REVIEW ARTICLE
Sentinel Lymph Node Biopsy and Nodal Ultrastaging in Colorectal Cancer Kelly T. Huynh, MD and Anton J. Bilchik, MD, PhD, FACS Abstract: The tumor status of the regional lymph nodes is the most important prognostic indicator in colorectal cancer (CRC), as it is in other solid tumors. Sentinel lymph node biopsy (SLNB), which has profoundly impacted the treatment of melanoma and breast cancer, has been applied in CRC in an attempt to improve nodal staging accuracy. The challenge lies in identifying patients who have tumor-negative nodes but are at high risk of regional or distant failure and therefore may benefit from adjuvant chemotherapy. Because standard pathological analysis of lymph nodes may incorrectly stage colon cancer, multiple studies have investigated nodal ultrastaging based on identification and immunohistochemical and/or molecular assessment of the sentinel node. This review focuses on the technique of SNLB, its feasibility and validity, and the controversies that remain regarding the clinical significance of nodal ultrastaging in CRC. Key Words: Colorectal cancer, micrometastases, sentinel lymph node, nodal ultrastaging, molecular biomarkers, prognosis (Cancer J 2015;21: 11–16)
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n the United States, colorectal cancer (CRC) remains the third most common cancer and the second leading cause of cancerrelated death.1 In the past 20 years, the death rate from CRC has decreased because of improved screening programs and treatment options.1 However, although surgical resection is often curative in early-stage disease, survival rates decrease when cancer metastasizes to the regional lymph nodes; the recurrence rate after clinically complete resection of stage III colon cancer can reach 70%.2 As with other solid tumors, staging in CRC is based on the TNM model. In 2010, the seventh edition of the American Joint Committee on Cancer (AJCC) staging manual introduced stage IIC CRC and reclassified stage IIIC CRC.3 Although this system considers new prognostic factors such as tumor regression grade, circumferential resection margin, presence of isolated tumor cells (ITCs), and other molecular markers, it still has limited prognostic accuracy. In fact, a recent study using the SEER (Surveillance, Epidemiology, End Results) database found that the seventh edition of the AJCC staging guidelines did not eliminate the survival discrepancies between patients with stage II and stage IIIA CRC.4 Rates of 5-year disease-specific survival (DSS) and overall survival (OS) were higher with stage IIIA disease than with stage IIA-IIC disease (DSS: 89% vs 86.3%, 79.4%, and 64.9% respectively [P < 0.001]; and OS: 79% vs 72.4%, 63.2%, and 54.6%, respectively [P < 0.001]).4 It is clear that additional From the Department of Surgical Oncology, John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, CA. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Cancer Institute or the National Institutes of Health. Conflicts of Interest and Source of Funding: Supported by grant 2R01 CA90848-05A2 from the National Cancer Institute and by funding from the California Oncology Research Institute, Los Angeles, CA. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Reprints: Anton Bilchik, MD, PhD, FACS, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 1528-9117
refinement of the staging system is needed to optimize prognostic stratification. In patients with distant metastasis or lymph node involvement, adjuvant therapies improve survival.5–7 Although adjuvant chemotherapy for stage III disease has been the standard of care for almost 20 years,2 its use in stage II disease is less clear. In 2004, the American Society of Clinical Oncology recommended consideration of its use in high-risk stage II patients (inadequate lymphadenectomy, T4 disease, colon perforation, or poorly differentiated histology) despite lacking direct evidence from randomized trials.8 The QUASAR collaborative study was the first randomized trial to demonstrate a small but significant absolute improvement in survival (3.6%) when patients with stage II CRC were treated with adjuvant chemotherapy (fluorouracil and folinic acid) instead of observation.9 The challenge lies in identifying the CRC patients whose nodes are tumor-negative by conventional pathological analysis but still have a high risk of recurrence. Up to 25% to 30% of these patients will ultimately have a recurrence,10,11 likely reflecting aggressive tumor biology, missed micrometastatic disease, inadequate nodal sampling, and/ or inaccurate pathologic staging. As adjuvant therapy is often dictated by stage of disease, which is predicated on regional nodal status, differentiating between stage II and III patients has significant impact on the selection of adjuvant chemotherapy, which may impact OS and at the same time reduce the unnecessary toxicity of chemotherapy in those patients cured by surgery alone.
Colorectal Cancer: Assessment of Nodal Metastases Surgical resection of CRC involves removal of the primary tumor with its associated mesentery at the level of the origin of its vascular supply. Although there is consensus that nodal metastasis is the most important prognostic factor in determining longterm survival, there is no agreement on the optimal number of harvested nodes or on the techniques used to evaluate these nodes. The importance of accurate pathological staging of the lymph nodes in CRC has led the AJCC and the College of American Pathologists to recommend examination of a minimum of 12 lymph nodes.12–14 Although patient factors such as age and sex, tumor factors such as grade and site, and surgical technique can affect the number of lymph nodes retrieved,15 if fewer than 12 lymph nodes are initially identified, it is recommended that the pathologist should reexamine the tissue and conduct a more extensive search for lymph nodes. In the Intergroup Trial INT0089, survival increased as more nodes were evaluated.16 Our group’s analysis of SEER data confirmed that increased nodal sampling was associated with improved survival.17 Subsequent studies supported this correlation between outcomes and adherence to the 12-lymph-node minimum,17–23 and findings in the Hari et al4 study confirmed that examination of at least 12 lymph nodes was associated with improved 5-year DSS and OS rates irrespective if staging was based on the sixth or seventh edition of the AJCC Cancer Staging Manual.
