Prognostic Value of Molecular Detection of Lymph Node Metastases After Curative Resection of Stage II Colon Cancer: A Systematic Pooled Data Analysis.

BACKGROUND: We aimed to clarify the prognostic value of guanylyl cyclase C (GCC) lymph node ratio (LNR) status as a predictor of recurrence in untreated stage IIA colon cancer on the basis of pooled individual data from previous studies. METHODS: Patients were classified according to predefined GCC LNR risk groups (low, LNR ≤ 0.1; intermediate, 0.1 < LNR ≤ 0.2; high, LNR > 0.2). Outcomes included time to recurrence, disease-free survival, and overall survival. Stratified log-rank tests and multivariate Cox models assessed the association between outcomes and GCC lymph node status. RESULTS: The final data set contained 553 patients with stage IIA colon cancer with a median of 18 lymph nodes examined after resection; 65 patients (11.8%) had recurrence. Overall, 109 patients (19.7%) were classified high risk on the basis of GCC LNR. In multivariate analysis, high GCC LNR value (> 0.2) was a significant predictor of cancer recurrence (hazard ratio [HR], 3.18; 95% confidence interval [CI], 1.77-5.71; P < .001) and lower disease-free survival (HR, 2.40; 95% CI, 1.60-3.62; P < .001) and overall survival (HR, 2.12; 95% CI, 1.35-3.33; P = .001). CONCLUSION: Patients considered at high risk on the basis of their GCC LNR status have significantly inferior outcomes compared to those with low GCC LNR values, particularly among those traditionally considered to be at low risk for recurrence.

Molecular testing for lymph node metastases as a determinant of colon cancer recurrence: results from a retrospective multicenter study.

PURPOSE: Recurrence risk assessment to make treatment decisions for early-stage colon cancer patients is a major unmet medical need. The aim of this retrospective multicenter study was to evaluate the clinical utility of guanylyl cyclase C (GCC) mRNA levels in lymph nodes on colon cancer recurrence. METHODS: The proportion of lymph nodes with GCC-positive mRNA (LNR) was evaluated in 463 untreated T3N0 patients, blinded to clinical outcomes. One site's (n = 97) tissue grossing method precluded appropriate lymph node assessment resulting in post hoc exclusion. Cox regression models tested the relationship between GCC and the primary endpoint of time to recurrence. Assay methods, primary analyses, and cut points were all prespecified. RESULTS: Final dataset contained 366 patients, 38 (10%) of whom had recurrence. Presence of four or more GCC-positive lymph nodes was significantly associated with risk of recurrence [hazard ratio (HR) = 2.46, 95% confidence interval (CI), 1.07-5.69, P = 0.035], whereas binary GCC LNR risk class (HR = 1.87, 95% CI, 0.99-3.54, P = 0.054) and mismatch repair (MMR) status (HR = 0.77, 95% CI, 0.36-1.62, P = 0.49) were not. In a secondary analysis using a 3-level GCC LNR risk group classification of high (LNR > 0.20), intermediate (0.10 < LNR ≤ 0.20), and low (LNR ≤ 0.10), high-risk patients had a 2.5 times higher recurrence risk compared with low-risk patients (HR = 2.53, 95% CI, 1.24-5.17, P = 0.011). CONCLUSIONS: GCC status is a promising prognostic factor independent of traditional histopathology risk factors in a contemporary population of patients with stage IIa colon cancer not treated with adjuvant therapy, but GCC determination must be performed with methodology adapted to the tissue procurement and fixation technique.

Evaluation of guanylyl cyclase C lymph node status for colon cancer staging and prognosis.

PURPOSE: The prognostic significance of guanylyl cyclase C (GCC) gene expression in lymph nodes (LNs) was evaluated in patients with stage II colon cancer who were not treated with adjuvant chemotherapy. We report a planned analysis performed on 241 patients. METHODS: GCC mRNA was quantified by RT-qPCR using formalin-fixed LN tissues from patients with untreated stage II colon cancer who were diagnosed from 1999-2006 with at least ten LNs examined and blinded to clinical outcomes. Lymph node ratio (LNR) is the number of GCC-positive nodes divided by total number of informative LNs. Risk categories of low (0-0.1) and high (>0.1) for LNR were chosen by significance using Cox regression models. The data were tested for association with time to recurrence. RESULTS: Twenty-nine patients (12%) had a disease recurrence or cancer death. The LNR significantly predicted higher recurrence risk for 84 patients (34.9%) classified as high risk (hazard ratio (HR), 2.38; P=0.02). The estimated 5-year recurrence rates were 10% and 27% for the low- and high-risk groups, respectively. After adjusting for age, T stage, number of nodes assessed, and MMR status, a significant association remained (HR, 2.61; P=0.02). In a subset of patients (n=181) with T3 tumor, ≥12 nodes examined and negative margins, a significant association between the GCC LNR and recurrence risk also was observed (HR, 5.06; P=0.003). CONCLUSIONS: Our preliminary results suggest that detection of GCC mRNA in LNs is associated with risk of disease recurrence in patients with untreated stage II colon cancer. A larger validation study is ongoing.

