Clinical importance of serum anti-p53 antibodies as tumor markers

Anti-p53 antibodies are autoantibodies induced by mutation of p53 cancer-suppressor gene, and are considered to be indirect markers for p53 gene mutations and abnormally high p53 gene levels. We evaluated the usefulness of the measurement of anti-p53 antibodies by enzymed-linked immunosorbent assay using serum samples from patients with various disorders and normal subjects. The anti-p53 antibody concentration was high in patients with lung, esophageal, gastric, hepatocellular, colonic, rectal or ovarian cancer and significantly differed between the group with neoplasms and those with non-neoplastic disorders. Particularly high concentrations were observed in patients with malignant tumors. The mean agreement rate between anti-p53 antibodies and conventional tumor markers was only 47.8% despite slight differences among disorders. The positive rate increased to 63.0% by their combination assay. In addition, anti-p53 antibodies were independent markers, not complimentary to conventional markers. The mean agreement rate between anti-p53 antibodies and tissue p53 was 70.0%. Though the anti-p53 antibody-positive rate was lower than the tissue p53-positive rate, anti-p53 antibodies may be useful new tumor markers because specimens from the affected tissue are not necessary.     

Clinical associations and prognostic significance of serum anti-p53 antibodies in Thai patients with hepatocellular carcinoma

Mutations of the p53 gene have been reported to be of prognostic significance in hepatocellular carcinoma (HCC). However, the clinical associations and prognostic value of anti-p53 antibodies, known to be products of the host immune response to these mutations, have been controversial. Serum anti-p53 antibodies were measured in 121 Thai patients diagnosed with HCC using a specific enzyme-linked immunosorbent assay (ELISA) kit. The clinical/pathological characteristics of the patients were compared with respect to the presence of serum anti-p53 antibodies. Cox regression analysis was performed to assess factor interaction and association with survival. Anti-p53 antibodies were detected in 13.2% (16 of 121) of our patients. There were no differences between groups with regard to age, sex, viral markers (HBsAg or anti-HCV), severity of liver disease and tumor advancement. The median survival rates for patients positive and negative for anti-p53 antibodies were 4.0 and 3.0 months, respectively (p = 0.443, by log-rank test). Multivariate analysis demonstrated that an advanced Okuda stage, lack of therapy and presence of portal vein thrombosis were independent factors related to the prognosis of the patients. Nonetheless, the presence of anti-p53 antibodies did not constitute a predictive variable associated with a poorer prognosis. Serum assay of anti-p53 antibodies, although rapid and easily performed, may not be suitable as an alternative to molecular detection of mutations in assessing tumor advancement and prognosis of patients with HCC.     

Detection of serum anti-p53 antibodies in patients with various types of cancer

The p53 tumor suppressor gene is altered in a variety of human cancers and mutated p53 protein was found to induce anti-p53 antibodies in sera of patients with various types of malignant neoplastic disease. We report here the presence of anti-p53 antibodies in sera from patients with various disorders as well as from healthy controls by enzyme-linked immunosorbent assay. The prevalence of anti-p53 antibodies in cancer was higher than neoplastic disease. High antibody concentrations were found in patients with 1 pharyngeal, 3 esophageal, 4 lung, 4 gastric, 1 pancreatic, 3 colonic and 4 ovarian cancer. Serological analysis does not require biopsy of the tumor. Therefore, determination of anti-p53 antibodies can be used for tumor screening and for early diagnosis.     

Use of specific ELISA for the detection of antibodies directed against p53 protein in patients with hepatocellular carcinoma.

