- Email: [email protected]
The impact on nodal staging of lymphatic mapping in carcinoma of the colon and rectum
The Impact on Nodal Staging of Lymphatic Mapping in Carcinoma of the Colon and Rectum D. Scott Johnson and Jan H. Wong Although the majority of individuals undergoing apparently curative resection for carcinoma of the colon rectum who are found to be without evidence of metastatic disease in the regional lymph nodes will remain free of disease, approximately 20% to 30% of these individuals will develop recurrent disease and die. This may, in part, be due to understaging of the disease. For this reason, there has been increasing interest in approaches to improving staging of all solid tumors, including carcinoma of the colon and rectum. Lymphatic mapping has revolutionized the management of patients with cutaneous melanoma and breast cancer and has been demonstrated to more accurately stage patients with solid tumors. Our investigations as well as others have taken a number of strategies to improving the staging of individuals of colorectal cancer patients with apparently node-negative disease, including the development of a novel ex vivo lymphatic mapping technique. This article summarizes our and other investigations into improved staging of colorectal cancer with an emphasis on the impact of lymphatic mapping on improving the accuracy of staging of apparently node-negative colorectal cancer patients. Semin Oncol 31:403-408. © 2004 Elsevier Inc. All rights reserved.
C
OLORECTAL CANCER represents a significant health problem and is the third leading cause of cancer mortality in the United States, exceeded only by lung and prostate cancer in men and lung and breast cancer in women.1 The single most important prognostic factor in apparently localized carcinoma of the colon and rectum is the presence or absence of regional node metastases. The 5-year survival rate for individuals with stage I or II colorectal cancer is approximately 80%. In contrast, in individuals with node-positive colorectal cancer, there is a substantial decrease in 5-year survival to approximately 50%. Accurate staging of the regional lymphatics is critical not only for its prognostic relevance, but also for its therapeutic implications. Substantial evidence now supports the efficacy of adjuvant chemotherapy in colorectal cancer.2 Currently, the presence of nodal metastases represents the primary indication for adjuvant chemotherapy It is apparent, however, that despite apparently curative surgical resection of histologically earlystage colorectal cancer, 20% to 30% of these individuals will develop recurrent disease and die. Although there are a number of potential reasons Seminars in Oncology, Vol 31, No 3 (June), 2004: pp 403-408
to explain the poorer outcome in these individuals, one of the potentially most important is inaccurate staging of the regional lymphatics. In this article, we will examine a number of approaches to improved staging of colorectal cancer and the potential impact of lymphatic mapping techniques to identify sentinel nodes on staging of colorectal cancer. LYMPH NODE HARVESTING AND STAGING ACCURACY
Resection of the primary tumor en bloc with the regional mesenteric draining lymphatics remains the primary curative therapy for apparently localized carcinoma of the colon and rectum. Resection based on anatomic structures provides not only for adequate radial and longitudinal margins, but also for clearance of the regional lymphatics. Despite well-recognized anatomic guidelines for the conduct of surgical resections, there may be substantial variations in the performance of resection of the colon and rectum,3 which may lead to substantial variations in the number of nodes, examined. Additionally, the number of nodes in a specimen may vary among patients4-6 and according to the diligence of the pathologic examination.7 Variations in the number of nodes examined can impact staging accuracy. In order to address this issue, numerous investigators have examined the number of nodes necessary to be harvested and/or examined to accurately stage the regional lymphatics in carcinoma of the colon and rectum with recommendations for adequate node harvesting to range from 6 to 17
From the Department of Surgery, John A. Burns School of Medicine; and the Program in Clinical Sciences, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI. Supported in part by the Surgical Oncology Research Fund at the University of Hawaii Foundation and a Grant from the Hawaii Community Foundation. Address reprint requests to Jan H. Wong, MD, Department of Surgery, John A. Burns School of Medicine, University of Hawaii at Manoa, 1356 Lusitana St, 6th Floor, Honolulu, HI 96813. © 2004 Elsevier Inc. All rights reserved. 0093-7754/04/3103-0014$30.00/0 doi:10.1053/j.seminoncol.2004.03.015 403
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nodes.8,9 The Working Party Report to the World Congress of Gastroenterology recommended that a minimum of 12 nodes be recovered and examined before the lymphatic basin could be considered free of metastatic disease.10 More recently, Joseph et al11 developed a mathematical model that suggested that there was less than a 25% probability of true node negativity in T1/T2 tumors when 18 nodes were removed. Tepper et al12 retrospectively reviewed 1,664 patients with rectal cancer from a prospective multimodality treatment study database. They determined that the minimal number of nodes necessary for staging purposes was 14. We examined the number of nodes needed to be retrieved and examined to accurately reflect the histology of the regional node basin in colorectal cancer and similar to Tepper et al, suggested that a minimum of 14 nodes needs to be examined.13 Additionally, a significant association between the number of nodes identified and both disease-free survival and overall survival was demonstrated in both of these patient populations,12,14 presumably the result of stage migration due to under staged individuals with fewer nodes that were examined. IMPROVING PATHOLOGIC ANALYSIS
The recognition of the importance of accurate staging not only for prognosis but for disease-free and overall survival has led to a number of strategies to improve not only the number of nodes examined but also the sensitivity of detecting disease in lymph nodes identified by routine gross examination.
