Serum anti-p53 antibodies in gastric adenocarcinoma patients are associated with poor prognosis, lymph node metastasis and poorly differentiated nuclear grade.

Mutation of the p53 tumour suppressor gene often leads to the accumulation of mutant p53 protein in tumour cells. Many cancer patients develop antibodies that recognize the overexpressed p53 protein. The presence of these antibodies is, in some tumour types, associated with poor prognosis. Gastric cancer is a highly prevalent disease associated with a high rate of mortality, there is a need for improved clinical and biological markers for disease behaviour. To investigate the clinical relevance of serum anti-p53 antibodies in patients with gastric adenocarcinoma, we have examined the sera of 501 gastric cancer patients for the presence of serum antibodies against the p53 protein. By immunoblotting analysis using a cell lysate containing overexpressed p53 protein as well as affinity-purified recombinant p53 protein as antigens, we have detected anti-p53 antibodies in 11.2% (61 of 501) of gastric cancer patients, but in none of 46 cancer-free individuals. The presence of anti-p53 antibodies was tightly associated with tumours of higher nuclear grade and lymph node metastasis, and a negative association was found between the presence of anti-p53 antibodies and survival. These results suggest that a preoperative test of serum anti-p53 antibodies in gastric cancer patients can be useful to identify subset of patients who may need gastrectomy with lymph node dissection and post-operative adjuvant therapy.     

Clinical significance of serum and ascitic p53 autoantibodies in epithelial ovarian carcinoma

BACKGROUND: Accumulation of mutated p53 in malignant cells can lead to the generation of anti-p53 autoantibodies in the serum and other body fluids of cancer patients. This retrospective study was performed to evaluate the prognostic significance of preoperative serum and ascitic anti-p53 antibodies in advanced ovarian carcinoma. METHODS: In 113 ovarian carcinoma patients who presented with significant amounts of ascites, anti-p53 autoantibodies were determined by a highly specific enzyme-linked immunosorbent assay of blood and ascites. Disease free and overall survival of study patients was estimated by the product limit method of Kaplan and Meier. Differences in survival were examined according to criteria of Mantel and Breslow. A multiple regression analysis based on the Cox proportional hazards model was used to determine the independence of prognostic variables. RESULTS: Serum and ascitic anti-p53 antibodies were found in 28 (25%) and 21 (19%) of the study patients, respectively. In univariate analysis, detection of anti-p53 antibodies in ascites but not in serum was found to be a sign of unfavorable disease free survival (P<0.003) and overall survival (P < 0.01). Multivariate analysis revealed that anti-p53 positivity in ascites retained independent significance only in the prediction of adverse progression free survival (P<0.01). CONCLUSIONS: The generation of a humoral immune response against p53 protein in the close tumor environment, as demonstrated by the occurrence of p53 autoantibodies in the ascitic fluid of ovarian carcinoma patients, is associated with poor disease free survival.     

The detection of serum anti-p53 antibodies from patients with gastric carcinoma in China

Background: Gastric carcinoma is one of the most frequently occurring cancers. The aim of this research was to increase the detection efficiency of anti-p53 antibodies in the sera of patients with gastric carcinoma and to improve the diagnosis for patients with gastric carcinoma. Methods: We prepared phage-displayed peptide DO7 and established an enzyme-linked immunosorbent assay method to detect the anti-p53 antibodies. We detected the anti-p53 antibodies of 61 patients with gastric carcinoma using the method and our previous ELISA method assisted by the recombinant wild-type human p53 protein to detect the anti-p53 antibodies. We studied the correlation between the anti-p53 antibodies and the clinicopathological data including sex, age, carcinoembryonic antigen, tumor size, tumor TNM staging, and lymph-node status. Results: The anti-p53 antibodies positive rate for patients with gastric carcinoma was increased (31.1%, 19/61) through the combination of p53-ELISA and phage-ELISA. We found that the positive anti-p53 antibodies correlated significantly with tumor size (P = 0.047). The combination of the anti-p53 antibodies and carcinoembryonic antigen could improve the diagnosis for patients with gastric carcinoma. Conclusions: This approach indicated an increased anti-p53 antibodies positive rate for patients with gastric carcinoma and provided a useful marker for clinical diagnosis for patients with gastric carcinoma.     

