Enzyme-Linked Immunosorbent Assay Detection For Malignancy Using Anti-P53 Antibodies

@#Tumour protein 53 (p53) plays an important role in the instruction of the cell cycle. In a variety of transformed cell lines, tumour protein is expressed in high amounts, and it is believed to contribute to transformation and malignancy. This research aimed to detect the anti-p53 antibodies in sera of patients with various malignant tumours and to evaluate the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA). A case-control study was conducted on samples from 49 patients with various types of malignant tumours at Sultanah Bahiyah Hospital, Alor Setar, Kedah, Malaysia, and 32 healthy control cases with non‐malignant disease collected from Universiti Sains Malaysia clinic, Penang, Malaysia. The antibodies against p53 protein in the serum samples were analysed using the commercial ELISA kit, Calbiochem® p53- ELISAPLUS. The results showed that the rate of anti-p53 antibodies in patients with various malignant tumours was 13 out of 49 (26.5 %), compared with only 2 out of 32 (6.25%) in healthy controls (p < 0.001). The sensitivity of this kit reached 28.6% and the specificity was 93.8%. In conclusion, these results suggest that the anti-p53 antibodies can be detected in different sera of malignant tumour patients and the ELISA kit is highly specific; nevertheless, its discrimination power is not perfect because of its low sensitivity to determine the anti-p53 antibodies.

Alterations of p53 gene in gallbladder cancer patients of North India.

Background: Mutations in p53 gene are found in a majority of human malignancies and usually occur in the exons 5, 6, 7 and 8. Mutated p53 protein is more stable and gets accumulated in the cells that induce the host to develop anti-p53 antibodies in sera of cancer patients. Aim: This study is aimed to observe the frequency and nature of mutations in exons 5-8 of p53 gene and to evaluate its correlation with prevalence of serum p53 antibodies in Indian patients with gallbladder cancer (GBC). Methods: Mutation studies were done in cancer tissues obtained from 62 patients with proven GBC (40 cytologically proven cases and 22 resected gallbladder cancer tissues) by polymerase chain reaction (PCR), restriction fragment length analysis (RFLP) and single strand conformation polymorphism (SSCP). Presence of serum p53 antibodies was determined using highly specific enzyme linked immunosorbent assay (ELISA) kit in 50 patients with GBC and 30 patients of cholelithiasis. Clinicopathologic characteristics of these patients were given attention. Results: Antibodies to p53 protein was present in the serum in 34% (17/50) of GBC patients and in 3.3% (1/30) patients with cholelithiasis (p<0.018). RFLP failed to detect common mutations in the exons 5- 8 of the p53 gene in 62 samples. Using SSCP analysis we could detect frameshift mutation in p53 gene in 2 of 22 (9.1%) GBC cases. Mutated samples were sequenced and found to have insertion of adenine at codon 271 (GAG) in exon 8 region. Conclusion: Our results show that 1/3rd of the north Indian patients with GBC have antibodies to p53 protein. The commonest identifiable alteration in the p53 gene was a frameshift mutation at codon 271.

Clinical Implication of Serum Anti-p53 Antibodies and Overexpression of p53 Protein in Myelodysplastic Syndrome and Acute Myeloid Leukemia / 대한혈액학회지

BACKGROUND: Alteration of p53 genes is rare, but it is related with progressive diseases in hematologic malignancies. The wild type of p53 protein is not usually detected, but mutated p53 proteins are accumulated in the nuclei of tumor cells, which can be detected by immunohistochemical stain. Anti-p53 antibodies are found in the sera of patients with various malignant tumors as the result of immune response to accumulation of mutated p53 protein in tumor cells. METHODS: The relation of serum anti-p53 anti-bodies and cellular p53 protein expression to clinical features in 36 cases of myelodysplastic syndrome (MDS) and 58 cases of acute myeloid leukemia (AML) was analyzed. Anti-p53 antibodies in the patient's sera were measured with enzyme immunoassay (p53 autoantibodies ELISA, Dianova, Hamburg, Germany). Immunohistochemical staining for p53 protein was performed with the streptavidin-biotin-peroxidase method (LSAB kit, DAKO, Denmark) and anti-p53 monoclonal antibody (DO-7, DAKO, Denmark) in paraffin embedded bone marrow biopsy sections. RESULTS: Anti-p53 antibodies were positive in one of 36 (2.7%) MDS cases, and in four of 58 (6.8%) AML cases. Overexpression of p53 protein was seen in five (13.9%) of MDS and in five (8.6%) of AML. Serum p53 antibodies and p53 protein overexpression were more frequently found in RAEB, RAEB-t and M6 sutypes. There was no relation between anti-p53 antibodies and p53 protein overexpression in MDS and AML. The patients of MDS with anti-p53 antibodies and p53 overexpression tended to have severe dyserythropoiesis, higher Bounemouth scores and poor prognostic karyotypes and associated with shorter survival duration as compared to those without anti-p53 antibodies and p53 overexpression (4+/-1 vs 26+/-4 months, P=0.007). The patients of AML with anti-p53 antibodies and p53 protein overexpression tended to have poor prognostic karyotypes and resistance to chemotherapy. CONCLUSION: Anti-p53 antibodies are rarely observed in sera of patients with MDS and AML, probably reflecting the relatively low incidence of p53 mutation. But the findings suggest that the presence of p53 alteration could be useful to predict resistance to chemotherapy and short survival in particular sutypes of MDS and AML.

Clinical Significance of p53 Protein Overexpression and Serum Anti-p53 Antibodies in Patients with Acute and Chronic Leukemia / 대한임상병리학회지

BACKGROUND: Mutations of p53 gene, rarely found in leukemia, result in accumulation of mutated p53 protein in the nuclei of tumor cells, which can be detected by immunohistochemistry. Lately, anti-p53 antibodies were found in the sera of patients who had solid tumors as a result of immune response to accumulation of mutated p53 protein in tumor cells. METHODS: For investigation of the clinical implication of cellular p53 protein overexpression and serum p53 antibody, immunohistochemical staining for p53 protein of B-5 fixed paraffin embedded bone marrow biopsies and enzyme immunoassay for the presence of anti-p53 antibodies of sera were performed simultanously; in 58 cases of AML, 34 cases of ALL, 11 cases of acute leukemia at relapse, 13 cases of CML in chronic phase and 5 cases of CLL. RESULTS: Overexpression of p53 protein was found in 9.1%(11/121) of all leukemias, with 8.6% of AML with predominance of M6, 5.9% of ALL, 18.2% of acute leukemia at relapse and 40% of CLL, but not found in CML. Serum anti-p53 antibodies were found in 5.8%(7/121) of all leukemias, with 6.9% of AML and 5.9% of ALL, 9.1% of acute leukemia at relapse, but not found in chronic leukemias. In AML and ALL, age, sex, hemoglobin, leukocyte count, platelet count and blast % were not related with p53 protein expression. The AML patients with p53 protein overexpression have more unfavorable karyotypes(complex karyotype, -5, -7 and t(10;11)), with shorter overall survival as compared to those without p53 protein overexpression. The presence of serum anti-p53 antibodies was not related with clinical findings of leukemias. CONCLUSIONS: The indications are that p53 gene alterations will contribute to disease development and progression in some specific patients with leukemia, due to the rare frequency of overexpression of p53 protein and serum anti-p53 antibodies in leukemia. Analysis of the p53 protein and serum p53 antibodies could screen p53 gene mutation and predict prognosis for some leukemias.