Melanoma antigen-encoding gene family member A1-6 and hTERT in the detection of circulating tumor cells following CD45− depletion and RNA extraction

Abstract

A total of 76 blood samples from patients without malignant disease and 107 blood samples from patients with malignant disease were investigated for the presence of circulating tumor cells (CTCs). To detect CTCs, hematopoietic cells were removed from the blood samples and different RNA extraction methods were used to amplify the melanoma antigen-encoding gene family member A1-family member A6 (MAGE A1-6) and the human telomerase reverse transcriptase (hTERT) gene as potential CTC markers. Comparison between four methods for extracting RNA from the blood was performed. The samples were enriched by cluster of differentiation 45 (CD45) antibody capturing, and the reverse transcription-quantitative polymerase chain reaction was used to amplify the MAGE A1-6 and hTERT genes. MAGE A1-6 and hTERT gene expression levels were also evaluated in 14 cancer cell lines, and the MAGE A1-6 and hTERT expression levels were 85.7 and 100%, respectively. The RNeasy method demonstrated the most sensitivity in the SNU1 cells mixed with blood, although the differences between methods was non-significant. The positive expression levels of MAGE A1-6 and hTERT was 11.8% in the control group and 58.9% in those with malignant disease. In the 70 patients with colorectal cancer, positive expression levels of MAGE A1-6 or hTERT were significantly higher in stages T3 and T4 compared with in stages T1 and T2. The CTC detection method involving CD45 antibody capture, RNA extraction and MAGE A1-6 and hTERT reverse transcription resulted in good rates of sensitivity and specificity. Thus, the present study concluded that MAGE A1-6 and hTERT genes may be potential and practical markers for CTCs in a clinical setting.

Detection of circulating tumor cells in patients with breast cancer using the conditionally reprogrammed cell culture method and reverse transcription‑PCR of hTERT and MAGE A1‑6

“…reverse transcriptase (hTERT) gene is also known to be expressed in most cancer cells (12). In our previous study, we used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of the MAGE A1-6 and the hTERT genes to detect CTCs in blood, and our results showed good sensitivity and specificity (13). It has been suggested that ex vivo culture and characterization of CTCs may be utilized in the study of cancer metastasis and patient-derived tumor models (7,9), and in recent years, several studies have shown the results of CTCs culture in different culture conditions (7,(14)(15)(16)(17).…”

“…To detect CTCs, we collected peripheral blood mononuclear cells (PBMC) after red blood cells (RBCs) lysis from the blood. CTCs were isolated by negative immunomagnetic separation technology (20) using the CD45 antibody capture system as previously described (13). Briefly, the RBCs were lysed with RBC lysis buffer (Roche Diagnostics GmbH).…”

“…RNA extracted from PBMC was then amplified for the hTERT and the MAGE A1-6 genes following the method described in previous studies (13,21). Briefly, extracted RNA was reverse transcribed using the ImProm-II Reverse Transcription System (Promega Corporation) according to the manufacturer's protocol.…”

Tissue Expression of Melanoma-associated Antigen A6 and Clinical Characteristics of Gastric Cancer

“…MAGEA6 is normally expressed in the testis and specifically overexpressed in cancer cells; however, its physiological and pathological functions are largely unknown (9,10,22). Moreover, MAGEA6 functions as a cancerspecific ubiquitin ligase which ubiquitinates and degrades AMP-activated protein kinase (AMPK), which plays a pivotal role in cell metabolism (11,23).…”

“…MAGEA6 resides on the human X chromosome and encodes a cancer-testis antigen which functions as a ubiquitin ligase physiologically (7,8). MAGEA6 is overexpressed in melanomas, head and neck squamous cell carcinomas, lung carcinomas, and breast carcinomas (9)(10)(11). However, to the best of our knowledge, there are no reports focusing on MAGEA6 expression in gastroenterological malignancies, including GC.…”