Testing for anti-p53 antibodies increases the diagnostic sensitivity of conventional tumor markers

The aim of this study was to determine whether anti-p53 antibodies are of clinical significance as a serological marker in the diagnosis and monitoring of malignancies. A total of 1874 serum samples from 591 patients with various types of cancer, esophageal, gastric, colorectal, pancreatic, hepatocellular, breast, and urogenital cancer, and 436 control individuals were analyzed by immunoblot for antibodies against p53. The anti-p53 antibody test was correlated with expression of conventional tumor markers, survival and the clinicopathological features of malignant disease. Anti-p53 antibodies were found in 23.4% (138/591) of the sera of patients with malignant disease (range 11.5-34%). The detection of anti-p53 serum antibodies had a specificity of 100% for malignancy (p<0.0001). The overall sensitivity of measuring established tumor markers was 62.9% (372/591). The elevation of conventional tumor markers and the presence of anti-p53 antibodies in the sera of patients with malignant disease turned out to be an independent variable (p<0.05). Combination of established tumor markers with the anti-p53 antibody test led to an increase in diagnostic sensitivity of 8% (49/591) (p<0.01). Thus, the independence of anti-p53 antibodies from established tumor markers allows the serological detection of additional tumor patients. Kaplan-Meier analysis revealed a trend toward a poorer prognosis in hepatocellular carcinoma and breast cancer patients who were anti-p53 serum positive. In conclusion, testing for anti-p53 antibodies can increase the diagnostic sensitivity when used in combination with measurement of conventional tumor markers. This increase is achieved without a parallel decrease in specificity.     

Clinical significance of circulating anti-p53 antibodies in European patients with hepatocellular carcinoma

p53 alterations are considered to be predictive of poor prognosis in hepatocellular carcinoma (HCC) and may induce a humoral response. Anti-p53 serum antibodies were assessed by enzyme-linked immunosorbent assay (ELISA) using purified recombinant human p53 on 130 European HCC patients before treatment and during the clinical course of the disease. p53 immunohistochemistry was performed on tumours from the 52 patients who underwent surgery, and DNA sequencing analysis was initiated when circulating anti-p53 antibodies were detected. Nine (7%) HCC patients had anti-p53 serum antibodies before treatment. During a mean period of 30 months of follow-up, all the negative patients remained negative, even when recurrence was observed. Of the nine positive patients, eight were still positive 12-30 months after surgery. The presence of anti-p53 serum antibodies was correlated neither with mutation of the p53 gene nor the serum alpha-fetoprotein levels and clinicopathological characteristics of the tumours. However, a greater incidence of vascular invasion and accumulation of p53 protein were observed in the tumours of these patients (P<0.03 and P<0.01 respectively) as well as a better survival rate without recurrence (P = 0.05). In conclusion, as was recently shown in pancreatic cancer, anti-p53 serum antibodies may constitute a marker of relative 'good prognosis' in a subgroup of patients exhibiting one or several markers traditionally thought to be of bad prognosis.     

Detection of serum p53 protein in patients with different gastrointestinal cancers

Overexpression of p53 has been found in many types of human malignancy. The present study aimed to detect preoperative serum p53 among 158 patients with different gastrointestinal cancers using ELISA technique based on mouse anti-p53 DO-7 monoclonal antibody and anti-p53 rabbit polyclonal antibody. A single band of 53kDa was detected in nuclear protein tissue extracts of selected cancer patients and in 96% of the corresponding sera using Western blot assay. The ELISA technique revealed that the serum p53 was detected in 100% of patients with cholangiocarcinoma, 76% of pancreatic carcinoma, 75% of hepatocellular carcinoma, 70% of colon cancer, 60% of esophagus carcinoma, and 35% of gastric carcinoma. The serum p53 concentrations of the positive patients were highly elevated (P<0.001) compared with healthy individuals. These results suggest that immunodetection of serum p53 could be valuable for post-operative monitoring during follow up in preoperatively positive patients with gastrointestinal cancers.     

