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© 2003 American Society for Clinical Oncology Effect of Lymphatic Mapping on the New Tumor-Node-Metastasis Classification for Colorectal Cancer
From the Division of Surgical Oncology, John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, CA; and the Division of Gastrointestinal Pathology, Armed Forces Institute of Pathology, Washington, DC. Address reprint requests to Anton J. Bilchik, MD, PhD, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; email: bilchika{at}jwci.org.
Purpose: Sensitive detection methods and accurate reporting are necessary to determine the prognostic significance of micrometastases (MM) and isolated tumor cells (ITCs) in lymph nodes that drain colorectal cancers (CRCs). This study examined the role of lymphatic mapping (LM) in the application of the new tumor-node-metastasis (TNM) classification for MM and ITC. Patients and Methods: All patients at the John Wayne Cancer Institute underwent LM immediately before standard resection of primary CRC between 1996 and 2001. Sentinel nodes (SNs) were identified using blue dye and/or radiotracer and were examined by hematoxylin-eosin (H&E) staining, cytokeratin immunohistochemistry, and multilevel sectioning. The comparison group comprised 370 patients whose primary CRCs were resected without LM during the same period at the same institution. Results: LM was successfully performed in 115 of 120 (96%) patients and correctly predicted the tumor status of the nodal basin in 110 of 115 (96%) patients. Thirty-seven patients (32%) were lymph node-positive by H&E; ITC and MM were found in 23 patients (29.4%) whose lymph nodes were negative by H&E. Tumor deposits were found in the SN only in 29 patients (50%). Nodal involvement was identified for 14.3%, 30%, 74.6%, and 83.3% of T1, T2, T3, and T4 tumors, respectively, in the study group, and for 6.8%, 8.5%, 49.3%, and 41.8% of T1, T2, T3, and T4 tumors, respectively, in the comparison group. The study group had a higher percentage of nodal metastases (53% v 36%; P < .01) and a higher incidence of MM and ITC (29.4% v 1.9%; P < .0001). The mean number of lymph nodes found in the study group (14) was also significantly more than the number found in the comparison group (10; P < .00001). Conclusion: Conventional examination of lymph nodes for CRC is inadequate for the detection of MM and ITC as described in the new TNM classification. Thus, LM and focused SN analysis should be considered to fully stage CRC.
THE PURPOSE of cancer staging and classification is to describe a tumor’s biophysical attributes and to provide information on its natural history. Staging allows clinicians to analyze and compare groups of patients and has a significant effect on therapeutic decision making, estimation of prognosis, and evaluation of treatment effect.1 For colorectal cancer (CRC), lymph node status largely dictates prognosis and choice of adjuvant therapy. Patients with tumor-positive lymph nodes have a 5-year survival rate of only about 50% to 60%.2 Chemotherapy confers a 33% survival benefit in these patients,3 but its use for patients without documented nodal metastasis has not been validated by randomized studies.4 Because the rate of postoperative recurrence in the latter group can reach 30%, accurate nodal staging becomes crucial to identify candidates for aggressive adjuvant therapies. Unfortunately, conventional methods for examining lymph nodes have specific limitations and are subject to sampling error that increases the risk of understaging. Pathologic techniques such as fat clearance, cytokeratin immunohistochemistry, and multilevel step sectioning can increase the number of nodes identified and reduce sampling error. In addition, our group has adapted intraoperative lymphatic mapping (LM) to identify the lymph nodes most likely to harbor any tumor cells metastasizing from a primary CRC. Focused analysis of these sentinel nodes (SNs) by serial sectioning and immunohistochemistry can identify isolated tumor cells (ITCs), tumor cell clusters, and micrometastases (MMs) that cannot be identified by conventional examination of nodes in a resected CRC specimen.5 Although the clinical significance of nodal MMs is unclear, they might increase the risk of relapse; if so, then adjuvant chemotherapy could be beneficial. In fact, the new tumor-node-metastasis (TNM) classification6,7 distinguishes MM and ITCs within lymph nodes and incorporates the SN concept. To determine the effect of focused SN analysis on the new TNM classification for CRC, we examined the results of LM in 120 consecutive patients with early CRC. We found that a significant proportion of SNs contained ITC and/or MM that were not identified by conventional examination. We therefore believe that focused analysis of the SN identified during LM is superior to conventional nodal assessment and should be considered in the application of the new TNM classification system.