Immunohistochemical Staining Numerous investigators have employed immunohistochemical staining techniques to identify micrometastatic disease that otherwise would not have been detected using conventional hematoxlyn-eosin staining. The incidence of immunohistochemically detected micrometastasis in apparently node-negative patients varied between 25% and 39%.7,17-27 Although some have suggested that these findings are prognostically relevant,7,24,27 others have not found that micrometastatic disease identified by immunohistochemical staining offers any significant prognostic information beyond that of routine hematoxlyn-eosin staining.17-19,22,25,28 Unfortunately, many of these investigations suffer from insufficient node harvesting, which could potentially compromise identifying any significant prognostic relevance. For this reason, the prognostic value of micrometastatic disease identified by immunohistochemical techniques remains controversial. In an effort to clarify this issue, we examined 100 node-positive patients to determine if micrometastatic disease (⬍0.2 mm in diameter) identified by routine hematoxlyn-eosin has the same prognostic impact as macrometastatic disease.29 Individuals with micrometastatic disease have similar outcomes to those with macrometastatic disease, suggesting that more sensitive assays may detect prognostically relevant disease. However, larger studies with adequate nodal harvesting and longer follow up are necessary to establish the biologic relevance of micrometastatic disease identified by immunohistochemical staining.
Fat Clearance Techniques The value of xylene and fat clearance techniques has been studied by a number of investigators in both colon6 and rectal cancer.15 In the colon and rectum, xylene and fat clearance techniques have significantly increased the number of pericolonic or perirectal nodes identified. Koren et al16 reported a threefold increase in the number of nodes retrieved using a lymph node–revealing solution in problematic specimens in which unsatisfactory lymph node yields were obtained by traditional methods. Although the fat clearing technique is simple and has been demonstrated to increase nodal yield, extended exposure of specimens to fixative is time-consuming and generally has not gained widespread acceptance.
Sentinel Node Staging of Solid Neoplasms In 1977, Cabanas suggested that penile carcinoma would drain to an anatomically defined inguinal node, which he termed the “sentinel lymph node.”30 In contrast, Morton et al31 hypothesized that the lymphatic drainage to a sentinel lymph node was based not on anatomic landmarks, but instead on unique lymphatic anatomy and therefore would required lymphatic mapping to be accurately detected. Wong et al32 developed an intraoperative technique to map the lymphatics and identified a sentinel node in a feline model, which has been validated now in both cutaneous melanoma31 and breast cancer.33 One of the most important benefits of lymphatic
LYMPHATIC MAPPING AND COLORECTAL CANCER
405
Table 1. Summary of In Vivo Lymphatic Mapping for Carcinoma of the Colon
Authors
No. of Patients
Saha et al35 Waters et al41 Tsioulias et al40 Joosten et al38 Merrie et al35
86 18 50 50 26
Feig et al37 Bendavid et al42 Esser et al43 Paramo et al44
48 20 31 55
Mapping Technique Isosulfan blue Isosulfan blue Isosulfan blue Isosulfan blue 99m colloid, isosulfan blue Isosulfan blue Isosulfan blue Isosulfan blue
Pathologic Analysis H&E, H&E, H&E, H&E, H&E,
IHC, LS IHC IHC, LS IHC IHC, PCR
H&E, IHC H&E, IHC H&E H&E, IHC, LS
Success Identifying Sentinel Node (%)
False Negative Rate (%)
98.8 100 94 70 88
9 0 — 60 45
98 90 58 82
38 5 6 3
Abbreviations: H&E, hematoxylin-eosin; IHS, immunohistochemistry; LS, level sectioning; PCR, polymerase chain reaction.
mapping is the ability to perform a focused pathologic examination on a relatively small amount of lymphatic tissue in a cost- and time-effective manner. Routine application of immunohistochemical staining of the sentinel node has demonstrated the potential to substantially upstage patients with both cutaneous melanoma31 and breast cancer34 who would otherwise have been considered node negative by routine hematoxlyn-eosin staining. Because of the critical importance of accurate nodal staging and the recognition of broad variability in the routine pathologic analysis of in colorectal cancer, we and others have initiated studies to evaluate the role and impact of lymphatic mapping in colorectal cancer. In Vivo (intraoperative) Lymphatic Mapping in Carcinoma of the Colon In vivo lymphatic mapping in carcinoma of the colon was first described in deteail by Saha et al.35 Initial feasibility studies involved a subserosal injection of isosulfan blue at the site of the primary tumor following mobilization of the tumor if located in the right or left colon, intraoperative lymphatic mapping and carefully following bluefilled lymphatic channels to blue-stained nodes. Blue-stained nodes were marked with a suture for future identification and a formal en bloc resection was then performed with the regional draining mesentery. In vivo lymphatic mapping has the potential advantage of providing a visual guide to the extent of mesenteric resection by allowing the operating surgeon to encompass aberrant lym-
phatic drainage. However, the technique may violate mesenteric fascial planes and possibly increase the risk of local recurrence36 and is technically difficult if not impossible to perform in the rectum. Experience gained from cutaneous melanoma and breast cancer suggested a potentially steep learning curve with lymphatic mapping and sentinel node identification in all solid tumors, including colon cancer. However, this has not proven necessarily to be the case. A number of investigators report the success rate in identifying the “blue” or “hot” node was, on average, 87%35,37-44 (Table 1). Paramo et al44 reported a near 100% sentinel node retrieval rate after only five cases among seven surgeons using in vivo lymphatic mapping. The accuracy of in vivo lymphatic mapping continues to be debated. Reported false-negative rates vary from 0 to 60% and have raised the issue of the reproducibility and reliability of the “blue” or “hot” node representing a biologic sentinel node. Reasons for these wide discrepancies are not apparent, but some have postulated that the presence of gross nodal or extranodal involvement might disrupt anatomical lymphatic drainage, leading to imprecise sentinel node identification. Another possible explanation may be attributable to the fact that the drainage pattern from peritumoral subserosal injections may not reproduce the lymphatic drainage pattern of the primary tumor. However, when comparing the results of Joosten et al,38 Merrie et al,39 and Feig et al35 to other studies
