Clinicopathological factors associated with HER2 FISH positivity in HER2 IHC 2 + breast cancer: a multicenter study

Abstract

HER2 immunohistochemistry (IHC) 2 + breast cancer represents a clinically challenging subgroup requiring confirmatory fluorescence in situ hybridization (FISH) testing. This multicenter retrospective study included 284 patients with HER2 IHC 2 + breast cancer from a regional cohort to evaluate the FISH positivity rate and identify associated clinicopathological factors. Overall, 52 patients (18.3%) were FISH-positive. Compared with FISH-negative patients, the FISH-positive group showed significantly higher Ki-67 levels, lower PR expression, and a higher proportion of histological grade III tumors. Multivariable analysis identified histological grade III as the only clinicopathological characteristic independently associated with FISH positivity (OR = 2.43, 95% CI: 1.26–4.71, P = 0.008). The multivariable model demonstrated moderate discriminatory ability, with an area under the curve of 0.703 (95% CI: 0.628–0.777). Exploratory categorical analysis showed higher FISH positivity rates in patients with Ki-67 ≥ 30% and histological grade III tumors, with a stepwise increase in FISH positivity according to the number of these risk factors.These findings suggest that histological grade and Ki-67 may provide supportive information for characterizing the likelihood of HER2 gene amplification in HER2 IHC 2 + breast cancer. However, these routinely available clinicopathological features should not replace guideline-recommended confirmatory FISH testing.

Introduction

Breast cancer remains the most commonly diagnosed malignancy among women worldwide and a leading cause of cancer-related mortality, accounting for substantial global disease burden¹. Molecular classification has become fundamental for guiding individualized treatment strategies, among which human epidermal growth factor receptor 2 (HER2) plays a pivotal role in tumor biology and therapeutic decision-making². HER2-positive breast cancer, characterized by HER2 protein overexpression and/or HER2 gene amplification, has historically been associated with aggressive clinical behavior; however, the introduction of HER2-targeted therapies has significantly improved survival outcomes³.

Accurate determination of HER2 status is therefore essential in clinical practice. Immunohistochemistry (IHC) is widely used as the initial screening method, and cases with an equivocal score of 2 + require confirmation by in situ hybridization (ISH), most commonly fluorescence in situ hybridization (FISH), to assess HER2 gene amplification^4,5. According to the updated American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines, standardized interpretation criteria have been established to improve diagnostic accuracy and reproducibility^5,6. Nevertheless, HER2 IHC 2 + tumors remain a clinically challenging subgroup, because they represent an equivocal category in which final HER2 classification depends on confirmatory ISH results.

The concept of HER2-low breast cancer has emerged as a novel therapeutic category, defined as tumors with IHC 1 + or IHC 2 + and negative ISH results^7,8. The pivotal DESTINY-Breast04 trial demonstrated that trastuzumab deruxtecan significantly improved progression-free survival and overall survival in patients with HER2-low metastatic breast cancer compared with standard chemotherapy⁷.These findings have further increased the clinical importance of accurately distinguishing HER2-amplified tumors from non-amplified tumors within the IHC 2 + population^7,9.

Despite its clinical relevance, the reported FISH positivity rate among HER2 IHC 2 + breast cancers varies considerably across studies, typically ranging from approximately 10% to 40%^10–12. This variability may be attributed to differences in patient populations, laboratory techniques, interpretation criteria, and institutional practices. In addition, several clinicopathological factors have been reported to be associated with HER2 gene amplification in this subgroup. Previous studies have suggested that HER2 amplification is more frequently observed in tumors with altered hormone receptor expression, higher histological grade, and increased proliferative activity as reflected by Ki-67^11,13,14. However, these associations remain inconsistent across studies, and their clinical value in real-world settings requires further validation.

