Abstract
Background/Aim: Tissue sample quality control has become increasingly crucial as these samples are integral to basic research and clinical practice, particularly in immunohistochemistry and gene panel testing. Standard PREanalytical Code (SPREC) was developed to standardize pre-analytical processes, including warm ischemia time (WIT), cold ischemia time (CIT), and fixation time (FT), which can influence the surgical specimen quality. This study investigated the impact of WIT, CIT, and FT on estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) expression in breast cancer (BC) surgical specimens. Patients and Methods: We enrolled 277 patients with first-time BC who underwent surgery at Kanagawa Cancer Center between May 2018 and April 2019. WIT, CIT, and FT were recorded using SPREC ver. 3.0, and their effects on ER, PgR, and HER2 expression were analyzed using immunohistochemistry. Surgical specimens were compared with preoperative needle biopsy samples from the same tumors to control for WIT, CIT, and FT variability. Results: The median WIT, CIT, and FT were 23 min, 37 min, and 43 h, respectively. Compliance with the American Society of Clinical Oncology/College of American Pathologists guidelines was 91.7% for CIT and 94.9% for FT. ER and PgR expression in surgical specimens decreased with prolonged CIT and FT, but differences were non-significant. However, HER2 expression increased significantly when WIT exceeded 30 min. Conclusion: WIT could significantly influence HER2 expression in BC surgical specimens, highlighting the need for meticulous WIT control during BC surgery to ensure accurate HER2 assessment, which is critical for guiding therapeutic decisions.
In recent years, tissue sample information has been utilized in both basic research and clinical practice, including immunohistochemical (IHC) staining and gene panel testing for diagnosis and decision-making regarding treatment. Therefore, quality control of samples has become increasingly crucial (1). The testing process using formalin-fixed paraffin-embedded samples is categorized into three stages: 1) pre-analytical, 2) analytical, and 3) post-analytical. Individual differences in samples at each stage can significantly impact the test results, with such cases most commonly occurring during the pre-analytic stage (2, 3).
Standardization methods, such as Standard PREanalytical Code ver.3.0 (SPREC) (4), Biospecimen Reporting for Improved Study Quality (BRISQ) (5), and Minimum Information About Biobank Data Sharing 2.0 Core (MIABIS) (6) have been employed for the pre-analytical management of biological samples. SPREC was developed in 2009 by the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group to provide a comprehensive and easy-to-implement tool to document the in vitro preanalytical (collection, processing, and storage) details of biospecimens. For solid samples, the following seven-element codes affecting the sample condition are recorded: 1) type of sample, 2) type of collection, 3) warm ischemia time (WIT), 4) cold ischemia time (CIT), 5) fixation type, 6) fixation time (FT), and 7) long-term storage.
The time from vascular clamp to resection (WIT), time from resection to fixation (CIT), and formalin FT are crucial factors that can significantly impact the results of solid surgical specimens. Therefore, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) has established methods for fixing, staining, and diagnosing the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) for determining the treatment strategy for breast cancer. The ASCO/CAP ER, PgR (7), and HER2 (8) guidelines recommend performing CIT within 60 min, and FT between 6 and 72 h. Although reports on the effects of CIT (9) and FT (10) on surgical specimens are available, no data exist on the effects of WIT because of the challenges in determining the vascular clamp time.
To address this, we first defined the vascular clamp time for breast cancer surgery in our previous study. Subsequently, we established a working group of surgeons, anesthesiologists, nurses, pathologists, and medical technologists in pathology and made efforts to produce quality control samples according to the SPREC ver.3.0 for solid samples. The median WIT, CIT, and FT were 23 min (3-116), 37 min (3-1,052), and 43 h (17-115), respectively. The compliance rates of CIT and FT with the ASCO/CAP ER, PgR, and HER2 guidelines improved to 89.3% and 95.2%, respectively, and the variation in the specimen processing time was smaller (11).
Here, to assess the effects of WIT, CIT, and FT on the quality of surgical specimens, we compared the differences in the IHC expression of ER, PgR, and HER2 between surgical samples and preoperative needle biopsy specimens from the same tumor tissue as controls.
Patients and Methods
Patients. The subjects were patients who underwent primary surgery for breast cancer under general anesthesia at the Kanagawa Cancer Center between May 2018 and April 2019. Cases in which paired samples from a preoperative needle biopsy and surgical specimens of the same tumor were not available, or in which there were missing values in the data, were excluded. Patients who received neoadjuvant therapy were excluded because the ER, PgR, and HER2 expression levels may differ before and after treatment (12). In addition, carcinoma in situ was excluded because ER and PgR expression was determined at the invasive site in 2020 (13). Partial mastectomy (Bp), total mastectomy (Bt), or nipple-sparing mastectomy (NSM) were selected for breast surgery procedures, and nine experienced breast surgeons performed all operations based on the same protocols of preoperative imaging and surgical procedures. This study was conducted in accordance with the relevant national regulations and institutional policies, as well as the tenets of the Helsinki Declaration. It was approved by the Ethics Committees of Kanagawa Cancer Center (28 research-24), and informed consent was obtained from all individuals enrolled.
