Pancreatic cancer is one of the most lethal malignant diseases, with the worst prognostic. It is the fourth leading cause of cancer-related death in men and women in USA(1).
Recent analysis of human pancreas genomes showed that 12 common signaling pathways involved in cellular repair mechanism, metabolism, cell-cycle regulation, genomic repair and metastasis are affected in over two thirds of the pancreatic cancer genome, including mainly point mutations(2).
2. Molecular genetics understanding of pathway in pancreatic cancer
The most frequent genetic abnormality in invasive pancreatic cancer is the mutation of the activating K-ras, which occurs in 75-90% of pancreatic cancers(3). The mutations in the K-ras gene are found in the earliest form of pancreatic intraepithelial neoplasia lesions and are considered to be one of the earliest genetic events to take place during pancreatic tumorigenesis(4).
There are several studies that focus on K-ras as a therapeutic target and have worked to develop treatments such as antisense therapy and RNA interference. In a phase II trial that included patients with locally advanced or metastatic pancreatic adenocarcinoma, the Ras family antisense inhibitor combined with gemcitabine showed a response rate of 10.4% and a median survival of 6.6 months(5).
RNA interference technology was studied in in vitro and in vitro trials published in 2011 that demonstrated that the knockdown of STAT3 gene using RNA interference technique may be a novel therapeutic option for the treatment of pancreatic cancer(6).
b. PI3K/AKT pathway
The PI3K/AKT pathway functions downstream of K-ras and it is activated during carcinogenesis by mutations. AKT is amplified and the PI3K-AKT pathway is activated in 20% and 59% of pancreatic cancers, according to a paper published in 2003 on the incidence, mechanism and prognostic value of activated AKT in pancreatic cancer(7).
HMGA1, an architectural transcription factor that has been implicated in several human cancers, correlates with poor prognosis, activates PI3K-AKT pathway and mediates resistance to gemcitabine(8).
In 2004, it was published an article on the inhibition of PI3K/AKT pathway through aberrant expression of PTEN (phosphatase and tensin homolog) which showed that persistent activation of signaling cascades resulted in dramatic consequences that included loss of cellular growth control and neoplastic transformation(9).
The vascular endothelial growth factor (VEGF) is a glycoprotein that promotes endothelial cell survival, mitogenesis, migration, differentiation and vascular permeability. Ras protein and hypoxia stimulates upregulation of VEGF expression.
Several trials are being conducted to examine bevacizumab in combination with other agents or treatment regimens; angiogenic inhibitors that target other non-VEGF pathways may be better to gain access to the tumour environment than an antibody(2).
The role of bevacizumab, a recombinant humanized monoclonal antibody directed against vascular endoltelial growth factor, in the treatment of pancreatic cancer remains unclear.
The objectives of the 2008 study were to determine the safety and efficacy in chemotherapy-naïve patients with metastatic pancreatic cancer receiving bevacizumab in combination with fixed dose rate of gemcitabine and low dose of cisplatin.
Of the 52 patients eligible for analysis, 19.2% had an unconfirmed response and 57.7% had stable disease. Median time to tumour progression was 6.6 months and median survival was 8.2 months.
In conclusion, this bevacizumab-containing study regimen is modestly effective in patients with metastatic pancreatic cancer, although occasional serious complications may occur(10).
3. Pancreatic cancer and the immune response
It is known that pancreatic cancer is characterized by a highly immunosuppressive environment, therefore there is great potential to target and reverse these mechanisms to create an environment that supports the infiltration of antitumour immune response and enables the generation of T cells capable of killing pancreatic tumour cells.
Further studies in this direction underlined that cytotoxic T-lymphocyte-associated protein-4 and B- and T-lymphocyte attenuator can increase immune response, further being confirmed that patients produce an immune response to the tumour(11).
Unfortunately, the antitumour immune response is ineffective because the tumour microenvironment is immunosuppressive and the tumour antigens are mutated self-antigens, so it does not result in the killing of the tumour.
