Abstract
Background/Aim: Advanced/recurrent breast cancer (ARBC) still has a poor prognosis; therefore, new treatment strategies are required. In this retrospective study, we aimed to investigate the efficacy of immune-cell therapy using T lymphocytes activated in vitro with or without dendritic cell vaccination in combination with standard therapies in terms of the survival of patients with ARBC. Patients and Methods: A total of 127 patients with ARBC were enrolled in this study. The correlation between overall survival and various clinical factors of each ARBC subset was examined by univariate and multivariate analyses. Results: Multivariate analysis demonstrated that performance status (PS) 0, the absence of prior chemotherapy, liver/pleural metastasis, and the presence of combined surgery in ARBC and PS 0 or the absence of liver metastasis in the HR+/HER− subset are indications for immune-cell therapy. Conclusion: A survival benefit could be potentially obtained by a combination of immune-cell therapy with other therapies in ARBC patients.
Although efforts have been made to improve the early diagnosis and treatment of breast cancer (BC), it remains the most common type of cancer and the increasing cause of cancer-related death in women (1, 2). Several targeted therapies, such as human epidermal growth factor receptor 2 (HER2)-targeting drugs, cyclin-dependent kinase 4/6 (cdk 4/6) inhibitor, and poly ADP ribose polymerase (PARP) inhibitor, have been developed and proven to be effective (3).
Genomics has improved our understanding of BC biology and revealed four intrinsic molecular subtypes: luminal A [resembling the histological phenotype: estrogen receptor (ER) +, progesterone receptor (PR) +, HER2−, Ki67−], luminal B (ER+, PR+, HER+/−, Ki67+), HER2 (ER−, PR−, HER2+), and basal-like subtype (ER−, PR−, HER2−) (4). The classification of BC into subtypes has clinical relevance. For instance, in the treatment of the hormone receptor (HR)+ subtypes (positive for ER and/or PR), endocrine therapeutics, including aromatase inhibitors or selective estrogen receptor mediators such as Tamoxifen, play an important role. HER2-overexpressing tumors are generally treated with HER2-targeting drugs such as trastuzumab and pertuzumab, whereas triple-negative BC (TNBC, largely resembling the basal-like BC subtype) is mostly treated with standard cytotoxic therapies. However, treatments for TNBC are limited, and the development of effective treatments against TNBC subtypes is required (5).
The immune system can protect the host from tumorigenesis through immune surveillance mechanisms (6). One of the mechanisms attributed to the occurrence or development of cancer is the deficiency of the immune system. Various strategies, which include the use of cytokines, cancer vaccines, checkpoint inhibitors, and adoptive cell transfer (ACT), have been developed to improve the immune function of cancer patients. Strategies to block the programmed death 1 (PD1) pathway have been substantially developed over the last 2-3 years, with novel agents already approved for various cancers, including lung cancer, renal cancer, gastric cancer, esophageal cancer, and melanoma, and other agents at different steps of clinical development (7).
Initially, BC was considered a poorly immunogenic tumor type and has therefore not been extensively investigated for its susceptibility to immune therapies. However, during the past years, it became evident that certain cases of BC are strongly infiltrated by immune cells and that the presence of these immune cells has significant prognostic value (8). Although immune therapies for BC are currently examined in many studies, still only a minority of patients appear to respond to such therapies, and little is known about the mechanisms underlying treatment efficacy. However, there are convincing data supporting their immunogenicity against BC, which may represent an ideal target for immunotherapy (9, 10).
ACT is a form of passive immunotherapy using immune cells that are exogenously produced or manipulated to promote an antitumor immune response (11). In ACT, cells from the blood or bone marrow are isolated from a patient, activated and expanded in vitro, and reinfused into the same patient (autologous) or a different patient (allogeneic). Several studies on ACT for BC in the advanced stage have shown encouraging results in some patients, but the number of patients enrolled in such studies was small and the efficacy of ACT for BC patients remains unclear (12-15).
In this study, we retrospectively analyzed patients with ARBC, who have been administered immune-cell therapy in combination with conventional therapy at the clinics of the Seta Clinic Group.
Patients and Methods
Patients. The database of patients administered immune-cell therapy at the clinics of the Seta Clinic Group was searched to identify patients with BC. As a result, 428 patients were identified and enrolled in this study. We retrospectively reviewed the medical records of those administered αβT cell therapy, dendritic cell (DC) vaccine therapy, or both between 1999 and 2015. The study protocol was approved by the Research Ethics Committee of the Seta Clinic Group. Available data on age, gender, performance status (PS) score on the Eastern Cooperative Oncology Group (ECOG) scale, metastasis sites, clinical stage, treatments, and vital status were extracted from the medical records of the patients.
