A specific autoantibody against a novel tumour-association antigen derived from human DNA-topoiomerase I is a potential biomarker for early diagnosis and favourable prognosis in patients with colorectal carcinoma.

Context: We previously reported a novel tumour associated antigen (TTA) with molecular weight around 48 kDa and identified the novel TTA as a fragment derived from human DNA-topoiomerase I (TOP1). We termed the novel TAA as TOPO48 and termed autoantibody against the TAA as anti-TOPO48 autoantibody.Objective: To explore the clinical significance of anti-TOPO48 autoantibody in patients with colorectal carcinoma (CRC).Materials and methods: Serum levels of the autoantibody in patients with CRC or benign tumours and healthy volunteers were measured with a specific ELISA.Results: CRC patients at early stage had higher frequency of positive levels of the autoantibody and CRC patients with positive autoantibody levels had higher overall survival rate than those with negative autoantibody levels.Conclusion: The autoantibody is a potential biomarker for early diagnosis and favourable prognosis of CRC.

A protein fragment derived from DNA-topoisomerase I as a novel tumour-associated antigen for the detection of early stage carcinoma.

BACKGROUND: The production of autoantibodies against tumour-associated antigens (TAAs) is believed to reflect greater immunologic reactivity in cancer patients and enhanced immune surveillance for cancer cells. Over the past few decades, a number of different TAAs and their corresponding autoantibodies have been investigated. However, positive frequency of autoantibody detection in cancer patients has been relatively low. Here we describe a novel TAA that was a fragment derived from human DNA-topoiomerase I and an autoantibody against the novel TAA with relatively high positive frequency in the sera of early-stage non-small-cell lung cancer (NSCLC), gastric cancer (GC), colorectal cancer (CRC) and oesophageal squamous cell carcinoma (ESCC). METHODS: Serologic enzyme-linked immunosorbent assay (ELISA) and western blot were used to discover a novel TAA with a molecular weight of 48 kDa separated by ion exchange chromatography. Autoantibody ELISA, immnohistochemistry and immunofluorescent staining, recombinant protein cloning/expression and western blot were used to identify the novel TAA. The association of the autoantibody against the novel TAA with early-stage carcinoma was explored by screening 203 stage I/II patients and 170 stage III/IV patients with NSCLC, GC, CRC or ESCC. RESULTS: We identified the novel TAA as a fragment derived from human DNA-topoiomerase I (TOP1). We found that the novel TAA induced specific autoantibodies with a high prevalence that ranged from 58 to 72% in some of the most common types of cancer. We observed that the immune response against the novel TAA was associated with early stage ESCC, GC, CRC and NSCLC. CONCLUSIONS: The findings in this study suggest that the autoantibody against the novel TAA may be a potential biomarker for use in the early detection and diagnosis of cancer.

HLA-G expression in human breast cancer: implications for diagnosis and prognosis, and effect on allocytotoxic lymphocyte response after hormone treatment in vitro.

OBJECTIVE: The aim of this study is to investigate clinical implications of human leukocyte antigen-G (HLA-G) expression in breast cancer. METHODS: HLA-G expression in 235 primary breast cancer tissues was investigated using immunohistochemistry, and plasma soluble HLA-G (sHLA-G) was measured in 44 breast cancer patients using a specific HLA-G enzyme-linked immunosorbent assay (ELISA). Effects of estradiol/progesterone and their antagonists tamoxifen/RU486 on HLA-G expression in cultured breast cancer MCF-7 cells were determined by real-time polymerase chain reaction (PCR) and the ELISA. Alterations of HLA-G expression by the hormone treatments on subsequent allocytotoxic lymphocyte (allo-CTL) response were also examined. RESULTS: In the study, approximately 66% of neoplasm lesions were identified to have positive HLA-G expression. This expression was significantly correlated with tumor size, nodal status, and clinical disease stage (P = 0.0001, 0.012, and 0.0001, respectively). Patients with positive HLA-G expression had a lower survival rate than those with negative expression (P < 0.028). Plasma sHLA-G levels were significantly higher in breast cancer patients than in healthy controls (P < 0.001), with the area under the receiver-operating characteristic (ROC) curve being 0.95. HLA-G expression in breast cancer MCF-7 cells was enhanced by estradiol/progesterone but reduced by their antagonists. Cytotoxicity studies showed that allo-CTL response in MCF-7 cells was inhibited by prior treatment with estradiol/progesterone, but was amplified by their antagonists. The effects could be restored or further strengthened by the addition of anti-HLA-G antibodies. CONCLUSION: Our findings suggest that HLA-G may have potential clinical implications in diagnosis, prognosis, and immunotherapy of patients with breast cancer.

