title: A validated quantitative assay to detect occult micrometastases by reverse transcriptase-polymerase chain reaction of guanylyl cyclase C in patients with colorectal cancer.
abstract: OBJECTIVE
  Guanylyl cyclase C (GCC), a receptor for bacterial diarrheagenic enterotoxins, may be a prognostic and predictive marker to detect occult micrometastases in patients undergoing staging for colorectal cancer. However, quantification of GCC expression in tissues by the quantitative reverse transcription-PCR (qRT-PCR) has not undergone analytic and clinicopathologic validation.
  
  
  METHODS
  A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC.
  
  
  RESULTS
  GCC qRT-PCR exhibited reaction efficiencies >92%, coefficients of variations <5%, linearity >6 orders of magnitude, and a limit of quantification of >25 copies of GCC cRNA. This assay confirmed that GCC mRNA was overexpressed by colorectal tumors from 41 patients, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses obtained with 164 lymph nodes from patients free of cancer and 15 nodes harboring metastases established a threshold for metastatic disease of approximately 200 GCC mRNA copies/mug total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (approximately 21%) nodes free of disease by histopathology from 6 of 23 (26%) patients, suggesting the presence of occult micrometastases.
  
  
  CONCLUSIONS
  Quantifying GCC mRNA in tissues by RT-PCR employing external calibration standards is analytically robust and reproducible, with high clinicopathologic sensitivity and specificity. This validated assay is being applied to approximately 10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schulz
authors.forename: Terry
authors.initials: T
authors.lastname: Hyslop
authors.forename: Janis
authors.initials: J
authors.lastname: Haaf
authors.forename: Christine
authors.initials: C
authors.lastname: Bonaccorso
authors.forename: Karl
authors.initials: K
authors.lastname: Nielsen
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Witek
authors.forename: Ruth
authors.initials: R
authors.lastname: Birbe
authors.forename: Juan
authors.initials: J
authors.lastname: Palazzo
authors.forename: David
authors.initials: D
authors.lastname: Weinberg
authors.forename: Scott A
authors.initials: SA
authors.lastname: Waldman
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002138:Calibration; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000201:enzymology* / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D006162:Guanylate Cyclase / Q000235:genetics*; D006801:Humans; D008207:Lymphatic Metastasis / Q000175:diagnosis* / Q000235:genetics; D008297:Male; D008875:Middle Aged; D018075:RNA, Complementary / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D018075:RNA, Complementary; D012333:RNA, Messenger; D018000:Receptors, Peptide; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled