Abstract
Background
Hormone receptor-positive, HER2-negative (HR + /HER2-) breast cancer generally has a favorable prognosis; however, early postoperative recurrence markedly reduces survival. Accurate prediction of early recurrence is crucial for personalizing treatment. This case–control study compared MammaPrint (MP) and BluePrint (BP) results between early recurrence patients and matched controls.
Methods
Patients were selected from our previous study, the WJOG15721B cohort (n = 2732). Those with recurrence within three years after surgery were randomly extracted, and controls matched for institution, clinical stage, and number of pathological lymph node metastases were included (n = 124). Tumor samples underwent MP and BP assays to classify recurrence risk and molecular subtypes.
Results
Of 115 submitted tumor samples, 85 were analyzed successfully (43 early recurrence, 42 no recurrence). High-risk MP classification was significantly more frequent in early recurrence patients (79.1% vs. 40.5%, p < 0.001), and Luminal B BP subtype was more common in early recurrence patients (79.1% vs. 38.1%, p < 0.001). High MP risk was associated with high Ki-67 levels and higher nuclear grade. Integrating clinical and genomic risk enhanced prognostic precision: patients with both clinical and genomic high risk had the highest recurrence rate (100%), those with low clinical and genomic risk had the lowest (28.1%), and patients with low clinical but high genomic risk showed an intermediate recurrence rate (57.5%).
Conclusions
Compared with patients without recurrence, those with early recurrence showed a significantly higher prevalence of high-risk MP results and Luminal B BP subtype. High-risk MP/Luminal B BP subtype suggested an association with early recurrence in patients with HR + /HER2- early breast cancer.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12282-026-01847-2.
Keywords: HR-positive HER2-negative breast cancer, Early recurrence, MammaPrint, BluePrint, Genomic risk
Introduction
Hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR +/HER2-) breast cancer has a good prognosis, but patients with early recurrence are difficult to treat and have a relatively poorer prognosis than those of with late recurrence. The 5-year survival of patients who experience recurrence within 3 years after initial treatment has been reported to be 29% [1]. As new treatment options for the postoperative treatment of HR +/HER2- breast cancer other than chemotherapy, such as cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, are emerging to reduce the risk of recurrence, it is important to estimate the risk of recurrence in detail to personalize treatment. We conducted a retrospective study to identify risk factors for early recurrence in patients with HR +/HER2- early breast cancer who received adjuvant endocrine therapy by evaluating the correlations among clinicopathological characteristics, perioperative treatment, and prognosis (WJOG15721B/RealisE, UMIN000047049). The results revealed that younger age, nuclear grade, vascular invasion, tumor size, and number of lymph node metastases were independent risk factors for early recurrence [2].
While clinicopathological factors are essential for predicting postoperative recurrence, a multigene assay for predicting recurrence has been clinically applied in HR +/HER2- breast cancer patients. MammaPrint, a multigene assay, classifies patients into high- and low-risk groups on the basis of the expression patterns of 70 genes and predicts recurrence. The MINDACT trial demonstrated that patients with high clinical risk and low genomic risk according to MammaPrint had excellent distant metastasis-free survival after endocrine therapy alone. As a result, adjuvant chemotherapy can be omitted in 46% of patients with high-risk breast cancer without affecting survival outcomes [3, 4]. Along with MammaPrint, BluePrint is a test to identify biological differences unique to individual breast cancers by capturing activated downstream molecular pathways on the basis of the expression of 80 genes and classifying them into four subtypes (Luminal A-type, Luminal B-type, HER2-type, and Basal-type) using formalin-fixed, paraffin-embedded specimens of the same primary tumor [5, 6].
Although MammaPrint can predict the risk of postoperative recurrence of early breast cancer and identify patients for whom adjuvant chemotherapy could be safely omitted, there have been no reports evaluating the results of MammaPrint and/or BluePrint in patients with HR +/HER2- breast cancer that recurred within 3 years after surgery. This study aimed to compare MammaPrint and BluePrint results between early recurrence cases and nonrecurrence controls from the WJOG15721B study.
Patients and methods
Patients
Patients aged ≥ 20 years with histologically confirmed HR +/HER2- clinical stage II-III invasive breast cancer who received adjuvant endocrine therapy between 2012 and 2017 (n = 2,732) were included in a retrospective cohort study (WJOG15721B study) across five institutions in Japan. The study methods and results were published previously [2]. Among the enrolled patients, we recruited patients for the WJOG16722B/RealisE-TR study (UMIN000050930) using the following method.