In 1996, we began using LM to stage early CRCs without clinical evidence of distant metastasis. All patients with biopsy-proven CRC were prospectively enrolled under an IRB (institutional review board)-approved protocol for LM. Although multiple surgeons were involved in this study, all LM procedures were performed by or under the direction of a single surgical oncologist (A.J.B.). For comparison, clinicopathologic data from the tumor registry were obtained after IRB approval for all patients who underwent resection for colorectal malignancy without LM during the same time period and at the same institution.
SN Mapping and Colectomy
Processing of CRC Specimen Non-SNs were bisected along the longest axis and fixed in paraffin. If they were less than 5 mm in length, the lymph node was processed whole. A single face section was stained with hematoxylin-eosin (H&E) and examined under light microscopy. SNs were measured and bisected along the longest axis. If the SN was smaller than 5 mm, it was processed whole. SN tissue was frozen and prepared for cryostatic sectioning. A face section was cut at a thickness of 5 µ for H&E staining. Six to eight further sections, representing approximately 72 µ of node, were preserved frozen for molecular analysis. The remaining nodal tissue was placed in 10% formalin and embedded in paraffin. Two additional 4-µ sections at approximately 200-µ intervals were taken for H&E and cytokeratin immunohistochemistry (CK-IHC) stains. CK-IHC was performed with a pan-specific cocktail of antibodies for human cytokeratin, AE1/AE3 (DAKO, Carpinteria, CA), and processed with an automated immunostainer (Ventana ES 320, Ventana Medical Systems, Tucson, AZ). A CK-IHC stain was considered positive if it demonstrated strongly positive cell clusters or individual cells with anatomic and cytologic features of tumor cells.
Classification and Staging of Tumors
Statistical Analysis All data were reviewed and analyzed by the Biostatistical Unit at the John Wayne Cancer Institute. Associations between categoric variables were examined using  2 analysis. For small sample sizes, Fisher’s exact test was used. Quantitative variables were compared using Student’s t test. A P value of less than .05 was considered statistically significant.
Average age was 70 years for the 120 study patients and 71 years for 370 comparison patients. Male to female ratio was 58:62 in the study group and 184:186 in the comparison group. Tumor characteristics and pathologic data are provided in Table 3  . The average length of the resection specimen (data not shown) and the distribution of the primary tumor’s histologic grade (Table 4) were similar between the two groups, but the average size of the primary tumor was significantly smaller in the study group (P < .001; Table 3).
In the study group, LM identified at least one SN in 115 (96%) patients (Table 5 ). The average number of SNs was 1.75 (range, 1 to 5), and the average number of sentinel plus nonsentinel lymph nodes examined was 14. Five patients had tumor-positive non-SNs despite the apparent absence of tumor in SNs. Sixty-three (53%) study patients had ITCs, MMs, or macrometastases in SNs and/or non-SNs (Fig 2). Twenty-nine patients (50%) had tumor deposits only in SNs (Table 5). Of 78 study patients whose sentinel and nonsentinel lymph nodes were negative for tumor by H&E, 23 (29.4%) had ITCs (n = 15) or MMs (n = 8) identified by CK-IHC examination of SNs.
In the comparison group, the average number of lymph nodes examined was 10, significantly less than the average number of lymph nodes examined in the study group (P < .00001). Of the 370 patients, 136 (36%) had N1 disease. Seven patients (1.9%) had MM within lymph nodes, but none had ITCs within lymph nodes. Thus, the incidence of nodal MM or ITCs was significantly lower in this group (P < .0001).
At any T category except T4, the number of tumor-positive lymph nodes was significantly higher for the study group (Table 6
These data demonstrate that conventional examination of randomly selected lymph nodes in a CRC specimen is less sensitive than focused examination of SNs identified during LM. LM was associated with an increased incidence of nodal metastasis overall and at any T category. When ITCs were included in the analysis, there was a significantly increased rate of tumor detection compared with conventional examination (P < .0001). Pioneered in melanoma by Morton et al,9 intraoperative LM has been successfully applied to a variety of solid tumors.10 LM can identify the primary lymph node that directly drains a tumor bed. Focused analysis of this SN can routinely identify small tumor cell deposits. In our series, CK-IHC detected tumor deposits in a significant proportion of lymph nodes that stained negative by H&E. This is in contrast to the few cases encountered using conventional methods of lymph node examination. Single-level sectioning and H&E staining of lymph nodes underestimates the true incidence of tumor cell metastases.