406
with false-negative rates less than 9%, level sectioning was not performed in these three studies, suggesting that nodal yield for micrometastatic disease may be significantly increased with a more thorough analysis of the node. Ex Vivo Lymphatic Mapping in Colorectal Cancer Although the feasibility of in vivo lymphatic mapping in colon cancer has been demonstrated in various studies, and refinements in technique and analysis have improved the success in sentinel node retrieval and improved detection of micrometastatic disease, this approach has potential problems that may limit broad applicability. These problems include not being applicable in rectal cancer due to anatomical constraints, that the subserosal injection of isosulfan blue may not reproduce the true lymphatic drainage of the primary tumor, that pericolic nodes adjacent to the injection site maybe difficulty to identify, and that the mechanical manipulation to increase lymphatic flow intraoperatively may potentially shed tumor cells into the lymphatics, facilitating spreading of disease prior to resection. Although rare, exposure to isosulfan blue places the patient at risk for potential adverse reactions.45 In an effort to address these issues, we developed an ex vivo lymphatic mapping approach to sentinel node staging of the colon and rectum.46 Ex vivo lymphatic mapping has a number of potential advantages over the in vivo approach to lymphatic mapping. Ex vivo lymphatic mapping allows the precise introduction of isosulfan blue into the submucosa, the site where lymphatics from the mucosa coalesce prior to penetrating the muscularis propria, avoids patient exposure to isosulfan blue, avoids the potential problem of shedding tumor cells or disrupting the fascial planes that may be critical in minimizing local recurrences, and is feasible in rectal tumors. Finally, ex vivo lymphatic mapping has the advantage that all specimens, when delivered in the fresh state to pathology, can be examined and mapped in a uniform fashion, which will facilitate standardization of technique and, potentially, improve quality control. The ex vivo technique, developed by the senior author, is performed within 30 minutes of resection. The fresh colorectal specimen is opened on the antimesenteric border longitudinally. For rectal tumors, the specimen is longitudinally opened
JOHNSON AND WONG
on the anterior border opposite the mesorectum. No attempt is made to avoid the tumor if it involves the antimesenteric wall. Four 0.25 mL submucosal injections of isosulfan blue are performed peritumorally, proximally and distally along the longitudinal axis of the specimen and at 90 degrees from these injection sites. If the tumor involves the antimesenteric wall, injections on both sides of the divided tumor are performed. Gentle massaging of the specimen at the injection site for 5 minutes follows, to induce lymphatic flow. The peritoneal reflection of the mesentery is then sharply incised, and with blunt dissection of the mesenteric fat, lymphatic channels stained blue identified and followed until blue-stained nodes are identified. These nodes are then harvested and submitted for histological examination. The rest of the specimen is then fixed in formalin overnight and the remaining nodes in the specimen are then harvested visually and/or with manual manipulation. The ndoes are then submitted for routine hematoxylin-eosin examination. The blue nodes harvested are initially embedded in paraffin and processed by routine hematoxylin-eosin staining. If the node is larger than 3 mm, it is bivalved before paraffin embedding. Blue nodes that are negative by conventional examination undergo additional level sectioning at 300 m and are further analyzed by hematoxylin-eosin and immunohistochemical staining. In a pilot study of 26 patients designed to demonstrate the feasability of ex vivo lymphatic mapping in colorectal cancer,46 the sentinel lymph node harbored metastatic disease either by conventional examination or with immunohistochemistry in 11 of 12 patients who were known to be node-positive by conventional pathological evaluation of the nonsentinel nodes. Although the sensitivity with hematoxlyn-eosin alone was only 58.3%, it improved to 93.7% with the addition of immunohistochemical staining. However, more importantly, with additional level sectioning and immunohistochemical analysis, four of 14 patients who were apparently node-negative by conventional examation were found to harbor metastatic disease, which potentially could represent an upstaging of 29%. In contrast to breast cancer and melanoma in which the sentinel node now determines the extent of the lymphadenectomy, lymphatic mapping in colorectal cancer is principally to improve staging. For this reason, we have fo-
LYMPHATIC MAPPING AND COLORECTAL CANCER
cused our attention on apparently node-negative colorectal cancer to determine the feasibility of ex vivo lymphatic mapping. To date, we have examined 124 patients with colorectal cancer. Sixty-six patients were nodenegative by conventional hematoxylin-eosin staining. A total of 2,177 nodes were analyzed from these 66 hematoxylin-eosin–stained nodenegative specimens (1,883 nonsentinel nodes and 294 sentinel nodes). Overall, sentinel lymph node metastases were identified in 13 of 278 sentinel nodes and in only five of 1,829 nonsentinel nodes (P ⬍ .001). Although five of 66 patients (7.5%) had evidence of metastatic disease in nonsentinel lymph nodes when the sentinel node was negative, 13 apparently node-negative patients (19.3%) were upstaged by inmmunohistochemistry of the sentinel lymph node (P ⫽ .04). In a similar fashion, Weise et al47 performed immunohistochemical staining with multilevel sectioning on all nodes identified in 25 patients undergoing in vivo lymphatic mapping and found that micrometastasis was rarely found when the sentinel node was negative. It is apparent that the most cost-effective method to upstage individuals is to focus on the sentinel lymph node. CONCLUSION