Clinical significance of detecting survivin-expressing circulating cancer cells in patients with non-small cell lung cancer.

We previously demonstrated that the detection of circulating cancer cells (CCC) expressing survivin mRNA could provide valuable information for predicting metastasis and recurrence in breast cancer. The objective of this study was to investigate the significance of detecting survivin-expressing CCC on the clinical outcomes of patients with non-small cell lung cancer (NSCLC). Peripheral blood samples collected from 143 NSCLC patients and 177 healthy volunteers were quantitatively evaluated using a technique developed in our laboratory that detected reverse transcription-polymerase chain reaction (RT-PCR) products based on a hybridisation-enzyme linked immunosorbant essay (ELISA), which we called RT-PCR ELISA. The presence of survivin-expressing CCC was detected in 63 cancer patients (44.1%) and was significantly associated with pathological T classification, nodal status, and disease stages (all P<0.001). During a follow-up period of 36 months, patients who had positive survivin expressions at the time of the initial assay test had a higher relapse rate and shorter survival time when compared to those who had negative survivin expressions (all P<0.001). Through multivariate analysis, the detection of survivin-expressing CCC was found to be an independent predictor for cancer recurrence (HR=43.5; 95% CI=2.67-70.9; P=0.008) and survival (HR=1.35; 95% CI=1.02-4.31; P=0.049). Thus, detection of survivin-expressing CCC could be used in the prediction of disease recurrence as well as in the prognosis of NSCLC.

Detection of survivin-expressing circulating cancer cells (CCCs) in peripheral blood of patients with gastric and colorectal cancer reveals high risks of relapse.

BACKGROUND: We previously demonstrated that the detection of circulating cancer cells (CCCs) expressing survivin mRNA could provide valuable information for predicting metastasis and recurrence in breast cancer. The objective of this study was to investigate whether or not the detection of survivin expression in the peripheral blood could also have significant effects on the clinical outcomes of patients with gastric and colorectal cancer. METHODS: Survivin-expressing CCCs in peripheral blood samples obtained from 55 gastric cancer patients, 86 colorectal cancer patients, and 87 healthy volunteers were quantitatively examined by using a RT-PCR ELISA. Its clinical significance was statistically evaluated. RESULTS: The CCCs in the peripheral blood samples were detected in 45.4% and 44.0% of gastric and colorectal cancer patients, respectively. The presence of survivin-expressing CCCs was found to be significantly associated with the degree of tumor penetration, nodal status, and disease stages for both types of cancers. During a follow-up period of 36 months, patients who had a positive detection at the time of the initial assay test had a higher relapse rate than those who had a negative detection. As well, survivin-expressing CCCs were statistically shown to be a significant and independent predictor for cancer recurrence. The detection of survivin-expressing CCCs was also demonstrated to be more accurate in terms of predicting recurrence than traditional detection methods such as plasma carcinoembryonic antigen (CEA) measurements. CONCLUSION: The detection of CCCs expressing survivin mRNA could be used to accurately identify gastric and colorectal cancer patients with high risks of relapse.

Serum anti-TOPO48 autoantibody as a biomarker for early diagnosis and prognosis in patients with esophageal squamous cell carcinoma.