AIMS: To analyse the significance of antibodies to p53 protein as a serological marker for changes in p53 gene expression in patients with hepatocellular carcinoma. METHODS: Thirty eight patients with hepatocellular carcinoma, 19 showing accumulation of p53 protein by immunohistochemistry and 19 having no accumulation, were studied. The presence of anti-p53 was tested using a novel ELISA utilising a recombinant p53 protein as a capture system and verified by western blotting. p53 gene mutations were sought by single strand conformational polymorphism and DNA sequencing analyses. RESULTS: Of 19 patients with p53 protein accumulation in tumour tissue, 10 (52%) had antibodies to p53 in serum by ELISA. Four patients with p53 negative immunohistochemistry also had detectable anti-p53. Western blot analysis confirmed the specificity of the ELISA positive serum samples. The presence of anti-p53 was independent of serum alpha-fetoprotein and was detected in 50% of small tumours while only 8% were alpha-fetoprotein positive. Mutations affecting exons 5 and 6 seem to be more frequently associated with development of anti-p53, than mutations in exons 7 or 8. CONCLUSIONS: The ELISA for anti-p53 is a convenient and specific tet for the detection of humoral response to alterations in p53 gene expression and could be of value in the diagnosis and characterisation of patients with hepatocellular carcinoma.     

Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. These are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.     

Autoantibodies to p53 in sera of patients with autoimmune thyroid disease

Mutations in the tumor suppressor gene, p53, lead to intracellular accumulation of abnormal p53 protein and are associated with p53 autoantibodies. p53 also accumulates in autoimmune diseases and Hashimoto's thyroiditis, but it is unknown if p53 autoantibodies occur in the latter. We measured p53 autoantibodies in the sera of 93 patients with thyroid disease and 19 patients without thyroid disease. Anti-p53 antibodies were detected in the sera from 4.2% (2/48) of patients with autoimmune thyroid disease, including one patient with Hashimoto's thyroiditis (3.7%, 1/27) and one with Graves' disease (4.8%, 1/21). A third patient with pseudohypoparathyroidism, but without thyroid disease, was also positive (1/19; 5.2%). None of 19 patients with differentiated thyroid cancer had anti-p53 antibodies. We conclude that anti-p53 antibodies can be detected in the sera from approximately 4% of patients with autoimmune thyroid disease. This finding suggests that increased DNA damage and apoptosis may be associated with autoimmune thyroid disease.     

Autoimmunity to the p53 protein is a feature of systemic lupus erythematosus (SLE) related to anti-DNA antibodies

The induction of anti-DNA autoantibodies in systemic lupus erythematosus (SLE) patients is problematic because mammalian DNA is poorly immunogenic at best. Here we demonstrate a chain of connected antibodies in SLE patient sera that could account for the induction of anti-DNA antibody, and possibly for some of the pathogenic features of SLE. We now report that SLE patients, in addition to anti-DNA, produce antibodies to the carboxy-terminal domain of the tumour suppressor molecule p53; this p53 domain recognizes damaged DNA. Hence, these anti-p53 antibodies could mimic damaged DNA immunologically. Indeed, SLE sera do contain anti-idiotypic antibodies to a prototypic anti-p53 antibody. Moreover, SLE anti-DNA antibodies also recognize this type of anti-p53 antibody. Indeed, binding of affinity-purified anti-DNA both to DNA and to the anti-p53 antibody could be blocked by a p53 peptide derived from the DNA-binding domain. This mimicry of the p53 DNA-binding domain by the SLE anti-DNA antibodies is functional: activation of the p53 molecule could be inhibited by such anti-DNA antibodies. Thus, anti-DNA antibodies may arise in SLE patients by a chain of idiotypic autoimmunity centered around p53 autoimmunity. The SLE anti-DNA and anti-p53 antibodies can functionally block p53 activation, and so could affect apoptosis.     

Serum anti-p53 autoantibodies in patients with type 1 diabetes

The presence of antibodies reacting with the p53 tumor suppressor protein has been described in patients with some autoimmune disorders. In this study we looked for serum anti-p53 antibodies in 64 patients with autoimmune type 1 diabetes mellitus within 4 mo of diagnosis. The presence of anti-p53 antibodies was observed in 6/64 (9.4%) subjects with type 1 diabetes, and in 1/44 (2.3%) subjects with other organ-specific autoimmune diseases (18 primary biliary cirrhosis, 10 autoimmune hepatitis, 16 thyroid diseases), but in none of 45 control subjects. No relationship was found between antibodies directed against islet- and non-islet-specific antigens and anti-p53 antibodies. These findings support a possible role for p53 in some autoimmune disorders.     

