Prognostic value of the 21-gene recurrence score in ER-positive, HER2-negative, node-positive breast cancer was similar in node-negative diseases: a single-center study of 800 patients

Abstract

Multi-gene assays have emerged as crucial tools for risk stratification in early-stage breast cancer. This study aimed to evaluate the prognostic significance of the 21-gene recurrence score (RS) in Chinese patients with pN0-1, estrogen receptor-positive (ER⁺), human epidermal growth factor receptor 2-negative (HER2^–) breast cancer. Among 800 patients recruited between 2009 and 2016, the median RS was 24 (0–69), with 27.4%, 46.8%, and 25.9% patients classified into low-, intermediate-, and high-risk groups. Cox regression analysis demonstrated that the high-risk category was associated with significantly higher odds of invasive disease-free survival (IDFS) and distant disease-free survival (DDFS) events compared with the low-risk category (IDFS: HR= 2.450, 95% CI 1.017–5.902, P= 0.046; DDFS: HR= 2.829, 95% CI 1.013–7.901, P= 0.047). No significant association between RS category and overall survival (OS) was found (intermediate vs. low: HR= 1.244, 95% CI 0.292–5.297, P= 0.768; high vs. low: HR= 2.933, 95% CI 0.759–11.327, P= 0.119). RS, as a continuous variable, was a highly significant predictor for IDFS (HR= 1.028, 95% CI 1.010–1.047, P= 0.002), DDFS (HR= 1.030, 95% CI 1.010–1.051, P= 0.003), and OS (HR= 1.034, 95% CI 1.007–1.063, P= 0.014). Our findings suggested that RS may predict IDFS in Chinese patients with ER⁺/HER2^– breast cancer with N0 or N1 disease.

Keywords

early breast cancer / 21-gene assay / recurrence score / prognosis

1 Introduction

Multigene assays have emerged as crucial tools for risk stratification in early-stage breast cancer. The 21-gene recurrence score (RS) assay (Oncotype Dx, Genomic Health, Redwood City, CA, USA) is a quantitative reverse-transcriptase polymerase chain reaction (RT-PCR)-based assay that measures 16 cancer-related genes and 5 housekeeping genes in formalin-fixed paraffin-embedded (FFPE) tumor tissue. Retrospective validation studies conducted in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 study population indicated that the RS category (low risk: RS less than 18; intermediate risk: RS 18 to 30; high risk: RS more than 30) can predict the likelihood of disease recurrence or breast cancer mortality in patients treated with tamoxifen alone [1]. Furthermore, the predictive value of RS for adjuvant chemotherapy has been verified in patients with estrogen receptor-positive (ER⁺), human epidermal growth factor receptor 2-negative (HER2^–), lymph node-negative (LN^–), or LN-positive (LN⁺) diseases through several retrospective analyses with the NSABP B-20 and Southwest Oncology Group (SWOG)-8814 study population [2,3]. In the recently published Trial Assigning IndividuaLized Options for Treatment (TAILORx) study, a prospective analysis indicated that patients with RS less than 11 can be safely spared from adjuvant chemotherapy, and patients with RS 11 to 25 will gain no significant benefit from adjuvant chemotherapy [4,5].

The 21-gene RS has been applied to guide adjuvant treatment decisions. Approximately 13%–34% of patients’ treatment decisions have changed to no chemotherapy after the RS was applied [6]. The 21-gene RS assay is now recommended by the American Society of Clinical Oncology (ASCO) guideline and the St. Gallen Consensus for patients with ER⁺/HER2^– early-stage breast cancer [7,8]. It has also been recently adopted in the 8th edition of the American Joint Commission of Cancer (AJCC) staging manual [9].

Although RS has been regularly recommended to patients with ER⁺/HER2^– early breast cancer, no studies have been conducted to evaluate its prognostic value in the Chinese population. Therefore, this study aimed to assess the prognostic significance of the 21-gene RS in Chinese patients with ER⁺/HER2^–, pN0, and pN1 breast cancer.

