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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Clin Cancer Res. Author manuscript; available in PMC Nov 15, 2009.
Published in final edited form as:
PMCID: PMC2586882
NIHMSID: NIHMS75743

Prognostic Relevance of Occult Nodal Micrometastases and Circulating Tumor Cells in Colorectal Cancer in a Prospective Multicenter Trial

Abstract

Purpose

Nodal micrometastasis and circulating tumor cells (CTC) detected by multimarker quantitative real-time RT-PCR (qRT) may have prognostic importance in patients with colorectal cancer (CRC).

Experimental Design

Paraffin-embedded sentinel lymph nodes (SLNs) from 67 patients and blood from 34 of these patients was evaluated in a prospective multicenter trial of SLN mapping in CRC. SLNs were examined by hematoxylin and eosin staining (H&E) and cytokeratin immunohistochemistry (CK-IHC). Both SLNs and blood were examined by a four-marker qRT assay (c-MET, MAGE-A3, GalNAc-T, CK20); qRT results were correlated with disease stage and outcome.

Results

In H&E SLN negative patients that recurred, CK-IHC and qRT detected metastasis in 30% and 60% of patients, respectively. DFS differed significantly by multimarker qRT upstaged SLN (p=0.014). qRT analysis of blood for CTC correlated with overall survival (p=0.040).

Conclusion

Molecular assessment for micrometastasis in SLN and blood specimens may help identify patients at high-risk for recurrent CRC, who could benefit from adjuvant therapy.

Keywords: RT-PCR, colorectal cancer, micrometastasis, sentinel lymph node

INTRODUCTION

Colorectal carcinoma (CRC) is a common gastrointestinal malignancy in the United States and remains the second most common cause of cancer mortality (1). The 5-year survival rate is approximately 90% for patients with localized disease and approximately 66% with regional disease determined at diagnosis (2). There is a 25% incidence of disease recurrence in the absence of regional node involvement, suggesting that conventional pathology may fail to detect occult nodal metastases (3). Adjuvant therapy improves survival in as many as one-third of patients with stage III disease (4); however, there is no consistent evidence that adjuvant therapy improves survival in node-negative (stage I/II) disease (5). Lymph node (LN) evaluation is essential for accurate staging and improves the selection of patients for adjuvant therapy (6).

Current techniques for nodal evaluation are inadequate for the detection of micrometastases; more sensitive techniques, such as multilevel step sectioning and intraoperative sentinel LN mapping (SLNM), have therefore been applied to solid malignancies. The SLN is the LN that has the highest probability of harboring metastatic tumor cells from a primary tumor (7-10); this concept has been validated in melanoma and breast cancers. SLN evaluation allows a focused examination for the detection of occult metastases not detected by conventional techniques. In colorectal cancer (CRC), we have demonstrated that the assessment of SLNs using multilevel sectioning and cytokeratin immunohistochemistry (CK-IHC), detects micrometastasis in 23% patients whose nodal specimens are tumor-free by routine H&E staining (11, 12). We have demonstrated the utility of molecular markers for the detection of occult metastasis in CRC histopathology-negative frozen SLNs (10, 13). Focused analysis of the SLN can therefore provide a more efficient approach for the detection of micrometastases and improve patient management (10, 13, 14) .

The development of a qRT assay has allowed rapid quantitative detection of occult metastatic tumor cells in LNs and blood (15-17). A multimarker assay is more accurate than single-marker assays for the detection of occult tumors (18-21). In this study, we evaluated four mRNA markers: c-MET(hepatocyte growth factor receptor), MAGE-A3(melanoma antigen gene-A3 family), GalNAc-T(β1→4-N-acetylgalactosaminyltransferase), and CK20(cytokeratin-20). c-MET is a proto-oncogene encoding the receptor for hepatocyte growth factor; c-MET mRNA overexpression is associated with tumor invasion and regional LN metastasis in CRC (15, 22). MAGE-A3 is commonly expressed tumor antigen in various tumors but not in normal tissue (23, 24). GalNAc-T is an enzyme involved in specific carbohydrate molecule synthesis (17, 25). CK20, a member of the CK family, has been used for detection of occult tumor cells in LNs and blood (26-28). We have previously demonstrated the utility of these biomarkers using frozen SLNs (10).

