Abstract
Background/Aim: In gastric cancer, accurate determination of human epidermal growth factor receptor type 2 (HER2) status is crucial for treatment decision-making. However, the optimal formalin fixation time of gastric cancer specimens for HER2 status determination remains unestablished. Here, we investigated real-world data on formalin overfixation and its effect on HER2 status determination in gastric cancer. Patients and Methods: We comprehensively analyzed HER2 testing results in 228 gastric cancer specimens, including those subjected to formalin overfixation. Subsequently, we divided 52 resected specimens of advanced gastric cancer into three groups and studied the effects of short-term (6-72 h) and long-term (1 and 2 weeks) fixation on HER2 status determination using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Results: A total of 21.5% (49/228) of the specimens were HER2-positive, whereas 78.5% (179/228) were negative. Among the HER2-negative specimens, no biopsies were overfixed, whereas 12.5% (9/72) of the surgical resection specimens were overfixed. The HER2 status of the 6-72-h group was 82.7% and 76.9% identical to that of the 1- and 2-week groups, when determined using IHC, and 73.1% and 36.5%, when determined using FISH, respectively. However, HER2 determination was not feasible in 26.9% and 63.5% of the specimens in the 1- and 2-week groups, respectively. Conclusion: Formalin overfixation may hinder the determination of HER2 status and should be avoided in gastric cancer sample preparation.
Gastric cancer, with over one million new cases and an estimated 769,000 deaths in 2020, still ranks fifth in terms of incidence and fourth in terms of mortality rate globally (1). Human epidermal growth factor receptor 2 (HER2) is a well-known key gene implicated in the growth of certain human solid tumors. In gastric cancer, HER2-positivity rates vary from 15% to 25% (2-4), and HER2-positivity is associated with aggressive disease, worse prognosis, and poor survival (2-5). Administering fluoropyrimidine plus platinum-based chemotherapy is currently the most widely accepted regimen for advanced gastric cancer worldwide. The additional benefit of the coadministration of trastuzumab with fluoropyrimidine/ platinum-based therapy was investigated in the Trastuzumab for Gastric Cancer (ToGA) trial, a large, randomized trial in patients with HER2-positive advanced gastric cancer (6). Consequently, combining trastuzumab with chemotherapy has become the new standard treatment for HER2-positive gastric cancer (7, 8). Additionally, compared to standard therapies, treatment with trastuzumab and deruxtecan considerably improves the response and overall survival in patients with HER2-positive gastric cancer (9).
The major testing methods for HER2 status include immunohistochemistry (IHC), in which protein expression is evaluated, and fluorescence in situ hybridization (FISH) analysis, in which gene amplification is evaluated. An accurate and reliable assessment of HER2 status is crucial for determining whether a patient has HER2-positive breast or gastric cancer. However, sample preparation conditions, including fixation procedures for HER2 testing, can vary considerably (10) and affect the results of HER2 testing (11). The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines, updated in 2018, recommend fixing HER2 testing specimens in 10% neutral-buffered formalin (NBF) for 6-72 h (12, 13). The relationship between HER2 testing results and duration of sample fixation has been reported by several studies on breast cancer (14-16). Although these findings may also be applied to gastric cancer, gastric cancer-specific data are required because the characteristics of HER2 over-expression (such as positivity and heterogeneity) differ between breast and gastric cancers (17-20). Furthermore, obtaining direct data on the effect of fixation time on HER2 testing for gastric cancer is important because there is insufficient evidence to interpret the results of HER2 testing performed on formalin-fixed tissues of resected gastric cancer.
In this study, we investigate whether formalin overfixation occurs in HER2 testing of gastric cancer using specimens collected from multiple centers in Yamaguchi Prefecture. We examined the effect of formalin overfixation on the results of HER2 testing using IHC and FISH of surgically resected gastric cancer specimens.
Patients and Methods
Patients and specimens. A retrospective, multicenter, observational study was conducted at Yamaguchi University Hospital, Shimonoseki Medical Center, and Kanmon Medical Center in Yamaguchi, Japan. A total of 1,945 patients were diagnosed with gastric cancer during 10 years from 2011 to 2020. Among these, 354 patients who had undergone HER2 testing were enrolled. Of these, 228 patients with unresectable or recurrent disease who received chemotherapy were included in the overall analysis population (Figure 1). Patient background, tumor characteristics, duration of specimen fixation for HER2 testing, HER2 test during treatment with or without trastuzumab, and survival rate were investigated.
Patient selection. HER2: Human epidermal growth factor receptor type 2.
