Introduction
Human epidermal growth factor receptor 2 (HER2), a receptor tyrosine-protein kinase, is encoded by the
HER2/neu gene in humans. Amplification or over-expression of this oncogene plays a crucial role in breast cancer development and progression by inducing downstream pathways, such as PI3K/Akt [
1]. Anti-HER2 drugs work by binding to HER2 expressed on the surface of cancer cells. HER2-positive breast cancers previously had poor prognosis [
2,
3], but the introduction of trastuzumab, a pioneer anti-HER2 drug, has dramatically improved patient outcomes [
4].
Attention is needed, however, in the definition of HER2-positive tumors. HER2 protein expression and
HER2/neu amplification are clinically assessed with immunohistochemistry (IHC) and fluorescence
in situ hybridization (FISH), respectively. According to the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines, a HER2-positive tumor is defined as either IHC(3+) (i.e., overexpressed) or FISH(+) (i.e., amplified) [
5]. In practice, however, most cases are first assessed with IHC, and only cases scored as IHC(2+), i.e., equivocal, are assessed with FISH for
HER2/neu amplification. Generally, HER2 overexpressed IHC(3+) tumors are considered to have
HER2/neu amplification, with a concordance rate of approximately 90% [
6,
7]. In IHC(2+) tumors, FISH is positive in around 10–20% of cases [
7‐
9].
While trastuzumab-based treatments have shown benefit in patients with IHC(3+) or FISH(+) tumors, the definition of HER2-positive tumors differed amongst clinical trials [
4,
6,
10‐
13]. In clinical practice, both IHC(3+) and IHC(2+)/FISH(+) tumors are treated as HER2-positive breast cancer, which is also the case for some new HER2-targeted drugs, such as pertuzumab. Numerous clinical trials have shown both IHC(3+) and IHC(2+)/FISH(+) tumors demonstrated significant benefit from additional trastuzumab, used either alone or in combination with chemotherapeutic drugs [
4,
6,
7,
10‐
14].
Anti-HER2 drugs may be less effective in IHC(2+)/FISH(+) tumors due to less HER2 protein expressed on the cell surface. In a large randomized phase III clinical trial (N9831, n = 1,888) investigating the benefit of additional trastuzumab to adjuvant chemotherapies, patients with IHC-negative and FISH(+) tumors showed no improvement in disease-free-survival with additional trastuzumab, while patients with IHC(3+)/FISH(-) tumors demonstrated disease-free-survival comparable to those with IHC(3+)/FISH(+) tumors, suggesting a key role of protein overexpression [
13]. The differences in patient outcomes and efficacy with anti-HER2 therapies between IHC(3+) and IHC(2+)/FISH(+) tumors, however, have not been well studied and are poorly understood.
Multi-gene panel tests have been recently introduced into clinical practice for patients with metastatic breast cancer. These tests evaluate gene status rather than protein expression in the tumor, and are being increasingly used to guide treatment decisions. Indeed, even if HER2 protein expression in the primary tumor is low, anti-HER2 therapy may be offered if the tumor is determined to be HER2-positive by gene panel tests.
The aim of this study was to determine if the therapeutic effects of anti-HER2 therapies and patient outcomes differed between IHC(3+) and IHC(2+)/FISH(+) tumors. We retrospectively investigated patients with HER2-positive invasive breast cancer treated at our hospital, focusing on the differences between patients with IHC(3+) and IHC(2+)/FISH(+) tumors.
Patients and methods
Patients
A total of 447 patients with HER2-positive invasive carcinoma underwent curative surgery at our institution from 2010 to 2019. HER2 status was assessed from surgical specimens or, for patients who had received neo-adjuvant chemotherapy (NAC) before surgery, biopsy specimens were assessed to avoid chemotherapy-related effects. Following surgery, standard adjuvant treatments were administered based on tumor characteristics. Details of adjuvant systemic treatments are shown in Additional file
1. Among the 318 patients who received chemotherapy, 171 (54%) patients were given an anthracycline-based regimen; epirubicin plus cyclophosphamide (EC), followed by taxanes (paclitaxel or docetaxel). Another 129 (41%) patients received EC only, while 18 (6%) were given a taxane only. In the 328 patients who received anti-HER2 therapy, trastuzumab was used alone in 289 (88%) patients, while pertuzumab was also used in a combination therapy in 38 (12%) patients. The current retrospective study includes patients who did not receive systemic treatments for some other reasons, such as refusal by the patient, or where the indication for chemotherapy was not clear. This study was performed with approval from the ethics committee of Juntendo University Hospital (H19-0289), and all data were collected after obtaining informed consent from the patients. All data were anonymized before use.
Pathologic assessment
Pathologic examinations were carried out at Juntendo University Hospital by two experienced pathologists. Tumor grade was judged based on the modified Bloom-Richardson histologic grading system. For patients who received NAC, a pathological complete response (pCR) was defined as the disappearance of invasive nest in the primary breast tumor, i.e., without any lymph node evaluation. Estrogen receptor (ER) and progesterone receptor (PgR) statuses were assessed semi-quantitatively with IHC, and reported as positive when > 1% of cancer cell nuclei showed staining. For the Ki67 labelling index, cells positive for nuclear Ki67 were evaluated semi-quantitatively within a selected hotspot microscopically under high magnification.