Approximately 70 patients who experienced recurrence within 3 years after surgery (early recurrence cases) were randomly selected from the patients enrolled in WJOG15721B study, within a retrospective cohort in which recurrence outcomes had already been determined. We defined early recurrence as recurrence occurring within 3 years after surgery because previous studies have used various definitions (e.g., 2.5 years from diagnosis or 5 years after surgery) without a clear consensus [7, 8], and considering that endocrine relapse within 2 years is regarded as primary endocrine resistance in international guidelines [9] and that the benefit of adjuvant abemaciclib persists beyond the 2-year treatment period [10]. Afterward, data from approximately 70 patients who did not experience recurrence (control) were identified 1:1 matching based on three factors: institution, clinical stage, and the number of pathologically positive lymph nodes. If matching was not possible, the case was excluded from the analysis. Importantly, the matching was performed by a biostatistician prior to sample testing and without knowledge of the outcome status. Furthermore, since the parent study (WJOG15721B) included approximately 2,700 cases, no missing data occurred due to an inability to identify matched controls for the selected cases.
Informed consent was obtained, but for cases where the patient had passed away or relocated with an unknown contact address, an opt-out procedure was conducted.
Molecular signature tests
Molecular signature testing was conducted using needle biopsy samples obtained at the time of diagnosis or tissue samples obtained at the time of surgery. MammaPrint® and BluePrint® (Agendia, Inc.) molecular signature tests were performed in a validated laboratory. For the MammaPrint® assay, each case was assigned to a recurrence risk category, along with a molecular subgroup determined by the BluePrint® assay." Tumors were classified with MammaPrint as low risk [0.000 < MammaPrint index (MPI) ≤ 1.000] or high risk (−1.000 ≤ MPI < 0.000). Among the low-risk patients, those with scores in the range of + 0.356 to + 1.000 were defined as being at ultralow risk. BluePrint categorizes tumors by subtype (Luminal, HER2, and Basal). Together, MammaPrint and BluePrint further stratified luminal-type tumors as Luminal A-type (MammaPrint low risk) or Luminal B-type (MammaPrint high risk) tumors.
Endpoints
The primary endpoint was the proportion of high-risk patients according to MammaPrint. The secondary endpoints were the proportions of BluePrint Basal-type and other molecular subtypes identified by BluePrint and the relationships between MammaPrint/BluePrint and clinicopathological factors.
Categorization into risk groups
Patients were classified into low or high clinical risk groups in an exploratory manner on the basis of the total nomogram score we developed in the WJOG1572B study to predict the risk of early recurrence [2]. The cutoff value was set at 250 points, which was based on the score corresponding to a 3-year invasive disease-free survival of 83.4% in the endocrine therapy-alone group in the monarchE trial [10, 11]. Additionally, MammaPrint high-risk and low-risk patients were categorized into high genomic high-risk and low genomic risk groups.
Statistical analysis
The MPI obtained via MammaPrint for the early recurrence and no recurrence groups was summarized and compared between groups using the Wilcoxon rank sum test. For each group, we estimated the proportion of MammaPrint high risk cases and the 95% confidence interval on the basis of the binomial distribution. To evaluate the association between high-risk MammaPrint results and recurrence, Fisher’s exact test was performed. Missing values were not imputed and were excluded from the analysis. BluePrint subtypes (Luminal A, Luminal B, HER2, and Basal types) were summarized for the early recurrence and no recurrence groups using Fisher's direct probability test. The associations between MammaPrint index values and clinicopathological factors were evaluated using t tests and analysis of variance (ANOVA), and the associations between the MammaPrint high-risk/low-risk results and clinicopathological factors were evaluated on the basis of odds ratios (ORs) and 95% confidence intervals (CIs) by logistic regression analysis. The associations between BluePrint subtype and clinicopathological factors were analyzed similarly. In this study, pair-matched analysis could not be performed due to the unavailability of some samples or the failure of genomic analysis after submission. All analyses were conducted with SAS (version 9.4; Cary, NC, USA).
Results
Patient clinical characteristics
A flow chart of patient selection is presented in Fig. 1. Among the 2732 patients included in the WJOG15721B study, 62 patients with early recurrence and 62 matched patients without recurrence were selected as candidates. MammaPrint/BluePrint testing was performed on 115 patients. Twenty-nine patients failed to pass the MammaPrint RNA quality check, 86 (74.8%) were successfully analyzed, and 85 were ultimately included in the study analysis, except one who was ineligible.
Fig. 1.