Cancer tissue specimens were obtained from a consecutive series of 52 patients who underwent curative resection for advanced gastric cancer at the Department of Gastroenterological, Breast, and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Japan, and the Department of Surgery, Shimonoseki Medical Center, Japan, between 2015 and 2019. The surgically resected tumor specimen of each patient was divided into three equal-sized pieces and immediately placed in 10% NBF (Muto Pure Chemicals, Fukuoka, Japan): one was fixed for 6-72 h, the second for 1 week, and the third for 2 weeks (Figure 2). Ethical, legal, and social implications were approved by the Ethics Committee of Yamaguchi University Hospital (H29-046 and H29-047) and Shimonoseki Medical Center. All samples were obtained with written informed consent of the patients.
Representative image of a resected advanced gastric cancer specimen. Gastric cancer specimens resected from the same site in all patients were cut into three equal-sized pieces and fixed in formalin solution for 6-72 h (group 1), 1 week (group 2), and 2 weeks (group 3), respectively.
IHC and FISH for HER2 testing. IHC and FISH for HER2 testing were performed using the iView detection kit (Ventana, Tucson, AZ, USA) and Path Vysion HER2 DNA probe kit (Abbott Molecular Inc., Des Plaines, IL, USA), respectively. The evaluation of IHC staining (scores: 0, 1+, 2+, and 3+) and FISH was based on ASCO/CAP guidelines (12, 13).
For the FISH analysis, at least 20 nonoverlapping nuclei of tumor cells were evaluated for HER2 and chromosome enumeration probe (CEP)17 signals. When this ratio was between 1.8 and 2.2, an additional 20 nuclei were counted, and the HER2/CEP17 ratio was estimated for 40 nuclei. A ratio of HER2 to CEP17 signal of ≥2.0 was considered positive, whereas that of <2.0 was considered negative.
Two blinded pathologists evaluated the scores of IHC and FISH. Finally, correlations between the HER2 status in IHC and FISH among the three sample groups were analyzed.
Statistical analysis. The relationship between HER2 expression and clinicopathological factors was analyzed using the chi-square test for categorical variables and Mann–Whitney U-test for continuous variables. The overall survival and median survival durations for each treatment group were estimated using the Kaplan–Meier method. Results with p<0.05 were considered statistically significant. For the IHC results, the Spearman’s correlation coefficient was calculated. The concordance rates of the staining scores between the 6-72-h and 1-week groups as well as between the 6-72-h and 2-week groups were estimated with 95% confidence intervals (CIs) using the Clopper–Pearson exact method. For the FISH results, the χ2 test was used. All statistical analyses were performed using the R language (R Core Team, Vienna, Austria).
Results
HER2 expression and overfixation status from a real-world clinical survey. A total of 228 HER2-tested patients with unresectable or recurrent gastric cancer treated with chemotherapy constituted the overall analysis population. The patient background, tumor characteristics, duration of specimen fixation for HER2 testing, HER2 test results, and trastuzumab treatment of the 228 patients are summarized in Table I. No correlations were observed between tumor localization, type, tumor size, or clinical stage and HER2 status. HER2-positivity was more common in patients with an intestinal type of adenocarcinoma than in patients with a diffuse type (p<0.02). We did not observe cases of overfixation among the 141 biopsy specimens; however, 9 of the 87 surgical resection specimens were overfixed, accounting for 12.5% (9/72) of HER2-negative cases. Moreover, trastuzumab was administered to 83.7% of the patients who tested positive for HER2 but not in those who tested negative for HER2. Overall, 3.9% (9/228) of the patient specimens were overfixed in clinical practice. However, we did not detect any difference in the survival rate between HER2-positive and -negative patients (Figure 3).
Characteristics of HER2-tested patients with unresectable/recurrent gastric cancer receiving chemotherapy (n=228).
Kaplan–Meier analysis of overall survival in human epidermal growth factor receptor type 2 (HER2)-tested patients with gastric cancer (unresectable/recurrent chemotherapy) (n=228). There was no difference in the survival rate between HER2-positive and -negative patients.
Clinicopathological features of resection specimens. The clinicopathological features of 52 patients (36 men and 16 women) with gastric cancer are summarized in Table II. The median age of all patients was 77 (range=48-92) years. Among them, 47 patients were clinically diagnosed with type (T) 2 or 3 tumors. However, based on pathological examination, 6 patients were diagnosed with T1, 7 with T2, 13 with T3, and 26 with T4 tumors according to TNM (tumor nodes metastasis) classification. A total of 24 specimens were classified as intestinal type, whereas 28 were classified as diffuse-type based on Lauren’s classification (21). The mean tumor size was 58.8 mm.
Clinicopathological characteristics of all resected specimens (n=52).
