Verification of the effects of calcium channel blockers on the immune microenvironment of breast cancer

All patients who visited the Osaka City University Hospital from February 2007 to March 2018 were screened to obtain their medical histories. In cases of suspected breast cancer, core needle biopsy or vacuum-assisted biopsy was performed with ultrasonography (US). When diagnosed pathologically with breast cancer, the subtype of breast cancer was determined by immunostaining and staging with computed tomography (CT), US, and bone scintigraphy. If evaluation of metastasis to lymph nodes was difficult using these imaging tests, lymph node biopsy was performed. For immunostaining of samples, the expression levels of estrogen receptor (ER), progesterone receptor (PgR), HER2, and Ki67 were evaluated. The cut-off value for Ki-67 staining was set at 15% [18]. We defined ER−/PgR−/HER2+ breast cancer as HER2BC, ER−/PgR−/HER2- breast cancer as TNBC, and breast cancer that was not HER2BC or TNBC as luminal breast cancer (luminal BC) [19]. In total, 338 patients with breast cancer, diagnosed with stage IIA (T1, N1, M0 or T2, N0, M0), IIB (T2, N1, M0 or T3, N0, M0), IIIA (T1–2, N2, M0 or T3, N1–2, M0), IIIB (T4, N0–2, M0), or IIIC (T1–4, N3, M0), received POC. During the first half of the POC regimen, all patients received four courses of FEC100 (500 mg/m² fluorouracil, 100 mg/m² epirubicin, and 500 mg/m² cyclophosphamide) every 3 weeks. During the second half of the POC regimen, 12 courses of 80 mg/m² paclitaxel were administered to all patients weekly, and weekly (2 mg/kg) or tri-weekly (6 mg/kg) trastuzumab was also administered in cases of HER2-positive disease [20‐22]. Antitumor effects were evaluated according to the Response Evaluation Criteria in Solid Tumors [23]. For analysis of the objective response rate (ORR), clinical partial response and complete response were defined as responders, and clinical stable disease and clinical progressive disease were defined as nonresponders. After confirming the therapeutic effects of POC, all patients were examined for continuation of AHTs before surgery; patients then underwent mastectomy or breast-conserving surgery [22]. Pathological complete response (pCR) was defined by the National Surgical Adjuvant Breast and Bowel Project B-18 protocol as “the complete disappearance of the invasive components of the lesion with or without intraductal components, including that in the lymph nodes” [24]. Standard postoperative radiotherapy was enforced if necessary, and postoperative adjuvant therapy suitable for the patient’s specific subtype was performed. As follow-up after surgery, all patients had physical examinations every 3 months, US every 6 months, and CT and bone scintigraphy annually. The median follow-up time was 1287 days (range, 13–3675 days) from operation.

Biopsy specimens before POC were used to evaluate TIL density. The definition and evaluation method of TILs were in accordance with the International TILs Working Group 2014 [25]. The average density of the infiltrating lymphocytes within the tumor stroma in five randomly selected fields was calculated. After categorization into four classes according to the TIL density (3: > 50%, 2: > 10–50%, 1: ≤ 10%, or 0: absent; Additional file 1: Figure S1), scores of 2 and 3 were defined as high, and scores of 0 and 1 were defined as low [26].

This study was conducted at Osaka City University Graduate School of Medicine, Osaka, Japan, according to the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelines and following a retrospectively written research, pathological evaluation, and statistical plan [27]. The study protocol was approved by the Ethics Committee of Osaka City University. Written informed consent was obtained from all patients (#926).

Three hundred thirty-eight patients received POC; the details of their clinicopathological features are summarized in Table 1. All patients were women, and the median age at operation was 55 years old (24–78 years old). The median tumor size was 28.7 mm (9.2–119.8 mm); the tumor size of 56 patients (16.6%) was 20 mm or less, and that of 44 patients (13.0%) was larger than 50 mm. Skin infiltration was observed in 50 patients (14.8%), and 224 patients (66.3%) were diagnosed with breast cancer having lymph node metastasis by imaging diagnosis. In classification by intrinsic subtype, 155 patients (45.9%) were classified as having luminal BC, 78 patients (23.1%) were classified as having HER2BC, and 105 patients (31.1%) were classified as having TNBC. Moreover, 298 patients (88.2%) were evaluated as responders in ORR. In the pathological examination of surgical specimens, 116 patients (34.3%) showed pCR. By evaluating the biopsy specimens before POC, 158 patients (46.7%) were classified into the high TIL density group, and 180 patients (53.3%) were classified in the low TIL density group.