Prevalence of autoantibodies to the p53 protein in autoimmune hepatitis

The target antigens of anti-nuclear autoantibodies in autoimmune hepatitis (AIH) are poorly characterised. Since antibodies to the p53 nuclear protein have been reported in various autoimmune diseases, we have assessed the prevalence of these antibodies in patients with AIH (n = 45), primary biliary cirrhosis (n = 60), hepatitis B (n = 22), hepatitis C (n = 55), and in a control group of subjects with various non-liver diseases (n = 56). A significant proportion of patients with AIH (31%) had elevated levels of autoantibodies to the p53 protein. In contrast, the prevalence of these antibodies in primary biliary cirrhosis (8%) and viral hepatitis (6%) was similar to that in the control group (4%). The clinical features of the anti-p53 seropositive AIH patients were similar to those of the seronegative ones. Thus, the prevalence of p53 autoantibodies in AIH is higher than in other forms of chronic hepatitis, and may be useful in differential diagnosis.     

Prevalence of Autoantibodies to the p53 Protein in Autoimmune Hepatitis

The target antigens of anti-nuclear autoantibodies in autoimmune hepatitis (AIH) are poorly characterised. Since antibodies to the p53 nuclear protein have been reported in various autoimmune diseases, we have assessed the prevalence of these antibodies in patients with AIH (n = 45), primary biliary cirrhosis (n = 60), hepatitis B (n = 22), hepatitis C (n = 55), and in a control group of subjects with various non-liver diseases (n = 56). A significant proportion of patients with AIH (31%) had elevated levels of autoantibodies to the p53 protein. In contrast, the prevalence of these antibodies in primary biliary cirrhosis (8%) and viral hepatitis (6%) was similar to that in the control group (4%). The clinical features of the anti-p53 seropositive AIH patients were similar to those of the seronegative ones. Thus, the prevalence of p53 autoantibodies in AIH is higher than in other forms of chronic hepatitis, and may be useful in differential diagnosis.     

Methods for generation of monoclonal antibodies to the very small drug hapten, 5-benzimidazolecarboxylic acid

Anti-p53 antibodies have been detected in the sera of patients with various types of cancers. In this report, we describe the development of a new ELISA aimed at detecting anti-p53 antibodies using two peptides belonging to immunodominant epitopes of the p53 N-terminal region. We first tested the reactivity of the sera by an indirect ELISA using the peptides as a capture system. Then, the specificity of the reaction was confirmed by an inhibition assay. Two systems of peptide presentation, phage display and the streptavidin/biotin system, were evaluated. Using a panel of sera from cancer patients, both systems were found to be equally reliable, demonstrating that both peptide-based ELISAs can be used for the specific detection of anti-p53 antibodies. The presence of anti-p53 antibodies was associated with p53 alteration whether it be mutation or accumulation.     

p53 as a mutagen test in breast cancer

The p53 gene is mutated in about half of all tumors. The p53 gene can be used as a "mutagen test," that is, the relative frequencies of the different types of mutation can be used as an epidemiological tool to explore the contribution of exogenous mutagens vs. endogenous processes in particular cancers. p53 has been used as a mutagen test in breast cancer. Surprisingly, the pattern of p53 mutations differs among 15 geographically and ethnically diverse populations. In contrast, mutation patterns in the human factor IX gene are similar in geographically and ethnically diverse populations. Diverse p53 mutation patterns in breast cancer are consistent with a significant contribution by a diversity of exogenous mutagens. Breast tissue may be uniquely sensitive to lipophilic mutagens because of its unique architecture, characterized by tiny islands of cancer-prone mammary epithelial cells surrounded by a sea of adipocytes. Mammary epithelial cells may be differentially susceptible to released lipophilic mutagens preferentially concentrated in adjacent adipocytes and originating in the diet. To test this hypothesis, we developed a method for measuring mutation load from ethanol-fixed, paraffin-embedded human tissues immunohistochemically stained with anti-p53 antibodies. Single cells staining positively for p53 overabundance are microdissected and the gene is sequenced. It is possible to identify individuals with a high mutation load in normal breast tissue and who are presumably at increased risk for breast cancer. In addition, analysis of the p53 gene with appropriate mutation detection methodology markedly improves the prediction of early recurrence, treatment failure, and death in breast cancer patients. Mutagen tests and mutation load measurements are useful tools to identify the role of mutagens in breast cancer.     