Effect of duration of formalin fixation on immunohistochemistry staining for HER2. We examined the effect of duration of formalin fixation on IHC staining for HER2. Following IHC staining, 16 of the 52 specimens (30.8%) were scored 0, 17 (32.7%) were scored 1+, 15 (28.8%) were scored 2+, and 4 (7.7%) were scored 3+ in the 6-72-h group. Whereas, in the 1-week group, 18 (34.6%) specimens were scored 0, 18 (34.6%) were scored 1+, 12 (23.1%) were scored 2+, and 4 (7.7%) were scored 3+. Finally, 15 (28.8%) specimens were scored 0, 23 (44.2%) were scored 1+, 10 (19.2%) were scored 2+, and 4 (7.7%) were scored 3+ in the 2-week group (Table III). Furthermore, 43 (82.7%, 95% CI=69.7%-91.8%) and 40 (76.9%, 95% CI=63.2%-87.5%) of the 52 specimens had a similar staining score between the 6-72-h and 1-week groups and between the 6-72-h and 2-week groups, respectively (Figure 4A-D). Thus, positive correlations existed between the 6-72-h and 1-week groups and between the 6-72-h and 2-week groups according to our IHC results (p<0.01, p<0.01) (Table IV). However, in five specimens (9.6%), HER2 expression was reduced following prolonged fixation (Figure 4E). Hence, 6-72-h fixation resulted in five specimens having an IHC score of 2+, which was incorrectly determined due to overfixation. Therefore, these specimens required reevaluation using FISH.
Complete immunohistochemistry data according to fixation duration.
Effect of fixation duration on immunohistochemistry (IHC). Representative IHC staining for human epidermal growth factor receptor type 2 (HER2) detection in specimens fixed for 6-72 h, 1 week, and 2 weeks; exhibiting the same score (A-D) or reduced score (E) with prolonged fixation. (A) HER2 IHC score of 0 for each duration of fixation. (B) HER2 IHC scores of 1+ for each duration of fixation. (C) HER2 IHC scores of 2+ for each duration of fixation. (D) HER2 IHC scores of 3+ for each duration of fixation. (E) HER2 IHC scores of 2+ for 6-72-h fixation and a score of 1+ for 1- and 2-week fixation.
Assessment results match the rate of immunohistochemistry data.
Effect of formalin-fixation duration on FISH results. We also investigated whether prolonged formalin fixation affected FISH results. We successfully determined the HER2/CEP17 copy number ratio in all specimens in the 6-72-h group, with 7 of the 52 specimens (13.5%) exhibiting gene amplification (copy number ratio ≥2.0). However, we could not detect the HER2/CEP17 copy number ratio in 14 (26.9%) and 33 specimens (63.5%) in the 1- and 2-week groups, respectively. Additionally, the not detected (ND) frequency was significantly higher in the 1- and 2-week groups than in the 6-72-h group (p<0.0001 and p<0.0001, respectively; Table V). We observed that 38 (73.1%) and 19 (36.5%) of the 52 specimens presented a similar HER2/CEP17 copy number ratio between the 6-72-h and 1-week groups and between the 6-72-h and 2-week groups, respectively (Table VI).
Complete fluorescence in situ hybridization data according to fixation duration.
Assessment results match the rate of fluorescence in situ hybridization data.
Ee observed that with prolonged fixation, the number of specimens whose assessment was challenging due to a weakened signal (Figure 5A), or increased background noise (Figure 5B) increased. Finally, 5 of 15 (33.3%) specimens with an IHC score of 2+, which were subjected to FISH testing, were incorrectly determined because of overfixation. This is an important finding in real-world practice.
Effect of fixation duration on fluorescence in situ hybridization results. (A) Highly amplified signals (red spots) were observed in the 6-72-h fixed (ratio >5) and 1-week fixed (ratio 2.62) specimens. However, signals were not detected after 2 weeks of fixation because of weakened amplification. (B) Low-amplification signals (red spots) were observed in specimens fixed for either 6-72 h (ratio 1.05) or 1 week (ratio 0.95). Signals were not detected after 2 weeks of fixing owing to cellular structure disruption.
Discussion
To the best of our knowledge, this is the first study on the frequency of overfixation of clinical specimens used for determining the HER2 status of patients with gastric cancer and examine the effect of formalin-fixation duration of surgically resected gastric cancer specimens on IHC staining and FISH results for HER2 testing. Our study confirmed that specimens used for HER status testing are sometimes overfixed in actual clinical practice. We detected formalin overfixation in 3.9% of all gastric cancer specimens whose HER2 status was clinically assessed and in 9 out of 72 (12.5%) HER2-negative specimens, limited to surgically resected specimens. Although the handling of specimens should be strictly controlled, real-world conditions lead to HER2 status determination using specimens that have been fixed longer than recommended. Furthermore, in this study, IHC staining results of the 52 advanced gastric cancer resection specimens revealed that 82.7% and 76.9% of all cases presented similar staining scores between the 6-72-h and 1-week groups and between the 6-72-h and 2-week groups, respectively. However, in FISH, only 73.1% and 36.5% of all specimens showed similar gene amplification between the 6-72-h and 1-week groups and between the 6-72-h and 2-week groups, respectively.
