Abstract
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. A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC. 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. 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.
Highlights
The sensitivity and specificity to detect tumor cells in lymph nodes employing Guanylyl cyclase C (GCC) reverse transcription-PCR (RT-PCR) was determined employing lymph nodes harboring metastases detected by histopathology and lymph nodes from patients with no evidence of colorectal cancer
It is tempting to speculate that those patients that harbor occult micrometastases detected by GCC quantitative reverse transcription-PCR (qRT-PCR) are at increased risk of developing recurrent colorectal cancer [23]
Recurrence in patients who are ostensibly disease-free presumably reflects the presence of occult micrometastases in tissues, including regional lymph nodes, undetected by current staging paradigms
Summary
Linear mixed model analysis of the relationship between Ct number and calibrator concentration (Fig. 1B) yielded an average intercept of 42.36 (95% confidence interval, 41.9442.79), an average slope of À3.53 (95% confidence interval, À3.62 to À3.44), and a mean amplification slope efficiency of 92%. GCC mRNA expression in 41 paired colorectal tumors and histologically normal adjacent mucosa was compared employing qRT-PCR and a GCC cRNA calibration curve. Omitting the ‘‘switched’’ lymph nodes yielded a threshold of 200 and a sensitivity and specificity of 100% and 97%, respectively Application of this threshold to 367 lymph nodes from colorectal cancer patients that were free of obvious metastases by histopathology suggests that f21% (76 of 367) of nodes or 26% (6 of 23) of patients might be harboring occult micrometastases by GCC qRT-PCR (Fig. 3A; Table 3). It is tempting to speculate that those patients that harbor occult micrometastases detected by GCC qRT-PCR are at increased risk of developing recurrent colorectal cancer [23]
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