Xue-Yuan Dong

Large Scale Identification of Human Hepatocellular Carcinoma-Associated Antigens by Autoantibodies

Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.

Identification of genes differentially expressed in human hepatocellular carcinoma by a modified suppression subtractive hybridization method

To identify differentially expressed genes in human HCC in China, we applied a modified SSH method for cDNA subtraction. Such modification has made the method more effective for subtraction. We have obtained 36 and 24 differentially expressed cDNA fragments after modified SSH from 4 paired samples of human HCC and non‐HCC tissues, respectively. Reverse Northern blotting analysis was performed to further identify the genes differentially expressed in the HCC and non‐HCC tissue samples. There were 25 genes really overexpressed in HCC, and their corresponding encoding molecules may reflect the events of cell accelerated metabolism, proliferation, angiogenesis, anti‐apoptosis, tumorigenesis (TLH107, TFH9) and the potential for metastasis. Of the 25 genes overexpressed in HCC, 5 were novel and their full‐length cDNAs were cloned. These 5 novel genes are functionally associated with the occurrence and development of HCC according to the Database analysis. In the paired non‐HCC tissues, there were 15 genes lowly or not expressed in HCC, and their encoding proteins function as tumor suppressors (TFA3, TFA11), acute‐phase reactive proteins, and the blood plasma proteins that are mainly or exclusively synthesized in the liver. The distinct profiles of the differentially expressed genes in HCC and the paired non‐HCC tissues have partially reflected the genetic alterations during HCC tumorigenesis. The novel HCC‐specific gene TLH6 and the CT antigen encoding gene TLH107 may have diagnostic and therapeutic potentials in HCC and/or other solid cancers.

PLAC1 is a tumor-specific antigen capable of eliciting spontaneous antibody responses in human cancer patients

Immunoselection and tumor evasion constitutes one of the major obstacles in cancer immunotherapy. A potential solution to this problem is the development of polyvalent vaccines, and the identification of more tumor‐specific antigens is a prerequisite for the development of cancer vaccines. To identify novel tumor‐specific antigens, suppression subtractive hybridization (SSH) was performed to isolate genes differentially expressed in human hepatocellular cancer (HCC) tissues. PLAC1 (PLACenta‐specific 1) was one of the genes identified highly expressed in HCC tissues but not in paired noncancerous tissues. Further analyses revealed its expression in several other types of cancer tissues as well as tumor cell lines, but not in normal tissues except for placenta. Among HCC samples tested, 32% (22/69) showed PLAC1 mRNA expression while the protein was detected in 23.3% (7/30). A serological survey revealed that 3.8% (4/101) of HCC patients had anti‐PLAC1 antibody response, suggesting the immunogenicity of PLAC1 in HCC patients. PLAC1 represents a new class of tumor associated antigen with restricted expression in placenta and cancer tissues, that may serve as a target for cancer vaccination.

Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients

FATE and TPTE genes were originally reported to be specifically expressed in the adult testis. We searched for the databases of Unigene and serial analysis of gene expression (SAGE) implying that these two gene transcripts might also be expressed in tumours. Herein, we demonstrated that FATE and TPTE mRNA transcripts were expressed in different histological types of tumours and normal testis. Both are cancer-testis (CT) antigens and renamed as FATE/BJ-HCC-2 and TPTE/BJ-HCC-5, respectively. Comparison at nucleotide sequence, the FATE/BJ-HCC-2 cDNA, was identical to that of FATE, whereas the TPTE/BJ-HCC-5 was found to have two isoforms in both cancers and testis: one was identical in cDNA sequence to TPTE, encoding a protein of 551 amino acids, and the other variant lacked an exon of 54 bp, encoding a protein of 533 amino acids. The mRNA expression was analysed by RT–PCR and real-time PCR. FATE/BJ-HCC-2 mRNA was detected in 66% (41 out of 62) in hepatocellular carcinoma (HCC) samples and 21% (three out of 14) in colon cancer samples, whereas the TPTE/BJ-HCC-5 mRNA was detected in 39% (24 out of 62) and 36% (five out of 14) in HCC and non-small lung cancer samples, respectively. The recombinant proteins were prepared and the reactivity of allogenic sera to these two antigens was screened. The frequency of antibody response against FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 proteins was 7.3% (three out of 41) and 25.0% (six out of 24), respectively, in HCC patients bearing respective gene transcripts. Therefore, FATE/BJ-HCC-2 and TPTE/BJ-HCC-5 are the novel CT antigens capable of eliciting antibody response in cancer patients.

Zinc-finger protein ZNF165 is a novel cancer-testis antigen capable of eliciting antibody response in hepatocellular carcinoma patients.

ZNF165 is a zinc-finger protein gene that was identified from human adult testis. Analysis of the origins of publicly available expressed sequence tags as presented in Unigene and SAGE databases revealed that ZNF165 mRNA was expressed in tumours of different tissues. RT–PCR, real-time PCR and Northern blotting analysis confirmed that ZNF165 mRNA was expressed in the hepatocellular carcinoma, gastric cancer, colon cancer and non-small-cell lung carcinoma. The nucleotide sequence of ZNF165 expressed in tumours is identical to that expressed in the testis. Humoral responses of hepatocellular carcinoma (HCC) patients against ZNF165 protein were determined by Western blotting using the recombinant ZNF165 protein. Antibodies against ZNF165 protein were detected in approximately 5% (four of 82) of the sera from HCC patients. These results suggest that ZNF165, a member of the ZNF family, is a novel CT antigen capable of eliciting humoral immune response and be involved in tumour biology.

Detection of antibodies against SARS-CoV in serum from SARS-infected donors with ELISA and Western blot.

Abstract Recombinant fragments of S proteins from the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARA-CoV) were generated and used in a Western blot (WB) assay that was compared to a commercial SARS ELISA method. In 85% of confirmed SARS cases (n = 20), the S2 recombinant fragment based WB was positive and this was comparable to the commercial ELISA using heat killed SARS-CoV. WB using the other four recombinant fragments in confirmed SARS cases generated lower rates of detection (S1—75%, S1-N—25%, S1-C—55%). Evaluation of sera from healthy controls (n = 60) resulted in two weakly positive ELISA results with the remainder being negative while the S2 protein WB demonstrated three positive results from the 20 controls with a history of SARS contact and no positive results in 40 noncontact controls. A discrepancy between the ELISA and S2 WB arose when evaluating per-2003 sera from individuals (n = 10) with SARS-like symptoms (ELISA—100% positive, S2 WB—30% positive). These data suggest that the S2 WB assay may be particularly useful in ELISA-negative SARS cases and in some ELISA-positive non-SARS cases.