Most existing evidence is derived from large tertiary cancer centers or highly specialized institutions, which may limit the generalizability of findings to broader populations^10–12,15.In contrast, multicenter data from regional hospitals remain relatively limited. Real-world data from such populations are useful for understanding local disease characteristics and for evaluating routinely available clinicopathological factors associated with HER2 FISH positivity¹⁵.

Furthermore, in the era of HER2-low disease and antibody–drug conjugates, improved characterization of HER2 IHC 2 + patients is increasingly important^8,16,17.Identifying clinicopathological features associated with FISH positivity may help clinicians better understand the pathological context associated with a higher likelihood of HER2 gene amplification. However, such features should be considered supportive risk indicators and should not replace guideline-recommended confirmatory FISH testing.

Therefore, the present study aimed to (1) determine the FISH positivity rate among breast cancer patients with HER2 IHC 2 + in a multicenter regional cohort, (2) compare clinicopathological characteristics between FISH-positive and FISH-negative groups, and (3) explore factors associated with HER2 gene amplification. Our findings are expected to provide real-world evidence for the evaluation of HER2 IHC 2 + breast cancer in regional clinical practice.

Materials and methods

Study design and population

This retrospective multicenter study was conducted using data from a regional cohort in the Sanming area of China. Clinical and pathological data were collected from multiple participating hospitals and cross-verified with a centralized laboratory database from Adicon Clinical Laboratories, Hangzhou, China. Consecutive patients diagnosed with primary invasive breast cancer between January 2020 and December 2025 were screened.

Eligible patients were those with an initial HER2 immunohistochemistry (IHC) score of 2 + who subsequently underwent fluorescence in situ hybridization (FISH) testing for HER2 gene amplification. HER2 IHC 2 + status was based on pathological reports from the participating centers according to routine institutional practice and ASCO/CAP guideline-based criteria^4,5. Patients were included in the primary analysis only if a recorded and verified FISH result was available. All FISH analyses were performed by a centralized certified laboratory to ensure consistency in the assessment of HER2 gene amplification.

Patients were excluded if they met any of the following criteria:

(1) non-invasive breast cancer, such as ductal carcinoma in situ;

(2) receipt of neoadjuvant therapy before pathological assessment;

(3) recurrent or metastatic disease at initial diagnosis;

(4) absence of a verified FISH result;

(5) missing key eligibility-related information required for cohort inclusion; or.

(6) inconsistent or ambiguous HER2 assessment records.

This study was approved by the Ethics Committee of Sanming First Hospital Affiliated to Fujian Medical University (Approval No. Ming Yi Lun [2025] No. 17), as well as the institutional review boards of the participating hospitals, including Sanming Second Hospital and Sanming Integrated Traditional Chinese and Western Medicine Hospital.

All procedures were conducted in accordance with the Declaration of Helsinki. The requirement for informed consent was waived due to the retrospective nature of the study.

Data collection

Clinicopathological data were retrieved from hospital medical records across the participating hospitals and cross-verified with the centralized laboratory database. The following variables were collected: demographic characteristics, including age at diagnosis; tumor characteristics, including tumor size, laterality, histological type, histological grade, and lymph node status; molecular markers, including estrogen receptor (ER), progesterone receptor (PR), Ki-67 proliferation index, and HER2 IHC status; and FISH results, including HER2 gene amplification status.

To ensure data accuracy, all data were independently reviewed and verified by trained investigators. Clinicopathological variables were matched with laboratory FISH results using unique patient identifiers. Histological grade was verified from the original pathology reports and was dichotomized as grade III versus grade I–II for analysis, because grade III tumors represent poorly differentiated tumors with more aggressive biological behavior.

HER2 assessment

HER2 status was initially assessed by immunohistochemistry (IHC) at each participating center according to routine institutional practice based on ASCO/CAP guideline criteria^4,5. Cases with an IHC score of 2 + were considered equivocal and were further evaluated by fluorescence in situ hybridization (FISH).

