Immunotherapy for patients with advanced pancreatic carcinoma: a promising treatment

There are limited data on the safety and efficacy of immunotherapy for patients with advanced pancreatic cancer (APC). A meta-analysis of single-arm trials is proposed to assess the efficacy and safety of immunotherapy for APC. Eighteen relevant studies involving 527 patients were identified. The pooled disease control rate (DCR), overall survival (OS), progression free survival (PFS), and 1-year survival rate were estimated as 59.32%, 7.90 months, 4.25 months, and 30.12%, respectively. Subgroup analysis showed that the pooled OS, PFS, and 1-year survival rate were significantly higher for autologous activated lymphocyte therapy compared with peptide-based vaccine therapy (OS: 8.28 months vs. 7.40 months; PFS: 6.04 months vs. 3.86 months; 1-year survival rate: 37.17% vs. 19.74%). Another subgroup analysis demonstrated that the pooled endpoints were estimated as obviously higher for immunotherapy plus chemotherapy compared with immunotherapy alone (DCR: 62.51% vs. 47.63%; OS: 8.67 months vs. 4.91 months; PFS: 4.91 months vs. 3.34 months; 1-year survival rate: 32.32% vs. 21.43%). Of the included trials, seven trials reported no treatment related adverse events , five trials reported (16.6 3.9) % grade 3 adverse events and no grade 4 adverse events. In conclusion, immunotherapy is safe and effective in the treatment of APC.


INTRODUCTION
Pancreatic cancer (PC) is the fourth most common cause of cancer death worldwide, which is characterized by an extremely poor survival rate [1]. Up to 80% deaths occur within the first year of diagnosis and the overall 5-year mortality rate is over 95% [2,3]. Although surgical resection is the only potentially curative approach, only 10-20% of pancreatic tumors are operable, about 40% are locally advanced, unresectable and 45% are with metastases [4,5]. Usually, patients with advanced pancreatic cancer (APC) are unable to resort to surgery, the treatment alternatives of whom are very limited, with gemcitabine as the current first-line treatment [6]. However, patients received gemcitabine had a overall survival (OS) of around 6 months and a one-year survival ≤ 20% [7]. Once APC patients are resistant to gemcitabine therapy, there is barely effective treatment. New treatment strategies are therefore urgently required.
Recently, immune cell-based cancer therapy has been attempted as an alternative treatment option for anticancer therapy [8]. It eliminates cancer cells by modulating the immune system to suppress cancer by active (potentiating the patient's intrinsic immune system against cancer cells ) and passive (administering extrinsic Research Paper man-made immune system components ) immunotherapy [9]. Immunotherapy has an advantage over chemo(radio) therapies due to its specificity against tumor without hurting normal tissue [10]. Immunotherapeutic methods to PC included the administration of antibodies [11], cytokines [12], peptide vaccines [6,[13][14][15][16][17][18], and autologous activated lymphocyte (e.g. dendritic cells, lymphokine activated killer cells, and cytotoxic T-lymphocyte) therapies [10,[19][20][21][22][23][24]. Despite the limited benefit of gemcitabine, it may enhance responses to specific vaccines or synergize immune system stimulators [22]. Hence, combining gemcitabine with immunotherapy for APC patients was used in most clinical trials. However, these published trials have been small with consistent results, precluding robust estimates of benefit.
To assess the benefit of immunotherapy in the treatment of patients with APC neutrally, we would like to perform a published data meta-analysis of all relevant single-arm trials.

Trials
A total of 18 trials involving 527 patients met the inclusion criteria were enrolled in this study ( Figure  1). It included seven trials (295 patients) of autologous activated lymphocyte therapies, eight trials (170 patients) of peptide-based vaccine therapy, one trial (34 patients) of monoclonal antibody plus gemcitabine, one trial (16 patients) of cytokine-induced killer (CIK) and one trial (12 patients) of gene-mediated cytotoxic immunotherapy (GMCI). There were 14 trials (454 patients) used immunotherapy plus chemotherapy, only four trials (73 patients) used immunotherapy alone. Table 1 shows the details of trial designs, publication year, number of patients, female/male ratio, treatment schedules, and study end points.

