Detection of CCND1 amplification using laser capture microdissection coupled with real-time polymerase chain reaction...

Several methods have been used to detect CCND1 amplification or overexpression in esophageal squamous cell carcinoma (ESCC), but problems remain, associated with heterogeneity of tumor tissue and quantification of gene copies. Laser capture microdissection coupled with real-time polymerase chain reaction (PCR) is a reliable method for the molecular analysis of gene profiles in specific tissues. All 35 specimens of ESCC studied were paraffin-embedded, cut into tissue slides, and stained by hematoxylin-eosin. The pure ESCC cell and normal squamous epithelia populations were separated by LCM and then genomic DNA was extracted from the dissected cells. CCND1 amplification was detected with real-time FQ-PCR and with PCR. Amplification was calculated by the formula X = 2(-DeltaDeltaCt) and R = (CCND1/ACTB) CANCER/(CCND1/ACTB) NORMAL. Twenty (57%) of primary ESCC cancer cell groups had a detectable CCND1 amplification (range, 2.06-fold to 25.9-fold) with real-time FQ-PCR, but only 2 of 15 primary ESCC cancer cell groups had detectable CCND1 amplification by PCR. CCND1 amplification was not correlated with age, sex, size of tumor, histological grade, and lymph node metastasis. In conclusion, LCM coupled with real-time fluorescence quantitative-PCR technique is more precise than PCR for the identifying amplified oncogenes; The role of CCND1 amplification in ESCC development and progression needs more extensive study.