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Are standard pathological techniques adequate for accurate assessment of the nodes in a CRC specimen? Conventional pathologic examination of lymph nodes involves microscopic evaluation of 1 or 2 sections stained with hematoxylin-eosin (H&E), which equates to analysis of less than 1% of the lymph node.24 However, as many as 70% of tumor-positive lymph nodes harbor metastases less than 0.5 cm in diameter,25 suggesting that these metastases can be easily missed by standard analysis. Furthermore, ITCs, defined as cell clusters of less than 0.2 mm, are even more challenging to detect without the use of serial sectioning or immunohistochemistry (IHC) (Fig. 1).24 Yet serial sectioning of all the lymph nodes submitted from regional lymphadenectomy is costly and impractical. Numerous studies therefore have focused on nodal “ultrastaging” based on sentinel lymph node biopsy (SLNB), specialized IHC stains, and molecular techniques such as quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR).24,26–28
Staging the Nodal Basin: The Sentinel Lymph Node Paradigm in CRC The technique of SLNB to focus analysis of the lymphatic drainage basin in solid tumors was pioneered by Morton et al29 in the 1990s starting with melanoma and adapted at the same institution to breast cancer30 and then applied to gastrointestinal malignancies.31 Conceptually and practically, the sentinel node represents the first tumor-draining node in the lymphatic basin and therefore the most likely site to harbor metastatic disease when regional nodes are involved.29 In melanoma and breast cancer, results of the SLNB can help avoid unnecessary complete lymph node dissection and its associated morbidities; however, in colon cancer, SLNB does not change the extent of lymph node dissection because all patients must undergo an oncologic en bloc lymphadenectomy with surgical resection of the
primary lesion. Instead, SLNB in CRC allows a more focused and extensive pathological analysis of a few lymph nodes that may harbor occult disease, thereby enhancing diagnostic accuracy and predictive value. Application of SLNB in CRC prompted a wave of studies that examined the feasibility of the technique, with considerable variability reported in the success rate, ranging from 58% to 100%.31–38 Most inconsistencies and failures were secondary to early surgical and pathological inexperience, lack of standardization of techniques, and inherent biological differences in assessing lymphatic drainage for low rectal cancers and nodal basins with extensive tumor burden.39 The proof of concept of the SLNB technique in gastrointestinal malignancies was demonstrated by Tsioulias et al40: lymphatic mapping was performed in 65 patients by injecting 0.5 to 1 mL of isosulfan blue dye around the periphery of the primary tumor, visualizing the afferent lymphatic channel and then following it to the first 1 to 3 blue-stained sentinel nodes, which were marked with a suture (Fig. 2A).41 Occasionally, aberrant lymphatic drainage may be identified,36 impacting the extent of surgery as the resection margins are then broadened to include the mesentery and blood supply associated with these lymph nodes. En bloc resection of the tumor and regional lymphadenectomy was then performed, and all sentinel nodes were serially sectioned at 2- to 3-mm intervals and analyzed by both H&E and IHC; nonsentinel nodes were evaluated by only H&E. In patients with large bowel adenocarcinoma (n = 50), sentinel lymph nodes (SLNs) were detected in 94%, and 20% of these patients were upstaged using the multisectioning and IHC technique.40 In a follow-up study, Wood et al36 validated SLNB for CRC by using in vivo, laparoscopic, and ex vivo approaches. At least 1 sentinel node was identified in 72 (96%) of 75 patients and occult micrometastases in 13 patients (17%),36 demonstrating that lymphatic mapping of sentinel nodes draining primary CRCs can be performed accurately. The feasibility of the ex vivo technique was
FIGURE 1. Definition of SLN metastases (“upstaging”) not detected by standard pathologic techniques. A and B, Isolated tumor cells defined as cell clusters of less than 0.2 mm. C and D, Micrometastases 0.2 to 2 mm. Reprinted with permission from Bilchik et al.24 Copyright © 2007, Wolters Kluwer Health.
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SLNB and Ultrastaging in Colorectal Cancer
lesser resistance to an uninvolved nonsentinel node, a phenomenon seen also in melanoma and breast cancer.29,30
Sentinel Lymph Node Biopsy: Validation Studies
FIGURE 2. Sentinel lymph node technique in CRC. A, After mobilization of the colon, isosulfan blue dye (0.5–1 mL) was carefully injected in 4 quadrants around the tumor. Within 30 to 60 minutes, the blue dye traveled from the primary site along the lymphatic channel (arrow) to the SLNs (arrowheads). Reprinted with permission from Bilchik et al.41 Copyright © 2001, American Society of Clinical Oncology. All rights reserved. B, Ex vivo identification of SLNs (arrows) in the mesentery after injection of isosulfan blue dye after the specimen had been removed from the body. Reprinted with permission from Wood et al.36 Copyright © 2001, Springer Science + Business Media.
examined as a primary approach, but also to address whether it might be useful in cases where in vivo lymphatic mapping failed to identify the sentinel node (Fig. 2B).36 Ex vivo mapping was performed after the specimen was removed from the body and was successful in 7 (88%) of 8 cases; 6 of the 7 cases were in low rectal tumors where in vivo mapping failed. The accuracy of this approach was confirmed by remapping the sentinel nodes in operative specimens from patients who had undergone successful in vivo mapping; in all cases, ex vivo injection of isosulfan blue dye intensified staining of nodes identified in vivo but not additional new nodes.36 The advantage of the ex vivo approach is that if the sentinel node is not identified intraoperatively by the surgeon, the specimen can be sent immediately to the pathologist for a more thorough dissection. After 100 consecutive cases of SLNB in early CRC, the sentinel node was found to accurately reflect the status of the nodal basin in 92 (95%) of 97 patients, with 24% upstaging of occult micrometastases missed by routine H&E.39 Review of the falsenegative cases demonstrated that the failures occurred during the first 30 cases with technical error playing a significant role. In addition, the only case in which a sentinel node was unable to be localized by both primary and salvage ex vivo lymphatic mapping occurred in a patient with rectal cancer with extensive nodal disease.39 When tumor completely replaces the lymphatic vessels leading to the true sentinel node, the blue dye can take a path of
A large phase II multicenter prospective trial by Bilchik et al10 assessed the accuracy of focused analysis of the SLN by multiple sectioning and IHC in stage I and II CRC. Between 2001 and 2005, 132 patients were evaluated at 4 tertiary centers with demonstrated surgical and pathological expertise in lymphatic mapping. Sentinel nodes were identified in vivo or ex vivo immediately after resection as described by Wood et al,36 and if the surgeon could not identify the SLN intraoperatively, the en bloc resected tumor and regional lymph node specimen were immediately sent to the pathologist, who conducted a more aggressive dissection to identify the SLN. Sentinel lymph nodes were processed and sectioned as previously described29 and analyzed by both H&E and IHC; in addition, further sections were also cut for molecular analysis by RT-PCR. Preliminary results reported in 2006 showed that the SLN was successfully identified in 96% of cases with a median number of SLNs and total lymph nodes examined being 3 and 14.5, respectively, which is compliant with even current recommendations on the number of nodes that should be removed for adequate pathologic staging. Metastases ranging from greater than 0.2 mm but less than 2 mm were considered micrometastases, whereas ITCs were single tumor cells or cell clusters less than 0.2 mm and usually detected by IHC. The false-negative rate (FNR), which was defined as the probability of not finding tumor in an SLN in the presence of nodal metastases, was 7.4% (6/81), which is comparable to the literature in melanoma and breast cancer.10,29,30 The US Military Cancer Institute Clinical Trials Group Study GI-01 was a prospective randomized multicenter trial conducted to determine if focused analysis of the sentinel node improves staging of the nodal basin.42 Patients were randomly assigned to standard pathological evaluation or ex vivo sentinel node mapping and ultrastaging with H&E and IHC, which defined SLN positivity. In this study, sentinel nodes were successfully identified in 82 (97.6%) of 84 patients,42 with a mean of 18.2 ± 0.9 nodes analyzed per patient. Significant nodal upstaging occurred using the SLN technique (57.3% vs 38.7%; P = 0.019),42 and although in the overall study population 5-year DFS only approached significance (61% control group vs 71% in SLN ultrastaging; P = 0.11),27 disease recurrence differed significantly between ultrastaged and conventionally staged node-negative patients (86% vs 71%, respectively; P = 0.04; Fig. 3).27 The authors concluded that focused analysis of the sentinel node improves the
FIGURE 3. Disease-free survival according to control versus TNA-us (targeted nodal assessment and ultrastaging) among nodenegative patients with colon cancer. Reprinted with permission from Nissan et al.27 Copyright © 2012, Wolters KLuwer Health. www.journalppo.com
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at least 20 to 30 SLNB procedures were required for adequate sensitivity and accuracy, a threshold of at least 20 cases appears to be appropriate. In addition, incorporating specialized cytokeratin immunohistochemical stains and, more recently, molecular analysis of the SLN with tumor-specific biomarkers, which were not included in the study of Bertagnolli et al,43 may have enhanced detection of micrometastatic disease in the sentinel node, although the prognostic significance remains debatable.