Recording method based on SPREC ver3.0. WIT, CIT, and FT were calculated for solid SPREC ver.3.0 (Figure 1) (4). The temperature conditions at each stage were approximately 37°C body temperature for WIT and 25°C room temperature for CIT and FT. A working group was established to record WIT, CIT, and FT. The vascular clamp and collection times for WIT were reported by the surgeon during flap formation and sample removal, and subsequently recorded by anesthesiologists and nurses. The removed specimen was immediately transported to the specimen room in the pathology department, where pathologists and medical technologists recorded the start and end times of fixation required for CIT and FT, respectively (11).
Seven-element-long Standard PREanalytical Code (SPREC) for solid samples. When the surgeon performs vascular clamp and resection, anesthesiologists and nurses mark the time by pressing the icon and log it in the record. Pathologists and laboratory technicians record the specimen fixation time.
Evaluation of ER, PgR, and HER2. ER, PgR, and HER2 fixation, staining, and diagnostic procedures were performed in compliance with the ASCO/CAP ER, PgR (13), and HER2 (14) guidelines in 2018-2019, when the specimens were collected. ER and PgR were recorded as the percentage of positive cells (0-100%), and HER2 was recorded as 0, 1+, 2+, or 3+.
Sample processing time and quality. We calculated the differences in IHC expression of ER, PgR, and HER2 between surgical samples and paired preoperative needle biopsy samples from the same tumor site. This was done under conditions of extremely short WIT and 100% compliance of CIT and FT, using the ASCO/CAP guidelines for ER, PgR, and HER2 as controls. We then compared the differences in the IHC expression of ER, PgR, and HER2 between surgical and needle biopsy specimens while controlling for WIT, CIT, and FT using multiple linear regression analysis. In these models, WIT, CIT, and FT were used for the adjustment of confounding factors. R statistical software from the R Foundation was used for the analyses, with p<0.05 considered statistically significant.
Results
Between May 2018 and April 2019, 393 patients (371 patients) underwent surgical treatment for first-time breast cancer [44 patients (22 patients) had synchronous bilateral breast cancer]. The final analysis included 277 cases, excluding carcinoma in situ (72 cases), neoadjuvant therapy (38 cases), no paired samples of needle biopsy and surgical specimens from the same tumor (seven cases), and any missing values of WIT, CIT, or FT (six cases). There was some overlap among these exclusion factors.
The patient characteristics are shown in Table I. The median patient age was 62 years (range=29-84 years); early breast cancer (stage I) accounted for 51.6% (143/277); the intrinsic subtype (luminal/luminal-HER2/HER2 enriched/triple negative) was present in 207, 26, 13, and 31 cases, respectively; and the surgical procedures consisted of breast-conserving surgery, mastectomy, and NSM in 50, 224, and three cases, respectively.
Patient characteristics.
Using SPREC ver. 3.0, the median WIT, CIT, and FT were 23 min (range=3-116 min), 37 min (range=20-1,052 min), and 43 h (range=17-115 h), respectively (Table II). The compliance rates for CIT (1 h or less) and FT (6-72 h) were 91.7% (254/277 cases) and 94.9% (263/277 cases), respectively, as recommended by the 2018 ASCO/CAP HER2 guidelines for breast cancer.
Results of Standard PREanalytical Code (SPREC) for breast cancer.
We investigated the differences in ER, PgR, and HER2 expression between surgical and needle biopsy specimens regarding WIT, CIT, and FT (Table III). As the CIT and FT were longer than those recommended by the ASCO/CAP guidelines, ER and PgR expression in surgical specimens tended to decrease, although the difference was not significant. In contrast, HER2 expression in surgical specimens increased significantly when the WIT was longer than 30 min (p=0.012) or 60 min (p=0.043).
Differences in estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) expression between surgical and needle biopsy specimens for warm ischemia time (WIT), cold ischemia time (CIT), and fixation time (FT).
We compared HER2 expression between needle biopsies and surgical specimens from the same tumor. The concordance, increase, and decrease rates of HER2 expression between needle biopsy and surgical specimens across all cases were 62.8% (174/277 cases), 23.8% (66/277 cases), and 13.4% (37/277 cases), respectively (Table IV). The concordance, increase, and decrease rates over 30 min in the WIT were 65.1% (53/91 cases), 30.8% (28/91 cases), and 11.0% (10/91 cases), respectively (Table V).
Comparison of human epidermal growth factor receptor 2 (HER2) expression between needle biopsy and surgical specimens from the same tumor.