Flow chart of patient selection. *Of the 124 eligible candidates, 10 were excluded, but one ineligible patient's specimen was submitted, for a total of 115 samples. HR hormone receptor, HER2 human epidermal growth factor 2
The patient background and clinicopathological characteristics of the study population are shown in Table 1 and Supplementary Table 1. The median age was 53.5 years in the no recurrence group and 47 years in the early recurrence group. Twenty-two patients (52.4%) in the no recurrence group and 26 (60.5%) in the early recurrence group were premenopausal. In the nonrecurrence group, 32 (76.2%) patients had clinical stage IIA disease, 4 (9.5%) patients had stage IIB disease, and 6 (14.3%) patients had stage III disease; in the early recurrence group, 31 (72.1%), 7 (16.3%), and 5 (11.6%) patients had clinical stage IIA, IIB and III disease, respectively. The number of pathological lymph node metastases was as follows: 16 (38.1%) had none, 19 (45.2%) had 1–3, and 7 (16.7%) had ≥ 4 in the no recurrence group and 15 (34.9%) had none, 19 (44.2%) had 1–3, and 9 (20.9%) had ≥ 4 in the early recurrence group. There was a trend toward a high proportion of nuclear grade 2 (n = 25, 58.1%) and grade 3 (n = 11, 25.6%) patients in the early recurrence group compared with grade 2 (n = 12, 28.6%) and grade 3 (n = 8, 19.0%) patients in the no recurrence group. There was a greater tendency for patients in the early recurrence group (23.3%) to receive neoadjuvant chemotherapy (no recurrence group: 9.5%), but the proportion of patients who received adjuvant chemotherapy did not differ between the no recurrence (39.5%) and early recurrence groups (40.5%) (Table 2).
Table 1.
Patient characteristics and clinicopathological features of the study population
| No recurrence (n = 42) |
Early recurrence (n = 43) |
Total (n = 85) |
|
|---|---|---|---|
| Age, years | |||
| Median (range) | 53.5 (32–78) | 47 (33–86) | 48 (32–86) |
| Performance status*1 | (n = 27) | (n = 23) | |
| 0 | 27 (100) | 23 (100) | 50 |
| 1 | 0 (0) | 0 (0) | 0 |
| Menopausal status | |||
| Premenopausal | 22 (52.4) | 26 (60.5) | 48 |
| Postmenopausal | 20 (47.6) | 16 (37.2) | 36 |
| Unknown | 0 (0) | 1 (2.3) | 1 |
| Clinical stage | |||
| IIA | 32 (76.2) | 31 (72.1) | 63 |
| IIB | 4 (9.5) | 7 (16.3) | 11 |
| IIIA | 3 (7.1) | 3 (7.0) | 6 |
| IIIB | 3 (7.1) | 2 (4.7) | 5 |
| IIIC | 0 (0) | 0 (0) | 0 |
| Nuclear grade | |||
| 1 | 22 (52.4) | 4 (9.3) | 26 |
| 2 | 12 (28.6) | 25 (58.1) | 37 |
| 3 | 8 (19.0) | 11 (35.6) | 19 |
| Unknown | 0 (0) | 3 (7.0) | 3 |
| Pathological tumor size (cm) | |||
| < 2 | 11 (26.2) | 5 (11.6) | 16 |
| 2 to less than 5 | 28 (66.7) | 29 (67.4) | 57 |
| ≥ 5 | 3 (7.1) | 7 (16.3) | 10 |
| Unknown | 0 (0) | 2 (4.7) | 2 |
| Number of pathological lymph node metastases | |||
| 0 | 16 (38.1) | 15 (34.9) | 31 |
| 1–3 | 19 (45.2) | 19 (44.2) | 38 |
| 4–9 | 4 (9.5) | 5 (11.6) | 9 |
| ≥ 10 | 3 (7.1) | 4 (9.3) | 7 |
*1 Data represent aggregated results from 50 cases obtained from two institutions
Table 2.
Perioperative treatment details for the study population
| No recurrence (n = 42) |
Early recurrence (n = 43) |
Total (n = 85) |
|
|---|---|---|---|
| Neoadjuvant chemotherapy | |||
| No | 38 (90.5) | 33 (76.7) | 71 |
| Yes | 4 (9.5) | 10 (23.3) | 14 |
| Anthracyclines | 4 (9.5) | 10 (23.3) | 14 |
| Taxanes | 4 (9.5) | 10 (23.3) | 14 |
| Adjuvant chemotherapy | |||
| No | 25 (59.5) | 26 (60.5) | 51 |
| Yes | 17 (40.5) | 17 (39.5) | 34 |
| Anthracyclines | 15 (35.7) | 16 (37.3) | 31 |
| Taxanes | 17 (40.5) | 17 (39.5) | 34 |
| Others | 2 (4.8) | 2 (4.7) | 4 |
| Adjuvant endocrine therapy | |||
| No | 0 (0) | 0 (0) | 0 |
| Yes | 42 (100) | 43 (100) | 85 |
Patient characteristics and clinicopathological findings comparing the successful analysis group (n = 85) and the unsuccessful analysis group (n = 29) are presented in Supplementary Table 3.