胃癌中CD44V6与p53蛋白的表达及其意义

 目的　分析胃癌研究中的两个热点基因蛋白CD44V6和p53与胃癌发生、发展及转移的关系。方法　运用免疫组化法,检测78例原发胃癌及癌旁良性病变的CD44V6及p53基因蛋白的表达。结果　30例胃癌出现CD44V6的阳性表达38.5%(30/78),癌旁良性病变中未检测到其表达;侵及浆膜或具有淋巴结转移及远距离转移者,V6表达率明显高于无浆膜受侵或无淋巴结转移及远距离转移者。肠型胃癌的V6表达率明显高于弥漫型胃癌的,但弥漫型胃癌中V6表达与淋巴结转移呈显著相关性,而肠型胃癌中则否。有28例胃癌检测到p53蛋白的积聚35.9%(28/78),p53阳性与各种临床病理因素均无关,但癌旁良性病变中却可见p53阳性。结论　CD44V6表达预示胃癌具有较强的侵袭转移能力,而p53基因蛋白的异常表达与细胞的早期癌变有关。     

p53 expression as a prognostic determinant in resected distal bile duct carcinoma.

AIMS: To determine the incidence and prognostic value of p53 immunopositivity in resectable distal bile duct carcinoma (DBDC). METHODS: Forty-seven paraffin-embedded archival tumour samples of patients with DBDC, who underwent subtotal pancreatoduodenectomy from 1985 to 1996, were immunohistochemically examined for p53 positivity, using the anti-p53 antibody D07. RESULTS: Nineteen (40%) of the 47 tumours demonstrated positive (>30%) p53 protein immunostaining. Focally positive or negative staining was seen in the remaining 28 (60%) cases. Patients in this low p53 category survived significantly longer than those in the high p53 category, with median survival durations of 29 and 13 months respectively (P=0. 039). p53 positivity was independent of age, sex, tumour size, radicality of resection, histopathological grading, lymph-node status, perineural invasion and vasoinvasive growth. CONCLUSIONS: This study indicates that low (0-30%) p53 expression is a favourable prognostic factor in patients with resected DBDC.     

Measurement and evaluation of serum anti-p53 antibody levels in patients with lung cancer at its initial presentation: a prospective study.

Anti-p53 antibodies in sera are known to be products of the host immune response to mutated p53 protein, and are present in some patients with various types of cancer. In this study, we measured serum anti-p53 antibody levels in 52 patients with lung cancer and 63 normal volunteers to determine the relationship between anti-p53 antibody level and clinical features of lung cancer patients. Anti-p53 antibody level was measured by an enzyme-linked immunosorbent assay and expressed as an anti-p53 antibody index, defined as the ratio of absorption of serum sample to that of p53-positive serum. The median anti-p53 antibody index was 6.6 for lung cancer patients, and higher than that in normal volunteers (1.7) (P = 0.0000). For lung cancer patients, significant differences in index levels were found by histology (4.3, n = 25, adenocarcinoma vs 8.7, n = 18, squamous cell carcinoma vs 64.8, n = 2, large-cell carcinoma vs 9.8, n = 7, small-cell carcinoma; P = 0.0109). High anti-p53 antibody index levels were observed for both large-cell carcinoma and small-cell carcinoma. When the cut-off level was set at 7.2, determined using the twice 95% specificity level for normal volunteers, the sensitivities of anti-p53 antibodies were 46.1% for all lung cancers, 28.0% for adenocarcinoma, 55.6% for squamous cell carcinoma, 100% for large-cell carcinoma and 71.4% for small-cell carcinoma. However, there were no significant differences in index level by gender, age, smoking index, presence of previous or concomitant cancer or disease stage. Multivariate analysis using a logistic regression model demonstrated that histological type of tumour was a dominant factor associated with elevation of anti-p53 antibody index level (P = 0.0184). These findings suggest that serum anti-p53 antibody index level might be independent of tumour burden and the presence of previous or concomitant cancer in our series of lung cancer patients, but is clearly strongly correlated with tumour histological type.     

Prognostic significance of CEA, CA 19-9 and CA 72-4 preoperative serum levels in gastric carcinoma.