2 Materials and methods

2.1 Study population

Patients with early stage breast cancer diagnosed and treated in Ruijin Hospital Affiliated Shanghai Jiao Tong University School of Medicine from January 2009 to January 2016 were retrospectively reviewed. The eligibility criteria included patients over 18 years of age with histopathologically confirmed invasive breast cancer, T1b-c, T2, or T3, ER⁺, HER2^–, and pN0–1. Patients with T1a or T4 tumors, de novo stage IV diseases, and a previous history of any malignant tumor, or who have received neoadjuvant treatment were excluded. After surgery, patients in our center underwent consultation for adjuvant treatment with a multi-disciplinary team [10,11] consisting of surgical oncologists, medical oncologists, radiation oncologists, radiologists, and breast cancer specialist nurses. For ER⁺/HER2^– patients with RS results, the adjuvant treatment recommendation was worked out on the basis of the patient’s traditional clinico-pathological factors, 21-gene RS, and preference, according to the St. Gallen Consensus, treatment guidelines, and results of 21-gene RS-related clinical trials. Radiation therapy was generally recommended to patients who had breast-conserving surgery (BCS), tumor(s) more than 5 cm in size, or nodal involvement. Detailed clinico-pathological characteristics, adjuvant treatment recommendation, and follow-up data were obtained from the Shanghai Jiao Tong University Breast Cancer Database (SJTU-BCDB). Tumors were classified into luminal A-like (ER⁺, progesterone receptor (PR)≥20% positive and Ki67<14%) and luminal B-like subtypes (ER⁺, PR<20% or Ki67≥14%).

The current study received approval from the Ethical Committees of Ruijin Hospital. All patients recruited signed the informed consent. The results of this study did not affect the adjuvant treatment recommendations.

2.2 Immunohistochemistry evaluation and 21-gene RS assay

Tumor clinico-pathological and immunohistochemistry (IHC) evaluations were independently conducted in the Department of Pathology, Ruijin Hospital, by two experienced pathologists. Tumors were defined as ER⁺ or PR⁺ when nuclear staining was observed in≥1% of invasive tumor cells [12]. HER2^– was defined as 0 to 1+ by IHC or 2+ by IHC with negative results by fluorescence in situ hybridization according to ASCO/CAP guidelines [13].

The 21-gene RS assay was conducted on a macro-dissected FFPE tumor specimen reviewed and selected by pathologist XC Fei. RS was tested and calculated as described in a previous work [14]. In brief, the expression of 16 cancer-related genes was measured by standardized quantitative RT-PCR reactions and normalized relative to five reference genes. According to previous retrospective studies, low-, intermediate-, and high-risk groups were defined as RS less than 18, 18 to 30, and more than 30, respectively [1].

2.3 Study endpoints

The standardized definitions for efficacy end points (STEEP) criteria were applied for the endpoint definitions [15]. The primary endpoint was the rate of invasive disease-free survival (IDFS), with an IDFS event defined as the first occurrence of local recurrence, ipsilateral invasive breast tumor, regional recurrence, distant recurrence, contralateral second primary invasive cancer, second primary non-breast invasive cancer, or death without evidence of recurrence. Secondary endpoints included the rate of distant disease-free survival (DDFS), with a DDFS event defined as the first occurrence of distant recurrence or death from any cause, and the overall survival (OS) rate, which is defined as the proportion of patients who were alive at the latest follow-up.

2.4 Statistical analysis

All statistical analyses were performed on STATA (version 14.0). The association of RS risk category and traditional clinico-pathological factors was evaluated by Chi-square test. IDFS, DDFS, and OS were estimated using the Kaplan–Meier estimator and tested with log-rank tests. Hazard ratio (HR), 95% confidence interval (CI), and the effects of clinico-pathological variables were estimated by multivariate Cox regression model. All statistical tests were two-sided and considered significant if P<0.05.

3 Results

3.1 Clinico-pathological features and distribution of RS

In summary, 800 patients with ER⁺HER2^–pN0–1 breast cancer were included in this study. The median age was 55 years, ranging from 27 years to 95 years. A total of 707 (88.3%) patients were diagnosed with invasive ductal carcinoma (IDC). Table 1 presents the pathology of the included population. T1 tumors composed 68.6% of all patients. Notably, 12.3% of them had 1–3 positive nodes, whereas the others were node-negative. Grade I, II, and III tumors were found in 15.6%, 50.4%, and 22.6% of all cases, respectively. A total of 328 (41.0%) patients received BCS. Adjuvant chemotherapy, radiotherapy, and endocrine treatment were administered in 49.3%, 56.5%, and 96.0% of all cases, respectively.

In all the patients, the median RS was 24, ranging from 0 to 69. A total of 219 patients (27.4%) were classified as low risk, 374 (46.8%) as intermediate risk, and 207 (25.9%) as high risk. The distribution of RS according to different clinico-pathological factors is shown in Table 2. Grade, pT stage, PR status, Ki67 expression level, and intrinsic molecular subtypes were significantly correlated to RS (all P<0.05, Table 2). A large proportion of high-risk RS was observed in patients with grade III, pT2–3, PR^–, Ki67>14%, or luminal B-like disease.

