Prognostic significance of free peritoneal tumor cells in the peritoneal cavity before and after neoadjuvant chemotherapy in patients with gastric carcinoma undergoing potentially curative resection.

BACKGROUND: Free peritoneal tumor cells (FPTCs) are an independent prognostic factor in patients undergoing curative resection for gastric carcinoma. Whether neoadjuvant chemotherapy (NAC) can eliminate FPTCs in the peritoneal lavage remains unclear. The aim of the study was to determine the effect of NAC on FPTCs. METHODS: From 1994 to 2000, data from a total of 61 patients with resectable gastric cancer were analyzed. Peritoneal cytology was performed before NAC at laparoscopy and at tumor resection. A minimum of 6 weeks of NAC, consisting of cisplatin, folinic acid, and fluorouracil, was administered. FPTCs were detected immunohistochemically with Ber-EP4 antibody. RESULTS: No FPTCs could be detected in 42 patients (69%), compared to 19 (31%) with FPTCs before NAC. During chemotherapy, 10 (24%) of 42 patients developed FPTCs, and 7 (37%) of 19 patients reverted from positive to negative. Patients who became FPTC negative (n = 7) showed an improved median survival (36.1 months) and a longer 2-year survival (71.4%) compared to FPTC-positive patients before and after NAC (n = 12), with a median survival of 9.2 months and a 2-year survival rate of 25%. In contrast, patients who reverted from FPTC negative to positive during NAC (n = 10) had a median survival of 18.5 months and a 2-year survival of only 20%. Multivariate analysis identified ypN category and FPTC change as independent prognostic factors. CONCLUSIONS: NAC for patients with positive cytology could lead to FPTC negativity in a subset of patients and improve their prognosis. However, NAC might be a risky strategy for almost one-quarter of patients whose disease develops positive cytology.

Reduced lymph node yield in rectal carcinoma specimen after neoadjuvant radiochemotherapy has no prognostic relevance.

BACKGROUND: In colorectal surgery UICC/AJCC criteria require a yield of 12 or more locoregional lymph nodes for adequate staging. Neoadjuvant radiochemotherapy for rectal carcinoma reduces the number of lymph nodes in the resection specimen; the prognostic impact of this reduced lymph node yield has not been determined. METHODS: One hundred two patients with uT3 rectal carcinoma who were receiving neoadjuvant radiochemotherapy were compared with 114 patients with uT3 rectal carcinoma who were receiving primary surgery followed by adjuvant radiochemotherapy. Total lymph node yield and number of tumor-positive lymph nodes were determined and correlated with survival. RESULTS: After neoadjuvant radiochemotherapy both total lymph node yield (12.9 vs. 21.4, p < 0.0001) and number of tumor-positive lymph nodes (1.0 vs. 2.3, p = 0.014) were significantly lower than after primary surgery plus adjuvant radiochemotherapy. Reduced total lymph node yield in neoadjuvantly treated patients had no prognostic impact, with overall survival of patients with 12 or more lymph nodes the same as that of patients with less than 12 lymph nodes. Overall survival of neoadjuvantly treated patients was significantly influenced by the number of tumor-positive lymph nodes with 5-year-survival rates of 88, 63, and 39% for 0, 1-3, and more than 3 positive lymph nodes (p < 0.0001). CONCLUSION: The UICC/AJCC criterion of a total lymph node yield of 12 or more should be revised for rectal carcinoma patients.

Prognosis of patients with colorectal cancer is associated with lymph node ratio: a single-center analysis of 3,026 patients over a 25-year time period.

OBJECTIVE: We examined the prognostic impact of lymph node ratio (relation of tumor-infiltrated to resected lymph nodes) in comparison to the pN category and other prognostic factors in patients with colorectal cancer. SUMMARY BACKGROUND DATA: Although the high prognostic impact of lymph node metastases and the total number of lymph nodes to be resected are well established, studies still report large differences in lymph node numbers. The lymph node ratios relevant for prognosis are not clearly defined and not routinely reported. METHODS: We analyzed the clinical and histopathological data of 3026 patients with colorectal cancer at a single surgical center over a 25-year time period (1982-2006). RESULTS: One thousand seven hundred sixty-three colon and 1263 rectal carcinomas were documented. The rate of curative resection was 77.4% and the median number of resected lymph nodes was 16. The optimal cut-off values for prognostic differentiation of LNRs were statistically calculated as 0.17, 0.41, and 0.69. The 5-year overall survival of patients without lymph node metastases was 87%. Patients with lymph node metastases had 5-year overall survival rates of 60.6%, 34.4%, 17.6%, and 5.3% with increasing LNRs (P < 0.001). Multivariate survival analysis identified both the LNR and the pN category, the number of resected lymph nodes, the patient's age, the tumor location (colon vs. rectum), the pT category, the pM status, the R status, the tumor grade, and the year of operation as independent prognostic factors. The LNR had better prognostic value than the pN category (P < 0.05). The analysis of the subgroup of patients separated into colon and rectal cancer patients confirmed the identified LNRs as independent prognostic factors (P < 0.001). CONCLUSIONS: The defined cut-off values of LNRs were strong independent prognostic factors for colorectal cancer patients and should be calculated for risk group stratification.

Microarray-based prediction of tumor response to neoadjuvant radiochemotherapy of patients with locally advanced rectal cancer.

BACKGROUND & AIMS: Neoadjuvant chemoradiotherapy has become a standard treatment of locally advanced rectal carcinomas, even though the responsiveness varies from complete response to resistance. The aim of the study was to evaluate the capacity of gene expression signatures to identify responders and nonresponders pretherapeutically. METHODS: By using microarray technology we generated gene expression profiles of 43 biopsy specimens of locally advanced rectal carcinomas. The transcription profile then was compared with histopathologic response and used to identify a set of genes discriminating responders from nonresponders. RESULTS: We identified a gene expression signature of 42 genes, mostly encoding proteins that either play a role in the nucleus, such as the transcription factor ETS2, or are associated with transport function, such as the solute carrier SLC35E1, or the regulation of apoptosis, such as caspase-1. In leave-one-out cross-validation the correct classification of a responder was 71%, the specificity of the analysis for a correct classification of a nonresponder was 86%. By applying an additional statistical method of 200 successive splittings into training and test data sets we generated an individual prediction accuracy measure for each predicted response. CONCLUSIONS: Our study shows that pretherapeutic prediction of response of rectal carcinomas to neoadjuvant chemoradiotherapy is feasible, and may represent a new valuable and practical tool of therapeutic stratification.

Correlation of CK-20-positive cells in peripheral venous blood with serum CEA levels in patients with colorectal carcinoma.

Tumor cell dissemination appears to be an early event in tumor progression, and tumor cells can be detected in peripheral venous blood at the time of the operation. Although cytokeratin 20 (CK-20) is not specifically expressed by colorectal carcinomas, it represents a widely used marker for the detection of colorectal tumor cells. We used the combination of density centrifugation and CK-20 real-time reverse transcription polymerase chain reaction to detect CK-20-positive cells in the peripheral venous blood of 37 patients with colorectal carcinoma. Detection rates were compared to serum levels of the tumor markers carcinoembryonic antigen (CEA), carbohydrate antigen CA 19-9, and cancer antigen CA 125. The prognostic impact was assessed by the overall survival and by univariate and multivariate analysis. Overall, CK-20-positive cells in peripheral venous blood were detected in 11 of 37 (29.7%) patients. CK-20-positive patients showed a significantly higher mean serum CEA level (90.3 ng/ml) than the 4.1 ng/ml found in the CK-20-negative group (p = 0.03). CEA levels also correlated with CK-20 copy numbers. No significant correlation was observed for CA 19-9 or CA 125. CK-20-negative patients showed a trend toward better survival (p = 0.08). In the univariate analysis, CA 19-9, CEA, tumor size, lymph node status, grading, the presence of distant metastases, and resection status reached significant prognostic levels, whereas the detection of CK-20-positive cells showed only a prognostic trend (p = 0.06). Multivariate analysis failed to identify independent prognostic parameters. Here we report the correlation of CK-20-positive cells in peripheral venous blood with the serum CEA level of patients with colorectal cancer, which may represent a potential marker of the tumor load.

Thymidine phosphorylase, dihydropyrimidine dehydrogenase and thymidylate synthase mRNA expression in primary colorectal tumors-correlation to tumor histopathology and clinical follow-up.

AIM: Evaluation of thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and thymidylate synthase (TS) mRNA levels in formalin-fixed, and paraffin-embedded tissues of patients with colorectal cancer and their prognostic and/or predictive value. MATERIALS AND METHODS: Total RNA was isolated from microdissected, formalin-fixed, and paraffin-embedded tissues (controls and tumor) and subjected to quantitative RT-PCR (QRT-PCR) in the LightCycler system. Resulting mRNA levels correlated to tumor histology (n=102) and the clinical follow-up in patients treated by resection alone (n=40) and by resection plus adjuvant 5-FU-based chemotherapy (n=52). RESULTS: Correlation to histopathological parameters revealed a significant association between tumor stage and the TP mRNA level (T and N category and UICC) as well as the TP:DPD (T and N category and UICC) and TS:DPD (T category) ratio. In addition, tumor differentiation was correlated to the TS mRNA level and the TS:DPD ratio. Finally, the TS:DPD ratio was a prognostic marker for overall survival in patients receiving resection alone (p=0.032). Moreover, a high TP:DPD ratio (>8.1; p=0.002) and, marginally, low DPD (<8.2; p=0.05) mRNA levels significantly correlated to disease-free survival. CONCLUSION: We present a novel, standardized approach for TP, DPD, and TS mRNA quantification in archival tissue specimens and applied this to a large series of primary colorectal tumors. Correlations to histopathological parameters and clinical follow-up revealed an association of TP, DPD and TS mRNA expression patterns with tumor stage and suggested new prognostic and predictive markers for patients with colorectal cancer.

Gene expression profiles of different clinical stages of colorectal carcinoma: toward a molecular genetic understanding of tumor progression.

BACKGROUND AND AIMS: Colorectal cancer is one of the leading causes of cancer deaths in the Western world. A better understanding of the development and progression of colorectal carcinoma is needed to define novel targets and strategies for treatment. PATIENTS/METHODS: Gene expression profiles were determined for primary tumors of 10 locally restricted (T3N0M0), 8 lymphatically metastasized (T3N+M0), 7 systemically metastasized (T3N+M1) colorectal carcinomas, and 6 specimens of normal colorectal tissue by histology-guided oligonucleotide microarray analysis. RESULTS: A total of 1,995 genes were differently regulated in primary tumors of colorectal carcinoma compared with normal colorectal tissue. Besides common features of dedifferentiation and different expression of genes involved in cell division, cell adhesion, angiogenesis, signal transduction and metabolism we observed a deregulation of genes with an as yet unclear function. We identified 126 genes that were subsequently up- and 204 genes down-regulated during tumor progression. Furthermore, we found a cluster of five genes exclusively up-regulated in primary tumors of systemically metastasized colorectal carcinomas. A comparison of locally restricted (T3N0M0) and systemically metastasized (T3N+M1) primary tumors showed 50 deregulated genes with a massive down-regulation of immune-modulatory genes in primary tumors of systemically metastasized carcinomas. Primary tumors of lymphatically (T3N+M0) and systemically metastasized (T3N+M1) carcinomas differed in the expression of 19 genes. CONCLUSION: These results provide an additional step toward the identification of crucial genes for the progression of colorectal cancer and the identification of novel treatment targets or strategies.

Serotonin excites neurons in the human submucous plexus via 5-HT3 receptors.

BACKGROUND & AIMS: Serotonin (5-hydroxytryptamine [5-HT]) is a key signaling molecule in the gut. Recently, the neural 5-HT3 receptor received a lot of attention as a possible target in functional bowel diseases. Yet, the 5-HT3 receptor-mediated changes in properties of human enteric neurons is unknown. METHODS: We used a fast imaging technique in combination with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl]pyridinium betaine to monitor directly the membrane potential changes in neurons of human submucous plexus from surgical specimens of 21 patients. An Ussing chamber technique was used to study 5-HT3 receptor involvement in chloride secretion. RESULTS: Local microejection of 5-HT directly onto ganglion cells resulted in a transient excitation of enteric neurons characterized by increased spike discharge. This response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, and blocked by the 5-HT3 receptor antagonist, tropisetron. The proportions of 5-HT-responsive nerve cells per ganglion ranged from 25.5% +/- 18.4% in the duodenum to 54.2% +/- 46.9% in the colon. Interestingly, 2-methyl-5-HT did not evoke chloride secretion in the human intestine but it did in the guinea-pig intestine. Specific 5-HT3A and 5-HT3B receptor subunit immunoreactivity as well as 5-HT3A and 5-HT3B receptor-specific messenger RNA were detected in the tissue samples. Based on co-labeling with the pan-neuronal marker HuC/D we conclude that submucous nerve cells potentially express heteromeric 5-HT3A/B receptors. CONCLUSIONS: We show that 5-HT excited human enteric neurons via 5-HT3 receptors, which may comprise both 5-HT3A and 5-HT3B receptor subunits.

Prognostic impact of CK-20-positive cells in peripheral venous blood of patients with gastrointestinal carcinoma.

Despite curative tumor resection, about 30%-50% of patients with locally advanced gastrointestinal (GI) carcinoma develop tumor recurrence which may be caused by pre- or intraoperative tumor cell dissemination. We examined the combination of optimized density gradient centrifugation with a CK-20 reverse transcriptase-polymerase chain reaction to detect and quantify circulating tumor cells in peripheral blood. Peripheral venous blood (20 ml) of patients with GI carcinomas was collected during primary tumor staging before and after the endoscopy procedure. CK-20 expression in peripheral venous blood was found in 22 of 82 patients (26.8%) with a nonsignificant difference between the upper GI tract (23.9%) and the lower GI tract (30.5%). The correlation with clinical outcome (24-month-survival) revealed a significantly worse prognosis (p < 0.05) of CK-20-positive patients with carcinoma of the upper GI tract and a trend toward a worse prognosis for patients with carcinoma of the lower GI tract. Quantification of CK-20 expression in peripheral blood showed a significantly higher circulating CK-20 copy number (median: 2816) in patients with metastatic tumors than in those with non-metastatic tumors (median: 983) (p < 0.05). For a subset of 42 primarily operated patients, we correlated the detection rate with UICC (International Union Against Cancer) staging categories. In contrast to the upper GI tract, the detection rate of patients with carcinoma of the lower GI tract showed a trend toward tumor size (pT) and a significant correlation with the presence of distant metastases (pM) (p < 0.01) and the postoperative residual tumor status (R) (p < 0.01). The endoscopy procedure did not lead to an increased detection of CK-20 expression.

Telomere length and human telomerase reverse transcriptase expression as markers for progression and prognosis of colorectal carcinoma.

PURPOSE: Maintenance of telomeres through reactivation of telomerase is a prerequisite for tumors to preserve their ability to proliferate. The purpose of this study was to evaluate telomere length and human telomerase reverse transcriptase (hTERT) expression as markers for progression and prognosis of colorectal carcinoma. PATIENTS AND METHODS: Telomere length and hTERT expression were analyzed in matched cancer and adjacent noncancer mucosa samples from 57 patients with R0-resected colorectal carcinoma. The median follow-up time was 76 months. RESULTS: Telomere length and hTERT expression correlated significantly in cancer tissues and adjacent mucosa samples (r = 0.52, P <.001; and r = 0.54, P <.001, respectively). Overall, cancer tissue had shorter telomeres than adjacent mucosa (P <.001). Only in noncancer tissue did telomere length decrease with age (r = 0.36; P <.01). Telomere length in cancer tissue was significantly correlated with tumor stage (P <.01), with longer telomeres in advanced tumors. Patients with ratios of telomere length in cancer to noncancer tissue greater than 0.90 had a significantly poorer overall survival compared with patients with smaller telomere length ratios (P <.002). In multivariate analysis, the telomere length ratio proved to be of independent prognostic value (P <.03). CONCLUSION: Telomeres in colorectal carcinoma tissue were significantly shorter compared with adjacent normal mucosa as an indication for extensive cell proliferation. The correlation with tumor stage and patient survival suggest that hTERT-mediated telomere stabilization may be critical for progression and prognosis of colorectal carcinoma.

Prognostic evaluation and review of immunohistochemically detected disseminated tumor cells in peritumoral lymph nodes of patients with pN0 colorectal cancer.