The criteria for HER2 assessment were revised slightly by the ASCO/CAP in 2018 [
5]. However, this study used the pre-revision criteria [
15,
16] since our cases were diagnosed before the 2018 revision. Employing rabbit monoclonal antibody (clone 4B5, Ventana), HER2 protein expression was judged as 0 (negative, no staining observed, or membrane staining in < 10% of tumor cells), 1+ (negative, faint focal membrane staining in > 10% of tumor cells), 2+ (equivocal, weak to moderate staining of the cell membrane in > 10%, or strong staining of the complete membrane in ≤ 10% of tumor cells), and 3+ (positive, strong staining of the complete membrane in > 10% of tumor cells). Patients diagnosed between 2010 and 2012 inclusive, used a 30% cut-off value for 3+, based on ASCO/CAP guidelines during that time [
15]. Representative images of IHC are shown in Additional file
2. FISH was conducted using a PathVysion HER2 DNA Probe Kit (Abbott Japan, Tokyo, Japan).
HER2/neu gene amplification was defined as being present when the FISH ratio was > 2.0. In rare cases, some tumors switched from HER2-negative to HER2-positive on IHC following NAC treatment (e.g., IHC 1 + to 3+). Such cases were excluded from the current study.
Statistical analysis
Statistical analyses were performed using JMP 11.2.1 statistical software (SAS Institute Inc., Cary, NC). For comparisons of mean values, such as age, examinations of unpaired data were performed employing the two-sided Student’s t-test. As a test of independence, the Pearson’s Chi-squared test was used. For evaluation of any independent prognostic effects of the variables, the Cox proportional hazard model was applied with a 95% confidence interval. For continuous variables, mean values were used as the threshold to distinguish between high and low groups. Mean values were 56 (age), 17 mm (pathological tumor size), and 48% (Ki67 labelling index). The full-model analysis selected variables according to their clinical significance, specifically; age, pathological tumor size, lymph node involvement, tumor grade, ER and HER2 status, and administration of chemotherapy. Kaplan-Meier curves were produced and the log-rank test was applied for comparisons of survival between the two populations. A P-value < 0.05 was considered statistically significant.
Discussion
In this study, we found patient outcomes did not differ between breast cancer patients with IHC(3+) and IHC(2+)/FISH(+) tumors. Even after stratifying patients into those that had or had not received adjuvant chemotherapy with anti-HER2 drugs, there was no difference in patient outcomes. To the best of our knowledge, there has been no other report comparing patient outcomes with these two tumors. To determine whether IHC(3+) and IHC(2+)/FISH(+) tumors differ in their intrinsic malignancy, it is necessary to compare patients who did not receive chemotherapy. However, our cohort was retrospectively collected and the application of chemotherapy was not randomized. Moreover, the sample size of the non-chemotherapy group was relatively small. Hence, we were not able to adequately examine this point. It is no longer ethical to employ an arm without anti-HER2 agents in prospective clinical trials. As such, more retrospective observational studies such as the present study need to be collated.
As to the effectiveness of anti-HER2 therapies, in the NAC setting, pCR rates were lower in IHC(2+)/FISH(+) tumors compared with IHC(3+), although the difference did not reach statistical significance. Small sample numbers might have affected the statistical analysis. Meanwhile, patients with IHC(2+)/FISH(+) tumors showed significantly shorter survival after developing distant metastases. Considering anti-HER2 therapies are given to patients with both IHC(3+) and IHC(2+)/FISH(+) tumors, we cannot exclude the possibility that IHC(2+)/FISH(+) tumors do not respond well to anti-HER2 therapies. In the N9831 clinical trial, patients with IHC-negative and FISH(+) tumors had no additional benefit, in terms of prolonging DFS, with receiving trastuzumab with adjuvant chemotherapies [
13]. The importance of HER2 protein overexpression merits further investigation. It would be relatively easy to conduct an additional analysis in recent clinical trials that include a variety of anti-HER2 drugs, to evaluate treatment effectiveness in IHC(3+) and IHC(2+)/FISH(+) tumors.
The IHC(2+)/FISH(+) group included significantly more HR-positive tumors. However, this phenomenon is probably caused by selection bias rather than biological difference, as ER-positive tumors were predominant among those in which FISH was examined (data not shown). ER can regulate and activate HER2 signaling [
9,
17,
18], thus crosstalk signaling may influence HER2 protein expression in FISH + tumors. However, to test this hypothesis, all FISH + tumors should be examined regardless of IHC results. We could not investigate this issue in the current study as FISH was conducted only in IHC(2+) tumors. Nevertheless, the fact that pCR rate did not differ in relation to ER status indicates chemotherapy with anti-HER2 treatment is effective in ER and HER2-positive tumors, as well as HER2 type (HR-negative and HER2-positive) tumors. Meanwhile, patients with ER-positive tumors had significantly longer DFS. Adjuvant endocrine treatments should, of course, contribute to these patients’ prognoses.
The major limitation of our study was that it was a retrospective observational study, thus systemic treatments, such as chemotherapy and anti-HER2 drugs, were not uniform. Further analysis with a larger sample size is required, especially to further examine patient outcomes of IHC(2+)/FISH(+) and HR-negative tumors, and to compare the effects of treatment after recurrence. In addition, while FISH was only performed in IHC(2+) tumors, it can be assumed that some IHC(0/1+) tumors are also FISH(+) [
19]. The biological behavior of FISH(+) tumors with none/little HER2 protein should be thoroughly assessed in the near future, particularly given that a large number of clinical trials of novel HER2 protein-anchored drugs are currently ongoing.
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