Circulating tumor cells (CTC) in the blood are essential for the formation of distant metastasis, and blood analysis can be used to depict real-time tumor spreading (16-18, 20, 21). We introduced the multimarker quantitative real-time RT-PCR (qRT) assay for detection of CTC in blood, and demonstrated the relation of CTC detection and disease stage and treatment outcome in various cancers (16-18, 20, 21, 29). In this study, we performed a multimarker qRT assay to detect metastasis, hypothesizing that a multimarker qRT assay for assessing SLN and/or blood would be a prognostic surrogate for micrometastasis and disease outcome.

PATIENTS AND METHODS

Patients

The 67 patients included in the qRT analysis were a subset of 148 patients enrolled in a prospective multicenter trial of SLNM for CRC. A more detailed description of the clinical study was recently reported (3). The SLN was identified in 146 (99%) patients. All patients with paraffin-embedded (PE) SLN available were included in the qRT analysis. Patients were seen at one of five participating centers (JWCI, CA; Century City Hospital, CA; University of Colorado Health Science Center, CO; Wake Forest University, NC; and Michigan State University, MI) and all had approval for specimen accrual in accordance with IRB guidelines.

SLNM and Histopathologic Examination

SLNM was performed during standard surgical resection, as previously described (3, 11, 12). The colon was mobilized, and isosulfan blue dye (Lymphazurin, U.S. Surgical Corporation) was injected subserosally around the tumor. Each blue-stained LN was identified as an SLN. A colectomy that included all blue-stained LNs was performed in standard fashion, and the en-bloc specimen was sent to pathology.

Each tagged SLN was harvested, PE individually in a tissue block cassette, and multiple sections of each PE specimen were the examined by routine H&E and CK-IHC (12). CK-IHC was performed with a pan-specific antibodies cocktail (AE1/AE3, DAKO).

Tumor deposits within LNs were classified and staged according to AJCC guidelines (30). Macrometastases were >2mm; micrometastases were ≤2mm; and isolated tumor cells(ITCs) were single tumor cells or cell clusters measuring ≤0.2mm that were almost always detected by IHC. Pathological examination was performed in a blinded fashion without clinicopathology knowledge.

CRC Cell Lines and Control Specimens for qRT

Eight CRC cell lines were used: HT-29, SW480, and SW620 were obtained from ATCC; cell lines CRC-A, B, C, D, and E were established at JWCI. Cells were grown and harvested for mRNA analysis, as previously described (16). Other specimens used for optimization of qRT assay included: 10 PE primary CRC tumors, six PE CRC-involved LNs, benign LNs from eight non-cancer patients; and peripheral blood leukocytes (PBL) from 47 healthy donors.

RNA Isolation and qRT Assay

All SLN and blood specimens were coded by a computer-generated number. Processing of SLN and blood specimens, RNA extraction, RT-PCR set-up, and post-RT-PCR product analysis was as previously described (15, 24).

For SLN multimarker qRT assay, 5−8 10-μm sections were cut from PE SLN; a sterile microtome blade was used for each specimen. Sections were deparaffinized and digested with proteinase K for 3hrs before RNA extraction using a modified RNAWiz protocol (Ambion) (31).

For CTC assay, peripheral blood specimens were collected in 2×4.5mL sodium citrate-containing tubes before surgery (16). Total cells in blood were collected by using Purescript RBC lysis solution (Gentra) and Tri-Reagent (Molecular Research Center) was used to isolate total cellular RNA (16). Total cellular RNA was quantified and assessed for purity by UV spectrophotometry and RiboGreen assay (Molecular Probes).