In conclusion, studies nowadays aim for a strategy to improve the clinical efficacy of immunotherapy and how to overcome immune suppression.
4. Active immunotherapy in pancreatic cancer
The main purpose of active immunotherapy is to induce long-term cellular immunity against cancer cells.
The goal of tumour specific vaccines is to present tumour-associated antigens to immune cells and produce potent and lasting cytotoxic effects against tumour cells. Antigen presenting cells such as dendritic cells and T cells are the targets and the effectors of this immune response(3).
4.1. Whole cell vaccines
Whole tumour cells used as a vaccine provide:
a) source of all potential antigens, eliminating the need to identify the most optimal antigen to target in a particular type of cancer;
b) multiple tumour antigens that can be targeted at once, generating immune responses to more than one tumour antigen;
c) immunized lymphocyte and serologic responses that can be explored to identify new tumour antigens(12).
Whole cell vaccines have been genetically modified to express cytokines, chemokines or costimulatory molecules to stimulate the immune response to the injected irradiated tumour cells. To develop the vaccines, there have been used allogenic or autologous tumour cells.
There are several studies that used GM-CSF (granulocyte macrophage colony-stimulating factor), a cytokine superior for its ability to activate an effective tumour targeted T cell response.
A phase II study tested the safety and efficacy of a granulocyte-macrophage colony-stimulating factor-based immunotherapy administered in patients with resected pancreatic adenocarcinoma. The first immunotherapy treatment was administered 8 to 10 weeks after surgical resection. Subsequently, patients received 5-FU based chemoradiation. Patients who remained disease-free after completion of chemoradiotherapy received treatments 2 to 4 each 1 month apart. A fifth, and final, booster was administered 6 months after the fourth immunotherapy. The median disease-free survival was 17.3 months. The administration of immunotherapy was well tolerated(13).
4.2. Peptide vaccines
Peptides corresponding to immunogenic tumour antigens or antigen epitopes have been administered as cancer vaccines in order to enhance T cell response, the most important response for the elimination of tumour. However, peptide vaccines showed limited clinical efficacy, influenced by the ability of tumour tumour cells to escape recognition by the immune system(14).
Since Sipuleucel-T was accepted as the first standard peptide vaccine for castration resistant prostate cancer, an increasing number of clinical trials have been conducted in many other cancer types.
Recently, the Wilms tumour gene peptide based vaccine in combination with gemcitabine was found to be more effective than gemcitabine alone for patients with advanced pancreatic cancer. Thirty-two HLA-A positive patients with advanced pancreatic cancer were enrolled. Patients received modified 9-mer Wilms tumour gene peptide 3 mg/body intradermally biweekly and gemcitabine 1000 mg/m2 on days 1, 8 and 15 of a 28 day cycle. Median survival time and 1-year survival rate were 8.1 months. The association between longer survival and positive delayed type hypersensitivity to Wilms tumour gene peptide was statistically significant. The vaccine in combination with gemcitabine was well tolerated for patients with advanced pancreatic cancer. In addition, the vaccine led to pain relief and alleviated distressing symptoms(15). The combination of chemotherapy with immunotherapy against cancer proved to be effective and synergistic.
4.3. VEGF vaccines
Vascular endothelial growth factor (VEGF) is overexpressed in pancreatic cancer and is associated with poor prognosis, larger tumour size and enhanced local spread.
A phase I trial combining of VEFGR2-169 an epitope peptide with gemcitabine was conducted for patients with metastatic and unresectable pancreatic cancer. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8 and 15, in a 28-day cycle. The vaccine was subcutaneously injected weekly in a dose escalation manner (doses of 0.5, 1 and 2 mg/body). 83% of the patients developed immunological reactions at the injection sites. The disease control rate in this study was calculated to be 67%. One patient achieved partial response, seven months later the tumour in the pancreatic body was reduced to 45% in comparison to that before the vaccination. Considering the evidence, it was believed that the combination of gemcitabine with immunotherapy might provide some clinical benefit to patients with advanced pancreatic cancer(16).
