Molecular detection of tumor cells in regional lymph nodes and blood from patients undergoing surgery for non-small cell lung cancer (NSCLC)

Abstract

The spread of metastases to regional lymph nodes in patients undergoing surgery for non-small cell lung cancer (NSCLC) is routinely detected by histopathological examinations of tissue sections from retrieved regional lymph nodes. If the patient’s lymph nodes do not show presence of metastases they are defined as LN- patients. In many cases the LN- patients get the disease back and die of it. This may be due to insensitivity of the routine investigation to detect micrometastases. The aim of the study was to find a group of patients where lymph node metastasis is not shown by routine investigation but they still have occult metastases in regional lymph nodes using more sensitive method that can be used on whole lymph nodes. Besides this the other aim was to detect circulating tumor cells (CTCs), the cells that are shed from primary tumor in to the circulation system, from blood. The 5 out of 7 (CK19, SFTPA, SFTPB, SFTPC, EPCAM, CEACAM and PVA) mRNA molecules have been selected as potential markers for detection of micrometastases by RT-PCR. The markers were selected from scientific research articles and cancer databases followed by their optimization and validation to ensure pure PCR product. A calibrator cDNA was included in each plate to compare the results from different runs. Certain cell lines were cultured to select the calibrator expressing all marker mRNAs included in the study. The verification of the PCR product was done by agarose gel electrophoresis and DNA sequencing. In the initial phase 7 makers were quantified in 16 tumors, 16 normal lymph nodes and 12 blood samples to select best candidate markers. Some of these markers (CK19, CEACAM and PVA) have already been quantified in many previous studies but SFTPA and SFTPC are new markers and quantification method has been established for these markers in this study. The markers selected in initial phase were quantified in tumors, lymph nodes and blood samples for a cohort of 55 patients operated for non-small cell lung cancer (NSCLC) at Norwegian Radium Hospital. The relative quantification of each marker was determined using 2 ΔΔCt method.