All FISH analyses were performed by a centralized certified third-party laboratory, Adicon Clinical Laboratories, Hangzhou, China, ensuring consistency and standardization in the assessment of HER2 gene amplification. HER2 gene amplification was determined based on the HER2/CEP17 ratio and/or average HER2 copy number in accordance with the 2018 ASCO/CAP focused update⁵. Specifically, cases were classified as HER2-positive if the HER2/CEP17 ratio was ≥ 2.0 with an average HER2 copy number ≥ 4.0 signals per cell, or if the average HER2 copy number was ≥ 6.0 signals per cell regardless of the ratio. Cases not meeting these criteria were considered HER2-negative.

For HER2 IHC assessment, the study relied primarily on the original pathological reports from the participating centers. Centralized re-review of all historical IHC slides was not performed. Therefore, potential inter-institutional variability in IHC interpretation could not be completely excluded and was acknowledged as a limitation.

Handling of missing data

Patients without a recorded and verified HER2 FISH result were excluded from the primary analysis, because FISH positivity was the primary outcome variable of this study. In the final study cohort, HER2 FISH status was available for all included patients, and the primary outcome was not imputed.

Baseline characteristics were summarized using observed data. The proportion of missing data was low for all baseline clinicopathological variables: age, 0% (0/284); tumor size, 1.1% (3/284); ER expression, 2.1% (6/284); PR expression, 2.1% (6/284); Ki-67 index, 2.1% (6/284); histological grade, 1.8% (5/284); lymph node status, 1.1% (3/284); and lymphovascular invasion, 1.1% (3/284).

Because the missing-data rates for baseline covariates were low, the primary analyses were performed using available observed data. No imputation was performed for HER2 FISH status.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics version 27.0 (IBM Corp., Armonk, NY, USA). A two-sided P value < 0.05 was considered statistically significant.

Continuous variables were tested for normality using the Shapiro–Wilk test. Normally distributed variables were presented as mean ± standard deviation and compared using the independent samples t-test. Non-normally distributed variables were expressed as median and interquartile range and compared using the Mann–Whitney U test. In particular, Ki-67 showed a non-normal distribution and was therefore analyzed using non-parametric methods.

Categorical variables were summarized as frequencies and percentages and compared using the chi-square test or Fisher’s exact test, as appropriate. Histological grade was dichotomized as grade III versus grade I–II for analysis, because grade III tumors represent poorly differentiated tumors with more aggressive biological behavior. Ki-67 was dichotomized using a threshold of 30% for exploratory categorical risk analysis.

Candidate variables were selected for multivariable logistic regression based on univariate findings and clinical relevance. Given the exploratory nature of this analysis, the model was not intended to establish a clinically applicable prediction tool. The multivariable model included tumor size, Ki-67, ER expression, PR expression, and histological grade. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to identify factors independently associated with FISH positivity.

The discriminatory ability of the multivariable logistic regression model was evaluated using receiver operating characteristic (ROC) curve analysis. The ROC curve was generated using the predicted probabilities derived from the multivariable model, and the area under the curve (AUC) with 95% CI was calculated. The optimal cut-off value was determined using the Youden index.

Exploratory categorical risk analyses were performed using Ki-67 ≥ 30% and histological grade III as clinically interpretable risk factors. A combined risk-stratification analysis was conducted according to the number of risk factors present. The trend in FISH positivity rate across increasing numbers of risk factors was evaluated using the chi-square test for trend.

Results

Patient characteristics

A total of 324 patients with primary invasive breast cancer from the Sanming regional cohort were initially screened. After exclusion of 40 patients due to non-invasive disease, receipt of neoadjuvant therapy, recurrent or metastatic disease at diagnosis, absence of verified FISH results, or ambiguous HER2 assessment records, 284 patients with HER2 IHC 2 + who underwent verified FISH testing were included in the final analysis. Among them, 52 patients were FISH-positive and 232 were FISH-negative, yielding an overall FISH positivity rate of 18.3%. The patient selection process is shown in Fig. 1.

