A phase II trial of personalized peptide vaccination in castration-resistant prostate cancer patients: prolongation of prostate-specific antigen doubling time
© Noguchi et al.; licensee BioMed Central Ltd. 2013
Received: 7 June 2013
Accepted: 3 September 2013
Published: 30 December 2013
Cancer vaccine is one of the attractive treatment modalities for patients with castration-resistant prostate cancer (CRPC). However, because of delayed immune responses, its clinical benefits, besides for overall survival (OS), are not well captured by the World Health Organization (WHO) and Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Several surrogate markers for evaluation of cancer vaccine, including prostate-specific antigen doubling time (PSADT), are currently sought. The purpose of this study was to assess prospectively the PSA kinetics and immune responses, as well as the efficacy, safety, and biomarkers of personalized peptide vaccination (PPV) in progressive CRPC.
One hundred patients with progressive CRPC were treated with PPV using 2–4 positive peptides from 31 candidate peptides determined by both human leukocyte antigen (HLA) class IA types and the levels of immunoglobulin G (IgG) against each peptide. The association between immune responses and PSADT as well as overall survival (OS) was studied.
PPV was safe and well tolerated in all patients with a median survival time of 18.8 months. Peptide-specific IgG and T-cell responses strongly correlated with PSADT (p < 0.0001 and p = 0.0007, respectively), which in turn showed correlation with OS (p = 0.018). Positive IgG responses and prolongation of PSADT during PPV were also significantly associated with OS (p = 0.001 and p = 0.004) by multivariate analysis.
PSADT could be an appropriate surrogate marker for evaluation of the clinical benefit of cancer vaccine. Further randomized trials are needed to confirm these results.
KeywordsProstate-specific antigen doubling time Personalized peptide vaccine Prostate cancer Surrogate marker Overall survival
Changes in serum prostate-specific antigen (PSA) can reflect the burden of disease and clinical benefit in patients with castration-resistant prostate cancer (CRPC) with cytotoxic chemotherapy or hormonal agents known to kill tumor cells; these changes can have practical utility by providing and updating prognostic information on an individual patient over time [1–4]. As observed in many clinical trials, however, immunotherapy can induce novel patterns of antitumor responses distinct from those of chemotherapy . For example, an autologous dendritic-cell-based vaccine (sipuleucel-T) is known to improve survival without having an impact on early PSA decline , whereas docetaxel's improvement in overall survival (OS) correlates for the most part with a PSA decline within the first 3 months of therapy [7, 8]. Thus, interpreting PSA decline in the context of novel immunotherapy must be carried out with caution on the basis of the mechanism of action, and may also depend on the time of sampling .
Personalized peptide vaccine (PPV) uses multiple peptides based on the pre-existing immunity. Under PPV treatment, each patient with human leukocyte antigen (HLA)-class IA types positive was tested for their immunological reactivity to 31 different peptides capable of inducing T-cell responses. The 31 peptides were derived from a number of tumor associated antigens: PSA, prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), multidrug resistance protein and a variety of other epithelial tumor antigens. We previously demonstrated that PPV was safe and improved OS with immune responses in phase I, I/II, and II clinical trials in patients with CRPC [10–16]. However, it was not addressed whether PSADT could be an appropriate surrogate marker for evaluation of the clinical benefit of cancer vaccine. To address this, we evaluated data from a phase II clinical trial for CRPC using PPV.
Peptide candidates for personalized peptide vaccination
Symbol for peptide
Position of peptide
Amino acid sequence
Study design and treatment
This was a single institution, single arm, open-label, phase II study. The endpoints of this study were primarily safety and feasibility of PPV in patients with CRPC. Secondary endpoints were to assess the PSA kinetics and immune responses. In addition, we identified potential factors for predicting OS and selecting suitable patients for this treatment. This study protocol was approved by Kurume University Ethical Committee. Written informed consent was obtained from all patients before any study procedures.
In this study, 31 peptides, whose safety and immunological effects had been confirmed in previously conducted clinical studies [10–18], were employed for vaccination [12 peptides for HLA-A2, 14 peptides for HLA-A24, 9 peptides for the HLA-A3 supertype (A3, A11, A31, or A33), and 4 peptides for HLA-A26] (Table 1). All peptides were prepared under conditions of Good Manufacturing Practice using a Multiple Peptide System (San Diego, CA). The selection of 2 to 4 peptides for vaccination to each patient was based on HLA typing and high titer level of peptide-specific IgG to candidate peptides. Each of the selected peptides was mixed with incomplete Freund’s adjuvant (Montanide ISA-51VG; Seppic, Paris, France) and emulsified in the 5 ml plastic syringe, and a maximum of four peptides of 1.5 ml emulsion (3 mg/peptide) were injected subcutaneously into the lateral thigh area once a week for 6 weeks. The peptides were re-selected according to peptide-specific IgG levels at every cycle of 6 vaccinations and administered at 2-, 3-, or 4-week intervals until withdrawal of consent or unacceptable toxicity.