Molecular Staging of the Sentinel Node: The Next Frontier
FIGURE 4. Kaplan-Meier curves for disease outcome according to detection of at least 1 qRT-PCR marker in SLNs. A, Overall survival by qRT-PCR(+) in SLNs (not significant). B, Disease-free survival by qRT-PCR(+) in SLNs (P = 0.014). Reprinted with permission from Koyanagi et al.47 Copyright © 2008, American Association for Cancer Research.
staging of colon cancer and thereby increases DFS by a clinically important absolute difference of 15%. Contrastingly, in a controlled prospective trial by Bembenek et al,37 the rate of SLN detection was only 85% (268/315 patients), with a seemingly high FNR of 46% (38 of 82 patients). Failure of the SLN to predict nodal status was reportedly 54% in a multicenter study by Bertagnolli et al.43 The low detection rate and high FNR reported in these 2 studies may be attributed to the learning curve phenomenon because few patients were accrued at multiple institutions, and pathological techniques were not standardized. The exact number of cases needed to achieve proficiency in SLNB for CRC is unknown. Based on our feasibility studies in CRC, in which falsenegative results occurred in the first 30 to 50 cases,39, 44 and taking into consideration that based on the Multicenter Selective Lymphadenectomy Trial for melanoma45 and American College of Surgeons Oncology Group Z0010 trial for breast cancer,46 in which
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The development of molecular assays such as RT-PCR for detection of micrometastases expanded our ability to detect occult tumor cells in primary and metastatic tissues, circulating blood, and other body fluids in cancer patients. Based on our multimarker RT-PCR assays in breast cancer and melanoma, we assessed 3 mRNA markers, β-hCG (β-chain human chorionic gonadotropin), c-Met (hepatocyte growth factor receptor), and uMAGE (universal melanoma-associated antigen A family), in paired primary tumors and SLNs of patients in our multicenter CRC SLN staging trial.41 In this pilot study, there was a 90% concordance between RT-PCR–positive and H&E-positive SLNs, and the addition of IHC and RT-PCR identified occult micrometastases in 53% of patients whose SLNs were negative by conventional staging. These initial molecular studies were conducted on frozen sections, but we subsequently switched to paraffin-embedded tissue, which provided the advantage of tissue preservation and easier comparison with histopathology slides. The updated analysis utilized a 4-marker qRT-PCR panel including c-MET, MAGE-A3 (melanoma-associated antigen A3), CK20 (cytokeratin 20), and GalNAc-T (β-1,4-N-acetylgalactosaminyltransferase) and showed no significant difference in OS by marker detection in the SLN but was significant for DFS (P = 0.014), with a mean DFS of 37 months for qRT-PCR– positive) versus 61 months for qRT-PCR–negative SLN (Fig. 4).47 Sentinel lymph node positivity by qRT-PCR maintained prognostic value for DFS independent of age, gender, and TNM staging (hazard ratio = 4.9; P = 0.027).47 An interim analysis of our multicenter SLN trial incorporating additional patients and the results of molecular analysis of the SLN by RT-PCR was published in 2007.24 Colorectal cancers were considered truly upstaged if focused analysis of the SLN specimen changed nodal status from N0 to N1mi (micrometastases) or N1 (macrometastases) but categorized as only possible upstaging if ITCs were detected by IHC (N0i+) and qRT-PCR (N0mol+). Of 92 node-negative colon cancer patients, true upstaging occurred in 7 patients (8%), and 18 additional patients (22%) had IHC ITCs (N0i+). At a mean followup of 25 months, 12 patients had a CRC-related recurrence (9.5%), 10 of which had a positive SLN. Excluding the rectal cancer patients, all patients with primary colon cancer in which the SLN was negative by H&E, IHC, or qRT-PCR did not have a recurrence,24 suggesting that surgical resection alone may have been curative in these patients. In 2012, Rahbari and colleagues48 published the results of a meta-analysis that included 39 studies of micrometastases or ITCs detected by IHC and/or RT-PCR in node-negative CRC. Molecular tumor-cell detection was associated with poor OS, DFS, and DSS. Subgroup analyses demonstrated that prognostic significance was independent of the detection method, molecular biomarker target, and number of lymph nodes retrieved.48 Critics point out that the value of RT-PCR assays for the detection of occult tumor cells in SLNs is dependent on using markers that are cancer specific, in order to maximize sensitivity
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and specificity and minimize false-positive or false-negative results. In addition, at this point in time, standardization of techniques for molecular assays is lacking, which hampers comparison of different studies.
