Ten-year distant-recurrence risk prediction in breast cancer by CanAssist Breast (CAB) in Dutch sub-cohort of the randomized TEAM trial

The parent trial for this study was TEAM, a global multicenter, open-label, randomized, phase III study of postmenopausal women with HR+breast cancer, conducted from 2002 to 2016 (Trial Registration: ClinicalTrials.gov NCT00279448, NCT00032136, and NCT00036270; Netherlands Trial Registry NTR267). The inclusion and exclusion criteria and registration information of TEAM were described previously [10]. Patients were randomly assigned (1:1) to receive exemestane (25 mg once a day, orally) or sequential therapy (tamoxifen of 20 mg once a day, orally for 2.5–3.0 years, and switched to exemestane therapy for a total duration of 5.0 years). Stratification factors included center, adjuvant chemotherapy (yes vs no) and time period between surgery and starting hormonal therapy (0–3 months vs 3–6 months vs > 6 months). In the original TEAM trial, 6120 patients were involved in the intention-to-treatment (ITT) analysis. The samples from a cohort of 592 patients were collected from the Dutch trial population of 2754 patients. Finally, CAB was successfully done on 480 patient samples. Patients with HER2-positive receptor status, TNM stage IV or missing lymph node status, and loss of follow-up were excluded from the final analysis of the present study (Additional file 1: Figure S1). The study has been carried out in accordance with the Declaration of Helsinki. All patients provided informed consent.

The primary endpoint of this study was the cumulative incidence of distant recurrence or metastasis/death due to breast cancer (DM), or the freedom from distant metastasis or death due to breast cancer (known as [DRFi]). DRFi was defined as the time from randomization to distant recurrence or death due to breast cancer if no recurrence was reported before death. Other endpoints were cumulative incidence of relapse or recurrence-free survival (RFS), cumulative mortality or overall survival (OS), and cumulative incidence of invasive breast cancer disease or invasive disease-recurrent free survival (iDFS). RFS was defined as the time from randomization to any breast cancer recurrence or death due to breast cancer if no recurrence was reported before death. OS was defined as the time from randomization to death due to any cause. iDFS was defined as the time from randomization to disease recurrence (locoregional or distant recurrence or new primary breast cancer, excluding ductal carcinoma in situ [DCIS] of any kind and new second primary non-breast cancer) or death from any cause.

Kaplan–Meier estimates of DM, cumulative incidence of relapse, cumulative mortality, and cumulative incidence of invasive disease were calculated for CAB risk groups for the total cohort, treatment groups or designated subgroups, compared by log-rank tests. Univariate and multivariate hazard ratios (HRs), as well as the interaction between subgroups and prognostic factors, were calculated by the Cox proportional hazard model. For multiple comparisons, the P value was adjusted using the method of Benjamini–Hochberg (BH). Data were collected in the Central Statistical and Data Centre (Leiden University Medical Center, LUMC, Leiden, the Netherlands). Statistical analyses were done with R (4.0.3 version) with the Survival, Survminer, Ggplot2, Ggpubr, Forestplot, Rms, and Rstatix packages. The endpoints were censored at 10.0 years. All tests were two-sided, and a P-value of less than or equal to 0.05 was considered significant.

Tumor samples were initially assessed for amount of tumor with the hematoxylin and eosin staining method. CAB is only performed in OncoStem’s CAP (College of American Pathologists) and ISO13485 accredited laboratory in Bangalore, India. Tumor samples with greater than 30% of tumor content were processed for immunohistochemistry (IHC) experiments for CAB markers. IHCs were performed on a Ventana Benchmark automated IHC stainer [8]. The IHC grading information along with clinical parameters (tumor size, histological grade, node status) was provided as inputs to the statistical algorithm. The algorithm predicts a risk score and based on the predefined threshold, patients with a risk score of below and equal to 15.5 are deemed to have a low risk for distant recurrence and patients with a risk score of above 15.5, are considered as a high risk for distant recurrence [16]. The team performing CAB were blinded to patient outcomes. 

Of the 592 Dutch patients enrolled in TEAM, 433 (73.1%), consistent with the inclusion criteria of the present study, underwent CanAssist Breast (CAB) test (Additional file 1: Figure S1). The two trial arms in the current study were well-balanced, with a median follow-up of 10.5 years (IQR 10.4–10.6). A total of 221 patients were randomized to the exemestane arm, and 212 patients were randomized into the sequential arm (Table 1). The median age was 65.0 years. The majority were T2 (51.3%), lymph node-positive (68.4%), TNM stage II (79.4%), and grade II-III (79.5%). Most of the patients (79.2%) received endocrine therapy alone. 404 (93.3%) patients had lymph node stage of pN0-1, defined as node-negative (31.6%) plus one to three nodes (61.7%) involved, while 6.7% had N2 disease. The distributions of the tumor stage, lymph node stage, TNM stage, age, and estrogen receptor (ER) were not significantly different compared to the Dutch population (n = 2754 patients), while in the current study, there were higher proportions of low-grade, progesterone receptor (PR)-positive and patients without receiving chemotherapy (Table 1).

CAB was analyzed as a categorical variable (low or high-risk). 32.5% of patients were classified as the high-risk group, while the rest of them were in the low-risk group. In Kaplan–Meier survival analysis, CAB significantly stratified patients for the primary endpoint, DM [11.5% (95% CI, 7.6–15.2) for low-risk, vs 30.2% (95% CI, 21.9–37.6) for high risk; HR, 2.90 (95% CI, 1.75–4.80); P < 0.001; Fig. 1A]. Other endpoints included cumulative incidence of relapse (HR, 1.99; 95% CI, 1.27–3.10; P < 0.001; Fig. 1B), cumulative mortality (HR, 1.38; 95% CI, 0.93–2.07; P = 0.09; Additional file 2: Figure S2A), and cumulative incidence of invasive breast cancer recurrence (HR, 1.40; 95% CI, 0.98–2.01; P = 0.05; Additional file 2: Figure S2B). In each of the endpoints barring cumulative mortality, CAB high-risk patients had significantly higher recurrences/worse outcomes than CAB low-risk patients.

