Jitian Li

Preferential Autoimmune Response in Prostate Cancer to Cyclin B1 in a Panel of Tumor-Associated Antigens

Previous studies have demonstrated that sera from patients with prostate cancer (PCa) contain autoantibodies that react with tumor-associated antigens (TAAs). Autoantibodies to cyclin B1 and fourteen other TAAs were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting in 464 sera from patients with PCa, benign prostatic hyperplasia (BPH), and other controls. Autoantibodies to cyclin B1 were detected in 31.0% of sera from randomly selected patients with PCa versus 4.8% in sera with BPH. In the further analysis, 31.4% of sera from PCa patients at the early stage contained anti-cyclin B1 autoantibody, and even 29.4% of patients who had normal prostate-specific antigen (PSA) levels in their serum samples were observed anti-cyclin B1 positive. The cumulative positive rate of autoantibodies against seven selected TAAs (cyclin B1, survivin, p53, DFS70/LEDGFp75, RalA, MDM2, and NPM1) in PCa reached 80.5%, significantly higher than that in normal control sera. In summary, autoantibody to cyclin B1 might be a potential biomarker for the immunodiagnosis of early stage PCa, especially useful in patients with normal PSA level. This study further supports the hypothesis that a customized TAA array can be used for enhancing anti-TAA autoantibody detection, and it may constitute a promising and powerful tool for immunodiagnosis of PCa.

Autoantibodies against tumor-associated antigens in the early detection of lung cancer

OBJECTIVES Autoantibodies against tumor-associated antigens (TAAs) identified in patients with advanced lung cancer may be detected in subjects with early lung cancer or even predate the diagnosis. The purpose of this study is to address the temporal relationship between lung cancer development and serum autoantibody response. MATERIALS AND METHODS Two cohorts of patients with newly diagnosed lung cancer were included. The first cohort included 90 sera from patients with lung cancer (Stages I-III) and 89 normal control sera. In the second cohort, 93 serial serum samples from 25 patients with CT-scan screen-detected stage I lung cancer were collected before the diagnosis of lung cancer (average 32 months) and 56 controls were matched on age, gender, and smoking. Autoantibody levels were measured by immunoassay. RESULTS Measurement of autoantibodies against seven TAAs (14-3-3ζ, c-Myc, MDM2, NPM1, p16, p53 and cyclin B1) individually could discriminate lung cancer patients from normal individuals in the first cohort and the area under curve (AUC) was 0.863 based on a panel of seven autoantibodies, with sensitivity of 68.9% and specificity of 79.5%. Autoantibodies in serial pre-diagnostic serum samples against the same panel of seven TAAs were detected prior to lung cancer diagnosis with sensitivity of 76.0% and specificity of 73.2% (AUC) (95%CI): 0.885 (0.797-0.973)). Elevated autoantibody levels could be detected greater than four years prior to lung cancer diagnosis. CONCLUSION A panel of seven TAAs may enhance the early detection of lung cancer, consistent with a humoral immune response to TAAs that can be detected months to years prior to the diagnosis.

Using Serological Proteome Analysis to Identify Serum Anti-Nucleophosmin 1 Autoantibody as a Potential Biomarker in European-American and African-American Patients With Prostate Cancer.

The prostate‐specific antigen (PSA) testing has been widely implemented for the early detection and management of prostate cancer (PCa). However, the lack of specificity has led to overdiagnosis, resulting in many possibly unnecessary biopsies and overtreatment. Therefore, novel serological biomarkers with high sensitivity and specificity are of vital importance needed to complement PSA testing in the early diagnosis and effective management of PCa. This is particularly critical in the context of PCa health disparities, where early detection and management could help reduce the disproportionately high PCa mortality observed in African‐American men. Previous studies have demonstrated that sera from patients with PCa contain autoantibodies that react with tumor‐associated antigens (TAAs).

Humoral autoimmune response to nucleophosmin in the immunodiagnosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a worldwide prevalent cancer with an exremely poor prognosis. Detection of serum α-fetoprotein (AFP) and liver imaging techniques are the conventional methods used clinically for the identification of this malignancy. However, these techniques are not reliable for early diagnosis, and particularly the sensitivity and specificity of AFP in HCC diagnosis are not optimal. Therefore, there is an urgent need for the development of more sensitive and specific methods that can improve AFP quantification in the early detection of HCC. In the present study, autoantibody responses to nucleophosmin (NPM1) in HCC patients were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting and indirect immunofluorescence. Immunohistochemistry (IHC) with tissue array slides was also performed to analyze protein expression of NPM1 in HCC and control tissues. The prevalence of autoantibodies against NPM1 was 22.4% (17/76) in HCC, which was significantly higher than that in sera from patients with liver cirrhosis (LC), chronic hepatitis (CH) and systemic lupus erythematosus (SLE) (P<0.01). The average titer of autoantibodies against NPM1 in HCC sera was higher compared to that in LC, CH, SLE and normal human sera (NHS) (P<0.01). In addition, anti-NMP1 autoantibodies were detected in sera from several HCC patients with serial bleeding samples. A stronger reactive band corresponding to NMP1 was visualized in the western blot analyses, utilizing sera from patients 3–6 months before the clinical diagnosis of HCC. Our data indicate that NPM1 and the anti-NPM1 system may have potential as an early-stage biomarker for HCC screening and diagnosis.