Warm ischemia time (WIT) >30 min.
Recently, in addition to the therapeutic agents for HER2 positivity (HER2 [3+] or HER2 [2+]/ISH [+]), antibody–drug conjugates (e.g., T-DXd) for low HER2 (HER2 [1+] or HER2 [2+]/ISH [−]) have appeared. Consequently, it has become important to assess low HER2 and HER2 positivity. When examining changes from HER2 negative in needle biopsy to HER2 positive in surgical specimens, as well as changes from HER2 negative in needle biopsy to low HER2 positivity in surgical specimens, no changes in HER2 positivity were observed (0 of 27 cases). However, a change to low HER2 positivity was observed in 63.0% of cases (17 of 27 cases).
Discussion
Regarding the quality control of samples, it has been reported that pre-analytical variables are responsible for more than 60% of the errors in the downstream analysis (15) and that the pre-analytical stage has the greatest impact on the test results (2, 3). Therefore, SPREC (4), BRISQ (5), and MIABIS (6) were developed as standardization methods for ancillary information in the pre-analysis phase of biological specimens, with the WIT, CIT, and FT as factors affecting specimen quality. This concept has also been introduced in the ASCO/CAP guidelines for IHC staining of ER, PgR, and HER2 in breast cancer.
It has been reported that CIT >1 h (16), or FT <6 h, or >72 h (17, 18) results in ER and PgR reduction by IHC staining. Based on these results, the ASCO/CAP guidelines for breast cancer recommend CIT of 1 h or less and FT of 6-72 h. In this study, 8.3% and 5.1% of patients exceeded the recommended time for CIT and FT, respectively; there was no significant difference between the compliant and non-compliant groups. The major outliers among the cases that failed to comply with the recommended times were a small number of cases (eight cases of >2 h for CIT and three cases of >100 h for FT).
HER2, ER, and PgR expression decreased over time (19); however, the expression of ER, PgR, and HER2 was analyzed over days or weeks and not over hours. The expression of Ki67, which has recently been reported to be important for treatment selection in breast cancer, was up-regulated for 5 h and then decreased (20). In our study, ER and PgR expression was lower in surgical specimens than in needle biopsy specimens, whereas HER2 expression was similar or higher. In particular, HER2 expression in surgical specimens increased with a WIT of 30 or 60 min or longer. The effect of WIT on surgical specimens has not been reported because of the difficulty in setting the vascular clamp time. To solve these problems, we set the definition of the vascular clamp time in breast cancer surgery and attempted quality control of samples according to SPREC (11). Furthermore, to the best of our knowledge, this is the first report to suggest that WIT affects HER2 expression in surgical specimens.
With the advent of antibody-drug conjugates, which are complex molecules composed of an antibody linked to a biologically active cytotoxic agent or drug, breast cancer cells with low HER2 IHC staining scores (score 1+ and 2+), not just high scores (3+), have become a therapeutic target. It has been reported that CIT and FT have little effect on HER2 IHC scores of 3+, while they have an effect on scores of 2+ or less (10, 21). Our findings that WIT affects the membrane expression of HER2 in breast cancer cells may have implications for breast cancer therapy against HER2.
Study limitations. Needle biopsy specimens from the same tumor, that is, WIT, CIT, and FT compliance, which have low variability, were used as controls to assess changes in ER, PgR, and HER2 expression due to differences in WIT, CIT, and FT. However, it is unclear to what extent factors, such as tumor heterogeneity, differences between observers making the pathological diagnosis, differences in the area of observation between needle biopsy specimens and surgical specimens, and the degree of tissue crushing affect the results. Additionally, the effect of WIT on HER2 expression remains unclear. We hypothesized that WIT affects HER2 expression due to hypoxia caused by blood flow interruption, and we intend to prove the results of this study from a basic study perspective.
Conclusion
This study investigated the effects of WIT, CIT, and FT on ER, PgR, and HER2 expression in surgically resected breast cancer specimens. Prolonged WIT was found to be associated with increased HER2 expression. This study emphasizes the significance of quality control of surgical specimens and provides new insights into the effect of WIT on HER2 expression. Future studies should aim to elucidate the underlying mechanisms using basic research techniques.
Acknowledgements
The Authors thank the anesthesiologists, pathologists, operating room nurses, and medical technologists in Pathology for their support in data recording. The Authors would also like to thank Editage (www.editage.com) for English language editing.
Footnotes
Authors’ Contributions
Conceptualization, N.S. and M.Y.; Original draft preparation, writing and review of the manuscript, N.S. and M.Y.; Resources and data curation, N.S. Y.M. A.T. T.Y. T.Y., and E.Y.; Statistical analysis, H.N. review and editing, D.H.; final review and supervision, M.Y. and A.S.; All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
- Received October 1, 2024.
- Revision received October 17, 2024.
- Accepted October 18, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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