Assessment of clinical activity
Patients were monitored at each visit by history and physical examinations. Serum PSA test and routine laboratory studies were performed every 6 vaccinations for any adverse effects. Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 (NCI-CTCAE Ver3).
All patients underwent relevant radiologic studies and bone scans every 6 months or at the progression of symptoms. PD was defined as radiographic progression evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) criteria  or clinical progression.
To assess the PSA response for each patient, percent PSA change from baseline was calculated for each phase of the study (pre- and during vaccination). In addition, PSA doubling time (PSADT) was calculated using all serum PSA values for a specified period, and using a minimum of three PSA values by the formula log2/b, where b denotes the least square estimate of the linear regression model of the log-transformed PSA values on time. For analytical purposes, negative PSADT estimates and high positive PSADT estimates (>36 months) were censored at 36 months.
To investigate biomarkers for OS that may allow patient selection and prediction of a response to PPV, serum amyloid A (SAA), C-reactive protein (CRP), and interleukin (IL)-6 in plasma at baseline were additionally examined by enzyme-linked immunosorbent assay (ELISA), respectively.
Measurement of humoral and T-cell responses specific to the vaccinated peptides
To study the humoral responses specific to the vaccinated peptides, peptide-specific IgG levels were measured by a Luminex system (Luminex, Austin, TX), as reported previously . If the total titers of selected peptide-specific IgG in any cycles of post-vaccination plasma were more than 2-fold higher than those in the pre-vaccination plasma, the changes were considered to be a positive response.
Although T-cell subsets using flowcytometry was not analyzed in this study, T-cell responses specific to the vaccinated peptides were evaluated by IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs), as reported previously . Peptide-specific T-cell responses were evaluated by the differences between the numbers of spots per 105 x PBMCs in response to the vaccine peptides and those to the control peptide at pre- and 6th vaccination; at least 2-fold more spots at the 6th vaccination than at pre-vaccination was considered positive.
All patients who received more than 6 vaccinations were considered evaluable for tumor response, and all patients entered were included in the survival analysis. Data were analyzed at the end of November, 2012 using commercially available computer software. The Student’s t-test and the chi-square test were used to compare quantitative and categorical variables, respectively. Survival was calculated from the date of first treatment until the date of any cause of death. Patients lost to follow-up were censored at the last known date of survival. The Kaplan-Meier method was used to estimate actuarial survival curves, and groups were compared using a log-rank test. Cox proportional hazards regression model was used for univariate and multivariate analyses to identify factors that had a significant impact on survival. All baseline parameters in the survival and proportional hazards regression analysis were analyzed as dichotomous variables using median or cut-off values. A two-sided significance level of 5% was considered statistically significant.
Characteristics of the patients
Patients (N = 100)
ECOG performance status
Baseline PSA, ng/ml
CRP, 3 μg/mL
SAA, 8 μg/mL
IL6, 2.4 pg/mL
Site of metastasis
Bone and nodal/organ
Adverse events during peptide vaccination
Injection site reaction
White blood cell count decreased
Blood triglycerides increased
There was no complete response or partial response in terms of measurable disease. The median time to disease progression, as defined by clinical and/or radiologic criteria, was 10.9 months (95% CI, 6 to 19 months). At the time of analysis with a median follow-up of 18 months (95% CI, 14.1 to 24 months), 64 deaths had occurred. Median survival time was 18.8 months (95% CI, 14.9 to 28.6 months) in all patients. Median survival time in chemotherapy naive patients and in patients after docetaxel chemotherapy were 21.6 months and 11.6 months, respectively.
The number of selected peptides were 4 peptides in 62 patients, 3 peptides in 17 patients and 2 peptides in 21 patients at the first screening. Same peptide at the first screening were only selected in 29 of 97 (30%) patients at second screening and in 10 of 66 (15%) patients at the third screening, remaining patients received at least 1 different peptide during the study. The most frequently selected peptides were Lck486 (40 patients), CypB129 (31 patients), PAP213 (24 patients), SART2-93 (21 patients), PSA248 (20 patients), Lck488 (17 patients) and WHSC2-123 (16 patients) at the first screening. All 31 peptides were selected at any screening in the study.