Substantial evidence now supports that the number of nodes examined influences staging accuracy and prognosis. Due to the inherent difficulties associated with routine nodal harvesting and the marked variability in the number of nodes examined, lymphatic mapping, by either in vivo or ex vivo techniques, offers a simple, cost- and timeeffective approach to improve staging in patients with carcinoma of the colon and rectum who apparently are node-negative following routine histological examination of the regional lymph nodes. Although some have questioned the sensitivity of lymphatic mapping in carcinoma of the colon and rectum, those studies in which the sentinel lymph node has been subjected to a thorough examination by a combination of immunohistochemical staining and multilevel sectioning have had false-negative rates consistent with those observed in melanoma and breast cancer. Although the prognostic significance and therapeutic relevance of micrometastatic disease remains a subject of ongoing debate and remains to be clearly established, lymphatic mapping and microstaging of the
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sentinel lymph node(s) offers an opportunity to further refine an understanding of the metastatic process in colorectal cancer and has the potential upstage significant number of individuals with apparently node-negative disease. REFERENCES 1. Jemal A, Thomas A, Murray T, et al: Cancer Statistics, 2002. CA Cancer J Clin 52:23-47, 2002 2. Moertel CG, Flemming TR, Macdonald JS, et al: Fluorouracil plus levamisole as effective adjuvant therapy after resection of stage III colon carcinoma: A final report. Ann Intern Med 122:321-326, 1995 3. Reinbach DH, McGregor JR, Murray GD, et al: Effect of the surgeon’s specialty on the type of resection performed for colorectal cancer. Dis Colon Rectum 37:1020-1023, 1994 4. Gilchrist RK, David VC: A consideration of pathologic factors influencing five year survival in radical resection of the the large bowel and rectum for carcinoma. Ann Surg 126:421428, 1947 5. Grinnell RS: Lymphatic metastases of carcinoma of the colon and rectum. Ann Surg 131:494-506, 1950 6. Scott KWM, Grace RH: Detection of lymph node metastases in colorectal carcinoma before and after fat clearance. Br J Surg 76:1165-1167, 1986 7. Greenson JK, Isenhart CE, Rice R, et al: Identification of occult micrometastases in pericolic lymph nodes of Dukes’ B colorectal cancer patients using monoclonal antibodies again cytokeratin and CC49. Cancer 73:563-569, 1994 8. Hernanz F, Revuelta S, Redondo C, et al: Colorectal adenocarcinoma: Quality of the assessment of lymph node metastases. Dis Colon Rectum 37:373-377, 1994 9. Goldstein NS, Weldon S, Coffey M, et al: Lymph node recovery from colorectal resection specimens removed for adencoarcinoma: Trends over time and a recommendation for a minimum number of lymph nodes to be recovered. Am J Clin Pathol 106:209-216, 1996 10. Fielding LP, Aresnault PA, Chapuis PH, et al: Clinicopathological staging for colorectal cancer: An international documentation system (IDS) and an international comprehensive anatomical terminology (ICAT). J Gastroenterol Hepatol 6:325-344, 1991 11. Joseph N, Sigurdson ER, Hanlon A, et al: Accuracy of determining nodal negativity in colorectal cancer on the basis of the number of nodes retrieved on resection. Ann Surg Oncol 10:213-218, 2003 12. Tepper JE, O’Connell MJ, Niedzwiecki D, et al: Impact of number of nodes retrieved on outcome in patients with rectal cancer. J Clin Oncol 19:157-163, 2001 13. Wong JH, Severino R, Honnebier MB, et al: Number of nodes examined and staging accuracy in colorectal carcinoma. J Clin Oncol 17:2896-2900, 1999 14. Wong JH, Bowles BJ, Bueno R, et al: Impact of the number of negative nodes on disease-free survival in colorectal cancer patients. Dis Colon Rectum 45:1341-1348, 2002 15. Cawthorn S, Gibbs N, Marks C: Clearance technique for the detection of lymph nodes in colorectal cancer. Br J Surg 73:58-60, 1986 16. Koren R, Siegal A, Klein B, et al: Lymph node-revealing
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solutions: Simple new method for detecting minute lymph nodes in colon carcinoma. Dis Colon Rectum 40:407-410, 1997 17. Adell G, Boeryd B, Franlund B, et al: Occurrence and prognostic importance of micrometastases in regional lymphnodes in Dukes’ B colorectal carcinoma: An immunohistochemical study. Eur J Surg 162:637-642, 1996 18. Broll R, Schauer V, Schimmelpenning H, et al: Prognostic relevance of occult tumor cells in lymph nodes of colorectal carcinomas: An immunohistochemical study. Dis Colon Rectum 40:1465-1471, 1997 19. Choi H, Choi Y, Hong S: Incidence and prognostic implications of isolated tumor cells in lymph nodes from patients with Dukes B colorectal carcinoma. Dis Colon Rectum 45:750-755, 1997 20. Clarke G, Ryan E, O’Keane J, et al: The detection of cytokeratins in lymph nodes of Duke’s B colorectal cancer subjects predicts a poor outcome. Eur J Gastroenterol Hepatol 12:549-552, 2000 21. Cote R, Houchens D, Hitchcock C, et al: Intraoperative detection of occult colon cancer micrometastases using 125 I-radiolabeled monoclonal antibody CC49. Cancer 77:613620, 1996 22. Cutait R, Alves VAF, Lopes LC, et al: Restaging of colorectal cancer based on the identification of lymph node micrometastases through immunoperoxidase staining of CEA and cytokeratins. Dis Colon Rectum 34:917-920, 1991 23. Davidson BR, Sams VR, Styles J, et al: Detection of occult nodal metastases in patients with colorectal