RT reactions were performed using Moloney murine leukemia virus reverse-transcriptase (Promega) with both oligo-dT and random primers (24). The qRT assay was performed to assess the presence of c-MET, MAGE-A3, GalNAc-T, and CK20 mRNA. The sensitivity and specificity of each marker for occult metastasis in SLN and blood have been previously described (10, 16, 24). ABI Prism 7900HT Detection System (Applied Biosystems) was used for detection of mRNA. Primer and probe sequences were designed for qRT assay. Fluorescence resonance energy transfer probe sequences were as follows: c-MET, 5’-FAM-TGGGAGCTGATGACAAGAGGAG-BHQ-1−3’; MAGE-A3, 5’-FAM-AGCTCCTGCCCACACTCCCGCCTGT-BHQ-1−3’; GalNAc-T, 5’-FAM-ATGAGGCTGCTTTCACTATCCGCA-BHQ-1−3’; CK20, 5’-FAM-ATCAGTTAAGCACCCTGGAAGAGAG-BHQ-1−3’; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5’-FAM-CAGCAATGCCTCCTGCACCACCAA-BHQ-1−3’. cDNA (200ng RNA) was added to a 384-well PCR microplate containing 0.5μmol/L primer, 0.3μmol/L probe, and 5μL of iTaq custom supermix with ROX (Bio-Rad). Samples were amplified with a precycling hold at 95°C for 10min, followed by denaturation at 95°C for 15sec. Annealing/extension was carried out for 1min at the following temperatures: 55°C for c-MET and GAPDH, 56°C for CK20, 58°C for MAGE-A3, and 62°C for GalNAc-T. The standard curve was generated with the threshold cycle (Ct) with plasmid template dilutions for each gene (106-101 copies). Sample Ct was interpolated from a standard curve to calculate mRNA copies.

Each qRT assay was performed at least twice and included marker-positive, -negative, and reagent controls (reagent without RNA or cDNA). If only one of the duplicates gave a positive result, we performed a third qRT to assay confirm the results. GAPDH gene was used as a housekeeping gene. Any specimen with inadequate GAPDH mRNA was excluded.

Statistical Analysis

Chi square analysis (Cohen's kappa) was used to assess agreement between any two of the four qRT markers and between marker detection in SLNs and marker detection in blood. Mantel-Haenszel chi square was used to assess the correlation between marker detection and the size of SLN metastasis, the tumor pathology stage, and AJCC stage. Age, gender and TNM staging were compared between the study sample and the remaining patients in the prospective multicenter trial using chi-square analyses and a Wilcoxon test.

Multinomial logistic regression was used to assess the ability of qRT detection in SLN to predict TNM staging after controlling for age and gender. The log-rank test was used to examine disease-free survival (DFS) and overall survival (OS) according to marker detection in SLN and blood. Survival curves were generated using the Kaplan-Meier method. Cox Proportional Hazards models were generated for OS and DFS in order to estimate the prognostic significance of marker detection in SLN and blood after controlling for age, gender and disease characteristics. Analyses were performed using SPSS statistical software and all tests were two-sided with significance level ≤0.05.

RESULTS

Patients for qRT Study

There were 74 patients from the trial who were eligible and consented for molecular studies based on SLN availability from participating investigators. Seven patients were excluded from the current analysis: six patients had benign tumors, and one patient's SLN was not identified. The 67 remaining patients included 33 males and 34 females with a median age of 74 years (range: 35−95). The tumor stage distribution was as follows: T1, 10 patients(15%); T2, 12(18%); T3, 45(67%). Blood samples from 34 of 67 patients were available for the CTC assay. Age, gender and TNM staging did not differ significantly between the 67 patients and the remainder of the patients in the prospective multicenter trial were not studied.