Immunohistochemical analysis of p53 protein overexpression in liver cell dysplasia and in hepatocellular carcinoma

We analysed p53 protein immunoreactivity in hepatocellular carcinomas (HCCs) and in liver cell dysplasia (LCD) of patients from an area in Northern China, using five anti-p53 protein antibodies recognizing different epitopes of the protein. In HCCs, the overall prevalence of p53 protein immunoreactivity was 78.3%. However, prevalence was strongly influenced by the type of antibody used, ranging from 67.5% for antibody PAb-1801 to only 10.8% for antibodies PAb-421 and DO-7. p53 protein immunoreactivity was not related to type or grade of HCC. In contrast to former reports, p53 protein staining was restricted to nuclei only when using the CM-1 antibody, whereas two other antibodies yielded both, nuclear and cytoplasmic or membrane staining, and no nuclear staining was observed with antibodies PAb-421 and DO-7, the latter two, however, demonstrating cytoplasmic and membrane staining. For LCD, three subtypes were morphologically and karyometrically defined. Nuclei of some LCD cells were p53 immunoreactive, but positivity was restricted to the small cell variant of LCD. Positivity was different for cirrhosis with or without associated HCC, amounting to 18.9% in the former and 39.4% in the latter. Interestingly, p53 protein immunoreactivity also occurred in a set of small hepatocytes not showing the typical features of LCD and therefore classified as simple regenerating liver cells.     

p24 antibody production in p24 antibody-negative HIV-infected subjects.

To determine if HIV-infected patients with no detectable serum antibodies to p24 are producing antibodies to p24 (anti-p24), blood was obtained from 49 HIV-infected patients at various stages of infection. Serum p24 antigen levels were measured and peripheral blood mononuclear cells were cultured for 1 week without mitogenic stimulation. The presence of anti-p24 in culture supernatants and sera was determined by radioimmunoprecipitation assays. Cells from 89% of the patients who had anti-p24 in their sera spontaneously synthesized anti-p24 in vitro. Similarly, cells from 83% of the HIV-infected patients who had no detectable anti-p24 in their sera spontaneously produced anti-p24 in vitro. Thus the absence of anti-p24 in serum did not reflect suppression in the ability of patients' cells to synthesize and secrete antibodies to p24. However, cells from patients whose sera contained anti-p24 spontaneously synthesized more anti-p24 than did cells from patients whose sera lacked anti-p24, suggesting that these two groups of patients may represent individuals with inherently high or low responses to p24 epitopes, respectively.     

Immunohistochemical analysis of biliary tract lesions.

The distinction among inflammatory, benign, and malignant lesions of the biliary tract can at times be difficult. Several methods have been used, including immunohistochemistry (IHC), with variable success. We evaluated a panel of IHC stains to determine their utility in discriminating between bile duct lesions. Formalin-fixed, paraffin-embedded 4-microm sections from 12 inflammatory lesions, 10 bile duct adenomas, and 13 bile duct carcinomas were immunostained using a modified avidin-biotin-complex technique after epitope enhancement using antibodies for p53, Ki-67, and bcl-2. For p53 and bcl-2, greater than 1% of cells staining positive was interpreted as positive. The proliferation index was calculated by determining the number of Ki-67-positive cells in a 1000 cell count. In the inflammatory group, 0 of 12 reacted with anti-p53, 2 of 12 were positive with anti-bcl-2, and the proliferation index with was 22.9% +/- 3.9%. Two of 10 bile duct adenomas showed reactivity with anti-bcl-2, and none were decorated with anti-p53 or Ki-67. In the carcinoma group, 6 of 13 were positive with anti-p53, 9 of 12 were positive with anti-bcl-2, and the proliferation index was 35.3% +/- 5.5%. The proliferation rates differed significantly between groups (P < 0.05). The presence of bcl-2 and p53 immunoreactivity coupled with a high proliferative rate in a biliary tract lesion suggests a malignant process. A panel using these antibodies may be useful in difficult cases.     