Immunoseroproteomic Profiling in African American Men with Prostate Cancer: Evidence for an Autoantibody Response to Glycolysis and Plasminogen-Associated Proteins

African American (AA) men suffer from a disproportionately high incidence and mortality of prostate cancer (PCa) compared with other racial/ethnic groups. Despite these disparities, African American men are underrepresented in clinical trials and in studies on PCa biology and biomarker discovery. We used immunoseroproteomics to profile antitumor autoantibody responses in AA and European American (EA) men with PCa, and explored differences in these responses. This minimally invasive approach detects autoantibodies to tumor-associated antigens that could serve as clinical biomarkers and immunotherapeutic agents. Sera from AA and EA men with PCa were probed by immunoblotting against PC3 cell proteins, with AA sera showing stronger immunoreactivity. Mass spectrometry analysis of immunoreactive protein spots revealed that several AA sera contained autoantibodies to a number of proteins associated with both the glycolysis and plasminogen pathways, particularly to alpha-enolase (ENO1). The proteomic data is deposited in ProteomeXchange with identifier PXD003968. Analysis of sera from 340 racially diverse men by enzyme-linked immunosorbent assays (ELISA) showed higher frequency of anti-ENO1 autoantibodies in PCa sera compared with control sera. We observed differences between AA-PCa and EA-PCa patients in their immunoreactivity against ENO1. Although EA-PCa sera reacted with higher frequency against purified ENO1 in ELISA and recognized by immunoblotting the endogenous cellular ENO1 across a panel of prostate cell lines, AA-PCa sera reacted weakly against this protein by ELISA but recognized it by immunoblotting preferentially in metastatic cell lines. These race-related differences in immunoreactivity to ENO1 could not be accounted by differential autoantibody recognition of phosphoepitopes within this antigen. Proteomic analysis revealed differences in the posttranslational modification profiles of ENO1 variants differentially recognized by AA-PCa and EA-PCa sera. These intriguing results suggest the possibility of race-related differences in the antitumor autoantibody response in PCa, and have implications for defining novel biological determinants of PCa health disparities.

Evaluation of serum autoantibodies against tumor-associated antigens as biomarkers in lung cancer:

We evaluated autoantibodies against nine tumor-associated antigens, including p62, p16, Koc, p53, Cyclin B1, Cyclin E, Survivin, HCC1, and RalA as serological markers in lung cancer. Enzyme-linked immunosorbent assay (ELISA) was used to detect autoantibodies in sera from 50 lung cancer patients and 42 normal controls. Then, four tumor-associated antigens of higher values were selected and validated in sera from validation group. Western blot and serum absorption test were used to confirm positive findings from ELISA. When cutoff values were set as mean optical density values plus 3 standard deviation of normal controls, the positive rate of autoantibodies against four tumor-associated antigens (Survivin, Cyclin B1, HCC1, and p53) reached 32%, 20%, 22%, and 18%, with area under the curve values of 0.653, 0.767, 0.622, and 0.623 in sera from 50 lung cancer, respectively (all p < 0.05). Results from the validation group confirmed the results. When lung cancer patients were divided by their clinicopathological characteristics into different subgroups, we have found that serum anti-Cyclin B1 and anti-HCC1 autoantibodies increased in stages 1, 2, and 3 lung cancer; anti-Survivin autoantibodies increased in stages 2 and 3 lung cancer; and anti-p53 autoantibody only increased in stage 1 when compared with their corresponding levels in controls (all p < 0.05). Serum anti-Cyclin B1 and anti-Survivin autoantibodies increased with disease histological grade 2 and 3 (both p < 0.05). And higher serum level of anti-p53 autoantibodies is positively associated with tumor size. Parallel utilization of these four anti-tumor-associated antigens (any positive) can increase sensitivity to 65.0% at 100% specificity with area under the curve of 0.908 (p < 0.001) in lung cancer detection in validation group. Our results suggest that autoantibodies against these four tumor-associated antigens have higher values in lung cancer detection, and serum anti-Cyclin-B1 has the potential to serve as novel non-invasive biomarkers in early-stage lung cancer.