BACKGROUND AND AIM: We previously reported a novel tumor associated antigen (TTA) with molecular weight around 48kDa that is a fragment derived from human DNA-topoiomerase I (TOP1). We termed the novel TAA as TOPO48 and termed autoantibody against the TAA as anti-TOPO48 autoantibody. The aim of this study is to further investigate the clinical applications of the autoantibody in patients with esophageal squamous cell carcinomas (ESCC). METHODS: Serum levels of the anti-TOPO48 autoantibody in 112 ESCC patients, 112 age- and gender-matched healthy controls and 75 patients with esophageal benign tumors were determined by using a specific anti-TOPO48 autoantibody ELISA. Then, we statistically evaluated its clinical significance. RESULTS: We found that serum anti-TOPO48 autoantibody levels in ESCC patients were significantly higher than that in healthy controls and benign tumor patients (P=0.001). The percentage of sera with a positive level of anti-TOPO48 autoantibody in early stages was significantly higher than that in advanced stages of the cancer patients when the maximum level of healthy control sera was taken as a cut-off value (P=0.001). The area under ROC curve was 0.863 (95% CI=0.797-0.928) for healthy controls vs. early stage ESCC. In addition, patients with positive anti-TOPO48 autoantibody had significantly higher survival rate and longer survival time than that with negative anti-TOPO48 autoantibody in cancer patients (P=0.038, 0.025 and 0.047 for all stages, early stage and advanced stage, respectively). CONCLUSIONS: Our results suggest that anti-TOPO48 autoantibody may be a potentially useful biomarker for early diagnosis and prognosis of ESCC.

An Autoantibody Against Human DNA-Topoisomerase I Is a Novel Biomarker for Non-Small Cell Lung Cancer.

BACKGROUND: We previously reported a novel tumor-associated antigen with a molecular weight of approximately 48 kDa that was a fragment derived from human DNA-topoisomerase I. The aim of this study is to further investigate the clinical significance of the autoantibody in patients with non-small cell lung cancer (NSCLC). METHODS: We determined serum levels of the autoantibody in 127 NSCLC patients, 127 age-, sex-, and smoking history-matched healthy control subjects, and 38 patients with pulmonary benign tumors by using a specific enzyme linked immunosorbent assay for the autoantibody. We then statistically evaluated its clinical application value. RESULTS: Serum levels of the autoantibody in NSCLC patients were significantly higher than in healthy control subjects and patients with benign tumors (p = 0.001). The percentage of sera with a positive level of the autoantibody was 71.8%, 65.6%, 41.9%, and 48.0% in stages I, II, III, and IV of the cancer, respectively (p = 0.049). The area under the receiver-operating characteristics curve was 0.971 (95% confidence interval: 0.953 to 0988) for healthy controls and patients with benign tumors versus early stage NSCLC patients. Moreover, the overall survival rate of the patients in stages I, II, and IV with negative levels of the autoantibody was significantly lower than that of patients with positive levels of the autoantibody (p = 0.013, 0.023, and 0.047 for stages I, II, and IV, respectively). CONCLUSIONS: Our results indicate that the autoantibody can be used as a novel biomarker for the early diagnosis and prognosis of NSCLC.

Expression of human leucocyte antigen G (HLA-G) is associated with prognosis in non-small cell lung cancer.

In a previous study, we demonstrated that human leucocyte antigen G (HLA-G) was aberrantly expressed in a majority of primary colorectal carcinomas, and that the detection of HLA-G expression had a strong and independent prognostic value in human colorectal cancer. In the current study, we look into whether the aberrant expression of HLA-G is also related to non-small cell lung cancer (NSCLC). The expression of HLA-G was investigated immunohistochemically in 106 patients with NSCLC. The correlation between HLA-G status and various clinicopathological parameters was analysed. As well, the level of HLA-G expression was also compared to the survival rate of patients with NSCLC. In total, we found that in 75% (79/106) of the primary site of NSCLC, an aberrant HLA-G expression was detected. However, this expression was not observed in the normal lung tissues. HLA-G expression in NSCLC was significantly correlated with lymph nodal metastasis, clinical stages of the disease, and host immune response (P = 0.0001, 0.0001, and 0.027, respectively). Patients with HLA-G positive tumours had a significantly shorter survival time than those with tumours that were HLA-G negative (P = 0.001). In addition, through multivariate analysis, HLA-G exhibited an independent prognostic factor (P = 0.01, relative risk 4.09; 95% confidence interval 1.40-11.9). All in all, our results indicate that the expression of HLA-G is a characteristic feature of NSCLC, and they suggest that immunostaining by anti-HLA-G antibodies may be a potentially useful prognostic indicator.

Expression of HLA-G is associated with prognosis in esophageal squamous cell carcinoma.

The detection of HLA-G expression might serve as a clinical marker in the diagnosis or prediction of clinical outcomes for certain types of carcinoma. The aim of this study was to determine whether the detection of HLA-G has any important clinical applications for patients with esophageal squamous cell carcinoma (ESCC) by using immunohistochemical methods. We observed that the HLA-G protein was expressed in 90.9% (110/121) of the primary sites of ESCC but not in the normal esophageal tissues. The expression of HLA-G in the tumors was significantly correlated with histologic grade, depth of invasion, nodal status, host immune response, and clinical stage of disease. Patients with positive HLA-G expression had a significantly worse prognosis. In multivariate analysis, HLA-G was an independent prognostic factor. Our results indicate that expression of HLA-G is a characteristic feature of ESCC and suggest that immunostaining by anti-HLA-G antibodies may be a potentially useful prognostic indicator.

Detection of Survivin-expressing circulating cancer cells in the peripheral blood of breast cancer patients by a RT-PCR ELISA.

Survivin mRNA expression was detected in 69.2%-93.8% of primary breast carcinomas, but is rarely expressed in normal breast tissues and hematopoietic cells. The objective of this study was to investigate the significance that the detection of Survinin-expressing circulating breast cancer cells in the peripheral blood has on clinical outcomes. The detection method was based on a RT-PCR ELISA technique developed in our laboratory. Sixty-seven breast cancer patients in various stages and 135 normal healthy women were investigated. Survivin-expressing circulating cancer cells were detected in the peripheral blood samples from 34 (50.7%) out of 67 breast cancer patients, but not in the healthy women that were used as controls. The presence of Survivin-expressing circulating breast cancer cells was found to be significantly associated with various clinicopathological parameters such as vessel infiltration, histological grade, tumor size, nodal status, ER/PgR status, Her-2 status and clinical stages of the disease (P < 0.01). During a follow-up period of 36 months, 9 out of 11 (81.8%) breast cancer patients that had a positive Survivin-expressing at the time of the initial assay test suffered a relapse of the disease, whereas recurrence was only found in 2 out of 6 (33.3%) breast cancer patients that had a negative Survivin-expression. Thus, the detection of circulating cancer cells expressing Survivin mRNA could provide valuable information for the prediction of metastasis and recurrence of breast cancer.

Using a sequence of estrogen response elements as a DNA aptamer for estrogen receptors in vitro.

Estrogen receptors (ERs) are overexpressed in approximately 70% of breast cancer cases, and they play an important role in tumorigenesis. ERs are strong predictive factors for measuring responses to hormonal therapies. Aptamers are short and single stranded oligonucleotides that are able to recognize target molecules with high affinity. In the present study, we selected and synthesized an oligonucleotide, which has a similar sequence to estrogen response element in the Xenopus Vitellogenin A2 gene. The synthesized oligonucleotide was evaluated by using immunostaining of paraffin-embedded breast cancer tissues and treating MCF-7 human mammary carcinoma cell line in vitro. We found that the synthesized oligonucleotide had a high binding affinity to ER similar to estradiol. Using a specific anti-ER antibody as a standard control, we showed that the synthesized oligonucleotide specifically recognized and immunostained tumor cells of breast cancer without cross-reaction with normal tissues. The overall agreement of ER detection between the anti-ER antibody and the ER aptamer was 97.1% (kappa value=0.943; 95% CI=0.879-1.006; p<0.002). Similar to tamoxifen or fulvestrant, the oligonucleotide also had an inhibitory effect on cell proliferation of MCF-7 cell line in a dose- and time-dependent fashion but had no cytotoxic effect on human normal mammary epithelial cells. Therefore, the synthesized oligonucleotide may be used as an aptamer for immunostaining of paraffin-embedded tissue sections for breast cancer diagnosis, as well as a potential ER antagonist in the treatment of breast cancer.

Detection of survivin-expressing circulating cancer cells in the peripheral blood of patients with esophageal squamous cell carcinoma and its clinical significance.