Comparison of histopathology and RT-qPCR amplification of guanylyl cyclase C for detection of colon cancer metastases in lymph nodes.

AIMS: In colorectal cancer (CRC), the presence of lymph node (LN) metastases is an important prognostic factor. Approximately 20% of patients diagnosed as having node-negative (pN0) CRC will relapse. Pathological nodal stage misclassification due to sampling error resulting from the small volume of tissue tested has been proposed to explain this recurrence rate in pN0 patients. The authors compared the assessment of node positivity by histopathology (HP) with a molecular method which can accommodate larger tissue volumes. METHODS: Detection rate of guanylyl cyclase C (GCC) mRNA was determined in 1,495 LNs from 99 CRC patients. Using a subset of 647 LNs, multiple levels of HP analysis were compared with GCC mRNA molecular detection. Finally, clinicopathological factors were correlated with the molecular detection of GCC and clinical outcome in 123 patients with pN0 colon cancer. RESULTS: GCC mRNA was detected in 8.0% of the 560 nodes initially identified as HP-negative, whereas two repeat HP examinations detected 3.0% of these cases. In HP-positive LNs, the GCC mRNA detection rate was 90% (78/87) when half-LN were tested. Testing the entire LN remaining after HP by GCC increased the detection rate of HP-positive LNs to 95% (p=0.027). In comparison, 75% (65/87) and 92% (80/87) of the LN positive by clinical HP remained positive when one or two subsequent sections were examined by HP. Finally, patients with pN0 disease who were GCC-positive exhibited an earlier time of recurrence (hazard ratio, 3.54; 95% CI 1.40 to 8.98; p=0.0077). CONCLUSIONS: Molecular detection of tumour cells in LNs may have prognostic value in identifying patients diagnosed as having pN0 colon cancer who will relapse following surgery.

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.

Identification of PCA3 (DD3) in prostatic carcinoma by in situ hybridization.

PCA3 is a specific marker of prostatic carcinoma. However, PCA3 has been detected only at RNA level and a corresponding PCA3 protein has never been identified. The aim of this study was to develop a technique capable of detecting PCA3 RNA on histology sections and to assess the cellular location of the molecule. Forty-eight formalin-fixed paraffin-embedded blocks of prostatectomy specimens were selected for PCA3 detection by in situ hybridization by both radioactive and chromogenic methods. Of the 48 sections, 28 contained prostatic adenocarcinoma and 20 had benign tissue located distant from the tumor. Using the radioactive detection method, 26 of 28 available cases (93%) of cancers presented at least focal cytoplasmic PCA3 expression. The benign glands located in proximity of the cancer presented PCA3 expression in eight (29%) cases, whereas those situated distant to the tumor showed focal expression in 2 of 20 (10%) cases only. High-grade prostatic intraepithelial neoplasia (HGPIN) expressed PCA3 in 25 of 26 (96%) cases. With the chromogenic detection method, 22 of the 24 interpretable cases (92%) of cancers had at least focal cytoplasmic staining. Benign glands located close to neoplastic glands expressed PCA3 in 8 (33%) cases, but none of those distant to the tumor expressed the marker. HGPIN was positive in 17 of 24 (71%) cases. The sensitivity, specificity, positive predictive value and negative predictive value for the detection of cancer were 93, 79, 71 and 95% for the radioactive detective method and 92, 80, 71 and 95% for the chromogenic detection method, respectively. Our study shows that PCA3 RNA is expressed by most prostate cancers and HGPIN. Normal glands rarely express the marker, except those located in immediate proximity of neoplastic glands, suggesting the presence of precursor molecular changes.