Fig. 1.

Flowchart of patient selection in the study.

A total of 324 patients diagnosed with primary invasive breast cancer in the Sanming region between January 2020 and December 2025 were initially screened. After excluding 40 patients due to non-invasive disease (n = 10), receipt of neoadjuvant therapy (n = 12), recurrent or metastatic disease (n = 8), and absence of verified FISH results or ambiguous HER2 assessment records (n = 10), 284 patients with HER2 immunohistochemistry (IHC) 2 + status and verified fluorescence in situ hybridization (FISH) results were included in the final analysis. Among them, 52 patients were HER2 FISH-positive and 232 were FISH-negative.

Baseline clinicopathological characteristics

The baseline clinicopathological characteristics of patients stratified by FISH status are summarized in Table 1.

Table 1.

Clinicopathological characteristics according to FISH status.

Variable	Total (n = 284)	FISH-positive (n = 52)	FISH-negative (n = 232)	P value
Age (years), median (IQR)	56 (48–62)	55 (50–60)	56 (47–62)	0.698
Tumor size (cm), median (IQR)	2.11 (1.50–2.50)	2.19 (1.50–3.00)	2.11 (1.50–2.50)	0.243
Ki-67 (%), median (IQR)	26.5 (15–40)	30 (28–40)	25 (15–35)	< 0.001
ER (%), median (IQR)	90 (85–95)	90 (85–95)	90 (83.7–95)	0.553
PR (%), median (IQR)	50 (15–80)	30 (6–60)	53.3 (20–80)	0.028
Histological grade III, n (%)	72 (25.4%)	22 (42.3%)	50 (21.6%)	0.003

Data are presented as median (IQR) or n (%).

Compared with the FISH-negative group, patients in the FISH-positive group demonstrated significantly higher Ki-67 levels (median, 30% vs. 25%; P < 0.001) and lower PR expression (median, 30% vs. 53.3%; P = 0.028). In addition, a higher proportion of histological grade III tumors was observed in the FISH-positive group than in the FISH-negative group (42.3% vs. 21.6%, P = 0.003).

No significant differences were observed between the two groups in age, tumor size, or ER expression. Analyses of lymph node metastasis and lymphovascular invasion were based on available cases and should therefore be interpreted with caution.

Comparisons were performed using the Mann–Whitney U test or chi-square/Fisher’s exact test, as appropriate.

Histological grade was dichotomized as grade III versus grade I–II for analysis, because grade III tumors represent poorly differentiated tumors with more aggressive biological behavior.

Analyses of lymph node metastasis and lymphovascular invasion were based on available cases.

Lymph node status and lymphovascular invasion were collected as baseline clinicopathological variables but were not included in the primary multivariable model. Their missing-data rates are reported in Sect. 2.4.

Multivariable analysis

Candidate variables were selected based on univariate findings and clinical relevance and were included in the multivariable logistic regression model (Table 2).

Table 2.

Multivariable logistic regression analysis for factors associated with FISH positivity.

Variable	OR	95% CI	P value
Histological grade III vs. grade I–II	2.43	1.26–4.71	0.008
Tumor size (cm)	1.27	0.91–1.77	0.155
Ki-67 (%)	1.01	1.00–1.03	0.155
ER (%)	0.99	0.98–1.00	0.259
PR (%)	0.99	0.98–1.00	0.124

Data are presented as odds ratios (OR) with 95% confidence intervals (CI).

After adjustment for tumor size, Ki-67, ER expression, PR expression, and histological grade, histological grade III remained the only clinicopathological characteristic independently associated with FISH positivity(OR = 2.43, 95% CI: 1.26–4.71, P = 0.008).

Other variables, including tumor size, Ki-67, ER expression, and PR expression, were not independently associated with FISH positivity in the multivariable model (all P > 0.05).

Multivariable logistic regression analysis was performed including variables selected based on univariate findings and clinical relevance.