3.2 Univariate and multivariate analyses of IDFS, DDFS, and OS

After a median follow-up of 37.1 (range: 10.8–105.1) months, 47 IDFS events, 38 DDFS events, and 19 OS events occurred. The overall rates of IDFS, DDFS, and OS were 94.1%, 95.3%, and 97.6%, respectively.

In the univariate model, pathologic T stage, Ki67 index, intrinsic molecular subtype, and RS category were significantly associated with IDFS (all P<0.05). The 4-year IDFS rates for patients with low-, intermediate-, and high-risk RS were 97.3%, 95.5%, and 89.5%, respectively (Table 3, Fig. 1). Cox regression analysis, including pathologic T stage, molecular subtype, and RS category, revealed that patients with high-risk RS were associated with significantly higher odds of IDFS event compared with those with low-risk RS (HR= 2.450, 95% CI 1.017–5.902, P = 0.046, Table 3). Pathologic T stage was the only factor remaining independently predictive for IDFS in the multivariate model (HR= 2.162, 95% CI 1.207–3.874, P = 0.010, Table 4).

Univariate analyses for DDFS suggested that pathologic T stage, Ki67 index, and RS category were significantly associated with DDFS (all P<0.05). The 4-year DDFS rates for patients with low-, intermediate-, and high-risk RS were 98.6%, 96.1%, and 92.6%, respectively (Table 3, Fig. 2). Cox regression analysis indicated that patients with high-risk RS were associated with significantly higher odds of DDFS event compared with those with low-risk RS (HR= 2.829, 95% CI 1.013–7.901, P = 0.047, Table 4). Pathologic T stage was capable of independently predicting DDFS according to multivariate analysis (HR= 2.117, 95% CI 1.105–4.053, P = 0.024, Table 4).

For the OS, no significant association between different pathologic T stages, molecular subtypes, or RS categories was found in either univariate or multivariate analyses, probably due to the limited number of events. For patients with low-, intermediate-, and high-risk RS, the 4-year OS rates were 98.7%, 98.6%, and 95.3%, respectively (Table 3, Fig. 3).

3.3 Association of RS as continuous variable and disease recurrence

Continuous RS was a highly significant predictor for IDFS (HR= 1.028, 95% CI 1.010–1.047, P = 0.002), DDFS (HR= 1.030, 95% CI 1.010–1.051, P = 0.003), and OS (HR= 1.034, 95% CI 1.007–1.063, P = 0.014). When a similar manner of calculation to the NSABP B-14 study [1] was applied in the current cohort, compared with 50 RS units, the HR was 3.978 (95% CI 1.645–9.939) for IDFS, 4.384 (95% CI 1.645–12.026) for DDFS, and 5.321 (95% CI 1.417–21.216) for OS.

3.4 Risk estimation in different subtypes of patients

Subgroup analyses revealed that RS consistently predicted invasive recurrence in all pT categories, tumor grade categories, and molecular subtypes (Fig. S1). Notably, patients with relatively favorable pathological factors, such as smaller tumors, grade I–II diseases, and luminal A subtype, were also seen in the high-risk RS group. Patients with pT1, grade I–II and luminal A diseases, and high-risk RS had an estimated risk of invasive recurrence at four years at 5.5%, 12.1%, and 8.4%, respectively.

4 Discussion

In summary, our study evaluated the prognostic significance of the 21-gene RS in 800 Chinese patients with ER⁺, HER2^–, pN0–1 breast cancer from a single institute. To our knowledge, this work has one of the largest cohorts for studying the prognostic value of RS in node-negative and node-positive populations. Consistent with previous reports [1–3,16], the current study managed to quantify the likelihood of invasive recurrence in these luminal-like patients with or without node involvement. The risk of invasive recurrence in patients with RS more than 30 was significantly higher than those with RS less than 30.