carcinoma. Cancer 65:967-970, 1990 24. Isaka N, Nozue M, Doy M, et al: Prognostic significance of perirectal lymph node micrometastases in Duke’s B rectal carcinoma: An immunohistochemical study by CAM5.2. Clin Cancer Res 5:2065-2068, 1999 25. Jeffers MD, O’Dowd GM, Mulcahy H, et al: The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma. J Pathol 172: 183-187, 1994 26. Oberg A, Stenling R, Tavelin B, et al: Are lymph node micrometastases of any clinical significance in Dukes stage A and B colorectal cancer? Dis Colon Rectum 41:1244-1249, 1998 27. Yasuda K, Adachi Y, Norio S, et al: Pattern of lymph node micrometastasis and prognosis of patients with colorectal cancer. Ann Surg Oncol 8:300-304, 2001 28. Tschmelitsch J, Klimstra DS, Cohen AM: Lymph node micrometastases do not predict relapse in stage II colon cancer. Ann Surg Oncol 7:601-608, 2000 29. Wong JH, Steinemann S, Tom P, et al: Volume of lymphatic metastases does not independently influence prognosis in colorectal cancer. J Clin Oncol 20:1506-1511, 2002 30. Cabanas RM: An approach for the treatment of penile carcinoma. Cancer 39:456-466, 1977
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31. Morton DL, Duan-Ren W, Wong JH, et al: Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg 127:392-399, 1992 32. Wong JH, Cagle LA, Morton DL: Lymphatic drainage of skin to a sentinel lymph node in a feline model. Ann Surg 214:637-641, 1991 33. Giuliano AE, Kirgan DM, Guenther JM, et al: Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg 220:391-401, 1994 34. Turner RR, Ollila DW, Krasne DL, et al: Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma. Ann Surg 226:271-278, 1997 35. Saha S, Wiese D, Badin J, et al: Technical detail of sentinel lymph node mapping in colorectal cancer and its impact on staging. Ann Surg Oncol 7:120-124, 2000 36. Heald RJ, Moran BJ, Ryall RDH, et al: Rectal cancer: The Basingstoke experience of total mesorectal excision, 19781997. Arch Surg 133:894-897, 1998 37. Feig B, Curley S, Lucci A, et al: A caution regarding lymphatic mapping in patients with colon cancer. Am J Surg 182:707-712, 2001 38. Joosten JJA, Strobge LJA, Wauters CAP, et al: Intraoperative lymphatic mapping and the sentinel node concept in colorectal carcinoma. Br J Surg 86:482-486, 1999 39. Merrie A, van Rij A, Phillips L, et al: Diagnostic use of the sentinel node in colon cancer. Dis Colon Rectum 44:410417, 2001 40. Tsioulias G, Wood T, Morton D, et al: Lymphatic mapping and focused analysis of sentinel lymph nodes upstage gastrointestinal neoplasms. Arch Surg 135:926-932, 2000 41. Waters G, Geisinger K, Garske D: Sentinel lymph node mapping for carcinoma of the colon: A pilot study. Am J Surg 66:943-945, 2000 42. Bendavid Y, Latulippe J, Younan R, et al: Phase I study on sentinel lymph node mapping in colon cancer: A preliminary report. J Surg Oncol 79:81-84, 2002 43. Esser S, Reilly W, Riley L, et al: The role of sentinel lymph node mapping in staging of colon and rectal cancer. Dis Colon Rectum 44:850-854, 2001 44. Paramo J, Summerall J, Poppiti R, et al: Validation of sentinel node mapping in patients with colon cancer. Ann Surg Oncol 9:550-554, 2002 45. Leong SPL, Donegan E, Heffernon W, et al: Adverse reactions to isosulfan blue during selective sentinel lymph node dissection in melanoma. Ann Surg Oncol 7:361-366, 2000 46. Wong JH, Steinemann S, Calderia C, et al: Ex vivo sentinel node mapping in carcinoma of the colon and rectum. Ann Surg 233:515-521, 2001 47. Wiese DA, Saha S, Badin J, et al: Pathologic evaluation of sentinel lymph nodes in colorectal carcinoma. Arch Pathol Lab Med 124:1759-1763, 2000
C
OLORECTAL CANCER represents a significant health problem and is the third leading cause of cancer mortality in the United States, exceeded only by lung and prostate cancer in men and lung and breast cancer in women.1 The single most important prognostic factor in apparently localized carcinoma of the colon and rectum is the presence or absence of regional node metastases. The 5-year survival rate for individuals with stage I or II colorectal cancer is approximately 80%. In contrast, in individuals with node-positive colorectal cancer, there is a substantial decrease in 5-year survival to approximately 50%. Accurate staging of the regional lymphatics is critical not only for its prognostic relevance, but also for its therapeutic implications. Substantial evidence now supports the efficacy of adjuvant chemotherapy in colorectal cancer.2 Currently, the presence of nodal metastases represents the primary indication for adjuvant chemotherapy It is apparent, however, that despite apparently curative surgical resection of histologically earlystage colorectal cancer, 20% to 30% of these individuals will develop recurrent disease and die. Although there are a number of potential reasons Seminars in Oncology, Vol 31, No 3 (June), 2004: pp 403-408
to explain the poorer outcome in these individuals, one of the potentially most important is inaccurate staging of the regional lymphatics. In this article, we will examine a number of approaches to improved staging of colorectal cancer and the potential impact of lymphatic mapping techniques to identify sentinel nodes on staging of colorectal cancer. LYMPH NODE HARVESTING AND STAGING ACCURACY
Resection of the primary tumor en bloc with the regional mesenteric draining lymphatics remains the primary curative therapy for apparently localized carcinoma of the colon and rectum. Resection based on anatomic structures provides not only for adequate radial and longitudinal margins, but also for clearance of the regional lymphatics. Despite well-recognized anatomic guidelines for the conduct of surgical resections, there may be substantial variations in the performance of resection of the colon and rectum,3 which may lead to substantial variations in the number of nodes, examined. Additionally, the number of nodes in a specimen may vary among patients4-6 and according to the diligence of the pathologic examination.7 Variations in the number of nodes examined can impact staging accuracy. In order to address this issue, numerous investigators have examined the number of nodes necessary to be harvested and/or examined to accurately stage the regional lymphatics in carcinoma of the colon and rectum with recommendations for adequate node harvesting to range from 6 to 17
