Feasibility study of personalized peptide vaccination for metastatic recurrent triple-negative breast cancer patients

Women with a histological diagnosis of metastatic recurrent breast cancer were eligible for inclusion in the present study. All patients were required to have evaluable recurrent and/or metastatic tumors at the time of entry. Patients were divided into three different intrinsic subtypes as follows: luminal (estrogen-receptor-positive)/HER2-negative type, HER2-positive type (immunohistochemical score 3+ or HER2 gene/chromosome 17 ratio >2.2 in fluorescence in situ hybridization) and TNBC (hormone-receptor-negative and HER2-negative). Most patients had failed standard chemotherapy, but a few patients who had failed hormonal therapy alone were also eligible for this study. All patients were required to show positive IgG responses to at least 2 of the 31 different vaccine candidate peptides, as reported previously [13‐17]. Other inclusion criteria were as follows: age between 20 and 80 years; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; positive status for human leukocyte antigen (HLA)-A2, -A24 or -A26 types, or HLA-A3 supertypes (A3, A11, A31 or A33); life expectancy of at least 12 weeks; and adequate hematologic, hepatic and renal function. Exclusion criteria included pulmonary, cardiac or other systemic diseases; an acute infection; a history of severe allergic reactions; pregnancy or nursing; and other inappropriate conditions for enrollment as judged by clinicians. Patients with a lymphocyte count of <1,000/μL were excluded from the study, since we previously reported that pre-vaccination lymphocytopenia (<1,000 cells/μL) is an unfavorable factor for overall survival (OS) in cancer patients receiving PPV [17]. The protocol was approved by the Kurume University Ethical Committee and registered in the UMIN Clinical Trials Registry (Registration, number UMIN000001844; Registration date, 5 April 2009). All patients were given a full explanation of the protocol and provided their informed consent before enrollment.

This was a phase II study to evaluate the safety and immunological responses in metastatic recurrent breast cancer patients under PPV. Thirty-one peptides, whose safety and immunological effects for other types of cancer were confirmed in previously conducted clinical studies [14‐17], were employed for vaccination (12 peptides for HLA-A2, 16 peptides for HLA-A24, 9 peptides for HLA-A3 supertypes (-A3, -A11, -A31, and -A33) and 4 peptides for HLA-A26) (Additional file 1: Table S1). These peptides were prepared under the conditions of Good Manufacturing Practice (GMP) by the PolyPeptide Laboratories (San Diego, CA, USA) and American Peptide Company (Vista, CA, USA). Peptides for vaccination of individual patients were selected in consideration of the pre-existing host immunity before vaccination, as assessed by the titers of IgG specific to each of the 31 different vaccine candidates.

A maximum of four peptides (3 mg/each peptide), which were selected based on the results of HLA typing and peptide-specific IgG titers, were subcutaneously administered with incomplete Freund’s adjuvant (Montanide ISA51; Seppic, Paris, France) once a week for six consecutive weeks. After the first cycle of six vaccinations, up to four antigen peptides, which were re-selected according to the titers of peptide-specific IgG at the sixth vaccination, were administered every two weeks. After the second cycle of six vaccinations, up to four antigen peptides, which were also re-selected, were administered every four to eight weeks according to the immune responses after PPV. These protocols were continued until remarkable disease progression or disease in remission was shown, according to the will of the individual patient. During the PPV, patients were allowed to receive combination therapies such as chemotherapy, hormonal therapy, anti-HER2 therapy and radiotherapy. Adverse events were monitored according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 (NCI-CTC Ver.-3.0). Complete blood counts and serum biochemistry tests were performed after every six vaccinations. The clinical responses were determined by the Response Evaluation Criteria in Solid Tumors (RECIST) in the vaccinated patients. The RECIST-based clinical responses were evaluated after nearly 12 vaccinations by radiological findings of computed tomography (CT) scan and/or magnetic resonance imaging (MRI), and the best overall responses during PPV treatment were shown. For the patients who did not complete the second cycle of 12 vaccinations, the newest radiological findings were evaluated, except in the case of patients who had died before the RECIST-based radiological evaluation.

Humoral immune responses specific to each of the 31 peptide candidates were determined by peptide-specific IgG levels using the Luminex system (Luminex, Austin, TX, USA), as previously reported [18]. If the titers of peptide-specific IgG to at least one of the vaccinated peptides in the post-vaccination plasma were more than two-fold higher than those in the pre-vaccination plasma, the changes were considered to be significant, as previously reported [14‐17]. Cellular immune responses specific to the vaccinated peptides were evaluated by interferon (INF)-γ ELISPOT using peripheral blood mononuclear cells (PBMCs) as previously reported [14‐17]. As a control, cellular immune responses specific to CEF peptides (MABTECH, Cincinnati, OH, USA), a mixture of virus-derived cytotoxic T lymphocyte (CTL) epitopes, were also examined.