Among the tumor characteristics that were evaluated, only tumor size was significantly different between the two groups. Interestingly, primary tumors were significantly smaller in the study group, even though the overall incidence of N1 disease was similar (Table 3 Although the length of the resected specimen was the same in both groups, the total number of harvested lymph nodes was significantly higher in the study group. This increase was probably the result of the pathologist’s meticulous search for all blue-stained lymph nodes. Techniques such as fat clearance that increase the yield of lymph nodes are not used routinely at our institution, largely because they are time consuming and costly and do not provide rapid diagnostic results.13 Certainly, the number of lymph nodes that should be examined is crucial to proper diagnosis.14,15 Intuitively, examining more lymph nodes increases the probability that a metastasis will be found. The recommended number ranges from as low as six nodes to as high as 14.16,17 The UICC recommends that a minimum of 12 lymph nodes is adequate for accurate pN staging.18 Our average number of lymph nodes after LM is on the high end of the spectrum. In a recent presentation, Joseph et al19 demonstrated that the accuracy of nodal status determination is highly dependent on the number of lymph nodes retrieved and examined by the pathologist. In an analysis of 1,535 patients, they concluded that the number of lymph nodes necessary to accurately predict nodal status with an 85% probability was more than 40 for T1/T2 lesions and at least 40 for T3 lesions. Although it is possible to harvest such a large number of lymph nodes in a properly resected specimen, it is neither cost- nor time-efficient for the pathologist. We believe that LM elegantly addresses this problem by focusing the attention of the pathologist on the node that has the highest probability of harboring metastasis because it is on the direct drainage pathway of the tumor bed. Almost half of our study patients with nodal metastasis had tumor deposits only in the SN. It has been our experience that although these nodes are involved with the tumor, they are still relatively small in size. Monig et al20 demonstrated that lymph node size is not a reliable indicator for lymph node metastasis. This conclusion was further supported by a study demonstrating that most metastases occur in lymph nodes less than 0.5 cm in size.21 These small nodes are often overlooked in a large mesenteric specimen, but are easily identified when they are mapped by the surgeon or the pathologist. Because face sections of a bisected lymph node represent less than 1% of the entire specimen, single-level sectioning and H&E staining can easily result in missed metastases, especially ITCs and MMs. Multilevel step sectioning and sensitive tests such as cytokeratin immunohistochemistry increase the identification of tumor deposits. In breast cancer, the minimum number of multilevel sections to accurately detect nodal involvement is two.22 Although multilevel sectioning is labor-intensive and can be cost-prohibitive if attempted for the large number of lymph nodes harvested from a CRC specimen, it is not impractical for the few tagged SNs. Clearly, the concept that the "more one looks, the more one will find" applies to nodal examination for CRC. LM with focused analysis of the SN simplifies this task for the pathologist. Although they are not yet readily available for general use, molecular techniques to search for tumor markers can further increase the sensitivity of detection. Unlike step sectioning and immunohistochemistry, reverse transcriptase polymerase chain action (RT-PCR) examines the bulk of the lymph node and is not dependent on where the metastases are located. In melanoma and CRC, RT-PCR analysis of SNs is positive for tumor markers in a significant percentage of patients whose nodes are otherwise tumor-free.23,24 However, the significance of nodal MM, ITCs within lymph nodes, and molecular markers of nodal disease remains undetermined. Early retrospective studies of nodal MM failed to demonstrate the prognostic significance of single tumor cells and cell clusters detected by immunohistochemical methods.25 More recent studies26 indicate that molecular detection of tumor markers within lymph nodes of patients with CRC is associated with increased recurrence and decreased overall survival. This controversy has not been resolved in a large, prospective clinical trial mainly because MM and ITCs previously have not been defined or uniformly reported.
The new TNM classification specifically outlines standards for the reporting of MM (Tables 1 With the advent of SN technology, MM and ITC are being reported at a much higher frequency in a variety of tumor types. For breast cancer, this is less of an issue, because the choice of adjuvant chemotherapy is dependent on a variety of primary tumor characteristics in addition to nodal status. In CRC, conventional methods of examination are subject to sampling error and are not sensitive enough to routinely identify these small tumor deposits. Molecular markers of the primary tumor such as k-ras mutations, loss of heterozygosity, Bcl-2, p27, and p53 may someday preclude assessment of the regional lymph nodes, but at present the AJCC recommends that these markers be used only in the setting of a clinical trial.27 Until these markers are of proven utility, nodal status must take precedence in the adjuvant management of patients with CRC. Because there is now a consensus for the classification and documentation of MM and ITCs, LM should be considered for staging early CRC.
Supported by grant CA090848 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and by funding from the Rogovin-Davidow Foundation, Los Angeles, CA, the Rod Fasone Memorial Cancer Fund, Indianapolis, IN, and the U.S. Surgical Corp, Norwalk, CT.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