BACKGROUND: The value of immunohistochemical detection of disseminated tumor cells in histopathologically tumor-free lymph nodes (pN0) of patients with colorectal cancer is still of uncertain prognostic value. We therefore evaluated the immunohistochemical detection rates and their prognostic value comparing three different monoclonal antibodies. METHODS: A total of 170 lymph nodes of 85 patients with curatively resected colorectal carcinoma at UICC stage I or II were evaluated for disseminated tumor cells. Frozen sections of each lymph node were immunohistochemically stained using three antibodies directed against CEA, CK20, and Ber-EP4. The detection rates were compared with histopathological tumor parameters and with the patient's survival. The median follow-up time was 86 months. RESULTS: CEA-, CK20-, and Ber-EP4-positive disseminated tumor cells were identified microscopically in lymph nodes of 23 patients (27%), 24 patients (28%), and 23 patients (27%), respectively. In 18 patients (21%) disseminated tumor cells were found in consecutive sections and stained positive for all three monoclonal antibodies. The lymph nodes of 10 of 18 patients (56%), which developed tumor recurrence, contained CEA- and CK20-positive disseminated tumor cells. Ber-EP4-positive cells were present in lymph nodes of 9 of 18 patients (50%) with tumor recurrence. The 5-year overall survival of the 23 patients with CEA-positive disseminated tumor cells was 72% compared to 91% of the patients without immunohistochemical evidence of tumor cells (p<0.01). While the identification of CK20-positive tumor cells was also correlated significantly with a worse overall patient survival (p<0.01), the application of Ber-EP4 failed to reach significance (p=0.057). Multivariate analysis identified the tumor site (colon versus rectal cancer) (p<0.006) and the presence of CEA-positive disseminated tumor cells (p<0.03) as independent prognostic factors. CONCLUSION: In colorectal carcinoma, the immunohistochemical detection of disseminated tumor cells in histopathologically pN0 peritumoral lymph nodes allows the identification of a subgroup with a significantly worse prognosis. Nevertheless, the prognostic value of immunohistochemically detected disseminated tumor cells remains controversial due to the nonuniform data in the literature.

Detection of circulating tumor cells in blood using an optimized density gradient centrifugation.

The aim of the study was to compare the new density gradient centrifugation system OncoQuick with the standard density gradient centrifugation system Ficoll for improved tumor cell enrichment in blood of tumor patients. Evaluation of OncoQuick and Ficoll density gradient centrifugation was performed by flow-cytometry and immunocytochemistry using 10 ml unspiked and tumor cell-spiked blood samples of tumor-free probands. From 10 ml blood, OncoQuick density gradient centrifugation separated a cell fraction which consisted of a mean cell number of 9.5x10(4) mononuclear cells compared to 1.8x10(7) cells by Ficoll. Density gradient centrifugation of tumor cell-spiked blood samples with OncoQuick and Ficoll led to similar tumor cell recovery rates, between 70% and 90% for both methods. The improved depletion of mononuclear blood cells by OncoQuick simplified further immunocytochemical evaluation of the enriched cell fraction, which could be spun onto 1-2 glass slides by cytocentrifugation. In comparison, the mononuclear cells separated by Ficoll had to be spun onto more than 50 glass slides for complete immunocytochemical evaluation. Consequently, tumor cell density on each cytospin was higher after OncoQuick preparation compared to Ficoll. Density gradient centrifugation with OncoQuick results in higher relative tumor cell enrichment than Ficoll density gradient centrifugation. This simplifies further immunocytochemical tumor cell detection and is a promising tool for the detection of circulating tumor cells in blood of tumor patients.

Telomere length and hTERT expression in patients with colorectal carcinoma.

The stabilization of telomere length by telomerase activation is an important step in carcinogenesis. Quantification of the catalytic telomerase subunit hTERT (human Telomerase Reverse Transcriptase) is a new indirect measure for telomerase. Telomere length and hTERT expression in cancer tissue and corresponding normal mucosa of 57 patients with completely resected colorectal carcinoma (UICC stage I-IV, R0) were determined for correlation with histopathological parameters and survival. Telomere lengths were measured using Southern Blot and hTERT-encoding mRNA was quantified by real-time RT-PCR. Telomere length and hTERT expression were significantly correlated in normal mucosa and cancer tissue (p<0.001). Telomere length and hTERT expression decreased with ageing only in normal mucosa. Cancer tissue had significantly shorter telomeres (p<0.001) and significantly lower hTERT expression levels (p<0.001) than corresponding normal mucosa. UICC stage I tumors showed significantly shorter telomeres than UICC stage II-IV tumors (p<0.002). Telomere length and hTERT expression were significantly correlated with overall survival. Telomere length and hTERT expression play an important role in ageing and carcinogenesis. Both parameters were identified as prognostic factors in patients with colorectal carcinoma.

Prognostic potential of the telomerase subunit human telomerase reverse transcriptase in tumor tissue and nontumorous mucosa from patients with colorectal carcinoma.