Evaluation and characterization of anti-RalA autoantibody as a potential serum biomarker in human prostate cancer

Autoantibodies against intracellular tumor-associated antigens (TAAs) are commonly found in human cancers. In this study, we characterized the serum autoantibody response to the RalA, Ras-like GTPase, in patients with prostate cancer (PCa). The autoantibodies were detected by immunofluorescence assay in PCa cell lines, ELISA, and immunoblotting in 339 serum samples from patients with PCa and benign prostatic hyperplasia (BPH), and in normal human sera (NHS). The expression of RalA in prostate tumor tissues was evaluated by immunohistochemistry (IHC) in tumor microarrays. The autoantibody level to RalA (median) in NHS was significantly lower than in PCa (0.053 vs 0.138; P < 0.001) and BPH (0.053 vs 0.132; P < 0.005) groups. The circulating anti-RalA autoantibody could distinguish PCa patients from normal individuals with the area under the receiver operating characteristic (ROC) curve (AUC) performing at 0.861, with sensitivity of 52.9% and specificity of 91.0%. Elevation in serum immunoreactivity was observed in PCa patients after radical prostatectomy. The combined use of both anti-RalA autoantibody and PSA showed a significantly higher discriminatory ability compared with either of those markers alone. RalA protein expression was detected by IHC in 85.3% of tumor tissues from PCa patients, but without significant difference compared to BPH or normal control tissues. Together, our study shows the additional benefits of anti-RalA autoantibody as a potential serological biomarker for PCa, particularly in patients with normal PSA, and further demonstrate the utility of biomarker combinations in the immunodiagnosis of PCa.

Identification of Osteosarcoma-Related Specific Proteins in Serum Samples Using Surface-Enhanced Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry

Osteosarcoma (OS) is the most common malignant bone tumor. To identify OS-related specific proteins for early diagnosis of OS, a novel approach, surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF-MS) to serum samples from 25 OS patients, 16 osteochondroma, and 26 age-matched normal human volunteers as controls, was performed. Two proteins showed a significantly different expression in OS serum samples from control groups. Proteomic profiles and external leave-one-out cross-validation analysis showed that the correct rate of allocation, the sensitivity, and the specificity of diagnosis were 100%. These two proteins were further identified by searching the EPO-KB database, and one of the proteins identified as Serine rich region profile is involved in various cellular signaling cascades and tumor genesis. The presence of these two proteins in OS patients but absence from premalignant and normal human controls implied that they can be potential biomarkers for early diagnosis of OS.

Serological proteome analysis approach-based identification of ENO1 as a tumor-associated antigen and its autoantibody could enhance the sensitivity of CEA and CYFRA 21-1 in the detection of non-small cell lung cancer

Purpose Lung cancer (LC) is the leading cause of cancer-related deaths for both male and female worldwide. Early detection of LC could improve five-year survival rate up to 48.8% compared to 3.3% of late/distant stage. Autoantibodies to tumor-associated antigens (TAAs) have been described as being present before clinical symptoms in lung and other cancers. We aimed to identify more TAAs to improve the performance for discovering non-small cell lung cancer (NSCLC) patients from healthy individuals. Methods Two independent sets were included in this study. Serological proteome analysis (SERPA) was used to identify TAAs from NSCLC cell line H1299 in a discovery set. In validation study, anti-ENO1 autoantibody was examined by immunoassay in sera from 242 patients with NSCLC and 270 normal individuals. Results A 47 KDa protein was identified to be alpha-enolase (ENO1) by using SERPA. Analysis of sera from 512 participants by ELISA showed significantly higher frequency of anti-ENO1 autoantibodies in NSCLC sera compared with the sera from normal individuals, with AUC (95%CI) of 0.589 (0.539-0.638, P=0.001). There was no significant difference in frequency of anti-ENO1 in different stages, histological or metastasis status of NSCLC. When anti-ENO1 detection was combined with other two tumor protein biomarkers (CEA and CYFRA 21-1), the sensitivity of NSCLC increased to 84%. Conclusions ENO1 can elicit humoral immune response in NSCLC and its autoantibody has association with the tumorigenesis of NSCLC. Furthermore, these intriguing results suggest the possibility of autoantibody against ENO1 serving as a potential diagnostic biomarker in NSCLC and have implications for defining novel histological determinants of NSCLC.

Identification of autoantibodies to ECH1 and HNRNPA2B1 as potential biomarkers in the early detection of lung cancer

ABSTRACT Identification of biomarkers for early detection of lung cancer (LC) is important, in turn leading to more effective treatment and reduction of mortality. Serological proteome analysis (SERPA) was used to identify proteins around 34 kD as ECH1 and HNRNPA2B1, which had been recognized by serum autoantibody from 25 LC patients. In the validation study, including 90 sera from LC patients and 89 sera from normal individuals, autoantibody to ECH1 achieved an area under the curve (AUC) of 0.799 with sensitivity of 62.2% and specificity of 95.5% in discriminating LC from normal individuals, and showed negative correlation with tumor size (rs = −0.256, p = 0.023). Autoantibody to HNRNPA2B1 performed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%, and showed negative correlation with lymph node metastasis (rs = −0.279, p = 0.012). By using longitudinal preclinical samples, autoantibody to ECH1 showed an AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3% in distinguishing early stage LC from matched normal controls, and elevated autoantibody levels could be detected greater than 2 y before LC diagnosis. ECH1 and HNRNPA2B1 are autoantigens that elicit autoimmune responses in LC and their autoantibody can be the potential biomarkers for the early detection of LC.