Treatment. For αβT cell therapy, activated lymphocytes were generated as previously described (16, 17). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from a patient’s peripheral blood using Vacutainer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The PBMCs were activated in a culture flask with an immobilized monoclonal antibody to CD3 (Jansen-Kyowa, Tokyo, Japan) in Hymedium 930 (Kohjin Bio, Saitama, Japan) containing 1% autologous serum. The PBMCs were then cultured for 14 days with 700 IU/ml recombinant interleukin-2 (IL-2) (Proleukin®; Chiron, Amsterdam, the Netherlands), after which, 3-10 × 109 cells were harvested and suspended in 100 ml of normal saline for intravenous injection. To prepare a DC vaccine, PBMCs were collected from the patients by leukapheresis and allowed to adhere to a plastic culture flask. The adherent cell fraction was used for DC culture for six days using a medium supplemented with 50 ng/ml IL4 (Primmune Corp., Osaka, Japan) and 5 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (Primmune Corp.) to generate immature DCs. The DCs were pulsed with antigenic tumor-specific peptides or an autologous tumor lysate and allowed to mature for 24 h. After the culture, 1-10×106 mature DCs were harvested and suspended in 1 ml of normal saline used for subcutaneous injection, and then cryopreserved until the day of administration. Immune-cell therapy consists of αβT cells or DC vaccine, or both, and is commonly administered six times, that is, every two weeks for three months, as one course.
Assessment. Overall survival (OS) was defined as the length of time from the initial administration of immune-cell therapy to death from any cause; it was calculated for every patient. The Kaplan–Meier analysis was used to calculate survival probabilities for all patients.
Statistical analyses. The OS of the patients was examined by the Kaplan–Meier analysis with the Log-rank test, and the hazard ratio was obtained by Cox regression methods in univariate and multivariate analyses. All statistical analyses were two-sided and performed using JMP, version 15.0.0 for Microsoft Windows 10 (SAS, Cary, NC, USA). Differences were considered statistically significant when p<0.05.
Results
Patient selection. A total of 428 patients with BC were enrolled in this study (Figure 1). Of the 428 patients, 127 had advanced or recurrent cancer (155 patients were excluded because of insufficient data and 146 were excluded because immune-cell therapy was performed as a prophylaxis against recurrence). Among the 127 patients with ARBC, 36 patients were HR+/HER2−, 32 patients were HR−/HER2+, 12 patients were HR+/HER2+, and 47 patients were TNBC.
Procedure for selection of patients enrolled in this study. BC: Breast cancer; ARBC: advanced/recurrent BC.
The patients’ characteristics are summarized in Table I. In this study, the correlations between OS and various factors including age, PS score, clinical stage, histology, surgery, chemotherapy, radiotherapy, and immune-cell therapy were evaluated by univariate analysis and multivariate Cox regression analysis.
Patient characteristics.
Overall survival. The median age of the patients with ARBC was 53 years old (127 patients; range=28-80 years) as shown in Table I. Sixty-nine patients (54.3%) who visited our clinic were PS 0 in full analysis sets of ARBC patients (Table I). Most of the patients’ clinical stage at diagnosis was more than II in each subset. Since the administration of immune-cell therapy has started, the median survival time (MST) of patients with ARBC was 33.7 months (Figure 2A). As for BC subsets, the MSTs of HR+/HER2−, HR−/HER2+, HR+/HER2+, and TNBC patients were 48.1, 20.7, 35.6, and 26.9 months, respectively (Figure 2B). As shown in Figure 2A, the 3- and 5-year OS rates of patients with BC were 47.6% and 35.2%, respectively. In TNBC patients, the 3- and 5-year OS rates were 37.0% and 21.1%, respectively. The OS rates of TNBC patients were significantly shorter than those of the other subsets (TNBC patients vs. HR+/HER2−, HR+/HER2+, and HR−/HER2+ patients, p=0.0348) (Figure 2B).
Kaplan–Meier estimates of overall survival in the full analysis set of ARBC (A) and BC subsets according to HR or HER2 expression (B). ARBC: Advanced or recurrent breast cancer; BC: breast cancer; N: number of patients; OS: overall survival (months); MST: median survival time; HR: hormone receptor; HER2: human epidermal growth factor receptor-2; TNBC: triple-negative breast cancer. (*p=0.0348, TNBC vs. HR+/HER2−, HR−/HER2+, HR+/HER2+).