CONCLUSIONS Despite ongoing debate, several recent meta-analyses of the SLNB in CRC cancer demonstrated that the detection rate ranges from 91% to 94% and concluded that it is a safe procedure that should be considered in addition to conventional resection in colon cancer patients.49,50 The limitations of the procedure include inconsistent results between institutions and surgeons suggesting that surgical and pathological techniques need to be standardized. Although both immunohistochemical and molecular analyses of the SLN have been shown to upstage conventional H&E, their prognostic value remains to be definitively established. Undoubtedly, application of the SLNB to CRC will continue to evolve and as we further elucidate the molecular pathways that drive tumorigenesis, we will be able to discover improved targeted and specific biomarkers for analysis. It is therefore likely that SLNB in CRC will continue to be investigated with enthusiasm. We still have much progress to make in determining the optimal method of staging CRC patients in order to appropriately stratify patients into subgroups that may benefit the most from adjuvant therapy while avoiding unnecessary toxicity for those that can be cured by surgery alone. Nevertheless, determining how to incorporate nodal ultrastaging techniques into clinical practice continues to provide a challenge for future studies. REFERENCES 1. Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. 2. MacDonald JS. Adjuvant therapy of colon cancer. CA Cancer J Clin. 1999;49:202–219. 3. Colon and rectum. In: Edge SB, Byrd SR, Compton CC, et al. eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer-Verlag; 2010:143–164. 4. Hari DM, Leung AM, Lee J-H, et al. AJCC Cancer Staging Manual 7th edition criteria for colon cancer: do the complex modifications improve prognostic assessment? J Am Coll Surg. 2013;217:181–190. 5. Andre T, Boni C, Navarro M, et al. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. J Clin Oncol. 2009;27:3109–3116. 6. Graham JS, Cassidy J. Adjuvant therapy in colon cancer. Expert Rev Anticancer Ther. 2012;12:99–109. 7. Kuebler JP, Wieand HS, O’Connell MJ, et al. Oxaliplatin combined with weekly bolus fluorouracil and leucovorin as surgical adjuvant chemotherapy for stage II and III colon cancer: results from NSABP C-07. J Clin Oncol. 2007;25:2198–2204. 8. Benson AB III, Schrag D, Somerfield MR, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol. 2004;22:3408–3419. 9. Quasar Collaborative Group, Gray R, Barnwell J, McConkey C, et al. Adjuvant chemotherapy versus observation in patients with colorectal cancer: a randomized study. Lancet. 2007;370:2020–2029. 10. Bilchik AJ, DiNome M, Saha S, et al. Prospective multicenter trial of staging adequacy in colon cancer. Arch Surg. 2006;141:527–534. 11. Weitz J, Koch M, Debus J, et al. Colorectal cancer. Lancet. 2005;365:153–165. 12. Compton CC, Greene FL. The staging of colorectal cancer: 2004 and beyond. CA Cancer J Clin. 2004;54:295–308. 13. Compton CC, Fielding LP, Burgardt LJ, et al. Prognostic factors in colorectal cancer. College of American Pathologists consensus statement. Arch Pathol Lab Med. 2000;124:979–994. 14. Sobin HL, Greene FL. TNM classification. Clarification of number of regional lymph nodes for pN0. Cancer. 2001;92:452. 15. Sari L, Bader G, Lusco D, et al. Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer. Eur J Cancer. 2005;41:272–279.
SLNB and Ultrastaging in Colorectal Cancer
16. Le Voyer TE, Sigurdson ER, Hanlon AL, et al. Colon cancer survival is associated with increasing number of lymph nodes analyzed: a secondary survey of intergroup trial INT-0089. J Clin Oncol. 2003;21:2912–2919. 17. Chen SL, Bilchik AJ. More extensive nodal dissection improves survival for stages I to III of colon cancer: a population-based study. Ann Surg. 2006;244:602–610. 18. Kim SH, Ha TK, Kwon SJ. Evaluation of the 7th AJCC TNM staging system in point of lymph node classification. J Gastric Cancer. 2011;11:94–100. 19. Johnson PM, Porter GA, Ricciardi R, et al. Increasing negative lymph node count is independently associated with improved long-term survival in stage IIIB and IIIC colon cancer. J Clin Oncol. 2006; 24:3570–3575. 20. Chen SL, Steele SR, Eberhardt J, et al. Lymph node ratio as a quality and prognostic indicator in stage III colon cancer. Ann Surg. 2011;253:82–87. 21. Hong KD, Lee SI, Moon YH. Lymph node ratio as determined by the 7th edition of the American Joint Committee on Cancer staging system predicts survival in stage III colon cancer. J Surg Oncol. 2011;103:406–410. 22. Wang J, Hassett JM, Dayton MT, et al. Lymph node ratio: role in the staging of node-positive colon cancer. Ann Surg Oncol. 2008;15:1600–1608. 23. Wong SL. Lymph node evaluation in colon cancer: assessing the link between quality indicators and quality. JAMA. 2011;306:1139–1141. 24. Bilchik AJ, Hoon DSB, Saha S, et al. Prognostic impact of micrometastases in colon cancer: interim results of a prospective multicenter trial. Ann Surg. 2007;246:568–577. 25. Rodriguez-Bigas MA, Maamoun S, Weber TK, et al. Clinical significance of colorectal cancer: metastases in lymph nodes < 5 mm in size. Ann Surg Oncol. 1996;3:124–130. 26. Iddings D, Ahmad A, Elashoff D, et al. The prognostic effect of micrometastases in previously staged lymph node negative (N0) colorectal carcinoma: a meta-analysis. Ann Surg Oncol. 2006;13:1386–1392. 27. Nissan A, Protic M, Bilchik AJ, et al. United States Military Cancer Institute Clinical Trials Group (USMCI GI-01) randomized controlled trial comparing targeted nodal assessment and ultrastaging with standard pathological evaluation for colon cancer. Ann Surg. 2012;256:412–427. 28. Wasif N, Faries MB, Saha S, et al. Predictors of occult nodal metastasis in colon cancer: results from a prospective multicenter trial. Surgery. 2010;147:352–357. 29. Morton DL, Wen DR, Wong JH, et al. Technical details of intraoperative lymphatic mapping for early stage melanoma. Arch Surg. 1992;127: 392–399. 30. Giuliano AE, Kirgan DM, Guenther JM, et al. Lymphatic mapping and sentinel lymphadenectomy for breast cancer. Ann Surg. 1994;220:391–398. 31. Bilchik AJ, Giuliano A, Essner R, et al. Universal application of intraoperative lymphatic mapping and sentinel lymphadenectomy in solid neoplasms. Cancer J Sci Am. 1998;4:351–358. 32. Evangelista W, Satolli MA, Malossi A, et al. Sentinel lymph node mapping in colorectal cancer: a feasibility study. Tumori. 2002;88:37–40. 33. Joosten JJ, Strobbe IJ, Wauters CA, et al. Intraoperative lymphatic mapping and the sentinel node concept in colorectal cancer. Br J Surg. 1999;86:482–486. 34. Kitagawa Y, Fujii H, Mukai M, et al.The role of the sentinel lymph node in gastrointestinal cancer. Surg Clin North Am. 2000;80:1799–1809. 35. Merrie AE, van Rij AM, Phillips LV, et al. Diagnostic use of sentinel node in colon cancer. Dis Colon Rectum. 2001;44:410–417. 36. Wood TF, Saha S, Morton DL, et al. Validation of lymphatic mapping in colorectal cancer: in vivo, ex vivo, and laparoscopic techniques. Ann Surg Oncol. 2001;8:150–157. 37. Bembenek AE, Rosenberg R, Wagler E, et al. Sentinel lymph node biopsy in colon cancer: a prospective multicenter trial. Ann Surg. 2007;245: 858–863. 38. Saha S, Seghal R, Patel M, et al. A multicenter trial of sentinel lymph node mapping in colorectal cancer: prognostic implications for nodal staging and recurrence. Am J. Surg. 2006;191:305–310. 39. Wood TF, Nora DT, Morton DL, et al. One hundred consecutive cases of sentinel lymph node mapping in early colorectal carcinoma: detection of missed micrometastases. J Gastrointest Surg. 2002;6:322–329. 40. Tsioulias GJ, Wood TF, Morton DL, et al. Lymphatic mapping and focused analysis of sentinel lymph nodes upstage gastrointestinal neoplasms. Arch Surg. 2000;135:926–932.