Before POC, 65 patients (19.2%) took AHTs. Patients who had been treated before the first visit but were not treated before POC were divided into groups excluding hypertensive patients. There were no untreated patients with hypertension before POC. The following AHTs were administered: CCBs, angiotensin-converting-enzyme inhibitor, angiotensin II receptor blockers, beta-blockers, and diuretics. Forty-one patients (12.1%) were taking CCBs, and CCBs were the most commonly used medication for hypertension. Twenty-four patients (7.1%) took several medications for hypertension. No patients started new AHTs or needed additional AHTs during POC. In three patients (0.9%), AHTs were discontinued during POC. Both of these patients were taking CCBs only, and the times of discontinuation were 1.5 months, 1 month, and 10 days before surgery, respectively.

Comparison of clinicopathological features based on intrinsic subtypes showed poorer pathological response in luminal BC than in HER2BC or TNBC (luminal BC: 18.1%, HER2BC: 55.1%, TNBC: 42.9%; Additional file 2: Table S1). For age, we set the median as the cutoff value. In luminal BC, the rate of patients in the high TIL density group was lower than those in patients with other subtypes (luminal BC: 30.3%, HER2BC: 67.9%, TNBC: 55.2%). There were no significant differences in other items by subtype.

We examined differences in clinicopathological features due to TILs (Additional file 3: Table S2). In the high TIL density group (n = 338), the expression levels of ER and PgR were significantly lower (p < 0.001 and p < 0.001, respectively), whereas the expression levels of HER2 and Ki67 were significantly higher than in the low TIL density group (p = 0.023, p < 0.001, respectively). Moreover, the TIL density was significantly lower in luminal BC and significantly higher in HER2BC and TNBC (p < 0.001, p < 0.001, p < 0.001, respectively). The ORR and pCR were significantly higher in the high TIL density group than in the low TIL density group (p < 0.001, p < 0.001, respectively). In 105 patients with TNBC and 78 patients with HER2BC, the same correlation between TILs and ORR or pCR was found (TNBC: p = 0.008, p = 0.042; HER2BC: p = 0.017, p = 0.019, respectively).

Notably, patients administered CCBs had significantly lower TIL densities (p = 0.040). Furthermore, in patients with TNBC, the TIL density was significantly lower in patients receiving hypertension treatment and patients receiving CCBs (p = 0.003, p = 0.009, respectively). In HER2BC, there were no correlations between AHTs and TILs.

The correlations between CCBs and clinicopathological features were examined in chi-squared tests and are shown in Table 2. In all patients and in patients with TNBC, patients administered CCBs were significantly older than patients without CCB administration (p < 0.001, p = 0.004, respectively). Moreover, patients with TNBC who were administered CCBs showed significantly lower response rates for POC (p = 0.040). No correlations between CCBs and pCRs was observed (p = 0.649). However, when we focused on patients with hypertension only, no relationship was found between CCBs and TILs (Additional file 4: Table S3).

In all patients receiving POC, no significant differences in DFS or OS were observed due to the use of CCBs, as determined using the Kaplan-Meier method and log-rank tests (p = 0.712, p = 0.478, log-rank tests, respectively; Fig. 1a, b). Furthermore, no significant differences were found, even in patients with TNBC (DFS: p = 0.441, OS: p = 0.727, log-ranks, respectively; Fig. 1c, d).

In patients with TNBC, a high TIL density significantly contributed to longer DFS using univariate analysis (p = 0.004, HR = 0.306; Table 3). Additionally, in multivariate analysis with DFS and OS, response in ORR was an independent factor (DFS: p = 0.004, HR = 0.258; OS: p = 0.001, HR = 0.143; Tables 3 and 4). Despite these results, there were no significant differences in univariate analysis with DFS or OS due to CCBs (DFS: p = 0.472, HR = 1.601; OS: p = 0.715, HR = 0.699). Similar analyses were carried out for all breast cancer and HER2BC, but no significant differences were found (Additional file 5, 6, 7, 8: Table S4–7).