Univariate and multivariate analyses. We performed univariate analysis to identify the prognostic factors for a full analysis set of ARBC patients and each BC subset. In the case of the full analysis set of ARBC patients, univariate analysis demonstrated that the patients whose PS was more than 1 showed a worse prognosis than those whose PS was 0 (HR=0.501, 95%CI=0.283-0.885, p=0.0172; Table II). In HR+/HER2− subsets, the patients with PS of 0 showed better prognosis than those with PS of ≥1 (HR=0.273, 95%CI=0.089-0.840, p=0.0235; Table II). However, there were no significant clinical factors that affected patients’ prognosis in the other BC subsets.
Univariate analyses of overall survival in relation to clinical background of breast cancer patients.
In terms of treatment strategy, univariate analysis demonstrated that the full analysis set of ARBC patients treated with immune cell therapy with prior surgery or prior chemotherapy showed worse prognosis than those without either prior treatment, and those with combined surgery showed better prognosis than those without it (prior surgery: HR=2.303, 95%CI=1.095-4.841, p=0.0278; prior chemotherapy: HR=2.131, 95%CI=1.053-4.312, p=0.0354; combined surgery: HR=0.228, 95%CI=0.071-0.731, p=0.0129; Table III). In HR−/HER2+ subsets, patients treated with combined surgery showed better prognosis than those without surgery (HR=0.122, 95%CI=0.016-0.945, p=0.0440; Table III). In TNBC subsets, univariate analysis demonstrated that patients treated with prior radiotherapy showed worse prognosis than those without radiotherapy, and those with combined radiotherapy showed better prognosis than those without the treatment (prior radiotherapy: HR=3.038, 95%CI=1.177-7.841, p=0.0216; combined radiotherapy: HR=0.322, 95%CI=0.107-0.969, p=0.0439; Table III). Regarding the type of immune-cell therapy (i.e., αβT or αβT with DC), we did not find any significant difference in survival between the types of immune-cell therapy. In relation to metastatic sites, univariate analysis revealed that the ARBC patients with liver or pleural metastasis showed worse prognosis than those without metastasis (liver: HR=2.332, 95%CI=1.424-3.819, p=0.0008; pleura: HR=4.197, 95%CI=1.281-13.754, p=0.0179; Table IV). In the HR+/HER2− and TNBC subsets, patients with liver metastasis showed poorer prognosis than those without metastasis (HR+/HER2−: HR=3.391, 95%CI=1.289-8.923, p=0.0134; TNBC: HR=3.175, 95%CI=1.428-7.058, p=0.0046; Table IV). Regarding the other BC subsets, we were unable to identify any specific metastatic sites that affect patients’ prognosis, probably owing to the small number of patients in the other BC subsets.
Univariate analyses of overall survival in relation to therapy in breast cancer patients.
Univariate analyses of overall survival in relation to metastatic site in breast cancer patients.
Finally, multivariate analysis showed that factors such as PS ≥1 (HR=2.411, 95%CI=1.302-4.465, p=0.0051), the presence of prior chemotherapy (HR=2.254, 95%CI=1.063-4.779, p=0.0340), and the presence of liver or pleural metastasis (liver: HR=2.034, 95%CI=1.185-3.491, p=0.0100; pleura: HR=4.003, 95%CI=1.178-13.600, p=0.0262) were poor prognostic factors, whereas the presence of combined surgery was a favorable factor (HR=0.283, 95%CI=0.083-0.958, p=0.0425) in the full analysis set of ARBC patients treated with immune-cell therapy (Table V). In the HR+/HER− subsets, multivariable analysis revealed that PS ≥1 and the presence of liver metastasis were unfavorable prognostic factors (PS ≥1, HR=3.661, 95%CI=1.162-11.536, p=0.0266; liver metastasis: HR=3.380, 95%CI=1.190-9.595, p=0.0222). In the case of TNBC patients, multivariate analysis demonstrated no significant clinical factors that affected the prognosis of these patients treated with immune-cell therapy, although patients with liver metastasis showed slightly worse prognosis than those without metastasis (HR=2.316, 95%CI=0.942-5.698, p=0.0674; Table VI).
Multivariate analyses of overall survival in relation to therapy and clinical background of full analysis set of breast cancer patients.
Multivariate analyses of overall survival in relation to therapy and clinical background of HR+/HER2− and TNBC subsets.