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41. Bilchik AJ, Saha S, Wiese D, et al. Molecular staging of early colon cancer on the basis of sentinel node analysis: a multicenter phase II trial. J Clin Oncol. 2001;19:1128–1136. 42. Stojadinovic A, Nissan A, Protic M, et al. Prospective randomized study comparing sentinel lymph node evaluation with standard pathologic evaluation for the staging of colon carcinoma: results from the United States Military Cancer Institute Clinical Trials Group Study GI-01. Ann Surg. 2007;245:846–857. 43. Bertagnolli M, Miedema B, Redston M, et al. Sentinel node staging for resectable colon cancer: results of a multicenter study. Ann Surg. 2004;4:624–628. 44. Bilchik AJ, Nora D, Tollenaar RA, et al. Ultrastaging of early colon cancer using lymphatic mapping and molecular analysis. Eur J Cancer. 2002;38:977–985. 45. Morton DL, Cochran AJ, Thompson JF, et al. Sentinel node biopsy for early-stage melanoma: accuracy and morbidity in MSLT-1, an international multicenter trial. Ann Surg. 2005;242:302–313.
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46. Posther KE, McCall LM, Blumencranz PW, et al. Sentinel node skills verification and surgeon performance: data from multicenter clinical trial for early-stage breast cancer. Ann Surg. 2005;242:593–599. 47. Koyanagi K, Bilchik AJ, Saha S, et al. Prognostic relevance of occult nodal micrometastases and circulating tumor cells in colorectal cancer in a prospective multicenter trial. Clin Cancer Res. 2008;14: 7391–7396. 48. Rahbari NN, Bork U, Motschall E, et al. Molecular detection of tumor cells in regional lymph nodes is associated with disease recurrence and poor survival in node-negative colorectal cancer: a systematic review and metaanalysis. J Clin Oncol. 2012;30:60–70. 49. van der Zaag ES, Bouma VH, Tanis PJ, et al. Systematic review of sentinel lymph node mapping procedure in colorectal cancer. Ann Surg Oncol. 2012;19:3449–3459. 50. van der Pas MHGM, Meijer S, Hoekstra OS, et al. Sentinel-lymph-node procedure in colon and rectal cancer: a systematic review and metaanalysis. Lancet Oncol. 2011;12:540–550.
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Sentinel Lymph Node Biopsy and Nodal Ultrastaging in Colorectal Cancer Kelly T. Huynh, MD and Anton J. Bilchik, MD, PhD, FACS Abstract: The tumor status of the regional lymph nodes is the most important prognostic indicator in colorectal cancer (CRC), as it is in other solid tumors. Sentinel lymph node biopsy (SLNB), which has profoundly impacted the treatment of melanoma and breast cancer, has been applied in CRC in an attempt to improve nodal staging accuracy. The challenge lies in identifying patients who have tumor-negative nodes but are at high risk of regional or distant failure and therefore may benefit from adjuvant chemotherapy. Because standard pathological analysis of lymph nodes may incorrectly stage colon cancer, multiple studies have investigated nodal ultrastaging based on identification and immunohistochemical and/or molecular assessment of the sentinel node. This review focuses on the technique of SNLB, its feasibility and validity, and the controversies that remain regarding the clinical significance of nodal ultrastaging in CRC. Key Words: Colorectal cancer, micrometastases, sentinel lymph node, nodal ultrastaging, molecular biomarkers, prognosis (Cancer J 2015;21: 11–16)
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n the United States, colorectal cancer (CRC) remains the third most common cancer and the second leading cause of cancerrelated death.1 In the past 20 years, the death rate from CRC has decreased because of improved screening programs and treatment options.1 However, although surgical resection is often curative in early-stage disease, survival rates decrease when cancer metastasizes to the regional lymph nodes; the recurrence rate after clinically complete resection of stage III colon cancer can reach 70%.2 As with other solid tumors, staging in CRC is based on the TNM model. In 2010, the seventh edition of the American Joint Committee on Cancer (AJCC) staging manual introduced stage IIC CRC and reclassified stage IIIC CRC.3 Although this system considers new prognostic factors such as tumor regression grade, circumferential resection margin, presence of isolated tumor cells (ITCs), and other molecular markers, it still has limited prognostic accuracy. In fact, a recent study using the SEER (Surveillance, Epidemiology, End Results) database found that the seventh edition of the AJCC staging guidelines did not eliminate the survival discrepancies between patients with stage II and stage IIIA CRC.4 Rates of 5-year disease-specific survival (DSS) and overall survival (OS) were higher with stage IIIA disease than with stage IIA-IIC disease (DSS: 89% vs 86.3%, 79.4%, and 64.9% respectively [P < 0.001]; and OS: 79% vs 72.4%, 63.2%, and 54.6%, respectively [P < 0.001]).4 It is clear that additional From the Department of Surgical Oncology, John Wayne Cancer Institute at Providence Saint John’s Health Center, Santa Monica, CA. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Cancer Institute or the National Institutes of Health. Conflicts of Interest and Source of Funding: Supported by grant 2R01 CA90848-05A2 from the National Cancer Institute and by funding from the California Oncology Research Institute, Los Angeles, CA. The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. Reprints: Anton Bilchik, MD, PhD, FACS, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 1528-9117
refinement of the staging system is needed to optimize prognostic stratification. In patients with distant metastasis or lymph node involvement, adjuvant therapies improve survival.5–7 Although adjuvant chemotherapy for stage III disease has been the standard of care for almost 20 years,2 its use in stage II disease is less clear. In 2004, the American Society of Clinical Oncology recommended consideration of its use in high-risk stage II patients (inadequate lymphadenectomy, T4 disease, colon perforation, or poorly differentiated histology) despite lacking direct evidence from randomized trials.8 The QUASAR collaborative study was the first randomized trial to demonstrate a small but significant absolute improvement in survival (3.6%) when patients with stage II CRC were treated with adjuvant chemotherapy (fluorouracil and folinic acid) instead of observation.9 The challenge lies in identifying the CRC patients whose nodes are tumor-negative by conventional pathological analysis but still have a high risk of recurrence. Up to 25% to 30% of these patients will ultimately have a recurrence,10,11 likely reflecting aggressive tumor biology, missed micrometastatic disease, inadequate nodal sampling, and/ or inaccurate pathologic staging. As adjuvant therapy is often dictated by stage of disease, which is predicated on regional nodal status, differentiating between stage II and III patients has significant impact on the selection of adjuvant chemotherapy, which may impact OS and at the same time reduce the unnecessary toxicity of chemotherapy in those patients cured by surgery alone.