Treatment-related adverse events
Adverse events were monitored based on the National Institute Common Terminology Criteria for Adverse Events version 3.0. Reports on treatment-related adverse events (TRAEs) were variable among trials. Of the 18 trials, 9 trials (7 using autologous activated lymphocyte therapy ± gemcitabine, 1 using peptide-based vaccine therapy, and 1 using monoclonal antibody + gemcitabine) reported no TRAEs, 7 trials (5 using peptidebased vaccine therapies, 1 using gene-mediated cytotoxic immunotherapy, and 1 using cytokine-induced killer cells) reported grade 3 TRAEs (e.g. anemia, lymphopenia, leukopenia, neutropenia, thrombocytopenia) with mean rate of 14.56% (ranging from 3.22% to 25.8%) and no grade 4 TRAEs. The remaining 2 trials did not report TRAEs.

Publication bias
Both the Egger's and Begg's tests revealed no publication bias for OS (P = 0.951 and 0.956, respectively) and for PFS (P = 0.085 and 0.754, respectively). Begg's test not Egger's test revealed no publication bias for 1-year survival rate (P = 0.815 and 0.000, respectively). Both the Egger's and Begg's tests revealed publication bias for DCR (P = 0.017 and 0.000, respectively).

DISCUSSION
The present meta-analysis gathers all currently available data from single-arm studies reporting immunotherapy for those patients with advanced pancreatic cancer. To our best of knowledge, ours is the first meta-analysis to analyze the efficacy and safety of immunotherapy using single-arm trials. The results showed immunotherapy is more effective than other previously reported standard treatmentin terms of OS, PFS, 1-year survival rate for advanced pancreatic carcinoma [28][29][30][31][32]. After stratifying trials according to the type of immunotherapy, we found that the benefits of autologous activated lymphocyte therapy were more   Since the early 1990s, many institutions have actively studied the use of autologous activated lymphocyte therapies, including lymphokine-activated killer lymphocytes (LAK), cytotoxic T-lymphocyte (CTL), and dendritic cell (DC) [33]. Among various immune cell types studied as potential candidates for effective immunotherapy, DCs are potent antigen-presenting cells that participate in the initiation of T-cell immunity and involved in the regulation of both innate and adoptive immune response [24,34]. Some previous clinical trials have used the combination antigen-pulsed or peptide-pulsed DC vaccine with LAK cells or CTLs in patients with advanced pancreatic cancer and observed significantly prolonged survival time [10,20,21,23,24]. As a matter of fact, all trials performed so far comparing immunotherapy with standard therapies failed to show the superiority of the former [35]. Obviously, cancer immunotherapy would not be able to take the place of chemotherapy and radiotherapy. Moreover, though recent clinical trials have investigated the sequential administration of immunotherapy and chemo (radio) therapy, the immunosuppressive effects of these standard therapies may weaken the efficacy of immunotherapy. Therefore, it will be essential to find an optimal way to integrate immunotherapy with chemo(radio) therapy. Subgroup analysis indicated that the benefits of combined therapy were more significant than that of immunotherapy alone (DCR: 62.51% vs. 47.63%; OS: 8.67 months vs. 4.91 months; PFS: 4.91 months vs. 3.34 months; 1-year survival rate: 32.32% vs. 21.43%). For chemotherapy, gemcitabine is currently one of the standard therapies for advanced pancreatic cancer, although many chemotherapeutic drugs have been used in clinical trials over the past two decades [35]. However, the effect of gemcitabine is limited and most patients received gemcitabine die within 6 months. Interestingly, some recent reports showed that gemcitabine may improve patients' response to immunotherapy [22]. Dauer et al have demonstrated in their in vitro study that human pancreatic cancer cell lines could be sensitized by gemcitabine against CTL-mediated lysis [36]. DC vaccine plus gemcitabine prolongs survival time in a animal model [37]. Although some trials of combined therapies including not only gemcitabine but also other cytotoxic drugs showed improved response rates compared with gemcitabine alone, they failed to gain survival benefits [38][39][40][41]. Thus, most trials included in this meta-analysis combined only gemcitabine with immunotherapy.
The importance of treatment-related adverse events (TRAEs) that related to immunotherapy must be emphasized. Reporting on TRAEs was variable across trials. Of the included 18 trials, no trails observed related to autologous activated lymphocyte therapy. Five trials using peptide-based vaccine therapies reported (16.6 ± 3.9)% grade III TRAEs and no grade IV TRAEs and one trial reported no TRAEs. Therefore, immunotherapy used in advanced pancreatic cancer patients is safe and no evidence of autoimmune disease was noted, especially for autologous activated lymphocyte therapy.
In summary, cancer immunotherapy was well tolerated and effective in the treatment of advanced pancreatic carcinoma. Currently, available clinical evidences suggest that the combination immunotherapy with standard therapies should be recommended as preferred therapy for those patients with advanced pancreatic carcinoma. For vaccine-based immunotherapy, autologous activated lymphocyte therapy was considered to be the more promising and encouraging treatment compared with peptide-based vaccine therapy. Further clinical investigations, especially randomized controlled trials are warranted to determine the effectiveness and tolerability of immunotherapy.

