Touch Imprint Cytology: A Simple Method To Enrich Tumor Cells for Molecular Analysis
S Dogan, J Becker, N Rekhtman, LH Tang, K Nafa, M Ladanyi, D Klimstra. Memorial Sloan-Kettering Cancer Center, New York, NY
Background: A relative excess of non-neoplastic cells in frozen carcinoma samples is often a cause of false negative results in molecular assays, necessitating painstaking techniques to enrich for tumor cells, such as laser-capture microdissection. Given the lower cohesiveness of epithelial tumor cells compared to non-neoplastic epithelium and mesenchymal stroma, we hypothesized that tumor procurement by touch imprint would provide a simple, cost-effective method to obtain enriched neoplastic cells, compared to frozen whole tumor samples.
Design: Six carcinomas with known KRAS mutations were tested: 2 primary colorectal carcinomas (CRCs), 2 liver metastases of CRCs, 1 adenocarcinoma of lung, and 1 adenocarcinoma of pancreas. Two sets of 8 touch imprint slides and 1 frozen whole tumor sample (without major areas of normal tissue), both with a corresponding H&E stained slide, were obtained from each tumor. Stained slides were evaluated by light microscopy to estimate the percentage of carcinoma cells. Extraction of DNA from unstained touch imprints and whole frozen samples was followed by KRAS Exon 2 PCR and mutation analysis by direct sequencing. The relative proportion of the mutant allele in a given sample was determined by calculating the height ratio between the mutant and wild type peaks on the sequencing electropherogram. Based on the calculated values we determined the fold increase in the mutant-enriched DNA in touch imprints versus frozen samples.
Results: By light microscopy, touch imprints showed 1.4-3.5 fold (mean 2.1 fold) enrichment in neoplastic cells compared with the frozen tissue. The mutant to wild type peak height ratio was 1.4-7.1 fold (mean 3.3 fold) higher in all 6 touch imprint samples compared with the corresponding frozen sample. The average amount of extracted DNA ranged from 145ng-7ug per touch imprint slide.
Conclusions: Procurement of carcinomas by touch imprint is rapid, simple, and inexpensive, consistently provides a tumor-enriched sample, and is an excellent source of high quality tumor DNA. Touch imprints are a useful method of tumor procurement for molecular analysis, particularly if mutation analysis by direct sequencing is to be performed, as a tumor-enriched sample could compensate for the relatively low sensitivity of direct sequencing.
Monday, March 22, 2010 1:00 PM
Poster Session II # 241, Monday Afternoon