Colorectal Cancer: Assessment of Nodal Metastases Surgical resection of CRC involves removal of the primary tumor with its associated mesentery at the level of the origin of its vascular supply. Although there is consensus that nodal metastasis is the most important prognostic factor in determining longterm survival, there is no agreement on the optimal number of harvested nodes or on the techniques used to evaluate these nodes. The importance of accurate pathological staging of the lymph nodes in CRC has led the AJCC and the College of American Pathologists to recommend examination of a minimum of 12 lymph nodes.12–14 Although patient factors such as age and sex, tumor factors such as grade and site, and surgical technique can affect the number of lymph nodes retrieved,15 if fewer than 12 lymph nodes are initially identified, it is recommended that the pathologist should reexamine the tissue and conduct a more extensive search for lymph nodes. In the Intergroup Trial INT0089, survival increased as more nodes were evaluated.16 Our group’s analysis of SEER data confirmed that increased nodal sampling was associated with improved survival.17 Subsequent studies supported this correlation between outcomes and adherence to the 12-lymph-node minimum,17–23 and findings in the Hari et al4 study confirmed that examination of at least 12 lymph nodes was associated with improved 5-year DSS and OS rates irrespective if staging was based on the sixth or seventh edition of the AJCC Cancer Staging Manual.
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Are standard pathological techniques adequate for accurate assessment of the nodes in a CRC specimen? Conventional pathologic examination of lymph nodes involves microscopic evaluation of 1 or 2 sections stained with hematoxylin-eosin (H&E), which equates to analysis of less than 1% of the lymph node.24 However, as many as 70% of tumor-positive lymph nodes harbor metastases less than 0.5 cm in diameter,25 suggesting that these metastases can be easily missed by standard analysis. Furthermore, ITCs, defined as cell clusters of less than 0.2 mm, are even more challenging to detect without the use of serial sectioning or immunohistochemistry (IHC) (Fig. 1).24 Yet serial sectioning of all the lymph nodes submitted from regional lymphadenectomy is costly and impractical. Numerous studies therefore have focused on nodal “ultrastaging” based on sentinel lymph node biopsy (SLNB), specialized IHC stains, and molecular techniques such as quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR).24,26–28
Staging the Nodal Basin: The Sentinel Lymph Node Paradigm in CRC The technique of SLNB to focus analysis of the lymphatic drainage basin in solid tumors was pioneered by Morton et al29 in the 1990s starting with melanoma and adapted at the same institution to breast cancer30 and then applied to gastrointestinal malignancies.31 Conceptually and practically, the sentinel node represents the first tumor-draining node in the lymphatic basin and therefore the most likely site to harbor metastatic disease when regional nodes are involved.29 In melanoma and breast cancer, results of the SLNB can help avoid unnecessary complete lymph node dissection and its associated morbidities; however, in colon cancer, SLNB does not change the extent of lymph node dissection because all patients must undergo an oncologic en bloc lymphadenectomy with surgical resection of the
primary lesion. Instead, SLNB in CRC allows a more focused and extensive pathological analysis of a few lymph nodes that may harbor occult disease, thereby enhancing diagnostic accuracy and predictive value. Application of SLNB in CRC prompted a wave of studies that examined the feasibility of the technique, with considerable variability reported in the success rate, ranging from 58% to 100%.31–38 Most inconsistencies and failures were secondary to early surgical and pathological inexperience, lack of standardization of techniques, and inherent biological differences in assessing lymphatic drainage for low rectal cancers and nodal basins with extensive tumor burden.39 The proof of concept of the SLNB technique in gastrointestinal malignancies was demonstrated by Tsioulias et al40: lymphatic mapping was performed in 65 patients by injecting 0.5 to 1 mL of isosulfan blue dye around the periphery of the primary tumor, visualizing the afferent lymphatic channel and then following it to the first 1 to 3 blue-stained sentinel nodes, which were marked with a suture (Fig. 2A).41 Occasionally, aberrant lymphatic drainage may be identified,36 impacting the extent of surgery as the resection margins are then broadened to include the mesentery and blood supply associated with these lymph nodes. En bloc resection of the tumor and regional lymphadenectomy was then performed, and all sentinel nodes were serially sectioned at 2- to 3-mm intervals and analyzed by both H&E and IHC; nonsentinel nodes were evaluated by only H&E. In patients with large bowel adenocarcinoma (n = 50), sentinel lymph nodes (SLNs) were detected in 94%, and 20% of these patients were upstaged using the multisectioning and IHC technique.40 In a follow-up study, Wood et al36 validated SLNB for CRC by using in vivo, laparoscopic, and ex vivo approaches. At least 1 sentinel node was identified in 72 (96%) of 75 patients and occult micrometastases in 13 patients (17%),36 demonstrating that lymphatic mapping of sentinel nodes draining primary CRCs can be performed accurately. The feasibility of the ex vivo technique was
FIGURE 1. Definition of SLN metastases (“upstaging”) not detected by standard pathologic techniques. A and B, Isolated tumor cells defined as cell clusters of less than 0.2 mm. C and D, Micrometastases 0.2 to 2 mm. Reprinted with permission from Bilchik et al.24 Copyright © 2007, Wolters Kluwer Health.
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SLNB and Ultrastaging in Colorectal Cancer
lesser resistance to an uninvolved nonsentinel node, a phenomenon seen also in melanoma and breast cancer.29,30
Sentinel Lymph Node Biopsy: Validation Studies
FIGURE 2. Sentinel lymph node technique in CRC. A, After mobilization of the colon, isosulfan blue dye (0.5–1 mL) was carefully injected in 4 quadrants around the tumor. Within 30 to 60 minutes, the blue dye traveled from the primary site along the lymphatic channel (arrow) to the SLNs (arrowheads). Reprinted with permission from Bilchik et al.41 Copyright © 2001, American Society of Clinical Oncology. All rights reserved. B, Ex vivo identification of SLNs (arrows) in the mesentery after injection of isosulfan blue dye after the specimen had been removed from the body. Reprinted with permission from Wood et al.36 Copyright © 2001, Springer Science + Business Media.