The prognostic value of preoperative serum levels of CEA, CA 19-9 and CA 72-4 tumor markers was investigated in 153 patients resected for gastric cancer. The positivity rates for CEA, CA 19-9 and CA 72-4 were 20.9, 34.6 and 28.1%, respectively. Multiple logistic regression analysis for positive levels of tumor markers indicates that CEA positivity is significantly related to the depth of invasion (p < 0.005) and the presence of distant metastasis (p < 0. 05), CA 19-9 positivity is related to nodal involvement (p < 0.05) and the depth of invasion (p < 0.05), whereas CA 72-4 positivity is influenced by tumor size (p < 0.005) and noncurative surgery (p < 0. 05). Positive levels of each tumor marker were associated with a worse prognosis if compared with negative cases using univariate analysis. Multivariate analysis of curatively resected cases identified depth in gastric wall (p < 0.0001), nodal status (p < 0. 0005), and tumor location in the upper third (p < 0.05) as significant prognostic variables; CEA, CA 19-9 and CA 72-4 serum positivity did not reach statistical significance. However, when the positivity of the three markers was associated, a p value < 0.05 was observed. The analysis of survival curves stratified by tumor stage revealed that marker positivity significantly affects survival in stages I, II and IV (p < 0.05). The combined assay of CEA, CA 19-9 and CA 72-4 preoperative serum levels provides additional prognostic information in patients resected for gastric cancer; patients with preoperative positivity for one of these tumor markers should be considered at high risk of recurrence even in early stages of gastric carcinoma.     

Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer

Antibodies reacting with the host protein p53 were found in the sera of patients with primary or secondary carcinoma of the breast. Fourteen out of the 155 sera from breast cancer patients tested were positive for anti-p53 antibodies (9%) and no positives were detected among 164 control sera from normal women tested. The locations of the first metastasis in patients with positive sera were unusual, with more lung metastases and fewer bone metastases than expected. The detection of anti-p53 antibodies indicates that p53 is altered in amount, type or presentation in breast tumors so that it becomes immunogenic.     

Detection of preoperative serum anti-p53 antibodies in gastric cancer.

The presence of circulating anti-p53 antibodies (p53Ab) was investigated in 81 gastric cancer patients by ELISA. Preoperatively, anti-p53Ab were revealed in 13 cases (16%), including 3 early gastric cancers. No significant correlation of p53Ab with patient age, sex, pathological parameters and tumor markers, such as CEA and CA19-9 was observed, although p53Ab positivity tended to be associated with a higher rate of lymph node metastases (p = 0.073). We have found no significant correlation between p53Ab and poor survival rate (p = 0.325). In conclusion, we have observed that p53Ab are detectable in gastric cancer patients, even at early stages of disease, however, p53Ab have not served as a predictor of poor prognosis in gastric cancer in this series.     

Detection of serum anti-P53 antibodies from patients with colorectal cancer in China using a combination of P53- and phage-ELISA: correlation to clinical parameters

Background: Colorectal cancer is one of the most common malignant tumors in China. The aims of this research were to increase the sensitivity of anti-p53 antibody detection in the sera of patients with colorectal cancer and to assist in their diagnosis. Methods: Sixty-seven non-selected Chinese with colorectal cancer were involved in this study. Anti-p53 antibodies in serum were detected by ELISA using recombinant human wild-type p53 protein and hybrid phage as the coating antigen. Correlations between the anti-p53 antibodies and clinicopathological parameters were also analyzed. Results: The detection efficiency of anti-p53 antibodies in the patients with colorectal cancer was increased (46.3%, 31/67) through the combination of the two ELISA methods compared with each method alone. The titer of serum anti-p53 antibodies was not associated with clinicopathological parameters, but there was a significant correlation between their presence, the CEA level, and the stage of the patient’s colorectal cancer. Conclusions: These results demonstrate that combination of the two ELISA methods increased the detection rate of anti-p53 antibodies in patients with colorectal cancer. This research may provide a useful method to complement conventional clinical diagnosis.     

Development of antibodies against p53 in lung cancer patients appears to be dependent on the type of p53 mutation.