Model performance

The discriminatory performance of the multivariable logistic regression model was evaluated using receiver operating characteristic (ROC) curve analysis. The ROC curve was generated using the predicted probabilities derived from the multivariable logistic regression model, which included tumor size, Ki-67, ER expression, PR expression, and histological grade.

After recalculation using the corrected dataset, the model demonstrated moderate discriminatory ability, with an area under the curve (AUC) of 0.703 and a 95% confidence interval of 0.628–0.777. The optimal cut-off value, determined using the Youden index, was 0.154, yielding a sensitivity of 76.9% and a specificity of 56.9%.

The ROC curve is presented in Fig. 2. Because this model was derived from a retrospective cohort and has not been externally validated, the cut-off value should be interpreted as exploratory rather than as a clinically validated decision threshold.

Fig. 2.

ROC curve of the multivariable model for estimating the likelihood of FISH positivity.

The model demonstrated moderate discriminatory ability, with an AUC of 0.703 (95% CI: 0.628–0.777). The optimal cut-off value was 0.154, with a sensitivity of 76.9% and a specificity of 56.9%.

Exploratory categorical risk analysis

Exploratory categorical risk analyses were performed using two clinically interpretable factors: Ki-67 ≥ 30% and histological grade III. These factors were selected because Ki-67 was significantly higher in the FISH-positive group in baseline comparisons, and histological grade III remained independently associated with FISH positivity in the multivariable model.

When evaluated separately, patients with Ki-67 ≥ 30% had a significantly higher FISH positivity rate than those with Ki-67 < 30% (27.5% vs. 9.6%, P < 0.001). Similarly, patients with histological grade III tumors had a higher FISH positivity rate than those with grade I–II tumors (30.6% vs. 14.2%, P = 0.003).

A combined risk-stratification analysis was then performed according to the number of risk factors present. The FISH positivity rate increased progressively from 6.8% in patients with neither risk factor to 23.0% in those with one risk factor and 36.4% in those with both risk factors (P for trend < 0.001). These findings suggest that the combination of increased proliferative activity and poor differentiation may help characterize patients with a higher likelihood of HER2 gene amplification, although this exploratory risk stratification should not replace confirmatory FISH testing. Table 3.

Table 3.

Exploratory categorical risk analysis according to Ki-67 and histological grade.

Analysis	Category	Total (n)	FISH-positive (n)	FISH positivity rate (%)	P value
Ki-67	< 30%	146	14	9.6
Ki-67	≥ 30%	138	38	27.5	< 0.001
Histological grade	Grade I–II	212	30	14.2
Histological grade	Grade III	72	22	30.6	0.003
Combined risk factors	0 risk factors	118	8	6.8
Combined risk factors	1 risk factor	122	28	23.0
Combined risk factors	2 risk factors	44	16	36.4	< 0.001

Data are presented as n (%). Risk factors were defined as Ki-67 ≥ 30% and histological grade III. The P values for Ki-67 and histological grade comparisons were calculated using the chi-square test. The P value for combined risk factors represents the trend across increasing numbers of risk factors.

Exploratory description of amplification-level parameters among FISH-positive cases

Among the 52 FISH-positive patients, numerical HER2/CEP17 ratio and/or average HER2 copy-number data were available for 15 patients. Therefore, amplification-level analyses were limited to this subset and were performed as descriptive exploratory analyses only (Table 4).

Table 4.

Exploratory description of HER2 amplification-level parameters among FISH-positive cases with available numerical data.

Variable	Category	n	Ki-67, mean (%)	Grade III, n (%)
HER2/CEP17 ratio	2–<2.5	10	30.0	1 (10.0%)
HER2/CEP17 ratio	≥ 2.5	5	31.1	2 (40.0%)
Average HER2 copy number	< 6	9	29.8	1 (11.1%)
Average HER2 copy number	≥ 6	6	31.5	2 (33.3%)

Data are presented as mean or n (%). Ki-67 is expressed as percentage. This exploratory analysis was limited to FISH-positive patients with available numerical HER2/CEP17 ratio and/or average HER2 copy-number data. Because of the small sample size and incomplete numerical FISH parameters, no formal statistical comparisons were performed.