Discussion
Many patients with BC have poor prognosis, especially those in the advanced stage, despite the development of combination chemotherapies and molecular targeting therapies that have prolonged the MST of patients with ARBC. Conventional treatments, including surgery, chemotherapy, and radiotherapy, may have various adverse effects and impair the patients’ antitumor immunity, resulting in residual tumor. In this retrospective study, we extracted 127 patients with ARBC from among the 428 patients who have visited our clinic and were diagnosed as having BC, and we analyzed the efficacy of immune-cell therapy combined with a standard therapy. As a result, we observed an increased efficacy of immune-cell therapy for patients with ARBC. The 3- and 5-year survival rates of ARBC patients were almost similar or higher than those of the historical control reported in the “Japanese Association of Clinical Cancer Centers” (18) since immune-cell therapy was administered in most of the patients several months or years after diagnosis (Figure 2). Furthermore, the survival rates of other subsets, such as HR+/HER2−, HR−/HER2+, HR+/HER2+, and TNBC patients, were also better than those of the historical control (19).
We have examined the effect of the clinical background of each BC subset on the prognosis, and found that PS affected the prognosis of the full analysis set and the HR+/HER2− subset, which is consistent with a previous report that a better PS is suitable for active treatment (Table II) (20).
Radiotherapy has been used to eradicate a localized disease or serve a palliative role; however, this therapy has recently been recognized as a potent immune response modulator that augments immune therapy (21, 22). Concerning the effect of treatment strategy on the prognosis of each BC subset, it was shown that the combination of immune-cell therapy with radiotherapy improved the prognosis of the TNBC subset (Table III).
Furthermore, the combination of surgical operation with immune-cell therapy improved patients’ prognosis in the full analysis set and the HR−/HER2+ subset (Table III). Solid tumors have a complex and inflamed microenvironment. The inflammation is induced via proinflammatory mediators secreted from tumors, tumor-infiltrating lymphocytes (TILs), cancer-associated fibroblasts, and myeloid-derived suppressor cells (MDSCs) (23-25). These cells have been shown to crosstalk with each other, resulting in the release of proinflammatory cytokines, chemokines, and growth factors that induce immune suppression (26, 27). It has been demonstrated that the inhibition of interaction between tumor cells and MDSCs might improve immune system dysfunction (28). From this viewpoint, tumor removal by surgical resection might result in the recovery from immune system dysfunction induced by MDSCs, leading to better prognosis in certain subtypes of BC, such as the HR−/HER2+ subset. Besides surgical operation, immune cell therapy can lead to the recovery of the immunosuppressive status and provide survival benefits for patients.
ARBC patients who develop distant metastasis to the liver have been reported to have a poor prognosis (19). We have also found that a combination of immune-cell therapy and standard therapy could not improve the prognosis of these patients with lung/liver metastasis, although the MST of these patients was longer than that of the historical control. These findings indicate that it might be difficult to restore the impaired immunological status in advanced-stage cancer patients by immune-cell therapy (Table V).
It has been demonstrated that some TNBC patients have high immune cell infiltration to eliminate continuously many immunogenic clones, resulting in lower clonal heterogeneity (10). In contrast, there were contradictory data that showed a weak positive relationship between the neoantigen load and the cytolytic immune gene expression pattern, indicating that some types of TNBC are immunogenic owing to the high tumor mutation burden (29). Although many promising outcomes have been seen with newer immunotherapies, such as immune checkpoint inhibitors (30), many unresolved issues still remain unclarified. Thus, when selecting treatment strategies, it is necessary to consider BC subsets (HR or HER2 status), TIL, and tumor mutation burden (9).
In conclusion, a better prognosis could be obtained by the combination of immune-cell therapy and other therapies with the patients’ normal immune-cell function preserved. However, to establish a comprehensive immunotherapy for BC, it is necessary to conduct a randomized trial to further elucidate the benefits of the combination of immune-cell therapy and various other treatments, such as chemotherapy, radiotherapy, and therapy with immune checkpoint inhibitors.
Footnotes
Authors’ Contributions
Conception and design: R. Takimoto, T. Kamigaki, and S. Goto; Administrative support: S. Okada, H. Ibe, and E. Oguma; Collection and assembly of data: S. Okada, H. Ibe, and E. Oguma; Data analysis and interpretation: R. Takimoto, S. Okada, T. Kamigaki, and S. Goto. Final approval of manuscript: All Authors.
Conflicts of Interest
The Authors affirm that there are no potential conflicts of interest in relation to this study.
- Received May 22, 2021.
- Revision received June 7, 2021.
- Accepted June 8, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