BACKGROUND: The stabilization of telomere lengths by telomerase activation is an important step in carcinogenesis and cell immortalization. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of this enzyme. The objective of this study was to evaluate the use of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for the quantification of hTERT in tumor and nontumorous tissue samples. METHODS: Matched samples of tumor and adjacent nontumorous mucosa samples from 57 patients with completely resected colorectal carcinoma (International Union Against Cancer Stage I-IV) who underwent complete resection (R0) were quantified for hTERT mRNA expression using real-time RT-PCR. The expression levels were correlated with histopathologic findings and with survival. The median follow-up was 76 months. RESULTS: hTERT mRNA was expressed in all tumor samples and in all samples of adjacent mucosa. In 12 patients (21%), there was higher hTERT expression in tumor samples compared with nontumorous samples. Compared with tumor samples, the expression of hTERT in samples of nontumorous mucosa decreased with age (P = 0.06). hTERT mRNA expression in both tumor tissue and adjacent mucosa was correlated significantly with the histologic grade of colorectal carcinoma (P < 0.04 and P < 0.05, respectively). Patients with hTERT expression in tumor tissue in relation to the adjacent mucosa of > 0.57 had a significantly poorer overall survival compared with patients with lower hTERT ratios (P < 0.02). In addition to the established prognostic factor lymphatic vessel invasion, the hTERT ratio proved to be of independent prognostic value (P < 0.05). CONCLUSIONS: The prognostic potential of hTERT in patients with colorectal carcinoma and the correlation of hTERT with tumor grade underlines the role of hTERT as a molecular marker for biologic tumor staging.

PET imaging with [11C]methyl- L-methionine for therapy monitoring in patients with rectal cancer.

In this study we evaluated whether positron emission tomography (PET) using the amino acid [11C]methyl- L-methionine (MET) may be used for therapy monitoring in patients with rectal cancer who are undergoing preoperative chemoradiotherapy. A total of 41 MET-PET scans were performed in 26 patients with locally advanced rectal cancers. All patients were examined prior to chemoradiotherapy. In 15 patients, MET-PET was repeated after preoperative chemoradiotherapy (45 Gy radiation dose, 250 mg 5-fluorouracil as continuous infusion). MET uptake prior to and after the completion of chemoradiotherapy was correlated with changes in T stage and histopathological regression. All tumours were visualised with high contrast and had a significantly higher SUV (5.7+/-2.2) than normal rectum (2.7+/-0.9) and all other organs in the field of view except the small intestine (3.9+/-1.7). In all tumours studied prior to and after chemoradiotherapy, MET uptake decreased during therapy (SUV before therapy, 6.2+/-2.3; SUV after therapy, 2.6+/-1.2; P=0.0007). However, the degree of change in MET uptake was not correlated with histopathological tumour response. In conclusion, primary rectal cancer can be imaged with MET-PET. However, for the studied chemoradiotherapy regimen, MET-PET did not allow an assessment of the response to therapy.

Prognostic significance of cytokeratin-20 reverse transcriptase polymerase chain reaction in lymph nodes of node-negative colorectal cancer patients.

PURPOSE: Approximately 20% to 30% of patients with curatively resected, node-negative (pN0) colorectal cancer die of tumor recurrence, which can be caused by minimal residual disease. To identify patients with an increased risk of tumor recurrence and evaluate the prognostic value of cytokeratin-20 (CK-20), we detected CK-20-positive cells in histopathologically tumor-free lymph nodes (pN0) of patients with colorectal cancer. PATIENTS AND METHODS: Two peritumoral lymph nodes each from 85 patients with completely resected (R0) colorectal cancer without lymph node metastases (pN0) by routine examination were analyzed using a CK-20-specific reverse transcriptase polymerase chain reaction (RT-PCR) and compared with CK-20-specific immunohistochemistry (IHC). The results were correlated with histopathologic findings and with survival. RESULTS: CK-20 RT-PCR was positive in 44 patients (52%) and detected 83% of cancer-related death. Positive RT-PCR was significantly correlated with poorer overall survival (P <.009). Comparing RT-PCR with IHC, 13 patients with positive RT-PCR were identified, where the CK-20 expression was caused by tumor cell contamination located exclusively outside the lymph node capsule and had no prognostic impact. Defining these 13 patients as RT-PCR negative improved specificity of the RT-PCR assay from 57% to 75%. The 5-year overall survival of the 31 RT-PCR-positive patients was 71%, compared with 96% in the 54 negative patients (P <.001). Multivariate analysis showed expression of CK-20 mRNA to be an independent prognostic factor with a relative risk of cancer-related death of 6.1. CONCLUSION: CK-20 RT-PCR in peritumoral histopathologic tumor-free (pN0) lymph nodes of colorectal cancer is an independent prognostic factor for overall survival. Additional CK-20 IHC improves the specificity and prognostic value of RT-PCR for cancer-related death.