From the Department of Surgery, John A. Burns School of Medicine; and the Program in Clinical Sciences, Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI. Supported in part by the Surgical Oncology Research Fund at the University of Hawaii Foundation and a Grant from the Hawaii Community Foundation. Address reprint requests to Jan H. Wong, MD, Department of Surgery, John A. Burns School of Medicine, University of Hawaii at Manoa, 1356 Lusitana St, 6th Floor, Honolulu, HI 96813. © 2004 Elsevier Inc. All rights reserved. 0093-7754/04/3103-0014$30.00/0 doi:10.1053/j.seminoncol.2004.03.015 403
404
JOHNSON AND WONG
nodes.8,9 The Working Party Report to the World Congress of Gastroenterology recommended that a minimum of 12 nodes be recovered and examined before the lymphatic basin could be considered free of metastatic disease.10 More recently, Joseph et al11 developed a mathematical model that suggested that there was less than a 25% probability of true node negativity in T1/T2 tumors when 18 nodes were removed. Tepper et al12 retrospectively reviewed 1,664 patients with rectal cancer from a prospective multimodality treatment study database. They determined that the minimal number of nodes necessary for staging purposes was 14. We examined the number of nodes needed to be retrieved and examined to accurately reflect the histology of the regional node basin in colorectal cancer and similar to Tepper et al, suggested that a minimum of 14 nodes needs to be examined.13 Additionally, a significant association between the number of nodes identified and both disease-free survival and overall survival was demonstrated in both of these patient populations,12,14 presumably the result of stage migration due to under staged individuals with fewer nodes that were examined. IMPROVING PATHOLOGIC ANALYSIS
The recognition of the importance of accurate staging not only for prognosis but for disease-free and overall survival has led to a number of strategies to improve not only the number of nodes examined but also the sensitivity of detecting disease in lymph nodes identified by routine gross examination.
Immunohistochemical Staining Numerous investigators have employed immunohistochemical staining techniques to identify micrometastatic disease that otherwise would not have been detected using conventional hematoxlyn-eosin staining. The incidence of immunohistochemically detected micrometastasis in apparently node-negative patients varied between 25% and 39%.7,17-27 Although some have suggested that these findings are prognostically relevant,7,24,27 others have not found that micrometastatic disease identified by immunohistochemical staining offers any significant prognostic information beyond that of routine hematoxlyn-eosin staining.17-19,22,25,28 Unfortunately, many of these investigations suffer from insufficient node harvesting, which could potentially compromise identifying any significant prognostic relevance. For this reason, the prognostic value of micrometastatic disease identified by immunohistochemical techniques remains controversial. In an effort to clarify this issue, we examined 100 node-positive patients to determine if micrometastatic disease (⬍0.2 mm in diameter) identified by routine hematoxlyn-eosin has the same prognostic impact as macrometastatic disease.29 Individuals with micrometastatic disease have similar outcomes to those with macrometastatic disease, suggesting that more sensitive assays may detect prognostically relevant disease. However, larger studies with adequate nodal harvesting and longer follow up are necessary to establish the biologic relevance of micrometastatic disease identified by immunohistochemical staining.
Fat Clearance Techniques The value of xylene and fat clearance techniques has been studied by a number of investigators in both colon6 and rectal cancer.15 In the colon and rectum, xylene and fat clearance techniques have significantly increased the number of pericolonic or perirectal nodes identified. Koren et al16 reported a threefold increase in the number of nodes retrieved using a lymph node–revealing solution in problematic specimens in which unsatisfactory lymph node yields were obtained by traditional methods. Although the fat clearing technique is simple and has been demonstrated to increase nodal yield, extended exposure of specimens to fixative is time-consuming and generally has not gained widespread acceptance.
Sentinel Node Staging of Solid Neoplasms In 1977, Cabanas suggested that penile carcinoma would drain to an anatomically defined inguinal node, which he termed the “sentinel lymph node.”30 In contrast, Morton et al31 hypothesized that the lymphatic drainage to a sentinel lymph node was based not on anatomic landmarks, but instead on unique lymphatic anatomy and therefore would required lymphatic mapping to be accurately detected. Wong et al32 developed an intraoperative technique to map the lymphatics and identified a sentinel node in a feline model, which has been validated now in both cutaneous melanoma31 and breast cancer.33 One of the most important benefits of lymphatic
LYMPHATIC MAPPING AND COLORECTAL CANCER
405
Table 1. Summary of In Vivo Lymphatic Mapping for Carcinoma of the Colon
Authors
No. of Patients
Saha et al35 Waters et al41 Tsioulias et al40 Joosten et al38 Merrie et al35
86 18 50 50 26
Feig et al37 Bendavid et al42 Esser et al43 Paramo et al44
48 20 31 55
Mapping Technique Isosulfan blue Isosulfan blue Isosulfan blue Isosulfan blue 99m colloid, isosulfan blue Isosulfan blue Isosulfan blue Isosulfan blue
Pathologic Analysis H&E, H&E, H&E, H&E, H&E,
IHC, LS IHC IHC, LS IHC IHC, PCR
H&E, IHC H&E, IHC H&E H&E, IHC, LS
Success Identifying Sentinel Node (%)
False Negative Rate (%)
98.8 100 94 70 88
9 0 — 60 45
98 90 58 82
38 5 6 3
Abbreviations: H&E, hematoxylin-eosin; IHS, immunohistochemistry; LS, level sectioning; PCR, polymerase chain reaction.