examined as a primary approach, but also to address whether it might be useful in cases where in vivo lymphatic mapping failed to identify the sentinel node (Fig. 2B).36 Ex vivo mapping was performed after the specimen was removed from the body and was successful in 7 (88%) of 8 cases; 6 of the 7 cases were in low rectal tumors where in vivo mapping failed. The accuracy of this approach was confirmed by remapping the sentinel nodes in operative specimens from patients who had undergone successful in vivo mapping; in all cases, ex vivo injection of isosulfan blue dye intensified staining of nodes identified in vivo but not additional new nodes.36 The advantage of the ex vivo approach is that if the sentinel node is not identified intraoperatively by the surgeon, the specimen can be sent immediately to the pathologist for a more thorough dissection. After 100 consecutive cases of SLNB in early CRC, the sentinel node was found to accurately reflect the status of the nodal basin in 92 (95%) of 97 patients, with 24% upstaging of occult micrometastases missed by routine H&E.39 Review of the falsenegative cases demonstrated that the failures occurred during the first 30 cases with technical error playing a significant role. In addition, the only case in which a sentinel node was unable to be localized by both primary and salvage ex vivo lymphatic mapping occurred in a patient with rectal cancer with extensive nodal disease.39 When tumor completely replaces the lymphatic vessels leading to the true sentinel node, the blue dye can take a path of
A large phase II multicenter prospective trial by Bilchik et al10 assessed the accuracy of focused analysis of the SLN by multiple sectioning and IHC in stage I and II CRC. Between 2001 and 2005, 132 patients were evaluated at 4 tertiary centers with demonstrated surgical and pathological expertise in lymphatic mapping. Sentinel nodes were identified in vivo or ex vivo immediately after resection as described by Wood et al,36 and if the surgeon could not identify the SLN intraoperatively, the en bloc resected tumor and regional lymph node specimen were immediately sent to the pathologist, who conducted a more aggressive dissection to identify the SLN. Sentinel lymph nodes were processed and sectioned as previously described29 and analyzed by both H&E and IHC; in addition, further sections were also cut for molecular analysis by RT-PCR. Preliminary results reported in 2006 showed that the SLN was successfully identified in 96% of cases with a median number of SLNs and total lymph nodes examined being 3 and 14.5, respectively, which is compliant with even current recommendations on the number of nodes that should be removed for adequate pathologic staging. Metastases ranging from greater than 0.2 mm but less than 2 mm were considered micrometastases, whereas ITCs were single tumor cells or cell clusters less than 0.2 mm and usually detected by IHC. The false-negative rate (FNR), which was defined as the probability of not finding tumor in an SLN in the presence of nodal metastases, was 7.4% (6/81), which is comparable to the literature in melanoma and breast cancer.10,29,30 The US Military Cancer Institute Clinical Trials Group Study GI-01 was a prospective randomized multicenter trial conducted to determine if focused analysis of the sentinel node improves staging of the nodal basin.42 Patients were randomly assigned to standard pathological evaluation or ex vivo sentinel node mapping and ultrastaging with H&E and IHC, which defined SLN positivity. In this study, sentinel nodes were successfully identified in 82 (97.6%) of 84 patients,42 with a mean of 18.2 ± 0.9 nodes analyzed per patient. Significant nodal upstaging occurred using the SLN technique (57.3% vs 38.7%; P = 0.019),42 and although in the overall study population 5-year DFS only approached significance (61% control group vs 71% in SLN ultrastaging; P = 0.11),27 disease recurrence differed significantly between ultrastaged and conventionally staged node-negative patients (86% vs 71%, respectively; P = 0.04; Fig. 3).27 The authors concluded that focused analysis of the sentinel node improves the
FIGURE 3. Disease-free survival according to control versus TNA-us (targeted nodal assessment and ultrastaging) among nodenegative patients with colon cancer. Reprinted with permission from Nissan et al.27 Copyright © 2012, Wolters KLuwer Health. www.journalppo.com
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at least 20 to 30 SLNB procedures were required for adequate sensitivity and accuracy, a threshold of at least 20 cases appears to be appropriate. In addition, incorporating specialized cytokeratin immunohistochemical stains and, more recently, molecular analysis of the SLN with tumor-specific biomarkers, which were not included in the study of Bertagnolli et al,43 may have enhanced detection of micrometastatic disease in the sentinel node, although the prognostic significance remains debatable.
Molecular Staging of the Sentinel Node: The Next Frontier
FIGURE 4. Kaplan-Meier curves for disease outcome according to detection of at least 1 qRT-PCR marker in SLNs. A, Overall survival by qRT-PCR(+) in SLNs (not significant). B, Disease-free survival by qRT-PCR(+) in SLNs (P = 0.014). Reprinted with permission from Koyanagi et al.47 Copyright © 2008, American Association for Cancer Research.
staging of colon cancer and thereby increases DFS by a clinically important absolute difference of 15%. Contrastingly, in a controlled prospective trial by Bembenek et al,37 the rate of SLN detection was only 85% (268/315 patients), with a seemingly high FNR of 46% (38 of 82 patients). Failure of the SLN to predict nodal status was reportedly 54% in a multicenter study by Bertagnolli et al.43 The low detection rate and high FNR reported in these 2 studies may be attributed to the learning curve phenomenon because few patients were accrued at multiple institutions, and pathological techniques were not standardized. The exact number of cases needed to achieve proficiency in SLNB for CRC is unknown. Based on our feasibility studies in CRC, in which falsenegative results occurred in the first 30 to 50 cases,39, 44 and taking into consideration that based on the Multicenter Selective Lymphadenectomy Trial for melanoma45 and American College of Surgeons Oncology Group Z0010 trial for breast cancer,46 in which
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The development of molecular assays such as RT-PCR for detection of micrometastases expanded our ability to detect occult tumor cells in primary and metastatic tissues, circulating blood, and other body fluids in cancer patients. Based on our multimarker RT-PCR assays in breast cancer and melanoma, we assessed 3 mRNA markers, β-hCG (β-chain human chorionic gonadotropin), c-Met (hepatocyte growth factor receptor), and uMAGE (universal melanoma-associated antigen A family), in paired primary tumors and SLNs of patients in our multicenter CRC SLN staging trial.41 In this pilot study, there was a 90% concordance between RT-PCR–positive and H&E-positive SLNs, and the addition of IHC and RT-PCR identified occult micrometastases in 53% of patients whose SLNs were negative by conventional staging. These initial molecular studies were conducted on frozen sections, but we subsequently switched to paraffin-embedded tissue, which provided the advantage of tissue preservation and easier comparison with histopathology slides. The updated analysis utilized a 4-marker qRT-PCR panel including c-MET, MAGE-A3 (melanoma-associated antigen A3), CK20 (cytokeratin 20), and GalNAc-T (β-1,4-N-acetylgalactosaminyltransferase) and showed no significant difference in OS by marker detection in the SLN but was significant for DFS (P = 0.014), with a mean DFS of 37 months for qRT-PCR– positive) versus 61 months for qRT-PCR–negative SLN (Fig. 4).47 Sentinel lymph node positivity by qRT-PCR maintained prognostic value for DFS independent of age, gender, and TNM staging (hazard ratio = 4.9; P = 0.027).47 An interim analysis of our multicenter SLN trial incorporating additional patients and the results of molecular analysis of the SLN by RT-PCR was published in 2007.24 Colorectal cancers were considered truly upstaged if focused analysis of the SLN specimen changed nodal status from N0 to N1mi (micrometastases) or N1 (macrometastases) but categorized as only possible upstaging if ITCs were detected by IHC (N0i+) and qRT-PCR (N0mol+). Of 92 node-negative colon cancer patients, true upstaging occurred in 7 patients (8%), and 18 additional patients (22%) had IHC ITCs (N0i+). At a mean followup of 25 months, 12 patients had a CRC-related recurrence (9.5%), 10 of which had a positive SLN. Excluding the rectal cancer patients, all patients with primary colon cancer in which the SLN was negative by H&E, IHC, or qRT-PCR did not have a recurrence,24 suggesting that surgical resection alone may have been curative in these patients. In 2012, Rahbari and colleagues48 published the results of a meta-analysis that included 39 studies of micrometastases or ITCs detected by IHC and/or RT-PCR in node-negative CRC. Molecular tumor-cell detection was associated with poor OS, DFS, and DSS. Subgroup analyses demonstrated that prognostic significance was independent of the detection method, molecular biomarker target, and number of lymph nodes retrieved.48 Critics point out that the value of RT-PCR assays for the detection of occult tumor cells in SLNs is dependent on using markers that are cancer specific, in order to maximize sensitivity
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and specificity and minimize false-positive or false-negative results. In addition, at this point in time, standardization of techniques for molecular assays is lacking, which hampers comparison of different studies.
