Multiple molecular markers MAGE-1, MAGE-3 and AFP mRNAs expression nested PCR assay for sensitive and specific detection of circulating hepatoma cells: Enhanced detection of hepatocellular carcinoma

Hepatocellular Carcinoma is a multifactorial, multistep and complex process. Its prognosis is poor and early diagnosis and monitoring of metastasis of HCC is of utmost importance. Circulating alpha-fetoprotien mRNA has been proposed as a marker of HCC cells disseminated into the circulation but the specificity of this molecular marker and its correlation with the main HCC clinico-pathological parameters remain controversial. In recent years; several different multi-marker assays have been developed for the detection of hepatoma cells in the peripheral blood of patients with HCC. In this study 58 patients and 15 matched healthy volunteers were included; the patients were divided into three groups; group A: patients with primary HCC [n = 32], group B: patients with cirrhosis with no evidence of HCC [n = 12], group C: patients with metastatic cancer in liver [n = 14]. Group D: 15 healthy volunteers age and sex matched. The staging of HCC was carried out according to the Tumor/Node/Metastasis [TNM] classification. Peripheral blood samples were obtained from all subjects; MAGE-1 and MAGE-3 and AFP mRNAs were detected by nested RT-PCR. The positive rates of MAGE-1, MAGE-3 and AFP mRNAs were 18/32 [56.3%], 15/32 [46.9%] and 19/32 [59.4%] respectively in the primary HCC patients. In the cirrhotic group only 4/12 [33.3%]patients were positive for AFP mRNA, whereas in the metastatic group 5/14 [35.7%] and 4/14 [28.6%] were positive to MAGE-1 and MAGE-3 mRNAs respectively. MAGE-1 and MAGE-3 mRNAs were correlated with TNM clinical stages; tumor number and tumor size [p < 0.05]. Our results indicate that a multi-marker nested RT-PCR assay with cancer-specific markers such as MAGE-1 and MAGE-3 in combination with a hepatocyte-specific AFP marker may be a promising diagnostic tool for monitoring hepatocellular carcinoma patients. Nested PCR exhibits higher sensitivity, stronger specificity and lower false-positive occurrence as compared to single RT

Development, characterization & potential clinical use of monoclonal antibodies against human alphafoetoprotein.

Four anti-alphafoetoprotein (AFP) monoclonal antibodies (MAb) were raised in the laboratory and characterized using ELISA and immunodot assays. The affinity constants of the MAbs, analysed by scatchard plots, ranged from 3.1 X 10(8) to 2.15 X 10(9) M/l. Epitope analysis using competition RIA indicated that MAb 5E2D7 and 5E2E3 recognize different epitopes on AFP. This combination was used to set up a two site sandwich ELISA with HRPO conjugated 5E2D7. AFP values in sera of hepatocellular carcinoma patients and pregnant women were quantitated using sandwich ELISA. The anti-AFP MAbs showed strong reactivity when tested on hepatoma tissue sections using immunoperoxidase technique.