Detection of p53 in inflammatory tissue and lymphocytes using immunohistology and flow cytometry: a critical comment.

AIMS: To analyse the expression of p53 in lymphatic cells found in inflammatory tissues and the peripheral blood by immunological methods. METHODS: Immunohistological analysis of synovial tissues from patients with rheumatoid arthritis and flow cytometric analysis of peripheral blood lymphocytes were performed with anti-p53 antibodies from different sources. RESULTS: The anti-p53 antibodies PAb240, PAb421, and PAb1801 from one supplier bound to the cytoplasm of lymphocytes, fibroblasts, and endothelial cells in rheumatoid synovial tissue, while the same anti-p53 antibodies from other sources and the p53 specific antibodies PAb1620 and DO1 were negative. Using flow cytometry, the antibodies that labelled cells in inflammatory tissues were shown to bind also to peripheral lymphocytes, while the antibodies that were negative in immunohistology did not react with peripheral blood lymphocytes. p53 expression could be confirmed by western blot in rheumatoid synovial tissue, but not in peripheral blood lymphocytes using PAb421 and PAb240 antibodies from our own laboratory, which had been negative in immunohistology. CONCLUSIONS: Demonstration of p53 by western blot is more sensitive and reliable than immunohistology and flow cytometry. Western blot is the gold standard for the demonstration of p53 expression and should be used, whenever possible, to confirm p53 expression in normal tissue shown by immunohistology or flow cytometry. All other reports on p53 expression, especially those obtained using antibodies with an unusual staining pattern must be interpreted with caution.     

Diagnostic and biological significance of anti-p41 IgM antibodies against Borrelia burgdorferi

We performed a prospective study to investigate the biological significance and diagnostic specificity of anti-p41 immunoglobulin (Ig)M antibodies against Borrelia burgdorferi. During a 1-year interval 2403 patients were referred to our department for B. burgdorferi serology. Sixty-three patients had repetitive positive tests for IgM anti-p41 antibodies and negative tests for anti-p41 IgG antibodies. Ten of the 63 patients recently had symptoms of erythema migrans. A confirmatory IgM Western blot gave a positive reaction in 5 patients out of 53 patients with little or no clinical evidence of B. burgdorferi infection. The remaining 48 patients were negative in this test and were considered as false-positives. Two whole cell enzyme-linked immunosorbent assay (ELISAs), two immunofluorescence assays and Western blotting were not useful as confirmatory tests. Sera from 330 blood donors and 72 cord sera were also screened for anti-p41 IgM. Five blood donor sera and five cord sera showed an IgM reactivity against p41. Based on our data we hypothesize that up to 1.5% of the population may have natural IgM antibodies against p41 in their sera. We observed that six out of nine sera with such antibodies could immobilize a B. afzelii reference strain in vitro. Whether anti-p41 IgM antibodies are capable of inactivating infective spirochetes and thereby prevent infection in vivo is, however, not yet clarified. The paradoxical conclusion that anti-p41 IgM antibodies may be a sign of resistance to infection rather than a sign of infection should be given consideration.     

CD44 and p53 immunoexpression patterns in NF1 neoplasms - indicators of malignancy and infiltration