mapping is the ability to perform a focused pathologic examination on a relatively small amount of lymphatic tissue in a cost- and time-effective manner. Routine application of immunohistochemical staining of the sentinel node has demonstrated the potential to substantially upstage patients with both cutaneous melanoma31 and breast cancer34 who would otherwise have been considered node negative by routine hematoxlyn-eosin staining. Because of the critical importance of accurate nodal staging and the recognition of broad variability in the routine pathologic analysis of in colorectal cancer, we and others have initiated studies to evaluate the role and impact of lymphatic mapping in colorectal cancer. In Vivo (intraoperative) Lymphatic Mapping in Carcinoma of the Colon In vivo lymphatic mapping in carcinoma of the colon was first described in deteail by Saha et al.35 Initial feasibility studies involved a subserosal injection of isosulfan blue at the site of the primary tumor following mobilization of the tumor if located in the right or left colon, intraoperative lymphatic mapping and carefully following bluefilled lymphatic channels to blue-stained nodes. Blue-stained nodes were marked with a suture for future identification and a formal en bloc resection was then performed with the regional draining mesentery. In vivo lymphatic mapping has the potential advantage of providing a visual guide to the extent of mesenteric resection by allowing the operating surgeon to encompass aberrant lym-
phatic drainage. However, the technique may violate mesenteric fascial planes and possibly increase the risk of local recurrence36 and is technically difficult if not impossible to perform in the rectum. Experience gained from cutaneous melanoma and breast cancer suggested a potentially steep learning curve with lymphatic mapping and sentinel node identification in all solid tumors, including colon cancer. However, this has not proven necessarily to be the case. A number of investigators report the success rate in identifying the “blue” or “hot” node was, on average, 87%35,37-44 (Table 1). Paramo et al44 reported a near 100% sentinel node retrieval rate after only five cases among seven surgeons using in vivo lymphatic mapping. The accuracy of in vivo lymphatic mapping continues to be debated. Reported false-negative rates vary from 0 to 60% and have raised the issue of the reproducibility and reliability of the “blue” or “hot” node representing a biologic sentinel node. Reasons for these wide discrepancies are not apparent, but some have postulated that the presence of gross nodal or extranodal involvement might disrupt anatomical lymphatic drainage, leading to imprecise sentinel node identification. Another possible explanation may be attributable to the fact that the drainage pattern from peritumoral subserosal injections may not reproduce the lymphatic drainage pattern of the primary tumor. However, when comparing the results of Joosten et al,38 Merrie et al,39 and Feig et al35 to other studies
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with false-negative rates less than 9%, level sectioning was not performed in these three studies, suggesting that nodal yield for micrometastatic disease may be significantly increased with a more thorough analysis of the node. Ex Vivo Lymphatic Mapping in Colorectal Cancer Although the feasibility of in vivo lymphatic mapping in colon cancer has been demonstrated in various studies, and refinements in technique and analysis have improved the success in sentinel node retrieval and improved detection of micrometastatic disease, this approach has potential problems that may limit broad applicability. These problems include not being applicable in rectal cancer due to anatomical constraints, that the subserosal injection of isosulfan blue may not reproduce the true lymphatic drainage of the primary tumor, that pericolic nodes adjacent to the injection site maybe difficulty to identify, and that the mechanical manipulation to increase lymphatic flow intraoperatively may potentially shed tumor cells into the lymphatics, facilitating spreading of disease prior to resection. Although rare, exposure to isosulfan blue places the patient at risk for potential adverse reactions.45 In an effort to address these issues, we developed an ex vivo lymphatic mapping approach to sentinel node staging of the colon and rectum.46 Ex vivo lymphatic mapping has a number of potential advantages over the in vivo approach to lymphatic mapping. Ex vivo lymphatic mapping allows the precise introduction of isosulfan blue into the submucosa, the site where lymphatics from the mucosa coalesce prior to penetrating the muscularis propria, avoids patient exposure to isosulfan blue, avoids the potential problem of shedding tumor cells or disrupting the fascial planes that may be critical in minimizing local recurrences, and is feasible in rectal tumors. Finally, ex vivo lymphatic mapping has the advantage that all specimens, when delivered in the fresh state to pathology, can be examined and mapped in a uniform fashion, which will facilitate standardization of technique and, potentially, improve quality control. The ex vivo technique, developed by the senior author, is performed within 30 minutes of resection. The fresh colorectal specimen is opened on the antimesenteric border longitudinally. For rectal tumors, the specimen is longitudinally opened
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on the anterior border opposite the mesorectum. No attempt is made to avoid the tumor if it involves the antimesenteric wall. Four 0.25 mL submucosal injections of isosulfan blue are performed peritumorally, proximally and distally along the longitudinal axis of the specimen and at 90 degrees from these injection sites. If the tumor involves the antimesenteric wall, injections on both sides of the divided tumor are performed. Gentle massaging of the specimen at the injection site for 5 minutes follows, to induce lymphatic flow. The peritoneal reflection of the mesentery is then sharply incised, and with blunt dissection of the mesenteric fat, lymphatic channels stained blue identified and followed until blue-stained nodes are identified. These nodes are then harvested and submitted for histological examination. The rest of the specimen is then fixed in formalin overnight and the remaining nodes in the specimen are then harvested visually and/or with manual manipulation. The ndoes are then submitted for routine hematoxylin-eosin examination. The blue nodes harvested are initially embedded in paraffin and processed by routine