4.4. Recombinant vaccines
Recombinant vaccines are based on bacterial and viral antigen carriers that increase dendritic cell activation and improve antigen presentation. Poxviral vectors have proven safety record and can be used to incorporate multiple transgenes. Carcinoembryonic antigen and MUC-1 are overexpressed in a substantial proportion of common solid carcinomas(17).
TRICOM vaccine is a poxvirus-based vaccination that uses three distinct T cell co-stimulatory molecules: B7-1, ICAM-1 and LFA-3.
In 2008, in a pilot study, 25 patients were treated with poxviral vaccine regimen consisting of the genes for carcinoembryonic antigen and MUC-1 along with TRICOM engineered into PANVAC-V as a prime vaccination and into PANVAC-F as a booster vaccination. Median overall survival was 6.3 months. Immune responses to MUC-1 and carcinoembryonic antigen were seen following vaccination in 9 of 16 patients tested. The vaccine was well tolerated, no grade >2 toxicity was seen in more than 2% of the cycles(17).
4.5. Antigen-pulsed dendritic cells
Dendritic cell therapy represents a new and promising immunotherapeutic approach for the treatment of advanced cancer, as well as for the prevention of cancer.
Human tumours express a number of protein antigens that can be recognized by T cells, thus providing potential targets for cancer immunotherapy. Dendritic cells are rare leucocytes that are uniquely potent in their ability to present antigens to T cells and this property has prompted their recent application to therapeutic cancer vaccines(18).
MUC-1 is an epithelial cell glycoprotein that is highly overexpressed and aberrantly glycosylated in pancreatic tumours. Phase I/II clinical trial used autologous dendritic cells pulsed with MUC-1 peptide and treated twelve pancreatic and biliary cancer patients following resection of their primary tumour. The vaccine was well tolerated and no toxicity was observed. Three patients had pre-existing MUC1 antibody responses that remained stable post vaccination. MUC1-specific T cell responses were difficult to evaluate due to the increase in activity of all CD8 and CD4 T cells following each vaccination. Vaccinated patients have been followed for over four years and four of the twelve patients were alive, all without evidence of recurrence(19).
5. Passive immunotherapy in pancreatic cancer
Administration of exogenous antibodies constitutes the passive humoral immunotherapy. The host immune’s response is initiated by external antibodies or other immune components such as checkpoint inhibitors and cytokines that are generated in a laboratory. This consequently enhances the preexisting immune response and delivers immunity against the tumour. Monoclonal antitumour antibodies may be conjugated with toxins or with radioisotopes, so that the antibodies deliver these toxic agents specifically to tumour cells.
5.1. Antibody-based therapies
There are several mechanisms through which monoclonal antibodies affect tumour cells:
antibody-dependent cell-mediated cytotoxicity;
antibody-dependent cellular phagocytosis that leads to apoptosis and blockade of cellular receptors growth factor.
Pancreatic cancer cells produce mesothelin. The mesothelin gene encodes a precursor protein proteolytically cleaved into an N terminus secreted form and a C terminus membrane bound form which is glycosylphosphatidylinositol-linked glycoprotein. Mesothelin has been shown to be overexpressed in several cancer types including mesothelioma, ovarian cancer and pancreatic cancer(20).
Further studies revealed a phase I trial in patients with advanced mesothelin expressing cancers treated with MORAb-009.
MORAb-009 is a chimeric monoclonal antibody that targets mesothelin. The patients included in the trial received MORAb-009 intravenously on days 1, 8, 15 and 22 at progressively increasing doses ranging from 12.5 to 400 mg/m2. Eleven subjects had stable disease. The adverse events were grade 4 transaminitis and grade 3 serum sickness. The conclusion of the study was that MORAb-009 is well tolerated and the maximum dose tolerated when administered weekly is 200 mg/m2(21).