Characteristics of MammaPrint and BluePrint
The MPI value was greater in the no recurrence group than in the early recurrence group, with a value of 0.086 (median, IQR: −0.1420.269; range: −0.9600.645) in the no recurrence group and −0.261 (median, IQR: −0.372–0.063; range: −0.8100.402) in the early recurrence group (p < 0.001) (Fig. 2a). In the nonrecurrence group, 17 patients (40.5%) were classified as high risk, 19 patients (45.2%) as low risk, and 6 patients (14.3%) as ultralow risk (Fig. 2b). In contrast, in the early recurrence group, 34 patients (79.1%) were classified as high risk, 7 patients (16.3%) as low risk, and 2 patients (4.7%) as ultralow risk (Fig. 2b). The proportion of patients at high risk on the basis of MammaPrint was 79.1% [95% confidence interval (CI) 64.0–90.0] in the early recurrence group and 40.5% (95% CI 25.6–56.7) in the no recurrence group, with a statistically significant difference (p < 0.001). Only one patient in the nonrecurrence group had the basal type (Fig. 2c). The percentage of Luminal B-type tumors was 79.1% (95% CI 64.0–90.0) in the early recurrence group and 38.1% (95% CI 23.6–54.4) in the no recurrence group, with a significant difference (p < 0.001) (Fig. 2c).
Fig. 2.
a Distribution of the MammaPrint index results in the early recurrence and no recurrence groups. b MammaPrint risk classification for the early recurrence and no recurrence groups. c Molecular subtyping based on the BluePrint index in the early recurrence and no recurrence groups
We performed the planned analysis to examine whether any subgroup exhibited high genomic risk, focusing on covariates that have been identified as prognostic or predictive factors for chemotherapy in previous studies (Table 3). There were significant associations of high-risk MammaPrint results with Ki-67 levels [≥ 30% vs. < 14%: odds ratio (OR) 26.43, 95% CI 3.03–230.16, p = 0.003] and nuclear grade (Grade 2 vs. Grade 1: OR 5.30, 95% CI 1.77–15.91, p = 0.003; Grade 3 vs. Grade 1: OR 12.26, 95% CI 2.84–52.87, p < 0.001). There were also significant interactions of Luminal B-type results in the BluePrint assay with Ki-67 levels (≥ 30% vs. < 14%: odds ratio (OR) 15.00, 95% CI 2.24–100.51, p = 0.005) and nuclear grade (Grade 2 vs. Grade 1: OR 5.30, 95% CI 1.77–15.91, p = 0.003; Grade 3 vs. Grade 1: OR 8.96, 95% CI 2.26–35.44, p = 0.002) (Table 4).
Table 3.
