We usually observe that the prognosis of children with B-ALL with the same risk at diagnosis is significantly different in clinical practice. The prognosis of B-ALL is not only related to various factors, such as age, sex, and molecular genetic characteristics,but also closely associated with host immune function.Reports have demonstrated that the autoantibodies produce in the transition to precancer20. Different from other markers, autoantibodies emerged in the early process of oncogenesis, and they are shown in serum before TAAs can be detected21;thus the examination of tumor autoantibodies using minimally invasive methods, has huge potentiality in early diagnosis, especially for asymptomatic patients.This can facilitate early detection and treatment, thereby protecting patients from early death.
Growing evidence has confirmed that there is a specific immune response in tumor patients,including patients with prostate cancer,hepatocellular carcinoma, breast cancer,lung cancer,pancreatic cancer and so on22.In the earliest stage of leukemia, the number of leukemic cells in the peripheral blood is very small, and the patients have almost no clinical symptoms.It is difficult to detect leukemia-associated antigens in peripheral blood due to their low expression, but pathological changes could be detected by the immune system from the earliest stages.Immune system responds to them quickly by producing a large number of specific autoantibodies against antigens. The discovery of novel B-ALL antigens not only helps to explain the molecular mechanism underlying the occurrence and development of B-ALL in children but also can be used as a target for immunotherapy of B-ALL, and the corresponding autoantibodies can be useful as serum biomarkers for early diagnosis, disease monitoring and assessment of B-ALL prognosis.
SERPA has been widely applied as a hopeful means for screening and identifying all components of immunoreactive proteins on the basis of 2-DE,immunoblotting, and MS23. Autoantigens were confirmed by SERPA in a variety of illnesses, containing gastric cancer24,colorectal cancer9,lung cancer11,gallbladdercarcinoma12, prostate cancer13 and primary open angle glaucoma15 in the past decade.In this study, mixed proteins from three B-ALL cell lines were dissolved by 2-DE, followed by Western blot analysis using mixed serum samples from children with B-ALL and healthy controls. As a result, 6 protein spots that were significantly and specifically recognized by serum from B-ALL patients were identified.In our SERPA analysis, aconitase,AIF, DLD,α-enolase, MCAD, and VDAC1 showed differential immunological reactions between children with B-ALL and healthy controls. To the best of our knowledge, we first showed that six proteins, aconitase,AIF,DLD,α-enolase, MCAD, and VDAC1, could induce the production of autoantibodies in B-ALL children. Based on literature searching of the identified proteins in patients with B-ALL or other cancers, we found that among these six proteins, AIF25,DLD26, and α-enolase27,28 can induce autoantibody production in other tumor patients, but there is no report about the autoantibody production induced by aconitase, VDAC1 and MCAD. Among the immunogenic proteins, α-enolase and VDAC1 (spot nos. 4 and 6) attracted our attention.In light of the above, we selected α-enolase and VDAC1 for clinical verification and analyzed the autoantibody levels in children with B-ALL by ELISA.We then verified the production of autoantibodies against α-enolase and VDAC1 in 30 children with B-ALL and 25 normal controls. In the validation stage, we proved that α-enolase and VDAC1 autoantibodies were new representative targets for distinguishing patients with B-ALL from normal controls. To the best of our knowledge,this study was the first report of the existence of autoantibodies against α-enolase and VDAC1 in serum from children with B-ALL by a proteomic approach,nevertheless, the α-enolase autoantibody was not all specific to B-ALL serum and were found in the serum of patients with other tumors,as well as α-enolase antibodies were found in some autoimmune diseases29–31.The mechanism which α-enolase and VDAC1 induce immune response in B-ALL is unclear.
One of these proteins, α-enolase,found on the surface of cells,is involved in several key biological processes of cancer, including proliferation, migration and invasion32,33.According to previous reports,α-enolase is overexpressed in multiple tumors, including lung cancer16,hepatocellular carcinoma34,pancreatic cancer19,and gastric cancer patients35, and has been proposed as a biomarker for early detection and prognosis36.Several reports have mentioned α-enolase as a tumor-associated antigen that induces autoantibody production in malignant tumors27,28.Autoantibodies against α-enolase have also been previously reported in acute leukemia37,chronic myeloid leukemia38, non-Hodgkin’s lymphoma39 and other solid tumors36.A recent study demonstrated that α-enolase and Annexin A1 autoantibodies could enhance diagnostic performance in lung cancers by combining CEA and CA12528.Another study suggested that α-enolase could accelerate metastasis of lung cancer cell through HGFR and WNTsignaling pathway and presented a new antibody targeting α-enolase in lung cancer33. In multiple myeloma, α-enolase expression negatively correlated with overall survival,and targeting α-enolase can enhance immunity and improve outcome40.Cui et al. found that positivity of α-enolase autoantibodies was observed in serum from 18 of 21 (86%) patients with acute leukemia(AL) and 20 of 22(90%) healthy controls37.However,we found that α-enolase autoantibodies were detected in serum from 8 of 30 (27%) children with ALL and 1 of 25(4%) healthy controls.The reasons for the differences described above may be related to subjects in the different age and leukemia groups.
Similarly, we also selected another protein, VDAC1, for verification. We demonstrated that positivity for autoantibodies against VDAC1 was observed in sera from 7 of 30 (23%) children with ALL, but no such activity was observed in 25 (0%) normal children. VDAC1,which mainly located in the outer membrane of mitochondria,can control cell growth,energy production and Ca2+ homeostasis41,42. Moreover, VDAC1 also regulates apoptosis by mediating the release of apoptotic proteins from mitochondria and interacting with antiapoptotic proteins17,41.The observation that VDAC1 is overexpressed in a variety of tumors shows that VDAC1 may be essential for cancer cell survival18,41,43.Accumulating studies have proved that abrogation of VDAC1 expression significantly inhibits tumor growth in cancers18,42−46,suggesting that VDAC1 may be a novel therapeutic target47–50. Furthermore, targeted drugs acting on VDAC1 against tumor growth and proliferation is a promising strategy for the treatment of cancer51.However, as far as I know, VDAC1 has never previously been reported as an autoantigen and may become a new target antigen. Antibodies against VDAC1 can also be found in the serum of B-ALL children, indicating that VDAC1 triggers autoimmunity,and leads to elevation of VDAC1 autoantibody. However, the exact mechanisms require further study.Our results proved that VDAC1 may be a promising antigen which could be immunogenic and autoantibodies against VDAC1 might serve as a potential biomarker for B-ALL.
Aconitase, one of the other four proteins, was reported that mitochondrial aconitase activity played an important role in the development of advanced metastatic prostate cancer52.AIF overexpression was found in a variety of malignant tumors,including uveal melanoma53, pancreatic cancer54,chronic lymphocytic leukemia55, and diffuse large B-cell lymphoma56. Otherwise,downregulation of AIF was reported in testicular germ cell tumors57 and renal cell carcinoma58. The relationship between AIF expression and disease prognosis has been reported in different kinds of tumors53,55−57.In addition,Li et al found that there were autoantibodies against AIF in the serum of hepatocellular carcinoma using SERPA25.A variety of evidences have shown that DLD is a mitochondrial enzyme which produces ROS and may be used an anticancer drug59. Yoneyama K et al found that DLD autoantibody may be a possible diagnostic marker for ovarian and endometrial cancer26,60.The last protein,MCAD was upregulated in glioblastoma compared to normal brain and could protect mitochondria from lipid peroxidation61.