Standard Curves and Specificity of Multimarker qRT Assay

The standard curves showed the expected linear increase of signal with logarithm of the copy number (data not shown). PCR efficiency assessed from the standard curves was between 90−100%. The correlation coefficients for all standard curves (Ct vs log copy number) in the study were ≥0.99. Each of the eight CRC lines expressed all four markers. Each of the 10 primary tumor specimens and tumor-involved LNs expressed the individual markers. No markers were detected in PBLs from 47 healthy donors or in control tissues under the optimized conditions. Individual marker sensitivity: detection in one to five CRC cells diluted in 107 healthy donors PBLs.

Metastasis Detection in SLN by qRT

Nodal qRT assay detected ≥1 marker in 27 of 67(40%) patients; 11(16%) patients had one marker and 16(24%) had more than one marker. Patients whose SLNs expressed a specific marker was 9(13%) for c-MET, 12(18%) for MAGE-A3, 13(19%) for GalNAc-T, and 13(19%) for CK20. The negative predictive value of the qRT assay was 90%; four recurrences(10%) were evidenced among 40 qRT(−) patients. Concurrent detection was significant for c-MET and CK20 (p<0.001), and for MAGE-A3 and GalNAc-T (p<0.001).

The prognostic utility of CK-IHC and qRT detection in the SLN, based on upstaging within those found negative by conventional pathological examination (H&E) is shown in Table 1. Of 12 total recurrences, 2(17%) were detected by H&E; 3 (30%) of the remaining 10 recurrences were detected by CK-IHC, 6 (60%) were detected by the qRT assay, and a total of 70% upstaging was evidenced using both CK-IHC and qRT detection methods.

Table 1
Upstaging by CK-IHC and qRT within H&E Negative SLN

To demonstrate the relationship between detection by the qRT assay and standard clinical indicators, we compared the number of markers detected by qRT with SLN metastasis size and TMN staging. The number of markers detected by qRT increased with the size of SLN metastasis (p<0.001)(Table 2). Of 13 patients with macrometastasis (n=10) or micrometastasis(3 patients), 11(85%) had ≥1 marker and 7(54%) had ≥2 markers. The marker detection rate was significantly higher for patients with AJCC N1 disease than patients with N0 disease (p<0.001). Table 3 shows the correlation between nodal stage (N) and the number of markers detected by qRT assay (p<0.001). Marker detection in SLNs significantly increased in patients with regional LN metastasis (p<0.001).

Table 2
Relation between qRT Multimarker Detection and Metastasis Size in SLN
Table 3
Relation between qRT Multimarker Detection in SLN and pN Stage

Next, we assessed the correlation between markers detected in SLN and AJCC stage (Table 4). Of 20 stage I patients, 16(80%) showed no markers and 2(10%) had ≥2 markers. In contrast, 14(70%) patients with stage III disease had ≥1 marker and 6(30%) had no markers. Marker detection in SLNs significantly increased with AJCC stage of disease (p<0.01), but was not correlated with pT stage. Finally, multivariate analysis indicated that after controlling for age and gender, qRT detection in SLN was significantly predictive of N stage (odds ratio for stage N1/N2 8.1, p<0.01) and AJCC stage (stage III odds ratio 13.0, p<0.01).

Table 4
Relation between qRT Multimarker Detection in SLN and AJCC Stage

CTC Detection in Blood by qRT

Of 67 patients, 34 had blood available for the multimarker qRT assay. Sixteen patients (47%) expressed ≥1 mRNA marker and 18(53%) expressed no markers. The number of specimens that expressed a marker was 6(18%) for c-MET, 4(12%) for MAGE-A3, 7(21%) for GalNAc-T, and 2(6%) for CK20. There was no significant concordance for any two markers.

Results of blood qRT showed no correlation with pN, pT, or AJCC stage (data not shown). The number of blood specimens that expressed a marker was 3(19%), 1(6%), and 12(75%) for patients with T1, T2, and T3 primary tumors, respectively. MAGE-A3 and CK20 were not detected in blood from patients with T1/T2 tumors. Three patients out of a total of six identified with primary tumor vascular invasion had markers detected in the blood.