Patients were descriptively stratified according to HER2/CEP17 ratio and average HER2 copy number. Among patients with available HER2/CEP17 ratio data, 10 had a ratio of 2 to < 2.5 and 5 had a ratio of ≥ 2.5. Among patients with available average HER2 copy-number data, 9 had an average copy number of < 6, and 6 had an average copy number of ≥ 6.

Numerically, patients with higher HER2/CEP17 ratios or higher average HER2 copy numbers appeared to have a higher proportion of histological grade III tumors, whereas Ki-67 values differed only slightly across amplification-level categories. However, because numerical FISH parameters were available only for a small subset of FISH-positive patients, no formal statistical comparisons were performed, and these findings should be interpreted as hypothesis-generating.

In some FISH-positive cases, HER2 amplification was confirmed by the centralized laboratory using qualitative or descriptive diagnostic interpretations, such as clustered signal patterns, but complete numerical ratio or copy-number values were not available in the research dataset. These cases were included in the primary binary FISH-positive analysis but were not included in this exploratory amplification-level analysis.

Discussion

Accurate assessment of HER2 status remains essential for treatment selection in breast cancer. HER2 IHC 2 + tumors represent a diagnostically challenging equivocal subgroup, because final HER2 classification depends on confirmatory in situ hybridization testing. In the present multicenter retrospective cohort, we evaluated clinicopathological factors associated with HER2 FISH positivity among patients with HER2 IHC 2 + breast cancer. The overall FISH positivity rate was 18.3%, indicating that most IHC 2 + tumors in this cohort were not HER2-amplified. This finding is consistent with the concept that HER2 IHC 2 + breast cancer represents a biologically and diagnostically heterogeneous group rather than a uniform disease category^1,5,6,10,12.

The clinical relevance of HER2 IHC 2 + disease has increased in the era of HER2-low breast cancer and antibody–drug conjugates. HER2-low breast cancer is commonly defined as IHC 1 + or IHC 2 + with negative ISH, and this subgroup has become clinically important because trastuzumab deruxtecan has demonstrated survival benefit in patients with HER2-low metastatic breast cancer^7,9,17. Therefore, accurate distinction between FISH-positive and FISH-negative cases within the IHC 2 + population remains critical. In this context, our observed FISH positivity rate of 18.3% provides real-world evidence from a regional multicenter cohort and supports the need for confirmatory FISH testing in IHC 2 + tumors.

In baseline comparisons, FISH-positive tumors showed higher Ki-67 levels, lower PR expression, and a higher proportion of histological grade III tumors than FISH-negative tumors. Ki-67 is a routinely assessed marker of tumor proliferation and has been associated with more aggressive clinicopathological features in breast cancer¹⁸. Previous studies of HER2 IHC-equivocal breast cancer have also suggested that higher proliferative activity may be associated with HER2 gene amplification¹¹. In our cohort, Ki-67 ≥ 30% was associated with a higher FISH positivity rate in categorical analysis. However, Ki-67 did not remain independently associated with FISH positivity when modeled as a continuous variable in the primary multivariable analysis. This finding suggests that the association between Ki-67 and FISH positivity may be partly influenced by tumor grade or other related biological characteristics.

Histological grade III was the only clinicopathological characteristic independently associated with FISH positivity in the multivariable model. Patients with grade III tumors had a higher probability of FISH positivity than those with grade I–II tumors.This result is biologically plausible, because high histological grade reflects poor differentiation and more aggressive tumor behavior, features that are commonly associated with HER2-positive breast cancer. From a practical perspective, histological grade is routinely available in pathological reports and may therefore serve as a simple clinicopathological indicator associated with an increased likelihood of FISH positivity among HER2 IHC 2 + tumors. Nevertheless, the effect size should be interpreted cautiously, and histological grade should not be used as a substitute for guideline-recommended confirmatory FISH testing.