Associations between high-risk MammaPrint classification and clinicopathological factors
| Factor | Level | n | Proportion of MammaPrint high risk |
OR (95% CI)*1 | P value*2 |
|---|---|---|---|---|---|
| Age | 20–39 years | 11 | 9 (81.8%) | 1 | (0.387) |
| 40–69 years | 62 | 35 (56.5%) | 0.34 (0.07–1.58) | 0.169 | |
| ≥ 70 years | 12 | 7 (58.3%) | 0.36 (0.06–2.29) | 0.278 | |
| Menopausal status | Premenopausal | 48 | 29 (60.4%) | 1 | 0.847 |
| Postmenopausal | 36 | 21 (58.3%) | 0.92 (0.38–2.21) | ||
| Bilateral breast cancer | No | 84 | 50 (59.5%) | 1 | 0.746 |
| Yes | 1 | 1 (100%) | 2.14 (0.02–211.12) | ||
| ER | < 1% | - | - | - | - |
| 1–9% | - | - | - | - | |
| ≥ 10% | 46 | 26 (56.5%) | 1 | 0.491 | |
| Unknown | 39 | 25 (64.1%) | 1.36 (0.57–3.26) | ||
| PgR | < 1% | 5 | 2 (40.0%) | 1 | (0.660) |
| ≥ 1% | 24 | 14 (58.3%) | 1.93 (0.27–13.65) | 0.508 | |
| Unknown | 56 | 35 (62.5%) | 2.31 (0.36–14.83) | 0.377 | |
| HER2 | 0 | 40 | 22 (55.0%) | 1 | (0.522) |
| 1 + | 34 | 23 (67.7%) | 1.68 (0.65–4.34) | 0.284 | |
| 2 + | 11 | 6 (54.6%) | 0.97 (0.25–3.71) | 0.966 | |
| Ki-67 | < 14% | 10 | 3 (30.0%) | 1 | (0.015) |
| 14–29% | 9 | 3 (33.3%) | 1.15 (0.17–7.79) | 0.883 | |
| ≥ 30% | 19 | 18 (94.7%) | 26.43 (3.03–230.16) | 0.003 | |
| Unknown | 47 | 27 (57.5%) | 2.87 (0.67–12.26) | 0.154 | |
| Nuclear grade | Grade 1 | 26 | 7 (26.9%) | 1 | (0.002) |
| Grade 2 | 37 | 25 (67.6%) | 5.30 (1.77–15.91) | 0.003 | |
| Grade 3 | 19 | 16 (84.2%) | 12.26 (2.84–52.87) | 0.001 | |
| Unknown | 3 | 3 (100%) | 18.21 (0.54–620.16) | 0.107 | |
| Histological grade | Grade1 | 5 | 3 (60.0%) | 1 | (0.514) |
| Grade2 | 6 | 2 (33.3%) | 0.40 (0.04–4.55) | 0.458 | |
| Grade3 | 2 | 2 (100%) | 3.57 (0.06–220.19) | 0.545 | |
| Lymphatic invasion | No | 34 | 17 (50.0%) | 1 | 0.134 |
| Yes | 51 | 34 (66.7%) | 1.97 (0.81–4.79) | ||
| Vascular invasion | No | 73 | 45 (61.6%) | 1 | 0.644 |
| Yes | 11 | 6 (54.6%) | 0.74 (0.21–2.65) | ||
| Pathological tumor size | < 2 cm | 16 | 8 (50.0%) | 1 | (0.646) |
| 2 cm to less than 5 cm | 57 | 34 (59.7%) | 1.47 (0.48–4.47) | 0.499 | |
| ≥ 5 cm | 10 | 7 (70.0%) | 2.14 (0.41–11.19) | 0.366 | |
| Number of pathological lymph node metastases | 0 | 31 | 18 (58.1%) | 1 | (0.450) |
| 1–3 | 38 | 21 (55.3%) | 0.90 (0.34–2.34) | 0.823 | |
| ≥ 4 | 16 | 12 (75.0%) | 2.03 (0.54–7.59) | 0.294 | |
| NAC | No | 71 | 39 (54.9%) | 1 | 0.061 |
| Yes | 14 | 12 (85.7%) | 4.11 (0.94–18.09) |
OR odds ratio, CI confidence interval, NAC neoadjuvant chemotherapy
*1 Odds ratios estimated via a logistic regression model using Firth's penalized maximum likelihood
*2 Wald test p values for odds ratios estimated via a logistic regression model
Table 4.