A recent phase II randomized trial looking at MORAb-009 with gemcitabine versus gemcitabine alone has now been completed and the results are pending. The purpose of the study was to investigate the activity of MORAb-009 when added to a standard regimen of gemcitabine in patients with previously untreated unresectable stage 3 or 4 pancreatic cancer(22).
Another trial investigated cetuximab and trastuzumab versus gemcitabine in the treatment of human pancreatic carcinoma xenografts. In first line therapy, mice survival was longer in the monoclonal antibody group compared with gemcitabine. In second line therapy, tumour regressions were observed after replacing gemcitabine by monoclonal antibodies treatment, resulting in significantly longer animal survival compared with mice receiving continuous gemcitabine injections. Therapeutic benefit of monoclonal antibodies was observed despite K-ras mutation. The study demonstrated a significant improvement of survival and tumour regression in mice treated with anti-EGFR/anti-HER2 monoclonal antibodies independently on the K-ras status(23).
6. Radioimmunotherapy in pancreatic cancer
Radioimmunotherapy is a type of targeted therapy that delivers radiation directly to cancer cells by combining a monoclonal antibody with radioactive material. When the monoclonal antibody binds to the cancer cell, the radiation kills the cell.
Currently radioimmunotherapy is used for the treatment of B cell non-Hodgkin lymphoma, but is being evaluated for the treatment of other types of cancer like pancreatic cancer, and is showing promising results.
A 2009 study investigated whether repeated cycles of 90Y-hPAM4 radioimmunotherapy plus radiosensitizing gemcitabine is efficient in treating advanced untreated unresectable pancreatic cancer.
90Yttrium clivatuzumab tetraxetan is a human monoclonal antibody targeting mucin antigen expressed in most pancreatic cancers. The monoclonal antibody 90Y-hPAM4 was given once weekly in weeks 2-4 in association with gemcitabine 200mg/m2 weekly for 4 weeks. Cytopaenias were the only significant toxicities. The conclusion of the trial was that dose escalation is continuing after fractionated radioimmunotherapy plus low dose gemcitabine demonstrated therapeutic activity at the initial dose levels of monoclonal antibody with minimal hematologic toxicity after four therapy cycles(24).
7. Immunotherapy targeting cancer stem cells
Cancers originally develop from normal cells that gain the ability to proliferate aberrantly and eventually turn malignant. These cancerous cells then grow clonally into tumours and eventually have the potential to metastasize.
A central question in cancer biology is which cells can be transformed to form tumours? Recent studies elucidated the presence of cancer stem cells that have the exclusive ability to regenerate tumours. These cancer stem cells share the same characteristics with normal cells, self-renewal and differentiation(25).
Pancreatic cancer cells resistant to chemoradiotherapy are rich in cancer stem cells fraction and they could be expanded during the acquisition of gemcitabine resistance. Targeted therapy against cancer stem cell fraction could be applied to overcome drug resistance in the treatment of pancreatic cancer(2).
Signaling through sonic hedgehog and mammalian target of rapamycin may be essential for cancer stem cell self renewal and could represent targets for novel treatment modalities. This 2009 study used in vivo and in vitro models of pancreatic cancer to examine the effects of this treatment on tumorigenic cancer stem cell population.
Neither rapamycine, nor cyclopamine alone or as supplements to chemotherapy were capable of effectively diminishing cancer stem cell population. Only the combined inhibition of both patways together with chemotherapy reduced the number of cancer stem cells to undetectable.
Further preclinical investigation of this promising approach may lead to the development of a novel therapeutic strategy to improve the prognosis of pancreatic cancer patients(26).
Immunotherapy in pancreatic cancer is still at the beginning of the road, but discovering new therapeutic targets and new modalities to deliver the treatment to the tumour gives us hope even if pancreatic cancer is a highly lethal disease. Although immunotherapy is not yet the standard of care in pancreatic cancer, with further studies in the field it may become the key treatment in the future.