Histopathologic assessment of SLNs identified macrometastasis, micrometastasis, and ITC in 4(12%), 1(3%) and 3(9%), respectively, of the 34 patients whose blood was assessed by qRT. Of 14 patients without nodal metastasis, 6(43%) had blood that expressed none of the markers and 8(57%) had blood that expressed ≥1 mRNA marker. Four of 6 (67%) patients with macrometastases and 1/3(33%) patients with micrometastases expressed ≥1 marker. The number of markers in blood showed no correlation with SLN metastasis size.

Of 16 patients whose blood expressed ≥1 marker, 7(44%) had SLNs that also expressed ≥1 marker: five of these patients had T3 tumors and CK-IHC(+), and the other two had stage I CRC and CK-IHC(−). Of 18 patients whose blood expressed no markers, 6(33%) had SLNs that expressed ≥ 1 marker. There was no correlation between marker expression in SLNs and blood.

Relation Between Marker Detection and Survival

At a median follow-up of 34 months, a log-rank test indicated no significant difference in OS curves by marker detection in SLN. The mean OS was 43 months (95% CI:37−50) for qRT(+) SLNs (n=27) and 57 months (95% CI:50−64) for qRT(−) SLNs (n=40). (Figure 1A). In addition, qRT(+) (n=27) in SLN was not found to be prognostic of OS after controlling for age, gender and TMN staging (HR=1.0, p>0.050). However, a log-rank test revealed a significant difference in DFS by marker detection in SLN (p=0.014) (Figure 1B). The mean DFS was 37 months (95% CI:29−45) for qRT(+) SLN and 61 months (95% CI:56−67) for qRT(−)SLN. Detection of qRT(+) in SLN was further shown to have prognostic value for DFS independent of age, gender and TMN staging (HR ratio=4.9, p=0.027). Of the four markers, two were also found to be independently prognostic (MAGE-A3 HR=5.1, p=0.013; GalNAc-T HR=4.6, p=0.012).

Figure 1Figure 1
Kaplan-Meier curves for disease outcome according to detection of at least one qRT marker in SLNs. (A) Overall survival by qRT(+) in SLNs (NS); (B) Disease-free survival by qRT(+) in SLNs (p=0.014).

A significant difference was found in OS curves by qRT detection of CTC in blood. (p=0.040) (Figure 2). The mean OS was 36 months (95% CI:26−46) for qRT(+) blood (n=16), as compared with 50 months (95% CI:44−56) for qRT(−) blood (n=19). A Cox Proportional Hazards model indicated that marker detection in blood has independent prognostic value for OS beyond age, gender and TMN staging (HR=12.3, p=.045).

Figure 2
Kaplan-Meier curves: qRT marker detection in blood. Overall survival by qRT(+) in blood (p=0.040).

DISCUSSION

Distant metastasis of node-negative CRC may be explained by failure to detect occult nodal metastasis in the resected CRC specimen (32, 33). This problem has been mitigated by introduction of SLNM, which targets a few tumor-draining nodes for focused analysis, thereby increasing the accuracy and decreasing pathology costs. Although SLMN does not decrease the extent of nodal resection in CRC, its use can improve nodal staging and identify patients likely to benefit from adjuvant chemotherapy (32).

Marker mRNA expression in the SLN was correlated with size of SLN metastasis, histopathologic status of the regional LNs, AJCC stage, and DFS. In contrast, mRNA marker CTC detection was correlated with stage of the primary tumor and with OS but not with nodal stage. The absence of a significant correlation between results of nodal and blood qRT assays suggests that metastasis from primary CRC may occur via lymphatic and/or hematogenous pathways. This suggests that qRT assessment of both nodal and blood specimens may have more clinical relevance. However, there was a 50% correlation between primary tumor vascular invasion and CTC detection.