Cox proportional hazards regression analysis of association between potential factors and death after PPV in the 100 CRPC patients
Positive vs. negative
ECOG performance status
0 vs. 1
Low (<3000 ng/mL) vs. high
Increase (2 times) vs. no
Low (<30 ng/mL) vs. high
Untreated vs. treated
Positive vs. negative
>50% PSA decline
Positive vs. negative
Number of lymphocytes
High (>1300/μL) vs. low
Low (<2.4 pg/mL) vs. high
Low (<69 years) vs. high
Low (<8) vs. high
Low (<8 μg/mL) vs. high
The factors showing p less than 0.05 in the univariate analysis were included in multivariate analysis of the model. Finally, positive IgG response (p = 0.001) and prolongation of PSADT (p = 0.004) during PPV, as well as baseline good performance status (p = 0.004), low CRP levels (p = 0.006), and low PSA levels (p = 0.008), were significantly favorable factors for OS (Table 4).
As observed in several clinical trials, immunotherapy can induce novel patterns of antitumor responses distinct from those of chemotherapy, which are consequently not captured by the WHO or RECIST criteria . On the other hand, there is debate regarding the utility of PSA changes, especially with immunotherapy, and the PSA Working Group 2 has advocated using radiographic progression-free survival as a preferred endpoint for phase II trials . Others have argued that changes in PSADT may be a marker of drug effect, understanding that shorter PSADT corresponds to worse prognosis and, thus, a favorable change in PSADT suggests drug activity [22, 23]. However, clinical trials of recently developed drugs, such as sipuleucel-T , cabazitaxel , and abiraterone acetate , for the treatment of progressive CRPC patients did not analyze the usefulness of PSADT as a surrogate marker of response in CRPC patients. In the current study, we attempted careful and stringent collection of multiple PSA values in order to calculate PSADT changes before and during PPV accurately. While delayed PSA responses were observed, we did see a statistically significant increase in PSADT. Importantly, patients with prolongation of PSADT showed statistically longer survival (p = 0.018). These results suggest that the development of late immune responses is associated with changes in PSADT.
The evaluation of T-cell immune responses to target self antigens after vaccine clinical trials presents several challenges. Antigen-specific T-cells can be evaluated by their peptide target specificity, proliferative capacity, cytokine secretion, cytolytic activity, and membrane markers of activation. At present, the best measure of antigen-specific T-cells is unknown, as is the optimal time to evaluate immune responses. In our current analysis, we evaluated both humoral responses determined by peptide-specific IgG levels using a Luminex system and antigen-specific CD8+ T-cell responses by using IFN-γ ELISPOT assays, to provide a more direct quantitative assessment after immunization. Delayed 50% PSA decline and prolongation of PSADT were observed in patients with positive IgG and T-cell respkonses, and these immune responses were associated with OS. These results suggest that further immunological analysis at multiple time points might be needed to determine whether T-cell response or the development of late immune responses is associated with clinical responses.
Cancer vaccinations do not always extract good immune and/or clinical responses in vaccinated patients. This study showed that IgG responses and prolongation of PSADT during PPV, along with baseline performance status, CRP, and PSA levels, were well correlated with OS in patients with CRPC treated by PPV. These results suggest that risk stratification based on these factors could be helpful for estimating the OS in patients with CRPC treated by immunotherapy.
Despite these encouraging observations, the current study must be interpreted as hypothesis-generating due to several limitations. This single-arm phase II study without a concurrent control arm did not allow estimation of the potential clinical or immune effects of this treatment. Another potential limitation of this study regarding OS is the lack of treatment data after the treatment phase of the trial. Imbalances due to chance may have occurred in treatments after progression. However, only docetaxel has been shown to affect survival in this population of patients, and only by a few months. The median survival of 18.8 months (95% CI, 14.1 to 24 months) observed in this study surpassed the survival that was observed from docetaxel-based clinical trials in a similar population by TAX-327 (median survival, 19.2 months) and South West Oncology Group 9906 (median survival, 17.5 months) [7, 8]. Thus, we think it unlikely that a potential imbalance in post-study treatments could explain the survival results.
This study showed that PPV in patients with CRPC was active and well tolerated, improving survival with immune responses, delayed PSA responses, and prolongation of PSADT. Further randomized trials are needed to confirm these preliminary results.
Castration-resistant prostate cancer
Cytotoxic T lymphocytes
Eastern cooperative oncology group
Human leukocyte antigen
- IFN- γ:
Peripheral blood mononuclear cells
Personalized peptide vaccination
Prostate specific antigen
Prostate specific antigen doubling time.
Details of all funding sources
This study was supported in part by Grants-in-Aid (KAKENHI) (no.22591782 to M.Noguchi), and by the grants from the Regional Innovation Cluster Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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