To the best of our knowledge, no other study has reported the effect of fixation duration of human gastric cancer tissues on IHC and FISH results. In this study, we investigated the effects of the duration of fixation on HER2 IHC staining and FISH results using paraffin-embedded specimens of resected gastric cancer.
Heterogeneity refers to intratumor variations in the genotype or gene expression. In gastric cancer, the term is used when a spotty positive result is detected using IHC staining or FISH. Ideally, the same block of HER2-positive tumor tissue should be used for IHC staining and FISH analysis to determine the area of maximum HER2 intensity, regardless of histology or grade. False positive reactions may be observed in areas of intestinal metaplasia adjacent to ulcer sites or in areas of high-grade dysplasia; such lesions should be avoided. Crush artifacts and necrotic tissue should also be avoided. Regions with the strongest IHC intensity may reveal gene amplification in heterogeneous tumors (6). Therefore, in this study, the resected tumor specimen of each patient was divided into three equal-sized pieces and fixed in formalin for different periods to analyze the effect of duration of fixation on IHC and FISH for HER2 determination. According to IHC staining, HER2 expression was almost identical among the three groups, even for longer fixation periods (1- and 2-weeks). Specimens showing HER2 over-expression (score 3+), evaluated using IHC after 6-72-h fixation, retained the high HER2 expression status (score 3+) even after 1 or 2 weeks of fixation. The National Comprehensive Cancer Network guideline (22) recommends states “specimens with 2+ expression of HER2 by IHC should be additionally assessed by FISH.” Therefore, precise evaluation of IHC and FISH results in HER2 testing using appropriate specimens is required for the correct diagnosis of gastric cancer. Among the 15 patients in the 6-72-h group who exhibited an HER2 score of 2+, 5 had HER2 scores of 1+ after 1 or 2 weeks of fixation. These patients might have been considered for HER2-targeted therapy, depending on the FISH results. However, FISH testing would not have been performed based on the IHC results following long sample storage.
Our study suggested that the duration of formalin fixation significantly affects FISH results. In 26.9% of the specimens fixed for 1 week and 63.5% of those fixed for 2 weeks, the determination of HER expression was not feasible. This non-feasibility may be attributed to the following reasons. FISH is a powerful molecular cytogenetic method that hybridizes specific DNA sequences to a target genome. Formalin fixation increases the complexity of the cell structure and chromatin condensation, causing DNA fragmentation and crosslinks due to chemical modification; thus, penetration and interaction of the probe with the target DNA might be challenging. These chemical modifications are likely the primary cause of amplification weakness and cellular structure disruption (23, 24).
Study limitations. In addition to the limited sample size, patients were enrolled from only two centers. Therefore, more studies with a larger sample are warranted to investigate the optimal duration of sample fixation in determining HER2 status in gastric cancer. Additionally, although specimens were obtained from the same part of the tumor, tumor heterogeneity could not be completely excluded, which may have affected HER2 status determination.
Conclusion
Our study indicated that the duration of formalin fixation affects HER2 IHC staining and FISH outcomes in gastric cancer, similar to that in breast cancer. Therefore, we recommend a sample fixation duration of no longer than 6-72 h, as indicated in the ASCO/CAP guidelines for breast cancer. To minimize the risk of false negatives that might reduce treatment options for the patients, it is essential to rigorously adhere to the guideline recommendations of CAP/ASCP/ASCO for gastric cancer when handling gastric cancer resection specimens.
Acknowledgements
The Authors thank Kenzo Ikemoto, Mihoko Setoguchi, and Kaori Kaneyasu for their technical assistance. The Authors also thank Yuki Nakagami for her timely assistance in statistical analysis. Finally, the Authors would like to thank Editage (www.editage.com) for English language editing.
Footnotes
Authors’ Contributions
Conceptualization: JK, SY, and HN; Writing - original draft: JK, SY, and HN; Resources: JK, SY, MI, ST, CN, YW, MN, YT, YS, TN, NS, and HN; Methodology: JK, YH, and HI; Formal analysis: JK, YH, and HI. All Authors have read and approved the final version of the manuscript and provided their consent for publication.
Conflicts of Interest
The Authors declare that they have no conflicts of interest in relation to this study.
Funding
No funding was received to assist with the preparation of this manuscript.
- Received October 26, 2023.
- Revision received December 19, 2023.
- Accepted December 20, 2023.
- 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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