Development of a high‐throughput membrane‐array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers

Abstract

Recently several noninvasive methods have been employed to detect circulating tumor cells (CTCs) in cancer patients. In this study, we have developed a highly sensitive, high-throughput colorimetric membrane-array method that was designed to detect a panel of mRNA markers including human telomerase reverse transcriptase (hTERT), , carcinoembryonic antigen (CEA) and mucin 1 (MUC1) mRNA for the presence of CTCs in the peripheral blood of patients with gastric cancer (GC). Digoxigenin-labeled cDNA targets synthesized following total RNA isolation from peripheral blood samples of 64 GC patients and 80 healthy individuals were subjected to membrane-array hybridization. The results showed that membrane array could positively detect 5 cancer cells/ml of peripheral blood in GC cell-dilution experiments. The sensitivity, specificity and diagnostic accuracy for hTERT, CK-19, CEA and MUC1 mRNA ranged from 78.1% to 82.8%, 76.3% to 85% and 81.3% to 83.3%, respectively. Both CEA and MUC1 mRNA expression was correlated significantly with all malignant biological properties of GC, such as macroscopic type, depth of tumor invasion, lymph-node metastasis, TNM stage and coexisting distant metastasis (all p < 0.05). Using these 4 markers in combination, the sensitivity, specificity and diagnostic accuracy of membrane array were raised to 89.1%, 91.3% and 90.3%, respectively. The expression of all 4 mRNA markers was an independent predictor for postoperative recurrence/metastasis. GC patients with the expression of all the 4 mRNA markers showed a poorer survival rate than those without the expression of any 1 mRNA marker (p 5 0.0223). These findings demonstrated that our membrane-array method could detect CTCs in the circulation of GC patients with considerably high sensitivity and specificity. The identification of CTCs in the peripheral blood may be useful in the auxiliary cancer diagnostics or postoperative surveillance of GC patients for recurrence/metastasis. ' 2006 Wiley-Liss, Inc.Key words: circulating tumor cell; membrane array; gastric cancer; molecular diagnosisThe degrees of tumor penetration in the stomach wall and lymph node metastasis have been used for many decades as the 2 major prognostic determinants for gastric cancer (GC) patients. Unfortunately, despite informative staging of patients with GC, some patients with apparently localized disease at diagnosis will subsequently develop recurrent or metastatic diseases. 1-4 One of the major causes is attributed to the presence of disseminated tumor cells shed from the primary carcinoma into circulation, prior to or during surgery. Even in patients with early GC, blood-born metastasis occurs. 2,5,6 Recent attempts to improve staging include sensitive detection of disseminated tumor cells in the blood or lymph nodes by molecular approaches. Evidence increasingly clarifies that primary cancers begin shedding neoplastic cells into the circulation at an early stage. [7][8][9] It is the dissemination of cancer cells into circulation that is widely accepted as the main cause l...

Prognostic role of telomerase activity in gastric adenocarcinoma: A meta-analysis

“…TA is thought to be a critical step in the evolution of most tumor types (34)(35)(36)(37), including GC (38,39). Considerable clinical research has been conducted with the aim of assessing the correlation between TA expression and clinicopathological outcome in patients with GC, but the Size Histo T N M TNM >5 cm P/U T3/T4 positive/ positive/ TIII/IV (<5 cm) (W/M) (T1/T2) (negative) (negative) (TI/TII) Liu et al (27) 2008 English China 26 (14) --20 (6) 22 (4) 11 (15) 24 (2) Yang et al (15) 2001 English China 40 (2) 26 (14) 30 (10) 26 (14) 20 (20) -17 (23) Wu et al (17) 2006 English China 52 (12) 23 (29) 49 (3) 40 (12) 37 (15) 14 (38) 33 (19) Hu et al (23) 2004 English China 18 (17) 10 (8) 14 (4) 7 (11) 14 (4) 10 (8) -Zhan et al (31) 1999 English China 81 (13) 46 (35) 43 (38) 68 (13) 51 (30) -60 (21) Hu et al (16) 2009 English China 41 (5) 28 (13) 33 (8) 21 (20) 5 (36) 30 (11) -Wang et al (18) 2004 English China 32 (9) 19 (13) 25 (7) (21) 43 (1) 18 (26) 43 (1) Positive, patient have T...…”

“…Finally, 34 studies lacked usable data that correlated telomerase or hTERT with lymph node status or TNM stage to create 2x2 tables. Thus, a total of 18 eligible studies related to GC patients were finally identified in our meta-analysis with good agreement between reviewers (κ=0.73) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32) (Fig. 1).…”

Multiple Molecular Markers as Predictors of Colorectal Cancer in Patients with Normal Perioperative Serum Carcinoembryonic Antigen Levels

“…The procedure for the design and preparation of the membrane array was in accordance with our recent work (16). Oligonucleotide probes were then synthesized according to the design sequences, purified, and controlled before being grafted onto the substrates ( Table 2).…”

Newly Identified Biomarkers for Detecting Circulating Tumor Cells in Lung Adenocarcinoma

“…hTERT has been included in CTC detection in peripheral blood by multi-marker qPCR analysis. Its role in diagnosis of metastasis and assessment of therapy efficacy and prognosis (Wu et al 2006;Uen et al 2007) has been illustrated in advanced biliary tract malignancies (Leelawat et al 2012), breast cancer (Strati et al 2011) and digestive tract tumors. In this study, the sensitivity and specificity of hTERT were 56.7% and 100.0%, respectively, indicating that hTERT was a suitable marker for combined diagnosis of CTCs in lung adenocarcinoma.…”