CONCLUSIONS Despite ongoing debate, several recent meta-analyses of the SLNB in CRC cancer demonstrated that the detection rate ranges from 91% to 94% and concluded that it is a safe procedure that should be considered in addition to conventional resection in colon cancer patients.49,50 The limitations of the procedure include inconsistent results between institutions and surgeons suggesting that surgical and pathological techniques need to be standardized. Although both immunohistochemical and molecular analyses of the SLN have been shown to upstage conventional H&E, their prognostic value remains to be definitively established. Undoubtedly, application of the SLNB to CRC will continue to evolve and as we further elucidate the molecular pathways that drive tumorigenesis, we will be able to discover improved targeted and specific biomarkers for analysis. It is therefore likely that SLNB in CRC will continue to be investigated with enthusiasm. We still have much progress to make in determining the optimal method of staging CRC patients in order to appropriately stratify patients into subgroups that may benefit the most from adjuvant therapy while avoiding unnecessary toxicity for those that can be cured by surgery alone. Nevertheless, determining how to incorporate nodal ultrastaging techniques into clinical practice continues to provide a challenge for future studies. REFERENCES 1. Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. 2. MacDonald JS. Adjuvant therapy of colon cancer. CA Cancer J Clin. 1999;49:202–219. 3. Colon and rectum. In: Edge SB, Byrd SR, Compton CC, et al. eds. AJCC Cancer Staging Manual. 7th ed. New York: Springer-Verlag; 2010:143–164. 4. Hari DM, Leung AM, Lee J-H, et al. AJCC Cancer Staging Manual 7th edition criteria for colon cancer: do the complex modifications improve prognostic assessment? J Am Coll Surg. 2013;217:181–190. 5. Andre T, Boni C, Navarro M, et al. Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial. J Clin Oncol. 2009;27:3109–3116. 6. Graham JS, Cassidy J. Adjuvant therapy in colon cancer. Expert Rev Anticancer Ther. 2012;12:99–109. 7. Kuebler JP, Wieand HS, O’Connell MJ, et al. Oxaliplatin combined with weekly bolus fluorouracil and leucovorin as surgical adjuvant chemotherapy for stage II and III colon cancer: results from NSABP C-07. J Clin Oncol. 2007;25:2198–2204. 8. Benson AB III, Schrag D, Somerfield MR, et al. American Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancer. J Clin Oncol. 2004;22:3408–3419. 9. Quasar Collaborative Group, Gray R, Barnwell J, McConkey C, et al. Adjuvant chemotherapy versus observation in patients with colorectal cancer: a randomized study. Lancet. 2007;370:2020–2029. 10. Bilchik AJ, DiNome M, Saha S, et al. Prospective multicenter trial of staging adequacy in colon cancer. Arch Surg. 2006;141:527–534. 11. Weitz J, Koch M, Debus J, et al. Colorectal cancer. Lancet. 2005;365:153–165. 12. Compton CC, Greene FL. The staging of colorectal cancer: 2004 and beyond. CA Cancer J Clin. 2004;54:295–308. 13. Compton CC, Fielding LP, Burgardt LJ, et al. Prognostic factors in colorectal cancer. College of American Pathologists consensus statement. Arch Pathol Lab Med. 2000;124:979–994. 14. Sobin HL, Greene FL. TNM classification. Clarification of number of regional lymph nodes for pN0. Cancer. 2001;92:452. 15. Sari L, Bader G, Lusco D, et al. Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer. Eur J Cancer. 2005;41:272–279.
SLNB and Ultrastaging in Colorectal Cancer
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41. Bilchik AJ, Saha S, Wiese D, et al. Molecular staging of early colon cancer on the basis of sentinel node analysis: a multicenter phase II trial. J Clin Oncol. 2001;19:1128–1136. 42. Stojadinovic A, Nissan A, Protic M, et al. Prospective randomized study comparing sentinel lymph node evaluation with standard pathologic evaluation for the staging of colon carcinoma: results from the United States Military Cancer Institute Clinical Trials Group Study GI-01. Ann Surg. 2007;245:846–857. 43. Bertagnolli M, Miedema B, Redston M, et al. Sentinel node staging for resectable colon cancer: results of a multicenter study. Ann Surg. 2004;4:624–628. 44. Bilchik AJ, Nora D, Tollenaar RA, et al. Ultrastaging of early colon cancer using lymphatic mapping and molecular analysis. Eur J Cancer. 2002;38:977–985. 45. Morton DL, Cochran AJ, Thompson JF, et al. Sentinel node biopsy for early-stage melanoma: accuracy and morbidity in MSLT-1, an international multicenter trial. Ann Surg. 2005;242:302–313.
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46. Posther KE, McCall LM, Blumencranz PW, et al. Sentinel node skills verification and surgeon performance: data from multicenter clinical trial for early-stage breast cancer. Ann Surg. 2005;242:593–599. 47. Koyanagi K, Bilchik AJ, Saha S, et al. Prognostic relevance of occult nodal micrometastases and circulating tumor cells in colorectal cancer in a prospective multicenter trial. Clin Cancer Res. 2008;14: 7391–7396. 48. Rahbari NN, Bork U, Motschall E, et al. Molecular detection of tumor cells in regional lymph nodes is associated with disease recurrence and poor survival in node-negative colorectal cancer: a systematic review and metaanalysis. J Clin Oncol. 2012;30:60–70. 49. van der Zaag ES, Bouma VH, Tanis PJ, et al. Systematic review of sentinel lymph node mapping procedure in colorectal cancer. Ann Surg Oncol. 2012;19:3449–3459. 50. van der Pas MHGM, Meijer S, Hoekstra OS, et al. Sentinel-lymph-node procedure in colon and rectal cancer: a systematic review and metaanalysis. Lancet Oncol. 2011;12:540–550.
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