HER2/neu immunohistochemical results significantly because immunohistochemical scores were predominantly 3+ with such fixatives (with the exception of Bouin solution), and scores of 2+ will trigger “reflex” FISH analysis in most institutions. For core samples that were scored as 2+, for the most part, this was due to a decrease in the percentage of cells with strong complete staining. Core samples that were scored as 1+, which were the 1-hour 10% NBF and Bouin samples, showed weaker and partial membrane staining Table 3.

Image 2

HER2/neu immunohistochemical stain of unfixed specimen (0 hours fixation) (×200).

Effect of Fixative Type and Fixation Time on HER2/neu FISH Results

All specimens fixed in 10% NBF showed successful hybridization in the first attempt, and the fixation duration had no significant effect on the HER2/CEP17 ratio (range, 8.25–13.97) Image 4 and Image 5. With Bouin solution fixation, all specimens, with the exception of the 1 fixed for 1 hour, failed to exhibit hybridization. The HER2/CEP17 ratio in the lone Bouin solution–fixed specimen with successful hybridization was 12.56, consistent with HER2/neu gene amplification. For the remaining 4 fixatives (15% NBF, SMF, Pen-Fix, and zinc formalin), the effect of fixation time on HER2/neu amplification was variable; in specimens with successful hybridization, there was unequivocal amplification of HER2/neu, with the HER2/CEP17 ratio ranging from 7.94 to 14.21 for 15% NBF, 11.04 to 15.31 for SMF, 10.01 to 13.83 for Pen-Fix, and 7.86 to 15.09 for zinc formalin Table 4. The unfixed specimen failed to hybridize.

Image 3

HER2/neu immunohistochemical stain after 168 hours of 10% formalin fixation (×400).

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Table 3
Image 4

Interphase nuclei showing HER2 amplification (red) and multiple copies of centromere 17 (green) after 1 hour (×1,000).


Hormone receptor studies, such as ER and PR analysis by immunohistochemical staining, along with the determination of HER2/neu status, are required in the assessment of all breast cancers for therapeutic purposes. Accurate testing and reporting of such predictive and therapeutic markers has been problematic, however, prompting several efforts to improve the situation, such as the publication of the ASCO/CAP and NCCN guidelines. These guidelines dictate that the failure to adhere to certain preanalytic conditions is grounds for the rejection of breast specimens for HER2/neu immunohistochemical and FISH testing. Specimens that should be rejected for HER2/neu analysis include tissue fixed in formalin for less than 1 hour and any specimen fixed in formalin for longer than 48 hours. The ASCO/CAP guidelines recommend that each laboratory document in the pathology report of each cancer case the total number of hours that samples are fixed in 10% NBF.3 Such guidelines were produced in response to studies that indicated that up to 20% of HER2/neu results obtained from small, local laboratories may be significantly discordant with those from large, central reference laboratories.8,9

Image 5

Interphase nuclei showing HER2 amplification (red) and multiple copies of centromere 17 (green) after 168 hours of formalin fixation (×1,250).

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Table 4

The CAP Laboratory Accreditation Program mandated that laboratories be in compliance with the ASCO/CAP guidelines starting in 2008 and participate in the CAP Proficiency Testing program. Documenting the length of breast specimen fixation time was an important issue addressed in the HER2/neu testing guidelines, with a recommendation for fixation of specimens in 10% NBF for at least 6 hours and not more than 48 hours. Laboratories are now expected to develop policies to address breast cancer cases that will require HER2/neu analysis. For example, breast cancer specimens from late Friday surgeries cannot be fixed in 10% NBF over the weekend, which would result in more than 48 hours of fixation, and cannot be left fresh in a refrigerator during the weekend, which would prolong the ischemic time to more than 1 hour. By using breast cancer tissue microarray blocks, Khoury et al14 studied the effect of delay to formalin fixation on ER and PR immunohistochemical results and on HER2/neu FISH results. They found that ischemic times greater than 1 hour negatively affected breast cancer biomarker results. Therefore, they concluded that breast specimens should not be stored fresh overnight at 4°C. Another part of laboratory policies now geared toward meeting the ASCO/CAP guidelines is the increased need for collaboration with radiologists and surgeons, whose teams will be required to document tissue fixation start times for breast core needle biopsy specimens (obtained in the radiology suite) and for excisional biopsy specimens (obtained in the operating room).

For some authors, the application of the ASCO/CAP guidelines has resulted in better breast biomarker testing. Middleton et al15 showed that after standardized fixation and adherence to the ASCO/CAP guidelines for HER2/neu testing, immunohistochemical and FISH had increased correlation, with decreased numbers of inconclusive FISH cases. Furthermore, following the ASCO/CAP guidelines led to decreased repeated ER requests and greater financial savings for their department.