Using immunoblotting techniques we studied the sera from small cell lung cancer and non-small cell lung cancer patients for antibodies directed against p53. We have also characterized the majority of these patients' tumors for p53 mutations. In the sera of 13% of the patients (4 of 40 small cell lung cancer and 2 of 6 non-small cell lung cancer) we found antibodies specific for the p53 tumor suppressor gene product. All of the antibody-positive patients tested had p53 missense mutations and expressed detectable p53 antigen in their tumor cell lines. No anti-p53 antibodies were detected in sera from patients whose tumor had p53 stop, splice/stop, splice, or frameshift mutations (n = 10). Thus, while we find that the ability of lung cancer patients to develop anti-p53 antibodies is correlated with the type of p53 mutation, many patients have tumors with missense p53 mutations and did not develop anti-p53 antibodies. The presence of p53 antibodies was not correlated to stage, prior treatment, sex, or survival. None of these lung cancer patient sera had measurable amounts of p53 antigen. By immunoblotting all six anti-p53 antisera we were able to detect a variety of mutant p53 proteins (including those from antibody-negative patients) and detected wild-type p53 protein. The development of anti-p53 antibodies represents an interesting model system for studying immune responses in cancer patients against mutant oncogene products.     

Serum anti-p53 antibodies in uterine and ovarian cancer: association with dna sequence copy number abnormalities.

The aim of the present study was to evaluate the clinical significance of the serum anti-p53 antibody in patients with uterine and ovarian cancer. Some of the ovarian patients were also evaluated for overexpression of p53 by immunohistochemistry and for cytogenetic alterations by comparative genomic hybridization (CGH). Serum anti-p53 antibodies were determined by an enzyme immunoassay kit. The antibody was detected in 8/30 (27%) of ovarian cancers, in 12/86 (14%) cancers of the uterine cervix, in 5/41 (12%) cancers of the uterine body, and 0/9 (0%) healthy women. The overall survival rate in patients with ovarian cancer was significantly worse in patients with anti-p53 antibody positivity than that in patients with anti-p53-antibody-negative cancers using the log rank test (p = 0.017). There was a significant correlation between the presence of anti-p53 antibody and tissue overexpression of p53 in ovarian cancers. CGH analysis showed that the aberrations in DNA sequence copy number in ovarian cancers were significantly increased in anti-p53-antibody-positive cases compared to antip53-antibody-negative cases including increased copy number on 20q and reduced copy number on 5q and 13q. Although the exact relationship between the presence of serum anti-p53 antibody (specific humoral response) and cytogenetic alterations is still unknown, these findings suggest that the measurement of serum anti-p53 antibody may be useful for the assessment of genetic instability and tumor biological aggressiveness.     

Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients

Antibody reactivity against survivin, a recently identified tumor-associated protein, was determined in sera from patients with lung (n = 51) or colorectal cancer (n = 49). The same collection of sera was tested for the presence of antibodies against p53. Eleven sera from lung cancer patients and four sera from colorectal cancer patients reacted with purified recombinant survivin in an ELISA (21.6% and 8.2%, respectively), whereas four sera from lung cancer patients and nine sera from colorectal cancer patients contained anti-p53 antibodies (7.8% and 18.4%, respectively). The increase in prevalence when anti-survivin and anti-p53 antibodies were determined in parallel was statistically significant (29.4% versus 7.8%, P = 0.005 in lung cancer population; 26.6% versus 8.2%, P = 0.015 in colorectal cancer population). The high prevalence of anti-survivin antibodies makes these antibodies an attractive novel marker for the diagnosis of lung and colorectal cancer, particularly in patients lacking anti-p53 antibodies.     

Serum anti-p53 antibodies in lung cancer: comparison with established tumor markers

As reported earlier, p53 antibodies are detected in the sera of patients with different types of cancer, including lung cancer. In contrast, in the serum of healthy subjects the presence of anti-p53 antibodies is extremely rare. We collected the venous blood samples of 109 patients affected with lung cancer (LC): 57 patients (46 M, 11 F) with non-small-cell carcinoma (NSCLC), 52 others (40 M, 12 F) with small-cell carcinoma (SCLC). Serum p53 antibodies were assayed using ELISA method and all positive sera were confirmed by Western-blot method. In addition, using IRMA methods we assayed serum CEA, TPA, CYFRA21-1 and NSE. Serum p53Ab are detectable (p53Ab-positive) in 35/109 (32.1%) patients with lung cancer. About 17/57 (29.8%) patients affected with NSCLC and 18/52 (34.6%) with SCLC were p53Ab-positive. CEA, TPA, CYFRA21-1 and NSE sensitivity in LC patients (NSCLC+SCLC) is 50.5%, 58.7%, 42.2%, 35.8%, respectively. The lower sensitivity (32.1%) of serum p53Ab is connected with the higher specificity and diagnostic accuracy (100% and 69%, respectively). Out of 35 patients p53Ab-positive, five (14.3%) exhibit only serum p53Ab, while serum values of the established tumor markers were lower than cut-off. Serum p53Ab assessment is a simple and a low-cost assay with a good specificity and diagnostic accuracy that in LC patients can be used at least in association with established tumor markers.     

