Copy-number analysis of topoisomerase and thymidylate synthase genes in frozen and FFPE DNAs of colorectal cancers.
BACKGROUND
Archived formalin-fixed, paraffin-embedded specimens represent an important resource for pharmacogenomic analysis in retrospective clinical studies but the quality of results from formalin-fixed, paraffin-embedded samples is of concern due to the fact of the degradation of DNAs and RNAs from formalin-fixed, paraffin-embedded tissues.


METHODS
In the present study, we used DNA from fresh frozen as well as formalin-fixed, paraffin-embedded tumor to detect copy-number changes in colorectal cancer, and our data shows that formalin-fixed, paraffin-embedded DNAs were able to deliver reliable copy-number data, and that quantitative PCR had the ability to detect copy-number changes from deletion to amplification, with high concordance of copy-number calls among formalin-fixed, paraffin-embedded and frozen DNAs.


RESULTS
The amplification of topoisomerase I and deletion of thymidylate synthase were found in 23% (12/52) and 27% (14/52) of colorectal cancers, but EGF receptor amplification was not common (5/52, <10%). Among 52 colorectal cancers, 31 tumors were both topoisomerase I and thymidylate synthase diploid, which may have a worse outcome for tumor chemotherapy; and there were five tumors with favorable genomics (topoisomerase I amplification and thymidylate synthase deletion). Furthermore, topoisomerase I-amplified tumors had a two-times higher RNA level and a nearly twofold higher protein expression level than did the diploid tumors (p < 0.001 and 0.01, respectively), but there were no correlations between copy-number status and RNA or protein level for thymidylate synthase.


CONCLUSIONS
Our study suggests a potential pharmacogenomic influence of topoisomerase I copy-number alteration on its RNA/protein expressions, which could be reflected on tumor response to chemotherapy in human colorectal cancer.
Washington University School of Medicine, Department of Pathology & Immunology, Saint Louis, MO, USA.
Jinsheng
J
Yu
Ryan
R
Miller
Wanghai
W
Zhang
Mala
M
Sharma
Vicky
V
Holtschlag
Mark A
MA
Watson
Howard L
HL
McLeod
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D005260:Female; D005557:Formaldehyde / Q000032:analysis; D005615:Freezing; D018628:Gene Dosage / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D016612:Paraffin Embedding; D013940:Thymidylate Synthase / Q000032:analysis* / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods
D004273:DNA, Neoplasm; D005557:Formaldehyde; D013940:Thymidylate Synthase; D004264:DNA Topoisomerases, Type I