Between January 2009 and April 2013, 79 metastatic recurrent breast cancer patients were enrolled in this study. The patient characteristics are shown in Table 1 for the overall patient group and each of the three subtypes. Among the 79 patients, 77 patients had been investigated to determine their intrinsic subtype before vaccination, while the remaining 2 patients had not. The HER2-positive group was associated with older median age (P = 0.019), restricted performance status (P = 0.046), higher positivity of HLA-A24 or -A2 (P = 0.039 or P = 0.015), higher frequency of visceral or brain metastasis (P = 0.009 or P = 0.002) and longer duration of previous chemotherapies (P <0.0001). Although the mrTNBC group had a shorter duration of previous chemotherapies (P <0.0001), most of the mrTNBC patients had received previous standard chemotherapy (anthracycline, P = 0.003; taxane, P = 0.0006).

The median number of peptide vaccinations was 14, with a range from 2 to 39 vaccinations (Table 1). Table 2 shows all adverse events during the PPV. As the vaccination-related adverse events, all patients showed grade 1 or 2 dermatological reactions to PPV at the injection sites, but no patients showed severe adverse events (grade 3 or more). Forty-one patients (52.0%) showed grade 3 or 4 adverse events strongly associated with combined chemotherapies and disease progression (Table 2).

During the PPV, 51 patients (64.6%; 32 cases with infusion chemotherapy and 19 cases with oral chemotherapy) received combined chemotherapies, while 23 patients (29.1%) and 11 patients (13.9%) received hormonal therapies and anti-HER2 therapies, respectively (Table 1). The mrTNBC patients received combined infusion chemotherapy more frequently than other breast cancer subtypes (P = 0.038). The most commonly used chemotherapy drug was capecitabine (fifteen cases; 19.0%), followed by gemcitabine (eight cases; 10.1%), eribulin mesylate (six cases; 7.6%), FEC (5-fluorouracil, epirubicin and cyclophosphamide), nab-PTX or vinorelbine (four cases each; 5.1%), paclitaxel (three cases; 3.8%), bevacizumab, irinotecan or S-1 (two cases each; 2.5%), and docetaxel, oral cyclophosphamide or tegafur (one case each; 1.3%). Eleven patients (13.9%) received combined anti-HER2 therapies including trastuzumab (six cases; 7.6%) and lapatinib (five cases; 6.3%); combined anti-HER2 therapy was the most used treatment for the HER2-positive group (P <0.0001). In addition, 23 patients (29.1%) received combined hormonal therapies using agents, such as aromatase inhibitor (16 cases; 20.3%), high-dose toremifene (5 cases; 6.3%) and fluvestrant (2 cases; 2.5%); combined hormonal therapy was the most used treatment for the luminal/HER2-negative group (P = 0.046).

Both humoral and cellular immune responses specific to the vaccinated peptides were analyzed in blood samples before and after vaccination. Plasma samples were collected from 79, 75, or 53 patients before vaccination, at the 6th vaccination, or at the 12th vaccination, respectively. For the monitoring of humoral immune responses, peptide-specific IgGs reactive to each of the 31 different peptides, including both vaccinated and non-vaccinated peptides, were measured by bead-based multiplex assay. The numbers of peptides employed for the first cycle of vaccinations were 2, 3, or 4 in 8, 6, or 63 patients, respectively (Additional file 2: Table S2, Additional file 3: Table S3, and Additional file 4: Table S4). Augmentation of IgG responses specific to at least one of the vaccinated peptides after 6 or 12 vaccinations was observed in 53/75 (70.7%) patients or 50/53 (94.3%) patients, respectively. Peptide-specific IgG responses after 6 or 12 vaccinations were augmented in 7/15 (46.7%) patients or 9/10 (90%) patients with mrTNBC (Additional file 2: Table S2). Such augmentation was seen in 28/40 (70.0%) patients or 29/31 (93.5%) patients in the luminal/HER2-negative group (Additional file 3: Table S3) and in 16/18 (88.9%) patients or 11/11 (100%) patients of the HER2-positive group (Additional file 4: Table S4), respectively.

Cellular immune responses to vaccinated peptides were assessed by IFN-γ ELISPOT assay. Antigen-specific CTL responses were detectable in 17/66 (25.8%) patients before vaccination (Additional file 2: Table S2, Additional file 3: Table S3 and Additional file 4: Table S4). In contrast, augmentation of the CTL responses specific to at least one of the vaccinated peptides after six vaccinations was observed in 34/63 patients (54.0%). Peptide-specific CTL responses after six vaccinations were augmented in 7/14 (50.0%) patients with mrTNBC (Additional file 2: Table S2), while such augmentation was seen in 18/31 (58.1%) patients and 7/16 (43.8%) patients in the luminal/HER2-negative group (Additional file 3: Table S3) and HER2-positive group (Additional file 4: Table S4), respectively. We also tested CTL responses to CEF peptides, a mixture of virus-derived CTL epitopes, as a control. CTL responses to CEF peptides were observed in 27/62 (43.5%) patients before vaccination and 15/58 (25.9%) patients after six vaccinations (Additional file 2: Table S2, Additional file 3: Table S3 and Additional file 4: Table S4).