hematoxylin-eosin staining. If the node is larger than 3 mm, it is bivalved before paraffin embedding. Blue nodes that are negative by conventional examination undergo additional level sectioning at 300 m and are further analyzed by hematoxylin-eosin and immunohistochemical staining. In a pilot study of 26 patients designed to demonstrate the feasability of ex vivo lymphatic mapping in colorectal cancer,46 the sentinel lymph node harbored metastatic disease either by conventional examination or with immunohistochemistry in 11 of 12 patients who were known to be node-positive by conventional pathological evaluation of the nonsentinel nodes. Although the sensitivity with hematoxlyn-eosin alone was only 58.3%, it improved to 93.7% with the addition of immunohistochemical staining. However, more importantly, with additional level sectioning and immunohistochemical analysis, four of 14 patients who were apparently node-negative by conventional examation were found to harbor metastatic disease, which potentially could represent an upstaging of 29%. In contrast to breast cancer and melanoma in which the sentinel node now determines the extent of the lymphadenectomy, lymphatic mapping in colorectal cancer is principally to improve staging. For this reason, we have fo-
LYMPHATIC MAPPING AND COLORECTAL CANCER
cused our attention on apparently node-negative colorectal cancer to determine the feasibility of ex vivo lymphatic mapping. To date, we have examined 124 patients with colorectal cancer. Sixty-six patients were nodenegative by conventional hematoxylin-eosin staining. A total of 2,177 nodes were analyzed from these 66 hematoxylin-eosin–stained nodenegative specimens (1,883 nonsentinel nodes and 294 sentinel nodes). Overall, sentinel lymph node metastases were identified in 13 of 278 sentinel nodes and in only five of 1,829 nonsentinel nodes (P ⬍ .001). Although five of 66 patients (7.5%) had evidence of metastatic disease in nonsentinel lymph nodes when the sentinel node was negative, 13 apparently node-negative patients (19.3%) were upstaged by inmmunohistochemistry of the sentinel lymph node (P ⫽ .04). In a similar fashion, Weise et al47 performed immunohistochemical staining with multilevel sectioning on all nodes identified in 25 patients undergoing in vivo lymphatic mapping and found that micrometastasis was rarely found when the sentinel node was negative. It is apparent that the most cost-effective method to upstage individuals is to focus on the sentinel lymph node. CONCLUSION
Substantial evidence now supports that the number of nodes examined influences staging accuracy and prognosis. Due to the inherent difficulties associated with routine nodal harvesting and the marked variability in the number of nodes examined, lymphatic mapping, by either in vivo or ex vivo techniques, offers a simple, cost- and timeeffective approach to improve staging in patients with carcinoma of the colon and rectum who apparently are node-negative following routine histological examination of the regional lymph nodes. Although some have questioned the sensitivity of lymphatic mapping in carcinoma of the colon and rectum, those studies in which the sentinel lymph node has been subjected to a thorough examination by a combination of immunohistochemical staining and multilevel sectioning have had false-negative rates consistent with those observed in melanoma and breast cancer. Although the prognostic significance and therapeutic relevance of micrometastatic disease remains a subject of ongoing debate and remains to be clearly established, lymphatic mapping and microstaging of the
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sentinel lymph node(s) offers an opportunity to further refine an understanding of the metastatic process in colorectal cancer and has the potential upstage significant number of individuals with apparently node-negative disease. REFERENCES 1. Jemal A, Thomas A, Murray T, et al: Cancer Statistics, 2002. CA Cancer J Clin 52:23-47, 2002 2. Moertel CG, Flemming TR, Macdonald JS, et al: Fluorouracil plus levamisole as effective adjuvant therapy after resection of stage III colon carcinoma: A final report. Ann Intern Med 122:321-326, 1995 3. Reinbach DH, McGregor JR, Murray GD, et al: Effect of the surgeon’s specialty on the type of resection performed for colorectal cancer. Dis Colon Rectum 37:1020-1023, 1994 4. Gilchrist RK, David VC: A consideration of pathologic factors influencing five year survival in radical resection of the the large bowel and rectum for carcinoma. Ann Surg 126:421428, 1947 5. Grinnell RS: Lymphatic metastases of carcinoma of the colon and rectum. Ann Surg 131:494-506, 1950 6. Scott KWM, Grace RH: Detection of lymph node metastases in colorectal carcinoma before and after fat clearance. Br J Surg 76:1165-1167, 1986 7. Greenson JK, Isenhart CE, Rice R, et al: Identification of occult micrometastases in pericolic lymph nodes of Dukes’ B colorectal cancer patients using monoclonal antibodies again cytokeratin and CC49. Cancer 73:563-569, 1994 8. Hernanz F, Revuelta S, Redondo C, et al: Colorectal adenocarcinoma: Quality of the assessment of lymph node metastases. Dis Colon Rectum 37:373-377, 1994 9. Goldstein NS, Weldon S, Coffey M, et al: Lymph node recovery from colorectal resection specimens removed for adencoarcinoma: Trends over time and a recommendation for a minimum number of lymph nodes to be recovered. Am J Clin Pathol 106:209-216, 1996 10. Fielding LP, Aresnault PA, Chapuis PH, et al: Clinicopathological staging for colorectal cancer: An international documentation system (IDS) and an international comprehensive anatomical terminology (ICAT). J Gastroenterol Hepatol 6:325-344, 1991 11. Joseph N, Sigurdson ER, Hanlon A, et al: Accuracy of determining nodal negativity in colorectal cancer on the basis of the number of nodes retrieved on resection. Ann Surg Oncol 10:213-218, 2003 12. Tepper JE, O’Connell MJ, Niedzwiecki D, et al: Impact of number of nodes retrieved on outcome in patients with rectal cancer. J Clin Oncol 19:157-163, 2001 13. Wong JH, Severino R, Honnebier MB, et al: Number of nodes examined and staging accuracy in colorectal carcinoma. J Clin Oncol 17:2896-2900, 1999 14. Wong JH, Bowles BJ, Bueno R, et al: Impact of the number of negative nodes on disease-free survival in colorectal cancer patients. Dis Colon Rectum 45:1341-1348, 2002 15. Cawthorn S, Gibbs N, Marks C: Clearance technique for the detection of lymph nodes in colorectal cancer. Br J Surg 73:58-60, 1986 16. Koren R, Siegal A, Klein B, et al: Lymph node-revealing
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