[413] DNA Qualitative and Quantitative Comparison of Alcohol Fixed Cytology Versus Formalin Fixed Tissue for PNA Clamp Real-Time PCR Detection of KRAS, EGFR and BRAF

Frida Rosenblum, Keith Tomaszewicz, Maryann St. Cyr, Andrew Fischer, Ediz F Cosar, Elizabeth M Kurian, Lloyd Hutchinson. University of Massachusetts Medical Healthcare System, Worcester, MA

Background: Formalin-fixed paraffin embedded tissue (FFPE) is the FDA-approved substrate for polymerase chain reaction (PCR) detection of mutations in BRAF and KRAS, and is commonly used for EGFR testing. We evaluate the quality of the desired DNA required for adequate molecular analysis in alcohol fixed cytology specimens, compared to FFPE.
Design: At UMASS, 20 surgical FFPE (8/2009-7/2012) and 20 alcohol fixed cytology cell blocks (1/2011–6/2012) from multiple organ sites were analyzed. Tumor was macrodissected from slides. DNA was extracted (∼10ng) and tested by spectrophotometry, multiplex endpoint QC-DNA assay and PNA clamp real-time PCR for BRAF, KRAS and EGFR (see Table 1). Peripheral blood DNA was used as the quality gold standard. The delta CT was calculated by subtracting the patient DNA CT value from the peripheral blood DNA.
Results: We evaluated the following parameters: DNA yield, OD 260/280 ratio and maximum amplicon size. The range and average values are shown:

Table 1. DNA results.
ParameterCytologySurgical
EGFR96
EGFR+KRAS11
EGFR+KRAS+BRAF01
KRAS03
KRAS+BRAF109
Average DNA yield (range)1.48 (0.37-4.35)1.48 (0.10-4.275)
Average OD260/280 (range)1.92 (0.94-2.37)1.98 (1.66-2.46)
Average amplicon size (range)448 (QNS/100-500)444 (QNS/300-500)
Average Delta CT-1.5238-2.82812


A delta CT equal to (or greater than) zero is equivalent to (or better than) the optimal DNA quality of the control peripheral blood. FFPE samples showed a slightly higher average delta CT by 1.3 PCR cycles compared to cytology cell blocks suggesting higher DNA quality for cytology samples. The OD260/280 is a measure of DNA purity with an optimal range of 1.8-2.2. No obvious differences between fixation methods were observed for quantity, average amplicon length, or OD 260/280.
Conclusions: The DNA purity and quantity of surgical and cytology specimens appear to be equivalent. DNA fragmentation based on PCR amplicon size shows the two fixation methods are virtually the same. The average deltaCT was larger for surgical specimens. The latter may be the result of a small cohort or, if considered real, may be attributable to formalin induced cross-linking with resultant retardation of PCR amplification. These findings validate the use of alcohol fixed cytology as a suitable and comparable specimen type for molecular testing.
Category: Cytopathology

Tuesday, March 5, 2013 1:00 PM

Poster Session IV # 106, Tuesday Afternoon

 

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