The exploratory categorical risk analysis further supported the clinical relevance of Ki-67 and histological grade. The FISH positivity rate increased from 6.8% in patients with neither Ki-67 ≥ 30% nor histological grade III to 23.0% in those with one risk factor and 36.4% in those with both risk factors. This stepwise pattern suggests that the coexistence of increased proliferative activity and poor differentiation may identify a subgroup with a higher likelihood of HER2 amplification. However, this analysis was exploratory and should be viewed as risk characterization rather than a validated prediction rule. These findings may help clinicians better understand the clinicopathological context in which HER2 amplification is more likely to occur, but they should not be used to omit FISH testing in eligible IHC 2 + cases.

The multivariable logistic regression model demonstrated moderate discriminatory ability, with an AUC of 0.703, indicating that routinely available clinicopathological features have limited ability to distinguish between FISH-positive and FISH-negative cases and are insufficient for use as a stand-alone clinical decision-making tool. Because histological grade III was the only independent factor identified in the multivariable analysis, no nomogram or decision curve analysis was further developed, in order to avoid overfitting and overinterpretation of the model. Therefore, the multivariable model and the derived cut-off value should be interpreted as exploratory and hypothesis-generating, and require external validation before potential clinical application.

PR expression was lower in FISH-positive tumors in baseline comparisons, whereas ER expression did not differ significantly between FISH-positive and FISH-negative groups. Neither ER nor PR remained independently associated with FISH positivity after adjustment. This may reflect the predominance of hormone receptor–positive tumors in the cohort and the complex relationship between hormone receptor expression, proliferation, tumor grade, and HER2 amplification. Previous HER2-low studies have shown that HER2-low disease is more common in hormone receptor–positive breast cancer, and that HER2 expression patterns may differ between hormone receptor–positive and hormone receptor–negative tumors^9,17. Therefore, hormone receptor status alone may have limited discriminatory value for predicting FISH positivity within an IHC 2 + population.

We also performed an exploratory description of amplification-level parameters among FISH-positive cases with available numerical HER2/CEP17 ratio and average HER2 copy-number data. In this limited subset, higher HER2/CEP17 ratios or higher average HER2 copy numbers appeared to be accompanied by a higher proportion of histological grade III tumors, whereas Ki-67 values differed only slightly across amplification-level categories. However, numerical amplification parameters were available for only a small subset of FISH-positive cases. Therefore, these findings should be regarded as descriptive and hypothesis-generating. The present data cannot support definitive conclusions that HER2 amplification level represents a continuous biological spectrum associated with tumor aggressiveness.

This study has several strengths. First, it provides real-world multicenter data from a regional cohort, complementing evidence from large tertiary cancer centers. Second, all included patients had HER2 IHC 2 + breast cancer with confirmed FISH status, allowing direct evaluation of clinicopathological factors associated with FISH positivity in this diagnostically equivocal subgroup. Third, the revised analysis used a corrected dataset and avoided imputation of the primary outcome, thereby improving the transparency and validity of the statistical approach. Fourth, the combined categorical risk analysis provides an easily interpretable description of how Ki-67 and histological grade jointly relate to FISH positivity.