Trials identification
We conducted separate PubMed (inception to June 2016), EMBASE (inception to June 2016) and the Cochrane Library (inception to June 2016) searches of all relevant English language articles using an extension of the Cochrane search strategy [25]. Potential studies were identified by using the following keywords: "immunotherapy" OR "immunotherapeutic" OR "immune-cell based cancer therapy" OR "vaccinebased immunotherapy" AND "pancreatic cancer" OR "pancreatic carcinoma" OR "pancreatic adenocarcinoma" AND "advanced" OR "unresectable" OR "metastatic" OR "refractory" OR "inoperable". Further information was manually searched reference lists from already retrieved studies and general medical journals (e.g., New England Journal of Medicine, The Lancet, British Journal of Medicine and JAMA) and journals in the cancer field (e.g., Journal of Clinical Oncology, Cancer Research, Cancer, Annals of Oncology, British Journal of Cancer, and Cancer Treatment Reviews). In particular, review articles were also examined for published results. By carefully checking the body of each publication and the name of all authors, we avoided duplications of data. The search strategy used is illustrated in Figure 1.

Selection criteria
The selection criteria was as follows: (1) Trials were in the English language and were limited to human trials; (2) Prospective or retrospective clinical trials with or without a control population were eligible for inclusion; (3) Trials assessed the efficacy and safety www.impactjournals.com/oncotarget of immunotherapy in the treatment of APC. (4) Trials reported endpoints without 95%CI or mean survival time were not included.

Primary and secondary endpoints
Data on disease control rate (DCR) (partial response [PR] + complete response [CR] + stable disease [SD]), overall survival (OS), progression-free survival (PFS), 1-year survival rate and treatment-related adverse events (TRAEs) were independently extracted by two reviewer, with any discrepancies resolved by consensus (and if necessary with a third reviewer). The primary endpoint evaluated was DCR. The secondary endpoints evaluated were OS, PFS, 1-year survival rate and TRAEs. OS and PFS were estimated from the date of the initial treatment to the date of death or final follow-up and the date of disease progression, respectively. These endpoints were analyzed for all included studies for which data were available. The subgroup analyses were conducted according to the type of immunotherapy (autologous activated lymphocyte therapies, peptide vaccine therapy, and other immunotherapies such as monoclonal antibodies), combination with or without chemotherapy.

Statistical analysis
We performed bootstrapping (number of samples = 1000) to calculate the 95%CI for OS, PFS, DCR and 1-year survival rate if study didn't report these endpoints' 95%CIs but reported individual participant data. In particular, we used metaprop command (metaprop DCR [or 1-year survival rate], random second (fixed) ftt cimethod (exect) label (namevar = Author, yearvar = Year) to summarize DCR and 1-year survival rate.
Considering the heterogeneity across studies, metaanalysis was performed using a random-effects model, using the DerSimonian-Laird method [26]. Heterogeneity across studies was assessed using the Cochran's Q and I 2 statistic with values ranging from 0% (no heterogeneity) to 100%; values > 50% indicated significant heterogeneity [27]. Univariate meta-regression using REML method was performed to find out sources of heterogeneity including study design, mean age, female/male (F/M) ratio, number of patients, publication year, the type of immunotherapy, and combination with or without chemotherapy. Additionally, pre-specified subgroup analyses were performed (i) by type of immunotherapy; (ii) by combination with or without chemotherapy. Publication bias was examined utilizing funnel plots by the Begg's and Egger's methods. All data were recorded in a Microsoft Excel spreadsheet (Microsoft Corp, Redmond, WA) and analyzed using Statistical Package for Social Sciences (SPSS) software version 23.0 (SPSS Inc., Chicago, IL, USA) and STATA 12.0 (Stata Corp LP, College Station, Texas, USA).

Abbreviations
APC Advanced pancreatic cancer DCR Disease control rate OS Overall survival PFS Progression-free survival TRAEs Treatment-related adverse events AALT Autologous activated lymphocyte therapy PBVT peptide-based vaccine therapy