Tumor size and molecular subtype are commonly considered as predictors of breast cancer relapse and are incorporated in modern treatment guidelines [17–19]. When RS was combined with data pertaining to tumor size and molecular subtype to predict the risk of invasive recurrence, only tumor size and RS maintained their statistical significance in multivariate analysis. Pan et al. also reported that in ER⁺ diseases, long-term distant recurrence rate and breast cancer mortality still depended on initial T-stage (T2N0 vs. T1N0: HR= 1.73, 1.53–1.95) besides grade and Ki67 index [20]. Given that RS is mainly adopted in patients with limited nodal involvement, tumor size will likely remain a predominant contributor of tumor burden. Furthermore, RS produces a quantitative and comprehensive portrait of tumor biology beyond traditional clinico-pathological characteristics. In this study, the molecular subtype no longer maintained its prognostic significance when combined with RS in multivariate analysis. Similarly, in the retrospective analysis of E2197 trial [16], high tumor grade was associated with a significantly increased recurrence risk only when RS was excluded in the multivariate model (P<0.001). However, RS was highly associated with recurrence (HR= 3.13, 95%CI 1.60–6.14, P = 0.0009), whereas tumor grade was not (P = 0.20) when they were both included in the multivariate model. We postulated that genomic risk assessment by RS, which is based on the integration of 16 cancer-related genes, provides additional prognostic information beyond traditional clinico-pathological factors for early stage ER⁺, HER2^– breast cancer in the Chinese population. Given the previous findings, the 21-gene RS assay should be part of routine tests for Chinese patients with ER⁺/HER2⁻ early breast cancer. For patients with clinical high risk, the assay may help us identify a certain subgroup of patients with good prognosis and can be safely spared from adjuvant chemotherapy.

The overall invasive and distant recurrence rates were relatively low in this study, with only 47 IDFS and 38 DDFS events. To note, the cumulative 4-year invasive recurrence rates were 2.7%, 4.5%, and 10.5%, while the 4-year distant recurrence rates were 1.4%, 3.9%, and 7.4% in patients with low-, intermediate-, and high-risk RS, respectively. Previous studies examining the prognostic value of RS have reported higher rates of distant recurrence [1]. For example, the Kaplan–Meier estimates of 10-year distant recurrence rate were 6.8%, 14.3%, and 30.5% in patients having low-, intermediate-, and high-risk RS in the NSABP B-14 study [1–3]. A possible explanation is that, in the B-14 study, patients with high-risk RS did not receive adjuvant chemotherapy as in our cohort (50.8%), likely contributing to the increased rate of distant recurrence. The NSABP B-20 study also showed that the distant relapse rate at 10 years for low-, intermediate-, and high-risk RS was 3.2%, 9.1%, and 29.5% in chemotherapy-naive patients and 4.4%, 10.9%, and 11.9% in chemotherapy-treated patients [2]. The adoption of chemotherapy in patients with high-risk RS was reduced but did not eliminate the difference in the rate of distant recurrence seen on the basis of RS.

On the basis of the retrospectively established RS category, 27.4%, 46.8%, and 25.9% patients were categorized into low-, intermediate-, or high-risk groups, respectively. Such distribution of the 21-gene RS was different from the previous results of the NSABP B-14 trial, which reported 51%, 22%, and 27% patients as low, intermediate, or high risk [1]. A discordant proportion of risk category was also reported in several studies [1,2,4]. Moreover, the distribution of RS observed by Genomic Health was different from the initial report [4]. One possible explanation is the bias of patient selection among institutions in clinical practice [19].

Our study had several limitations. First, the retrospective design might have drawn selection bias, leading to a differently distributed RS category. Second, considering the pattern of recurrence and treatment effect in ER⁺, HER2^– breast cancer, the follow-up time was relatively too short to observe any survival difference between different risk groups. Furthermore, the selection of the cutoff value was based on the results from NSABP B-14 and NSABP B-20 studies instead of the TAILORx parameters. However, in the exploratory analysis, we found that when using 11 and 25 as the cutoff values for risk classification, the new risk category displayed similar prognostic performance compared with the model using cutoffs of 18 and 30 in terms of LR-c (cutoff 11 and 25: 16.48; cutoff 18 and 30: 20.18) and C-index (cutoff 11 and 25: 0.6635; cutoff 18 and 30: 0.6663). Nevertheless, our findings were consistent with the results of previous studies. With the recent integration of the RS in prognostic staging systems of ER⁺, HER2^– breast cancer by the AJCC [9], the RS assay is likely to continue playing an important role in disease prognosis prediction and treatment decision guidance.

In conclusion, our study delineated the prognostic significance of RS in Chinese patients with ER⁺, HER2⁻, pN0–1 breast cancer for the first time. Further prospective studies with long-term follow-up are warranted to verify the prognostic and predictive value of RS in the Chinese population.

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