Although the CAP has mandated that accredited laboratories be in compliance with the ASCO/CAP guidelines, a study by Nakhleh et al,16 in which survey questionnaires were sent to 757 laboratories, showed that there are still gaps in HER2/neu testing validation. Their study demonstrated that only 27% of the surveyed laboratories reported breast specimen fixation times in the pathology reports. In addition, their data showed that when comparing HER2/neu immunohistochemical with FISH, 81% and 73% of laboratories reached the 95% concordance level that the ASCO/CAP guidelines recommend for negative and positive test results, respectively.

In our study, after much delay in fixation (ischemic time of 4 days, such as would be encountered during a long weekend, including a holiday weekend), the tumor still showed no degradation of histomorphologic features and no significant differences in HER2/neu immunohistochemical and FISH results. Exceptions to this statement are as follows: (1) For HER2/neu immunohistochemical testing, after fixation for 1 hour with 10% NBF or Bouin solution, erroneous results occurred with a score of 1+ (no overexpression). However, it is interesting that no fixation (0 hours) resulted in an immunohistochemical score of 3+. (2) For HER2/neu FISH testing, fixation with Bouin solution led to failure to hybridize except for 1 condition (after 1 hour fixation).

Our results show that while Bouin solution may work for HER2/neu immunohistochemical studies, this fixative is not ideal for FISH testing. With regard to all other fixatives and at all fixation time lengths, the FISH tests showed amplification whenever there was hybridization. HER2/neu gene amplification as assessed by FISH was seen not only with fixation durations between the ASCO/CAP-recommended 6 to 48 hours but also with fixation times of 1 to 5 hours (Image 4), 48 hours, and even up to 168 hours (1 week; Image 5). For the most part, there was also no significant change in HER2/neu immunohistochemical results after 2 hours of fixation and up to 168 hours fixation (Image 3). The optimal fixative for HER2/neu FISH analysis seems to be 10% NBF, followed by 15% NBF, zinc formalin, Pen-Fix, and SMF, in decreasing order of desirability.

Our results regarding HER2/neu FISH analysis were similar to those of Selvarajan et al,17 who studied 35 breast cancer cases using the PathVysion HER2 (Vysis, Downers Grove, IL) FISH test, and found that with 10% NBF, fixation periods from 2 hours to 1 week did not affect FISH results. Beyond 1 week of fixation, however, there was no signal detection, with the conclusion that breast specimens fixed for a period shorter than 1 week are suitable for FISH analysis.

We found ultimately that changing preanalytic factors such as ischemic time, fixative type, and fixation time (ranging from 0–168 hours) did not significantly alter HER2/neu immunohistochemical and FISH results, except under the 2 aforementioned conditions. In fact, our study shows that FISH is a robust assay, which is less sensitive to prolonged ischemia, variable fixation time, and variable fixatives. We reported similar findings with regard to ER and PR immunohistochemical results for breast cancer cases.11

Of note, we did not evaluate indeterminate immunohistochemical cases or equivocal FISH cases; doing so may lead to significantly altered results with varying preanalytic factors. The core samples used in this study were deliberately not handled in a manner similar to clinical samples. The study was confined to a single HER2/neu+ case. No conclusions or extrapolations can be made about how these testing conditions would affect negative or equivocal cases.

Moreover, we did not take into account the possibility of heterogeneity within our tumor specimen. Intratumoral heterogeneity occurs in breast cancers and may lead to inaccurate HER2/neu test results.18

In terms of future directions, additional studies need to be completed to verify our findings before concluding definitively that variation in preanalytic factors does not significantly alter HER2/neu immunohistochemical and FISH results. Other variables, such as analytic and postanalytic factors, should also be investigated to determine the reasons for discordance between HER2/neu immunohistochemical and FISH results obtained from low-volume, small laboratories and those from high-volume, reference laboratories.


In our study, altering the preanalytic variables of fixative type, fixation time (ranging from 0 to 168 hours), and ischemic time (with a delay in fixation of 4 days at 4°C) may still produce accurate results on HER2/neu immunohistochemical and FISH analysis in breast core needle biopsy–sized specimens.


Upon completion of this activity you will be able to:

  • list preanalytical factors that may alter the accuracy of HER2/neu breast biomarkers by immunohistochemical stains and fluorescence in situ hybridization.

  • compare the preanalytical factors affecting HER2/neu breast biomarker analysis by consensus recommendations and guidelines for American Society of Clinical Oncology/College of American Pathologists with our findings.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. 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 818. Exam is located at www.ascp.org/ajcpcme.


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