The Cox regression model was adjusted for T stage, lymph nodal status, grade, and CAB risk in multivariate analysis. The result showed that CAB risk score (HR, 2.54; 95% CI, 1.67–3.85; P < 0.001) was an independent prognostic factor for DM (Table 2).

The CAB risk score was prognostic for DM in the subset of patients with lymph node-positive (N+; HR, 3.11; 95% CI, 1.76–5.48; P < 0.001; Fig. 1C), or in the subset of patients with N0-1 (HR, 2.63; 95% CI, 1.48–4.68; P < 0.001; Fig. 1D). During the first five years, CAB was also prognostic for early DM in N+ patients (HR, 2.60; 95% CI, 1.24–5.46; P = 0.008; Additional file 3: Figure S3A). The significant interaction between node status and CAB was recorded (P_interation < 0.001), but node status was not an independent risk factor in the multivariate regression model.

In the exemestane arm, patients were significantly classified into low or high-risk groups by CAB for DM (HR, 4.76; 95% CI, 2.26–10.04; P < 0.001; Fig. 2A). In the sequential arm, CAB could also predict the prognosis for DM (HR, 2.00; 95% CI, 1.01–3.97; P = 0.03; Fig. 2B). The stratification ability of CAB was better in the exemestane arm than in the sequential arm.

For patients with N+, there was significant association between CAB risk category and DM at ten years, in exemestane arm (HR, 4.74; 95% CI, 2.03–11.03; P < 0.001; Additional file 3: Figure S3B) and sequential arm (HR, 2.17; 95% CI, 1.01–4.65; P = 0.04; Additional file 3: Figure S3C).

CAB provided statistically significant prognostic information on DM in patients who received endocrine therapy alone (HR, 3.30; 95% CI 1.83–5.94; P < 0.001; Fig. 2C). For patients with N+ who did not receive chemotherapy, CAB could significantly stratify patients for DM as well (HR, 3.05; 95% CI, 1.58–5.88; P < 0.001; Fig. 2D). Furthermore, CAB could stratify patients into low or high-risk groups for DM in both arms; exemestane alone (HR, 4.59; 95% CI, 2.11–10.00; P < 0.001; Additional file 2: Figure S2C) or sequential regimen (HR, 2.32; 95% CI, 0.94–5.71; P = 0.03; Additional file 2: Figure S2D).

CAB statistically significantly stratified three groups at ten years (log-rank P < 0.001; Fig. 3A), including CAB high-risk with a DRFi of 69.8% (95% CI, 62.4–78.1), CAB low-risk in the exemestane arm with a DRFi of 92.7% [95% CI, 88.5–97.2; vs CAB high-risk, HR, 0.21 (95% CI, 0.11–0.43), P < 0.001], and CAB low-risk in the sequential arm with a DRFi of 84.2% [95% CI, 78.2–90.7; vs CAB high-risk, HR, 0.48 (95% CI, 0.28–0.82), P = 0.009].

In CAB low-risk group (Additional file 4: Figure S4A), for patients who received endocrine therapy alone, exemestane arm had better overall survival (OS; HR, 1.79; 95% CI, 1.16–2.78; P = 0.03), iDFS (HR, 1.92; 95% CI, 1.29–2.87; P = 0.007), and RFS (HR, 2.05; 95% CI, 1.17–3.58; P = 0.04) than the sequential arm, but this survival benefit was not observed for DRFi (HR, 1.51; 95% CI, 0.77–2.96; P = 0.31). In addition, in low-risk group (Additional file 4: Figure S4A), patients who received chemo-endocrine therapy did not have an advantage of improved survival benefit for either OS (HR, 1.99; 95% CI, 1.03–3.83; P = 0.09), iDFS (HR, 1.42; 95% CI, 0.85–2.38; P = 0.24), RFS (HR, 0.99; 95% CI, 0.54–1.82; P = 0.98), or DRFi (HR, 0.93; 95% CI, 0.44–1.97; P = 0.88), when compared with endocrine therapy alone.

In high-risk group, for patients who received endocrine therapy alone (Additional file 4: Figure S4B), there was no significant difference in the outcomes between two randomized arms for all the end points: OS (HR, 0.79; 95% CI, 0.45–1.40; P = 0.50), iDFS (HR, 0.81; 95% CI, 0.48–1.35; P = 0.50), RFS (HR, 0.80; 95% CI, 0.44–1.45; P = 0.54), and DRFi (HR, 0.77; 95% CI, 0.41–1.43; P = 0.48). Moreover, in high-risk group (Additional file 4: Figure S4B), the patients who received chemotherapy had an advantage of OS (HR, 2.38; 95% CI, 1.21–4.68; P = 0.04), and iDFS improvement (HR, 2.40; 95% CI, 1.31–4.38; P = 0.02) over patients received endocrine therapy alone; however, this survival advantage was not observed in either RFS (HR, 1.80; 95% CI, 0.97–3.34; P = 0.12) or DRFi (HR, 1.59; 95% CI, 0.85–2.96; P = 0.22). Our data showed chemotherapy may have benefits in CAB high-risk patients of exemestane arm alone, but due to small sample size, this cannot yet be convincingly shown.