Collectively, 30/63 (47.6%) patients showed both increased CTL and IgG responses to the vaccinated peptides, 23/63 (36.5%) patients showed either increased CTL or IgG responses, and the remaining 10 (15.9%) patients showed neither CTL nor IgG boosting. In patients treated with PPV alone (n = 27), IgG responses were more frequently increased than those in patients treated with combined chemotherapies (n = 47) (P = 0.002), although there was no significant difference in the increase in CTL responses (P = 1.000).

The RECIST-based clinical responses were evaluated in 64 patients by radiological findings. There were 3 complete response (CR), 6 partial response (PR), 27 stable disease (SD) and 28 progressive disease (PD). The overall response rate of PPV was 14%, including three CR and six PR cases. Among the responsive patients, combined chemotherapy was used in eight cases and hormonal therapy in one case. The intrinsic subtypes showed one mrTNBC and two luminal/HER2-negative types in the CR cases and one mrTNBC and five luminal/HER2-negative types in the PR cases. Computed tomography findings of each of the mrTNBC cases showing PR or SD are shown in Figure 1. The PR case (case 2 in Additional file 2: Table S2) was a 63-year-old woman with a recurrent lung mass treated with a combination of gemcitabine and PPV. At four months after the first vaccination, the lung mass was remarkably reduced in size (Figure 1a and Figure 1b). She survived 32 months after the first vaccination and died due to disease progression. The SD case (case 18 in Additional file 2: Table S2) was a 34-year-old woman with a recurrent lung mass treated with a combination of eribulin mesylate and PPV. At three months after the first vaccination, the lung mass was slightly decreased in size (Figure 1c and Figure 1d). She was subsequently treated by radical resection of the lung mass and pathological evaluation. The lung mass was metastatic TNBC with a high Ki-67 labeling index (42.0%). It expressed epidermal growth factor receptor (EGF-R) and squamous cell carcinoma antigen recognized by T-cells 2 (SART2) antigens which were vaccinated antigens (Figure 2a and Figure 2b), and peritumoral infiltration of T lymphocytes was confirmed (Figure 2c and Figure 2d). She is still alive at 13 months following the first vaccination.

Figure 3 shows survival curves for the three intrinsic subtypes. The median progression-free survival time (MPFST) and median overall survival time (MST) of mrTNBC patients were 7.5 and 11.1 months, while those of luminal/HER2-negative patients were 12.2 and 26.5 months, and those of HER2-positive patients were 4.5 and 14.9 months, respectively. For each intrinsic subtype, the survival curves were compared for patients treated with PPV plus concurrent chemotherapies and those treated with PPV alone (Figure 4). There was no significant survival advantage of concurrent chemotherapies in each intrinsic subtype, compared with treatment with PPV alone (mrTNBC: PFS and OS, P = 0.467 and P = 0.347, respectively; luminal/HER2-negative type: PFS and OS, P = 0.220 and P = 0.850, respectively; and HER2-positive type: PFS and OS, P = 0.296 and P = 0.957, respectively).

Survival analyses along with analyses of the immune responses to PPV were also conducted in the three subtypes. Figure 5 shows the survival curves for patients with or without increased IgG responses after PPV in each intrinsic subtype. IgG boosting was a significant prognostic factor for OS and PFS in HER2-positive patients, whereas there was no significant difference between increased IgG responses and these prognoses in mrTNBC and luminal/HER2-negative patients (mrTNBC: PFS and OS, P = 0.274 and P = 0.152, respectively; luminal/HER2-negative type: PFS and OS, P = 0.732 and P = 0.571, respectively; HER2-positive type: PFS and OS, P = 0.0001 and P = 0.001, respectively). Figure 6 shows the survival curves for patients with or without increased CTL responses after PPV in each intrinsic subtype. CTL boosting was suggested to be a potential prognostic factor for OS but not for PFS in mrTNBC patients, whereas there was no significant difference between CTL boosting and these prognoses in luminal/HER2-negative and HER2-positive patients (mrTNBC: PFS and OS, P = 0.345 and P = 0.053, respectively; luminal/HER2-negative type: PFS and OS, P = 0.272 and P = 0.740, respectively; HER2-positive type: PFS and OS, P = 0.714 and P = 0.758, respectively).