Because metastatic tumors are heterogeneous in marker expression, CK-IHC staining may have limited sensitivity for the detection of occult tumor cells. As we previously demonstrated, the combination of markers can compensate for variations in individual marker expression, increasing the detection of tumor cells (18). qRT positivity based on four markers in nodal or blood specimens was higher than qRT positivity based on any single marker. These findings confirm the limited clinical utility of a single CTC-marker assays (18, 34, 35).

Results of nodal qRT assay revealed expression of ≥1 marker in 29(41%) of 71 patients. Of the 14 patients with histopathologic evidence of SLN metastasis, 12 had positive nodal qRT results, and the number of markers increased with the size of SLN metastasis. In 17 patients with ITC, only 6(35%) had positive qRT markers. Negative results in the remaining 11 cases may have reflected a failure of ITC to express any of the four mRNA markers; alternatively, the portion of the node submitted for molecular analysis might not have been the portion that contained ITC.

The qRT assay may be of value when CK-IHC fails to detect SLN micrometastasis. Although the clinical significance of IHC-detected micrometastases remains subject to question, we found that results of qRT were correlated not only with pN and AJCC stage but also with DFS. The number of qRT(+) SLN patients who have not recurred may reflect the median duration of follow-up and/or the clinical impact of early resection. For this stage of disease, a longer follow-up may be needed to obtain the true value of the molecular upstaging as shown in the melanoma SLN studies (24).

Recent studies suggest the promise of qRT assay for assessment of subclinical disease and response to treatment (16, 36). In general, vascular invasion of the primary tumor is the first step in the metastatic cascade, and the degree and intensity of vascular invasion usually depend on characteristics of the primary lesion. This would explain our demonstrated correlation between qRT evidence of CTC and pathologic stage of the primary tumor. Results of blood qRT also confirmed recent reports of a correlation between CTC and disease outcome (17, 36). CTC had a significant inverse correlation with OS, suggesting that assessment of blood may have prognostic importance. Larger studies with long-term follow-up are needed to determine the clinical significance of CTC in early-stage CRC.

In conclusion, qRT assessment of SLNs that drain primary CRC can detect nodal micrometastases missed by conventional pathological examination. In addition, the multimarker qRT assay has independent prognostic significance for CTC detection in blood of patients with CRC. The concurrent assessment of blood and SLN may help identify candidates for adjuvant therapy. Subsequently, serial sampling and assessment of blood could prove useful for monitoring the response to therapy.

ACKNOWLEDGEMENTS

We thank members of the Department of Molecular Oncology, administrative staff of JWCI and John Wayne Clinic, Saint John's Health Center, and Doug Iddings, D.O., for helping in this study. This study was supported in part by NIH NCI CA090848; Roy E. Coats Research Laboratories, The Leslie and Susan Gonda (Goldschmied) Foundation, and The Martin H. Weil Foundation.

Footnotes

STATEMENT OF CLINICAL RELEVANCE

The study described provides, for the first time in colorectal cancer, sentinel lymph node (SLN) analysis that molecular upstaging has significant prognostic value in disease outcome. The novelty of the study is that the molecular upstaging was performed by multimarker mRNA markers on paraffin-embedded tissue sections of SLN. The study also demonstrated that detection of circulating tumor cells (CTC) in SLN patients by multiple mRNA markers had prognostic utility for disease outcome. The importance of the study is that molecular upstaging with specific mRNA markers is of clinical utility in both the SLN and blood of colorectal cancer patients. Our approach demonstrates that molecular diagnosis of colorectal cancer patients SLN may be of significance in improving staging. Identification of CTC in colorectal cancer patients during primary tumor removal may be of utility in determining those patients likely to have distant disease recurrence. The diagnosis of early stage occult disease in SLN by molecular approaches may improve overall management of colorectal cancer patients and identify patients not likely to recur.

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