Several limitations should also be acknowledged. First, this was a retrospective study and was therefore subject to selection bias and information bias. Second, although this was a multicenter study, all participating hospitals were located within the Sanming region, and FISH testing was performed by a single centralized certified laboratory. Therefore, the generalizability of the findings to broader populations or other laboratory settings may be limited. Third, although centralized FISH testing improved consistency in the assessment of HER2 gene amplification, HER2 IHC 2 + classification was based primarily on original pathological reports from participating centers, and centralized re-review of all historical IHC slides was not performed. Potential inter-institutional variability in IHC interpretation may therefore have influenced patient selection. Fourth, baseline covariate missingness may have affected the analysis; however, patients without verified FISH results were excluded from the primary analysis, and the primary outcome was not imputed. Fifth, detailed numerical FISH parameters, including HER2/CEP17 ratio and average HER2 copy number, were unavailable for a substantial proportion of FISH-positive cases, limiting the amplification-level analysis. Sixth, the model demonstrated only moderate discrimination and lacked external validation, which limits its generalizability and precludes clinical use as a prediction tool. In addition, the number of FISH-positive events was limited relative to the number of variables included in the multivariable model, and the event-to-variable ratio was borderline. Therefore, the model should be interpreted as exploratory and requires external validation.

Conclusion

In conclusion, this multicenter retrospective study demonstrated that the FISH positivity rate among patients with HER2 IHC 2 + breast cancer was 18.3%. Histological grade III was independently associated with a higher likelihood of HER2 FISH positivity, while Ki-67 showed clinical relevance in baseline comparison and categorical risk analysis.

The combined exploratory risk analysis showed that patients with both Ki-67 ≥ 30% and histological grade III had a higher FISH positivity rate than those with neither risk factor. These routinely available clinicopathological features may help characterize the likelihood of HER2 gene amplification in HER2 IHC 2 + breast cancer.

However, these findings should be interpreted as supportive and exploratory risk indicators only. They should not be used to replace guideline-recommended confirmatory FISH testing. The amplification-level analysis was limited by incomplete numerical FISH parameters and should be regarded as descriptive and hypothesis-generating. Further large-scale prospective studies with external validation and complete FISH parameter reporting are warranted to validate these findings.

Clinical implications

The findings of this study have practical clinical relevance because histological grade and Ki-67 are routinely available pathological parameters in breast cancer. In patients with HER2 IHC 2 + tumors, histological grade III was independently associated with a higher likelihood of HER2 FISH positivity, while Ki-67 ≥ 30% showed clinical relevance in exploratory categorical risk analysis.

These findings suggest that poor differentiation and increased proliferative activity may help clinicians better understand the clinicopathological context associated with a higher likelihood of HER2 FISH positivity.In particular, patients with both Ki-67 ≥ 30% and histological grade III showed a higher FISH positivity rate than those with neither risk factor.

However, these clinicopathological features should be considered supportive risk indicators only. They should not be used to omit or replace guideline-recommended confirmatory FISH testing. The exploratory amplification-level analysis was limited by incomplete numerical FISH parameters and should not be interpreted as evidence that HER2 amplification represents a continuous biological spectrum.

Therefore, the main clinical implication of this study is that routinely available pathological features may assist in risk characterization of HER2 IHC 2 + breast cancer, while confirmatory FISH testing remains necessary for definitive HER2 classification.

Author contributions

W.Y. and H.D. collected and analyzed the data and drafted the manuscript. W.G., Q.L., and G.H. contributed to data collection and interpretation. S.Z. and S.D. performed statistical analysis and assisted in data visualization. W.C. conceived and designed the study, supervised the research, and critically revised the manuscript. All authors reviewed and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Fujian Province of China (Grant No. 2021J011387 and 2025J011549) and the Sanming Science and Technology Project (Project Code: 2024-S-084; Approval Document No.: Ming Ke Cheng [2024] No. 15).

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request, subject to institutional and ethical restrictions.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Sanming First Hospital Affiliated to Fujian Medical University (Approval No. Ming Yi Lun [2025] No. 17), as well as the institutional review boards of the participating hospitals, including Sanming Second Hospital and Sanming Integrated Traditional Chinese and Western Medicine Hospital.

All procedures were conducted in accordance with the Declaration of Helsinki. The requirement for informed consent was waived by the Ethics Committee of Sanming First Hospital Affiliated to Fujian Medical University and the institutional review boards of the participating hospitals due to the retrospective nature of the study and the use of anonymized clinical data.