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

Molecular detection of circulating thyroid specific transcripts [TSHR/Tg-mRNAs] in thyroid cancer patients: their diagnostic significance

Thyroid cancer is the most prevalent endocrine malignancy. The preoperative diagnosis of differentiated thyroid cancer [DTC] that relies solely on fine-needle aspiration [FNAC] biopsy, sometimes possesses conflicting results. New molecular markers for thyroid cancer have been investigated with most of them based on the detection in thyroid nodules or tumor tissue specimens. Recently, it was possible to detect thyroid cancer cells in the circulation by measuring the mRNA of thyroid specific genes. Among these, thyroglobulin and more recently thyroid stimulating hormone receptor mRNAs, TSHR/Tg-mRNAs in peripheral blood might serve as cancer-specific markers. These have become promising new circulating markers for thyroid cancer. The purpose of this study is to assess TSHR/Tg-mRNAs as diagnostic molecular markers for thyroid cancer and if they can be used preoperatively in synergy with FNAC. This study was performed on 60 subjects; 20 healthy volunteers and 40 patients; including 16 patients with benign thyroid diseases, 24 patients with thyroid cancer; 18 patients with newly diagnosed [DTC] and 6 patients with recurrent thyroid cancer. Diagnosis of cancer was based on FNAC and histopathology of surgical specimens. All subjects had TSHR/Tg-mRNAs in peripheral blood measured by reverse transcriptase [RT]-PCR. Based on cytology/pathology; 18 patients had newly diagnosed DTC and 11 had benign thyroid disease. Preoperative FNAC was performed on 29 of 40 patients; FNAC was diagnostic in 11/18 of malignant lesions [61.1%], in 8/11 of benign lesions [72.7%], while 10/29 [34.5%] were indeterminate. TSHR/Tg-mRNAs correctly diagnosed DTC in 20/24 and 19/24 [sensitivity 83.3% and 79.1%] and benign disease in 14/16 and 13/16 [specificity 87.5% and 81.3%], respectively. With indeterminate FNA, TSHR/Tg-mRNAs correctly diagnosed DTC [follicular type] in 5/7 and benign disease in 2/3 [combined sensitivity 71.4%; specificity 66.7%]. There was high concordance between RT-PCR results for TSHR-mRNA and Tg-mRNA. Of the controls 19/20 [95%] and 16/20 [80%] were negative for both TSHR- and Tg-mRNAs. With the use of a carefully selected primer pair and qualitative RT-PCR; our results indicate that TSHR/Tg-mRNAs in peripheral blood are both equally sensitive and specific markers for detection of thyroid cancer cells. Combining TSHR/Tg-mRNAs and FNAC and ultrasound enhances the preoperative detection of cancer in patients with thyroid nodules, reducing unnecessary surgeries and correctly classified most follicular cancers and could have spared surgery in patients with benign disease

Detection of circulating tumor cells by nested RT-PCR targeting EGFR/CEA/CK20mRNAs in colorectal carcinoma patients

EGFR is involved in the epidermal growth factors pathway that regulates cellular processes and is associated with the development of many types of cancer including colorectal cancer. Molecular methods with high sensitivity such as nested polymerase chain reaction [PCR] based assays have been used to search for tumor cell specific markers. This study aimed to detect the circulating EGFRmRNA expressing tumor cells and its diagnostic value in colorectal cancer compared with that of known markers of circulating cancer cells CEA and CK20. This study included 36 patients diagnosed as having colon cancer of different stages and 18 matched healthy controls. The staging was carried out according to the TNM classification. We used nested RT-PCR-based reverse transcription PCR assay for the detection of circulating cancer cells in the peripheral blood. The blood samples from the colon cancer patients showed detection of EGFR in 15/36 patients [41.7%]; CEAmRNA in 22/36 patients [61.1%] and CK20mRNA in 24/36 patients [66.7%]. No evidence of EGFR mRNA expression in any of the samples used as controls. 3/18 [16.7%] and 4/18 [22.2%] of healthy controls gave a positive result of CEA/CK20mRNAs. There was a statistically significant difference in the prevalence of EGFR/CEA and CK20mRNAs expression between the early disease group [stage I and II] and the advanced disease group [stage III and IV] [P < 0.01]. Colon cancer patients with a high level of serum CEA exhibited detectable concentrations of EGFR and CEA and CK20mRNAs more often than those with a low serum CEA level, there is significant difference [P < 0.01]. EGFR assay might represent a suitable marker for detection of circulating tumor cells in colon cancer patients. CEA and CK20mRNAs are significantly more frequently detected in colon cancer patients than in healthy controls supports the hypothesis that they are promising complementary markers for CRC diagnosis. The assessment of multiple molecular tumor markers improved the sensitivity in detecting circulating tumor cells but due to limited specificity; identification and validation of genes and proteins implicated in metastatic processes need to be further investigated