Neurofibromatosis type 1 (NF1) provides a unique system to evaluate the complete range of neoplastic expressions, from encapsulated benignity to invasiveness and malignancy. This study was aimed at determining whether CD44 and p53 may serve as indicators of malignant progression of neurofibroma. CD44, a transmembrane glycoprotein receptor for hyaluronic acid, and participates in cell-extracellular matrix interactions and migration. CD44 may play a vital role, either through under or overexpression, with invasion and metastases of tumors, altering their ability to infiltrate the adjacent tissue. The tumor suppressor gene, p53, has also been implicated in malignant progression of various human tumors including malignant peripheral nerve sheath tumors (MPNST). A total of 44 tumors from 33 patients with NF1 were evaluated with an anti-human CD44H, CD44 splice variant v6 and anti-p53 monoclonal antibodies. Morphologic expression patterns of expression were evaluated for CD44 while semiquantitative criteria were applied to assess, p53 nuclear positivity. Immunoexpression of p53 was markedly higher in 12 of 16 MPNST (75%). Thirteen of 28 (46%) benign neurofibroma also had some expression of p53 above ‘normal level'', although much lower than the MPNST. Plexiform neurofibroma did not differ from other benign lesions in their expression of p53. Our results suggest that p53 mutation as evidenced by immunohistochemical overexpression is a factor in malignant transformation and progression of neurofibroma. 70% of benign neurofibroma demonstrated some, usually focal, CD44 positivity. The pattern of CD44 expression in plexiform neurofibroma was revealing, as it was maximal in the ‘nonencapsulated’ portions of the tumors. Eight of 11 (72%) locally infiltrative cutaneous neurofibroma and 13 of 16 (81%) MPNST exhibited diffuse CD44 positivity. CD44v6 expression was positive in control tissues but was not identified in any of tumor samples. Also, within the confines of encapsulated tumors CD44 expression is limited, while in poorly circumscribed neurofibroma CD44 expression is upregulated. This is interpreted as a reflection of the interaction of CD44+ tumor cells with extracellular matrix, hence facilitating infiltrative behavior.     

p53, c-erbB2, and PCNA status in benign, proliferative and malignant ovarian surface epithelial neoplasms: a study of 75 cases.

Low malignant potential tumors of the ovary are believed to behave in a manner intermediate to their benign and malignant counterparts. However, recent evidence suggests these lesions are in fact benign and better classified as proliferative. Based on our previous work and evaluating p53, c-erbB2, and PCNA status in a full spectrum of ovarian surface epithelial tumors, with emphasis on low malignant potential tumors, we tested this hypothesis. Immunohistochemical stains with monoclonal antibodies were used on 75 archival ovarian neoplasms. The results demonstrated anti-p53 reactivity in 30 carcinomas (40%), 2 of which were proliferative, and no reactivity in the benign tumors. Overexpression of c-erbB2 was seen in 31 malignant neoplasms (64.5%), 4 of which were proliferative (22.1%), and none in benign tumors. The PCNA proliferative index showed means of 42.8%, 22.8%, and 14.9% with benign, low malignant potential, and malignant tumors, respectively. Predicting immunoreactivity in carcinomas for anti-PCNA (Student t test), anti-p53, and anti-c-erbB2 (Pearson chi2 test) versus a lack of immunoreactivity in proliferative tumors indicate P values of .001, <.001, and <.001, respectively. These data show significant differences in the expression of these markers in ovarian tumors and suggest a possible role for these oncogenes as supplemental tools in diagnostic pathology. Further, our findings also support the designation of proliferative as opposed to the current nomenclature of low malignant potential tumors.     

Identification of p53 peptides recognized by CD8(+) T lymphocytes from patients with bladder cancer

In many types of cancer, p53 frequently accumulates in tumor cells and anti-p53 antibodies can be detected. However, only four CD8(+) T-cell epitopes from p53 have been identified in humans so far. To further analyze the development of a T-cell response against p53, peptides having binding motifs specific for HLA-A1, -A2, -A3, -A24, -B7, -B35, -B44, and -B51 molecules have been defined. The HLA-binding capacity of those peptides was tested, and the stability of formed complexes was defined. Thirteen peptides that bound to HLA-A24 and -B44 molecules are presented. The positive peptides were then used to detect the anti-p53 response of CD8(+) T lymphocytes from patients with bladder cancer. Six peptides, presented by HLA-A2, -B51, or -A24, were able to stimulate T cells from two patients (among 16) with tumor cells that strongly accumulated p53. On the contrary, p53 peptides systematically failed to stimulate T cells from healthy donors or patients with low or undetectable levels of p53 in their tumor cells. These results have led to the identification of four new potential T CD8(+) epitopes from p53: 194-203 associating with HLA-B51 and 204-212, 211-218, and 235-243 associating with HLA-A24.