A validated quantitative assay to detect occult micrometastases by reverse transcriptase-polymerase chain reaction of guanylyl cyclase C in patients with colorectal cancer
A validated quantitative assay to detect occult micrometastases by reverse transcriptase-polymerase chain reaction of guanylyl cyclase C in patients with colorectal cancer
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DOI：
10.1158/1078-0432.ccr-06-0865
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发表时间：
2006-08-01
影响因子：
11.5
通讯作者：
Waldman, Scott A.
中科院分区：
文献类型：
--
作者：
Schulz, Stephanie;Hyslop, Terry;Waldman, Scott A.
目的：鸟苷酸环化酶C（GCC）是一种细菌致腹泻肠毒素的受体，可能是检测结直肠癌分期患者隐匿性微转移的预后和预测标志物。然而，通过定量逆转录聚合酶链反应（qRT - PCR）对组织中GCC表达的定量尚未经过分析和临床病理验证。
实验设计：开发了一种利用RT - PCR定量组织中GCC mRNA的技术，并使用与GCC互补的RNA外部校准标准进行了验证。
结果：GCC qRT - PCR的反应效率>92%，变异系数<10%，线性动态范围跨越6个数量级，GCC cRNA的定量限>25拷贝。该检测证实41例患者的结直肠肿瘤中GCC mRNA过度表达，这与免疫组化定量的GCC蛋白增加相关。对无癌患者的164个淋巴结和15个有转移的淋巴结进行分析，确定了转移性疾病的阈值约为每微克总RNA含200个GCC mRNA拷贝，灵敏度为93%，特异性为97%。在23例患者中的6例（26%）经组织病理学检查无疾病的367个淋巴结中的76个（约21%）检测到高于该阈值的GCC mRNA，提示存在隐匿性微转移。
结论：使用外部校准标准通过RT - PCR定量组织中的GCC mRNA在分析上稳健且可重复，具有较高的临床病理灵敏度和特异性。在一项前瞻性试验中，这种经过验证的检测方法正应用于约10,000个淋巴结，以确定GCC qRT - PCR对结直肠癌患者分期的灵敏度。
Purpose: 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.Experimental Design: 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 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 similar to 200 GCC mRNA copies/mu g total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (similar to 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 similar to 10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.