Serum anti-p53 antibodies in patients with squamous cell carcinoma of the esophagus

Immunoassay of serum anti-p53 antibodies was performed in a series of 63 patients with squamous cell carcinoma of the esophagus. p53 alterations were also analyzed with DGGE to detect gene mutations (n=53) and by immunohistochemistry to assess overexpression of p53 (n=43). An immune response was observed in 16 sera (25%). The corresponding biopsies all had a p53 gene mutation or overexpression of protein p53. We were unable to demonstrate any significant relationship between habitual tumor parameters (localization, cell differentiation, TNM stage) and development of p53 alterations. However, none of the patients with a localized tumor developed an immune response, while some of them had a muted gene or overexpressed p53.     

Clinical significance of serum p53 antibody detection in a chemosensitivity assay in cases of human colorectal cancer

Alteration of the p53 gene product occurs frequently during the progression of colorectal cancer. Recently, mutated p53 protein was found to induce the production of anti-p53 antibodies in the serum of patients. The purpose of this study was to evaluate the relationship between p53 status in serum and chemosensitivity in resectable colorectal cancer patients. A total of 35 patients with primary colorectal cancer who underwent surgical treatment were examined by chemosensitivity test with the viable tumor samples using Histoculture Drug Response Assay (HDRA). Serum samples of these patients to test for p53 antibodies were obtained before tumor resection, and assayed in duplicate by using an enzyme-linked immunosorbent assay (ELISA) kit. The inhibition index of 5-FU and CDDP, determined by the HDRA method, in the sero-positive group was significantly lower than that of the sero-negative group (p < 0.01). Furthermore, significant statistical differences in chemosensitivity to 5-FU and CDDP were revealed depending on the presence of serum p53 antibodies. There was no relationship between chemosensitivity assay and tumor marker positivity or clinicopathological features in these patients. Detection of serum p53 antibodies, which reflects p53 mutations in tumor tissue, is a simple method which correlates with chemosensitivity, and may contribute to the selection of favorable chemotherapeutic strategies of colorectal cancer.     

Detection of serum anti-p53 antibodies from patients with ovarian cancer in China: correlation to clinical parameters

Background: Ovarian cancer has been one of the most common malignant tumor among women in China. The aims of this research were to increase the detection efficiency of anti-p53 antibodies in the sera from patients with ovarian cancer and to assist the diagnosis for patients with ovarian cancer. Methods: The hybrid phage displaying the immunodominant epitope SQAMDDLMLS in p53 N-terminal region was constructed and the fusion protein was prepared and purified. Ninety-two nonselected Chinese women with ovarian cancer were involved in this study. Tumor p53 overexpression was assessed by immunohistochemical analysis of tissue sections. Serum antibodies to p53 were also detected by enzyme-linked immunosorbent assay (ELISA) using recombinant human wild-type p53 protein and the hybrid phage as the coating antigen respectively. Furthermore, the correlations between the anti-p53 antibodies and clinicopathological parameters were analyzed. Results: The positive rate of anti-p53 antibodies in the patients with ovarian cancer was increased (39.1%, 36/92) through the combination of the two ELISA methods compared with each method. The anti-p53 antibodies were not associated with the clinicopathologic parameters, while there was a significant correlation between the presence of p53 Abs and tissue overexpression of p53 in ovarian cancer. Conclusion: These preliminary results demonstrate that the combination of the two ELISA methods increased the positive rate of anti-p53 antibodies in patients with ovarian cancer and provided a useful marker to complement routine clinical diagnosis for patients with ovarian cancer.