Association between Luminal B-type disease identified by BluePrint and clinicopathological factors
| Factor | Level | n | Proportion of BluePrint Luminal B-type | OR (95% CI)*1 | P value*2 |
|---|---|---|---|---|---|
| Age | 20–39 years | 11 | 9 (81.8%) | 1 | (0.346) |
| 40–69 years | 62 | 34 (54.8%) | 0.32 (0.07–1.48) | 0.145 | |
| ≥ 70 years | 12 | 7 (58.3%) | 0.36 (0.06–2.29) | 0.278 | |
| Menopausal status | Premenopausal | 48 | 28 (58.3%) | 1 | 0.996 |
| Postmenopausal | 36 | 21 (58.3%) | 0.10 (0.42–2.40) | ||
| Bilateral breast cancer | No | 84 | 49 (58.3%) | 1 | 0.732 |
| Yes | 1 | 1 (100%) | 2.23 (0.02–218.41) | ||
| ER | < 1% | - | - | - | - |
| 1–9% | - | - | - | - | |
| ≥ 10% | 46 | 26 (56.5%) | 1 | 0.650 | |
| Unknown | 39 | 24 (61.5%) | 1.22 (0.51–2.91) | ||
| PgR | < 1% | 5 | 2 (40.0%) | 1 | (0.720) |
| ≥ 1% | 24 | 14 (58.3%) | 1.93 (0.27–13.65) | 0.508 | |
| Unknown | 56 | 34 (60.7%) | 2.15 (0.34–13.75) | 0.420 | |
| HER2 | 0 | 40 | 22 (55.0%) | 1 | (0.681) |
| 1 + | 34 | 22 (64.7%) | 1.48 (0.58–3.78) | 0.413 | |
| 2 + | 11 | 6 (54.6%) | 0.97 (0.26–3.71) | 0.966 | |
| Ki-67 | < 14% | 10 | 3 (30.0%) | 1 | (0.021) |
| 14–29% | 9 | 3 (33.3%) | 1.15 (0.17–7.79) | 0.883 | |
| ≥ 30% | 19 | 17 (89.5%) | 15.00 (2.24–100.51) | 0.005 | |
| Unknown | 47 | 27 (57.5%) | 2.87 (0.67–12.26) | 0.154 | |
| Nuclear grade | Grade 1 | 26 | 7 (26.9%) | 1 | (0.004) |
| Grade 2 | 37 | 25 (67.6%) | 5.30 (1.77–15.91) | 0.003 | |
| Grade 3 | 19 | 15 (79.0%) | 8.96 (2.26–35.44) | 0.002 | |
| Unknown | 3 | 3 (100%) | 18.21 (0.54–620.04) | 0.107 | |
| Histological grade | Grade1 | 5 | 3 (60.0%) | 1 | (0.514) |
| Grade2 | 6 | 2 (33.3%) | 0.40 (0.04–4.55) | 0.458 | |
| Grade3 | 2 | 2 (100%) | 3.57 (0.06–220.19) | 0.545 | |
| Lymphatic invasion | No | 34 | 17 (50.0%) | 1 | 0.188 |
| Yes | 51 | 33 (64.7%) | 1.81 (0.75–4.38) | ||
| Vascular invasion | No | 73 | 44 (60.3%) | 1 | 0.707 |
| Yes | 11 | 6 (54.6%) | 0.78 (0.22–2.80) | ||
| Pathological tumor size | < 2 cm | 16 | 7 (43.8%) | 1 | (0.428) |
| 2 cm to less than 5 cm | 57 | 34 (59.7%) | 1.86 (0.61–5.70) | 0.277 | |
| ≥ 5 cm | 10 | 7 (70.0%) | 2.72 (0.52–14.23) | 0.237 | |
| Number of pathological lymph node metastases | 0 | 31 | 17 (54.8%) | 1 | (0.409) |
| 1–3 | 38 | 21 (55.3%) | 1.02 (0.39–2.64) | 0.971 | |
| ≥ 4 | 16 | 12 (75.0%) | 2.30 (0.62–8.59) | 0.215 | |
| NAC | No | 71 | 39 (54.9%) | 1 | 0.141 |
| Yes | 14 | 11 (78.6%) | 2.70 (0.72–10.16) |
OR odds ratio, CI confidence interval, NAC neoadjuvant chemotherapy
*1 Odds ratios estimated via a logistic regression model using Firth's penalized maximum likelihood
*2 Wald test p values for odds ratios estimated via a logistic regression model
Comparison of genomic risk and clinical risk
Among the 85 patients analyzed in this study, 7 patients were excluded because they were not subjected to multivariable analysis in the WJOG15721B study due to missing values and thus could not have their total points in the nomogram appropriately calculated. The remaining 78 patients were categorized into four groups: clinical high-risk/genomic high-risk, clinical high-risk/genomic low-risk, clinical low-risk/genomic high-risk, and clinical low-risk/genomic low-risk (Table 5). Among patients with both high clinical and genomic risk, the rate of early recurrence was 100% (5/5). In contrast, those with both low clinical and genomic risk had a low risk of early recurrence, at 28.1% (9/32). Only one patient had high clinical risk and low genomic risk, and this patient did not experience early recurrence. Notably, among patients with low clinical risk but high genomic risk, 57.5% (23/40) experienced early recurrence, which is numerically higher than the 28.1% (9/32) recurrence rate observed in those with both low clinical risk and low genomic risk. Furthermore, the 85 patients were divided into an early recurrence group and a no recurrence group, and the breakdown of genomic risk was illustrated using a swimmer plot (Fig. 3). Remarkably, of the 43 patients with early recurrence, 15 (34.9%) were negative for lymph node metastasis, and 13 (30.2%) of them were classified as having low clinical risk.
Table 5.
Clinical risk and genomic risk in the early recurrence and no recurrence groups
| Clinical high risk/Genomic high risk n = 5 (%) |
Clinical high risk/Genomic low risk n = 1 (%) |
Clinical low risk/Genomic high risk n = 40 (%) |
Clinical low risk/Genomic low risk n = 32 (%) |
|
|---|---|---|---|---|
| Early recurrence | 5 (100) | 0 (0) | 23 (57.5) | 9 (28.1) |
| No recurrence | 0 (0) | 1 (100) | 17 (42.5) | 23 (71.9) |
Fig. 3.
Swimmer plot of MammaPrint genomic risk in early and no recurrence groups (n = 85). In addition to the swimmer plot, the clinical scores according to the nomogram, age, nuclear grade, pathological invasive tumor size, and number of pathological lymph node metastases are shown in the table
Discussion
Previous studies have shown that combining clinical and genomic information allows for a more precise prognostic assessment than using either type of information alone in patients with breast cancer [12, 13]. This study focused on early recurrence within 3 years after surgery and revealed that clinical risk classification, when combined with a MammaPrint risk score, provided a more accurate assessment of the likelihood of early recurrence in HR +/HER2- early breast cancer patients. The study findings suggested that patients with both high clinical and genomic risk were at the highest risk for early recurrence, whereas those with both low clinical and genomic risk were at the lowest risk. Interestingly, patients with low clinical risk but high genomic risk appear to have an intermediate risk for early recurrence.
MammaPrint identifies genes related to distant metastasis through comprehensive genomic analysis rather than selecting genes known to be associated with prognosis [14, 15]. The genes in the MammaPrint assay are involved in early metastatic invasion processes, which consist of seven steps: growth and proliferation, angiogenesis, local invasion, intravasation, survival in circulation, extravasation, and microenvironmental adaptation at the metastatic site [16]. MammaPrint results had the greatest prognostic value for identifying patients at high risk for recurrence within 5 years after surgery in a validation study [16]. These findings aligned with the results of the current study, in which high-risk MammaPrint status was more frequently observed in patients with early recurrence.
In addition, a significantly greater percentage of patients in the early recurrence group than in the no recurrence group had Luminal B-type disease according to the Blueprint assay; basal-type disease was observed in only one patient in the no recurrence group, and the HER2-type disease was not observed in either group. In the MINDACT trial, prognostic outcomes varied by molecular subtype, with the Basal-type having the poorest prognosis, followed by the Luminal B, HER2, and Luminal A subtypes [5-year distant metastasis-free survival (DMFS): 90.4%, 92.7%, 94.3%, and 96.7%, respectively] [17]. In the NBRST trial, which aimed to demonstrate the utility of MammaPrint and BluePrint in the neoadjuvant setting, approximately 20% of the 426 cases pathologically classified as HR + HER2- were reclassified into non-luminal subtypes (85 cases as Basal-type and 2 cases as HER2-type) [18]. Furthermore, 80% of the cases classified as Basal-type experienced recurrence events within three years of diagnosis [18]. We hypothesized that the proportion of Basal-type cases would be the highest among patients with early recurrence. However, possibly due to the small sample size, Basal-type cases were rarely observed.
In this study, an association was observed between MammaPrint high risk results, Ki-67 levels, and nuclear grade. Previous reports have also shown a correlation between MammaPrint high risk results and a high Ki-67 index, as well as an association between a high Ki-67 index and the Nottingham histological grade [19]. On the other hand, when these clinicopathological factors were excluded, a MammaPrint high risk results was considered an independent prognostic marker.
In the present study, we did not evaluate whether clinical or genomic factors are predictive of systemic treatment outcomes. In the FLEX registry trial, patients with MammaPrint high-2–risk, BluePrint Luminal B, HR +/HER2- early breast cancer had a higher 3-year recurrence-free survival rate with anthracycline plus a taxane and cyclophosphamide than with a taxane and cyclophosphamide therapy in a nonrandomized cohort, and this tendency was not observed in patients with MammaPrint high-1–risk disease [20]. Although this study primarily evaluated prognostic factors, identifying predictive markers for the efficacy of individual therapies, including molecularly targeted agents, remains essential.
Although there are several commercially available gene expression profiling assays including Oncotype DX, we didn’t compare the performance of different assays in the present study. MammaPrint/BluePrint provides both prognostic risk stratification and molecular subtyping based on tumor biology, and we anticipate that the precise identification of tumors prone to early recurrence by MammaPrint/BluePrint may guide patient selection for intensified therapeutic strategies, including molecularly targeted agents.
Early recurrences were also observed among patients with both clinical low-risk and genomic low-risk disease, underscoring the challenge of identifying such cases and guiding appropriate treatment strategies. Further clinical studies are needed to optimize treatment, particularly for patients with clinical low-risk and genomic high-risk disease. More accurate identification of patients at elevated risk of early recurrence may enable better selection of candidates for adjuvant treatment intensification, such as CDK4/6 inhibitors. In addition, whether MammaPrint and BluePrint can serve not only as prognostic tools but also as predictive markers for the efficacy of specific therapies warrants evaluation in future prospective clinical trials. Such investigations will advance precision medicine and help refine therapeutic decision-making for patients with HR +/HER2 − breast cancer.
The primary limitations of this study are its retrospective nature and the small sample size. The availability of tissue specimens from the WJOG15721B trial was limited, and although 115 samples were submitted for MammaPrint and BluePrint testing, successful measurement was achieved in only 86 cases, resulting in an analysis success rate of approximately 75%. One possible reason for the low success rate is that the tissue samples used in this study were collected between 2012 and 2017, during which time the cold ischemia time and formalin fixation time were not as strictly controlled as they are today. As a result, the samples may not have been in an optimal condition for RNA analysis. Additionally, variations in the type and concentration of formalin used across different institutions and time points of sample collection may have influenced the results. Although a matched analysis was originally intended, only 86 samples were successfully measured, resulting in a number of unmatched cases and necessitating an unmatched analysis. This reduction in the number of evaluable samples represents sample attrition from the original study population and may have decreased the statistical power of the analysis. Furthermore, because the analysis was ultimately restricted to cases with successfully measured specimens, selection bias cannot be excluded, as these cases may not fully represent the overall study population. Therefore, the loss of the original matched design and the reduced sample size should be taken into account when interpreting the robustness and generalizability of our findings, and the results should be interpreted with appropriate caution. Despite these limitations, our study demonstrated meaningful results for current and future personalized medicine approaches.
Conclusions
In this study, patients with early recurrence had a significantly greater prevalence of MammaPrint high-risk results and BluePrint Luminal B-type results than patients with no recurrence did. A potential association with the genetic risk score, molecular subtype, and early recurrence was suggested in patients with HR +/HER2- clinical stage II–III breast cancer.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We would like to thank all the patients who participated in this study and their families. We also thank members of the data center at WJOG.
Author contributions
All the authors contributed to the study conception and design. Data collection was performed by Rurina Watanuki, Yuri Kimura, Atsushi Yoshida, Yukinori Ozaki, Akemi Kataoka, Natsue Uehiro, Hidenori Kamio, and Atsushi Fushimi. Statistical analysis was performed by Masashi Wakabayashi and Takashi Ikeno. Data interpretation was performed by Rurina Watanuki, Hitomi Sakai, and Junji Tsurutani. The first draft of the manuscript was written by Rurina Watanuki and Hitomi Sakai, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.
Funding
Open Access funding provided by SHOWA Medical University. This study was funded by Eli Lilly Japan, NE.
Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
Hitomi Sakai has received personal honoraria and institutional research grant from Eli Lilly, and honoraria from Daiichi Sankyo, Gilead Sciences, and Astra Zeneca. Atsushi Yoshida has received honoraria from Daiichi Sankyo, Pfizer, Chugai Pharmaceutical Co., Ltd., Limited, Exact Sciences Corporation, AstraZeneca PLC, and Eisai Co., Ltd. Yukinori Ozaki has received honoraria for lectures from Daiichi Sankyo, Pfizer, Kyowa Kirin, Eli Lilly and Chugai Pharmaceutical Co., Ltd. Mayumi Iida is an employee of Eli Lilly Japan, and minor stockholders of Eli Lilly and Company. Tsutomu Kawaguchi is an employee of Eli Lilly Japan, and minor stockholders of Eli Lilly and Company. Masashi Wakabayashi has received honoraria from Nihon Medi-Physics Co., Ltd and Nihon Medi-Physics Co.,Ltd. Junji Tsurutani has received honoraria, research grant and advisory board member fee from Eli Lilly. Toshimi Takano has received honoraria for lectures from Eli Lilly, Daiichi Sankyo, and Gilead Sciences.
Ethical approval and consent to participate
This study was performed in accordance with the principles of the Declaration of Helsinki. Approval was granted by the Showa University Research Ethics Review Board (July 18, 2023/2023–051-B). Written informed consent was obtained, but for cases where the patient had passed away or relocated with an unknown contact address, an opt-out procedure was conducted.
Footnotes
Publisher's Note
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.