Research On Serum Proteome Of Allogeneic Cellular Immunjotherapy For Non Small Cell Lung Cancer

Objective:To evaluate the safety, immunologic, clinical activity,feasibility and serum polypeptidome and proteome of granulocyte macrophage colony stimulating factor(GM-CSF) secreting allogeneic cancer cell vaccine(GMV) in patients with stageⅢandⅣnon-small-cell lung cancer(NSCLC).Methods:Patients were required to have completed or refused conventional therapy and enrolled at Tianjin Cancer Hospital between November 2006 and May 2008, after institutional review board approval and written informed consent was obtainded. StageⅢandⅣNSCLC patients (n=64) received GMV treatment. Each injection contains inactivated 3 NSCLC tumor cell more than 1×107 and GM-CSF secreting cell which can secret GM-CSF more than lug/10/24h. Vaccines were administered subcutaeous every week for a total six vaccinations. End points were changes in immunology, progression free survival, overall survival and changes in serum polypeptidome and proteome.DTH were administered intradermal on the first and fifth vaccinations. Collected 10ml peripheral venous blood at pre- and post-treatment, check the percentage of CD4+ T cell, CD8+ T cell,Treg cell,NK cell by using flow cytometry.The levels of INF y,IL-4,TGFβ1 were evaluated by using the ELISA. Serum polypeptidome detected by functional magnetic bead based sample fractionation method combinded with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) technology.Serum proteome used a label-free quantitative proteomic approach based on nanoscale high performance liquid chromatography (HPLC) electro-spray ion tandem mass spectrum (LC-ESI-MS/MS).Results:Part I:Vaccination was safe, and all the patients completed the treatments. The most common toxicity was a local injection site reactions and fatigue;no autoimmune toxicities were seen.Post treatment patients showed no statistically significant in serum cytokines level (INF y,IL-4,TGFβ1) compared with prevaccination.Immune response showed that Treg cell had a significantly decrease at posttreatment compared with pretreatment(p=0.028). No patients achieved a partial or complete tumour responses. After a median follow-up of 18.6 months, median overall survival(OS) for all patients is 13.2 months,with corresponding estimates of 1-year,2-year,3-year survival of 45.4%,21.8%,7.8%. patients with DTH (+) response (n=43) had a significantly longer progression free survival(PFS) and OS than DTH (-) (n=21)(log-rank tests P=.031 and P=.041, respectively). The estimated median OS was 14.2 months(range,3.5 to 42.0 months) and 10.8 months (range,3 to 36.0 months) for DTH (+) responders and DTH (-) ones, respectively.PartⅡ:Serum polypeptidome showed that 7 differential expression peptide peaks of patients before and after the treatment from a total of 151 protein peaks were obviously different.Most of them were complement C3 and acute phase reactive protein fragment.PartⅢ:Serum proteome showed that 14 protein were significantly highly expressed in the serum of GMV post-treatment patients compared with prevaccination, while 3 protein were lowly expressed. The increased proteins could be divided into 5 categories according to their functions:(1)acute phase reactive protein;(2)immunoglobulin;(3)complement protein; (4)cell damage release protein; (5)others.The decreased expressed proteins could be divided into 3 categories: (1)transport protein;(2)metabolic protein; (3)anchoring protein.Conclusions:These results suggested that this GMV immunotherapy is well tolerated and may have clinical activity in NSCLC patients. Patients with DTH (+) response was associated with prolonged survival. Serum proteome showed that GMV treatment may correlated with inflammatory reaction, immune reaction, cell damage, and these differentially expressed proteins could provide clues for the study and discovery of new protein targets for anticancer drugs.