We previously demonstrated that the detection of circulating cancer cells (CCC) expressing survivin mRNA could provide valuable information for predicting recurrence in patients with breast, lung, gastric and colorectal carcinoma. The purpose of this study is to investigate whether the detection of survivin-expressing CCC in the peripheral blood is also useful for predicting recurrence in patients with esophageal squamous cell carcinoma (ESCC). Blood samples obtained from 108 ESCC patients and 75 healthy volunteers were quantitatively investigated by a technique that detected reverse transcription-polymerase chain reaction products based on a hybridization-enzyme linked immunosorbent essay. Not all of the patients were available for the follow-up study. Only 48 patients who were treated with similar adjuvant therapy regimens were available and followed-up for 33 months after the initial assay test. Survivin-expressing CCC were detected in 51 (47.2%) patients. The presence of survivin-expressing CCC was found to be significantly associated with depth of invasion, vascular invasion, nodal status, and disease stages (P = 0.032, 0.019, 0.018, and 0.001, respectively). During the follow-up period, patients who had positive survivin expressions had a higher relapse rate and a shorter survival time than those who had negative survivin expressions (P = 0.002 and 0.016, respectively). Examination of survivin-expressing CCC could provide valuable information in the prediction of haematogenous recurrence as well as in the prognosis of ESCC.

Expression of human leukocyte antigen G (HLA-G) correlates with poor prognosis in gastric carcinoma.

OBJECTIVE: We had previously demonstrated that human leukocyte antigen G (HLA-G) was expressed in a majority of primary colorectal carcinomas and that the detection of HLA-G expression had a strong and independent prognostic value for that cancer. Currently, we investigate whether or not HLA-G is also expressed in patients with gastric carcinoma and whether the expression has any clinical application value. METHODS: The expression of HLA-G was investigated immunohistochemically in 160 patients with gastric carcinoma. The correlation between HLA-G status and various clinicopathological parameters was analyzed with the levels of HLA-G expression used to compare the survival length amongst patients. RESULTS: HLA-G protein expression was observed in 71% (113 of 160) of the primary site of gastric carcinomas, but not in the normal stomach tissues. HLA-G expression in the tumors was significantly correlated with the tumor location, histological grade, depth of invasion, lymph nodal metastasis, clinical stages of the disease, and host immune response (P = .012, .008, .001, .038, .030, and .016, respectively). Patients with HLA-G positive tumors had a significantly shorter survival time than those patients with tumors that were HLA-G negative (P = .001). As well, in multivariate analysis, HLA-G demonstrated an independent prognostic factor (P = .0001, relative risk 9.08; 95% confidence interval, 3.44-24.0). CONCLUSIONS: Overall, our results indicated that the expression of HLA-G is a characteristic feature of gastric carcinoma and that immunostaining by anti-HLA-G antibody may be a potentially useful prognostic indicator.

Human leukocyte antigen G expression: as a significant prognostic indicator for patients with colorectal cancer.

Aberrant expression of human leukocyte antigen G (HLA-G) has been proposed to be involved in tumor escape mechanisms. It has been also proposed that detection of HLA-G might service as a potential biomarker for diagnosis or prediction of the clinical outcomes in ovarian and breast cancers, carcinoma of the lung and endometrial cancer. The aim of this current study is to determine if HLA-G is expressed in colorectal carcinomas and if the expression is associated with clinicopathological and prognostic data. The expression of HLA-G was investigated immunohistochemically in 201 patients with colorectal carcinomas. The correlation between HLA-G status, clinicopathological factors and the overall survival rate was analyzed. In this prospectively study, HLA-G protein expression was observed in 64.6% (130/201) of the primary site colorectal carcinomas, but not in the normal colorectal tissues or benign adenomas. HLA-G expression in the tumors was significantly correlated with the depth of invasion, histological grade, host immune response, lymph nodal metastasis and clinical stages of the disease (P=0.001, 0.0001, 0.002, 0.001 and 0.031, respectively). Patients with HLA-G positive tumors had a significantly shorter survival time than those patients with tumors that were HLA-G negative (P=0.0001). As well, in multivariate analysis, HLA-G demonstrated an independent prognostic factor (P=0.021, relative risk 3.14; 95% confidence interval, 1.34-8.10). Therefore, it can be gathered that HLA-G might serve as an independent prognostic factor for colorectal cancer patients.