Analytical performance of a qRT-PCR assay to detect guanylyl cyclase C in FFPE lymph nodes of patients with colon cancer.
Up to 30% of patients with stage II (pN0) colon cancer develop recurrences, suggesting that the presence of lymph node (LN) metastases escaped detection at histopathologic staging. A simple way to overcome this limitation and to improve staging accuracy is to use reverse transcription-polymerase chain reaction (RT-PCR) to examine a larger fraction or an entire specimen. The Guanylyl cyclase C (GCC) gene is uniquely expressed in apical cells of the gastrointestinal tract. Its expression in colon cancer cells and metastases is conserved. Therefore, detection of GCC mRNA in LNs has been shown to be indicative of the presence of colon cancer metastases. As the current processing of LNs involves formalin fixation and paraffin embedding, we developed a method for extracting RNA from formalin-fixed paraffin-embedded LN specimens and detecting GCC mRNA by quantitative RT-PCR. The assay has a dynamic range of 5 logs, an average amplification efficiency of 98.4% (95% confidence interval, 96.6-100.3), a reaction linearity of 0.998 (95% confidence interval, 0.997-0.999), and also intraplate and interplate CVs of <1% and <5%, respectively. The test specificity was 98% with LNs collected from patients affected by conditions other than colon cancer (n=380). Sensitivity was 97% for patients with stage III colon cancer (n=34), whereas 35% of patients with stages I and II disease (n=51) had at least 1 GCC mRNA-positive LN. The high specificity of GCC mRNA suggests that routine utilization of the quantitative RT-PCR test has the potential to improve the detection of colon cancer metastases in LNs.
Beaulieu
Martin
M
DiagnoCure Inc., 2050 René-Lévesque Blvd. West, Québec, QC, Canada.
Desaulniers
Marie
M
Bertrand
Nicolas
N
Deschesnes
Réna G
RG
Beaudry
Guillaume
G
Garon
Geneviève
G
Haince
Jean-François
JF
Houde
Michel
M
Holzer
Timothy J
TJ
D003110:Colonic Neoplasms / Q000175:diagnosis* / Q000556:secondary*; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000235:genetics*; D006801:Humans; D008198:Lymph Nodes / Q000473:pathology*; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods*; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
D005404:Fixatives; D018000:Receptors, Peptide; D005557:Formaldehyde; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled