Skip metastases in colon cancer: Assessment by lymph node mapping using molecular detection

Skip metastases in colon cancer: Assessment by lymph node mapping using molecular detection Arend E. H. Merrie, MBChB, PhD, Lynne V. Phillips, NZCS, Kankatsu Yun, MD, PhD, and John L. McCall, FRACS, MD, Dunedin and Auckland, New Zealand

Background. Colon cancer has been assumed to spread sequentially through the regional lymphatic bed, with skip metastases occurring in only 1% to 3% of cases. Molecular techniques allow the detection of occult metastases, but to date have not been applied to assess the pattern of regional lymphatic spread of colon cancer. Methods. Fifty-five tumors from 54 patients with colonic adenocarcinoma were studied. Lymph node mapping was performed on fresh colonic specimens recording the position of each node on an anatomical diagram. Half of each lymph node was submitted for routine histology examination and half assayed for keratin 20 gene expression by reverse transcription-polymerase chain reaction. Logistic regression was used to analyze the distribution of histologic and occult metastases. Results. A total of 1084 lymph nodes were dissected (median, 19 nodes; range, 4-52). Sixty-four lymph nodes from 20 tumors had histologically evident metastases and 76 lymph nodes from 13 tumors had occult metastases. There was no difference in the distribution of either histologic or occult metastases among paracolic, intermediate, and apical node groups. Ten patients had evidence of anatomical skip lesions after lymph node mapping and molecular analysis, only 1 of which was histologically detectable. Conclusions. This study demonstrates a higher incidence of skip metastases in colon cancer assessed by molecular techniques than has previously been reported, challenging the concept of sequential development of early lymph node metastases.(Surgery 2001;129:684-91.) From the Departments of Surgery and Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand, and the Department of Surgery, Faculty of Health Science, University of Auckland, Auckland, New Zealand

UNIDENTIFIED METASTATIC DISEASE accounts for a high rate of tumor recurrence after potentially curative resection of colorectal cancer.1-3 The detection of such occult disease and the development of prognostic markers have been the subject of considerable research.4 Both the presence and number of histologically evident lymph node metastases have been shown repeatedly to be independent prognostic indicaSupported by grants from the Anderson Telford Charitable Trust and the Otago Medical Research Foundation and by the Royal Australasian College of Surgeons Richard Stewart Scholarship (A. E. M.) and the New Zealand Gastroenterological Society–Glaxo/Wellcome Research Fellowship (A. E. M.). Accepted for publication December 8, 2000. Reprint requests: Dr Arend Merrie, Department of Surgery, Northshore Hospital, PO Box 503, Takapuna, Auckland, New Zealand. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/56/113887 doi:10.1067/msy.2001.113887

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tors of survival.5-9 Therefore, to maximize the detection of lymph node metastases and occult disease, both the number of nodes examined and the examination technique are of great importance. There have been several studies of more sensitive techniques to detect occult metastases in colorectal cancer.4 These have involved the enhancement of histopathologic analysis with either fat clearance,10 serial sectioning,11,12 or immunohistochemistry13-15 or molecular analysis by polymerase chain reaction (PCR).16-20 All of these assays have been based on the assessment of mesenteric lymph nodes. However, none of these studies has used its technique to describe the anatomic distribution of locoregional metastatic spread in detail. Histologic methods of detecting occult metastatic disease all appear to improve the detection of lymph node metastases. Some studies have shown a benefit in terms of prognostic accuracy,13-15 but many have been inadequate.4 Only 2 studies have assessed the prognostic importance of occult

Surgery Volume 129, Number 6 metastatic disease detected by PCR. Hayashi et al16 reported a high recurrence rate in patients with occult lymph node metastases detected by K-ras or p53 mutations analysis. By using a reverse transcription-PCR (RT-PCR) assay for carcinoembryonic antigen, Liefers et al17 found a significant difference in survival between patients with and those without occult metastatic disease. In clinical practice, the use of a more sensitive test would be an adjunct to histopathologic analysis. However, the assessment of all dissected lymph nodes is time consuming and expensive. In addition, PCR-based techniques have the advantage of being able to assess the whole lymph node, whereas routine histopathology and immunohistochemistry examine only a small portion of the lymph node. If a lymph node group within the regional lymphatic bed could be identified that was predictive of the presence of histologic and occult metastases, then it would be possible to limit the examination with a more sensitive test to this group. For the purposes of this paper, such a lymph node group has been referred to as an index lymph node group. To select an index lymph node group for detailed analysis, knowledge of the locoregional spread of colorectal cancer at the submicroscopic level is required. Systems automating RT-PCR and specimen analysis are available.21 These allow the rapid return of results in a cost-effective manner. The combination of analysis of an index lymph node group and automation of RT-PCR would increase the potential of molecular assays to be used as diagnostic tests. Lymphatic drainage of the colon and rectum is believed to occur in an orderly progression from the submucosal lymphoid follicles through the bowel wall to epicolic, paracolic, intermediate, and para-aortic lymph nodes.22 This is based on the original work of Dukes and Gabriel.23,24 Their anatomic dissections demonstrated that locoregional lymphatic metastases from rectal cancer occurred in a sequential pattern, but found that 1% of metastases appeared to bypass, or skip, tiers of lymph nodes. These metastases have been referred to as skip metastases. Other studies of the distribution of lymph node metastases have supported this with the rate of histologically detected skip metastases in colorectal cancer occurring in 1% to 3% of the cases.25,26 The aims of this study were to describe the pattern of locoregional lymphatic spread in colonic cancer at both the histologic and submicroscopic level and to identify the existence of an index lymph node group within the regional lymphatic

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bed whose lymph node status was representative of the lymph node status of the entire lymphatic bed. MATERIALS AND METHODS Patients. The protocol was approved by the regional ethics committee and written informed consent was obtained from patients undergoing potentially curative operation for colonic cancer. Patients undergoing anterior resection or abdominoperineal resection were excluded because the dissection of lymph nodes interfered with histologic analysis of circumferential margins. Fifty-four patients were recruited for the study. Patient management was not altered by the results of this study; in particular, adjuvant therapy was not offered to those with evidence of occult metastatic disease. Procedures. Nine surgeons participated in the study, performing between 1 and 21 procedures each (median, 6). The aims of the study and the need for maximal mesenteric tissue were discussed with the surgeons before commencement of the study and before each procedure. The anatomic boundaries for each specific operation were determined by the tumor location. No attempts were made to dissect porta hepatic nodes or nodes outside the resection area. Detection of occult metastases. To detect occult lymph node metastases, we have used an RT-PCR assay for cytokeratin 20 (K20). K20 is an intermediate filament of gastrointestinal epithelial cells. It has been reported to be tissue-specific27-30 and have potential for the detection of occult metastatic disease in colorectal cancer.18-20,31,32 We have previously shown that K20 RT-PCR is highly tissue-specific, demonstrating expression in only gastrointestinal mucosa and tumors arising from it.33 The molecular detection of lymph node metastases in colorectal cancer with this method is both sensitive and specific.18,33 A large prospective clinical trial has been undertaken to assess the prognostic significance of occult metastases detected by K20 RTPCR. Preliminary results, based on an interim analysis at a median of 25 months follow-up, show that K20 is a highly significant predictor of overall survival (P = .0002) and disease-free survival (P = .0003) on univariate analysis with an independent prognostic significance for overall survival (P = .0294; hazard ratio, 3.162) on multivariate analysis (unpublished data). These data will be the subject of a separate report after a longer follow-up period; however, the preliminary results indicate that detection of K20 in lymph nodes does correlate with outcome. Lymph node mapping. Resected specimens were obtained fresh from the operating room. Bowel

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Fig 1. Anatomic classification of lymph node distribution according to Japanese General Rules for Clinical and Pathological Studies on the Colon, Rectum and Anus.34 Light gray shading, n1: pericolic ≤ 5 cm from tumor. Dark grey shading, n2: intermediate nodes along named arteries or > 5 cm and ≤ 10 cm from tumor. Black shading, n3: main nodes at root of superior mesenteric artery or inferior mesenteric artery or > 10 cm from tumor.

ends were tied to prevent epithelial cell contamination of the mesentery before lymph node dissection. Dissection of the mesentery was then undertaken in a designated area in the surgical laboratory before further histopathologic processing and analysis. Specimens were dissected on an UV irradiated chopping board by using RNA- and DNA-free instruments and a sterile surgical blade. After orientation of the specimen and identification of named mesenteric vessels, the peritoneum was carefully dissected off the mesentery by using a combination of sharp and blunt dissection. Commencing at the apex of the resected specimen, lymph nodes were then individually dissected, initially following vessels in a retrograde fashion, and then dissecting the intervascular spaces. Lymph nodes were identified by visual characteristics, palpation, and transillumination. The position of each lymph node was mapped on an anatomic diagram showing the lymph node in relation to the site of the tumor and the mesenteric vessels.

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The determination of optimum tissue size and RNA extraction technique were performed on the first 10 patients and confirmed with 8 more patients. In this group of 18 patients, lymph nodes were weighed before RNA extraction and the calculation of RNA yield; and analysis of the effect of overnight incubation in guanidine thiocyanate solution or repeated chloroform extraction was performed. Lymph nodes were bisected along the longitudinal axis. For smaller nodes (less than 100 mg), half of the node was submitted for routine histopathologic evaluation and half for RNA extraction. For large lymph nodes, a central biopsy was taken for RT-PCR, and the remainder of the node was submitted for a histopathologic evaluation. Samples of the normal colonic mucosa and colon tumor were taken from each patient for RNA extraction. RNA extraction and reverse transcription. Lymph nodes and tissue specimens were individually homogenized by using sterile DNA-free techniques. Lymph nodes too small to be homogenized were incubated overnight in guanidine solution. Total RNA was extracted by using a modification of the Chomczynski and Sacchi protocol as described previously and resuspended in 40 µL autoclaved distilled deionized water.18 RNA quantitation and yield were calculated by spectrophotometry at 260 nm. All specimens were accompanied by a reagentonly negative control. Physical separation of the specimen dissection, RNA extraction, complementary DNA (cDNA) synthesis and PCR and PCR product electrophoresis were done to prevent specimen contamination. A maximum of 5 µg of total RNA from each sample was treated with RNase-free DNase 1 (Gibco BRL, Gaithersburg, Md) in a total volume of 10 µL. cDNA was synthesized from 5 µL DNA-free RNA primed with random hexamers (Boehringer Mannheim, Mannheim, Germany) by using 100 U Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, Md) by the manufacturer’s method in a total volume of 10 µL. The other half of the DNA-free RNA was used as a reverse transcriptase–negative control. Polymerase chain reaction. One-twentieth of the cDNA synthesized (0.5 µL) was used in each PCR assay with 150 µmol/L of each deoxyribonucleoside triphosphate, 1 µmol/L of each primer, 1 × PCR buffer (Qiagen), and 0.5 U Taq DNA polymerase (Qiagen) in a total volume of 10 µL. PCR for K20 consisting of an initial denaturation at 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 62.5°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes was performed on all cDNA samples in a Hybaid

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Surgery Volume 129, Number 6 Table I. Distribution of tumors by anatomic location

Table II. Dukes’ staging on analysis of all lymph nodes submitted for histologic examination

Site

No.

Dukes’ stage

Cecum Ascending colon Hepatic flexure Transverse colon

23 15 3 3

Splenic flexure

1

Descending colon Sigmoid colon

1 9

Procedure Right hemicolectomy Right hemicolectomy Right hemicolectomy Transverse colectomy (2) Extended right hemicolectomy (1) Extended right hemicolectomy Left hemicolectomy Sigmoid colectomy

Touchdown thermal cycler (Hybaid, Middlesex, United Kingdom). β-Actin PCR was carried out under the following conditions for all samples as a control for cDNA synthesis and RNA extraction: 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. Five µL of PCR product was electrophoresed through 2% agarose/1 × Tris-acetate gels containing 0.5 µg/mL ethidium bromide. All PCR reactions were accompanied by 1 or more no-template negative controls and a DNA-positive control. Analysis. Histopathologic analysis was performed on single 3-µm thick hematoxylin-andeosin stained sections of fixed and embedded lymph nodes in keeping with the standard practice of the laboratory. Disease stage was determined with the classic Dukes’ classification of colorectal cancer,23 with subclassification of Dukes’ C depending on the apical node status into C1 and C2.24 RT-PCR results were scored on the presence or absence of a 298 base pair (bp) PCR product for K20 and a 200 bp product for β-actin after electrophoresis and UV transillumination. The anatomic distribution and patterns of lymph node metastases for both histologic and occult metastases were examined by using the Japanese General Rules lymph node classification.34 This was used because it is a solely anatomically based classification of lymph node metastases from colorectal cancer (Fig 1). A data plot of the lymph node weight and yield data revealed a nonparametric distribution. Spearman’s correlation coefficient was used to determine the relationship between lymph node weight and RNA yield. The Mann Whitney U test was used to examine the relationship between lymph node weight and β-actin expression. Logistic regression, adjusted for repeated measures, was used to assess the distribution of metas-

A B C

Patients (No.) 4 27 23

tases. The chi-square test was used to assess differences in the rate of skip metastases between histologic and molecular analyses. Statistical analysis was performed with the SAS statistical package (SAS Institute Inc, Cary, NC) and Statview 4.51 (Abacus Concepts, Inc, Berkeley, Calif). RESULTS Demographics. Fifty-four patients with 55 tumors were enrolled in the study (21 men), with a median age of 74 years (range, 39 to 92 years). There was a preponderance of right-sided colonic tumors, in keeping with the exclusion of low anterior and abdominoperineal resections (Table I). A total of 1084 histologically confirmed lymph nodes were harvested from the 55 specimens, ranging from 4 to 52 per specimen with a median value of 19 lymph nodes dissected fresh from each specimen. When adjusted for the operation site, the number of lymph nodes harvested was no different between surgeons. A further 253 lymph nodes were harvested after formalin fixation (median, 2 per patient; range, 0-38). These were submitted for separate histopathologic analysis. All patients with histologically evident Dukes’ C adenocarcinoma were offered chemotherapy. Histopathologic characteristics. An analysis of all the lymph nodes submitted for histopathologic evaluation staged 4 tumors as Dukes’ A, 28 tumors as Dukes’ B, and 23 tumors as Dukes’ C (Table II). The patient with a synchronous tumor had a Dukes’ C carcinoma of the cecum and a Dukes’ B carcinoma of the sigmoid. Ten patients had poorly differentiated tumors, 4 with evidence of vascular invasion, 2 with K-ras mutations, and 1 with microsatellite instability (MSI). Forty-five patients had well- or moderately differentiated tumors, 5 with evidence of vascular invasion, 12 with K-ras mutations, and 10 with MSI. Nine of the 10 patients with MSI had right-sided colonic tumors. Molecular analysis. There were 377 lymph nodes from 18 patients that were assessed for the relationship between tissue weight, yield, and β-actin expression (median, 21 per specimen; range, 1137). The median weight was 26.9 mg (range, 0.5311.6 mg), and the median yield was 1.28 µg

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Table III. Lymph node status of tumors by histologic evaluation and K20 RT-PCR Histology

Positive Negative Total

K20 RT-PCR (tumors) Positive 20 14 34

Negative 0 21 21

Total 20 35 55

Table IV. Analysis of β-actin–positive dissected lymph nodes by histologic evaluation and K20 RT-PCR Histology Positive Negative Total

K20 RT-PCR (lymph nodes) Positive 54 76 130

Negative 10 841 851

Total 64 917 981

RNA/mg tissue (range, 0.04-145.87 µg/mg). βActin expression was detected in 346 nodes (92%). There was an inverse linear relationship between tissue weight and yield (P = .014), and β-actin expression was significantly associated with a lesser weight (mean weight difference, 40.6 mg; 95% CI 24.3-58.8 mg; P < .001). Overnight incubation in guanidine thiocyanate consistently produced successful RNA extraction with smaller nodes, and no difference in yield was observed with a double chloroform extraction. RNA was successfully extracted and reverse transcribed in 981 of the 1084 (90%) lymph nodes and all 55 tumors as determined by β-actin or K20 expression. Fifty-four of the tumors expressed K20. Sixty-four of the freshly dissected lymph nodes from 20 tumors were histologically positive (Tables III and IV). In 34 tumors, 130 lymph nodes showed evidence of occult metastases detected by molecular techniques (Tables III and IV). Of the 35 histologically node-negative tumors, 76 lymph nodes from 14 tumors were positive as determined by molecular techniques (34%). Anatomic distribution of metastases. Ten of the 55 tumors exhibited anatomic skip metastases (18%) (Table V). One tumor demonstrated an anatomic skip metastasis detected by histology (1.8%). The remaining skip metastases were detected only with molecular techniques (P = .032 for difference) (Table V, Fig 2). Four of the patients with no histologically evident metastases had occult anatomic skip metastases involving the

Fig 2. Lymph node map and agarose gel of a patient with Dukes’ B adenocarcinoma of the sigmoid colon. One to 12, Lymph nodes dissected and mapped; N, normal epithelium; T, tumor; B, reagent only blank; ddH2O, PCR blank. βActin expression, controlling for RNA extraction, and cDNA synthesis is evident in all lymph nodes except lymph node 5. Lymph nodes 8 and 11 show K20 expression, demonstrating the presence of occult metastatic disease. The pattern of spread is a skip lesion involving the apical node (11) and missing the intermediate group of draining nodes.

apical nodes (Table V: patients 4, 6, 10, 12). In addition, 7 other patients were restaged by the use of molecular techniques. All remaining patients showed concordant staging by the Japanese General Rules, TNM, and Dukes’ staging systems. The initial pilot study of 20 patients demonstrated a progressive increase of occult metastases from the paracolic to the apical areas (P = .043 for trend) with occult metastases more prevalent in apical than paracolic nodes (P = .034).35 However, an analysis of all 55 tumors did not reveal a statistically significant difference between the anatomic distribution of histologic or occult metastases (Table VI). Follow-up. A median follow-up of 28 months has been undertaken (range, 12-36 months). During this time, 11 deaths have occurred: 7 Dukes’ C patients, 2 Dukes’ B patients with occult metastases, and 2 Dukes’ B patients with no evidence of occult lymph node metastases. Hepatic metastases devel-

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Surgery Volume 129, Number 6

Table V. Patients with skip metastases or different lymph node status by 3 staging systems after molecular analysis Histologic stage Patient

Anatomic skip lesion

1 4 5 6 10 12 14 15 16 25 29 30 31 33 41 45 54

√ √ — √ √ √ √ — √ — √ — — — √ — √

Dukes’*

Molecular stage

TNM pN

JGR n

0 0 1 0 0 0 0 1 0 1 0 0 0 1 0 0 2

0 0 1 0 0 0 0 1 0 1 0 0 0 1 0 0 2

B B C1 B B B B C1 B C1 B B B C1 B B C2

Dukes’*

TNM pN

JGR n

2 3 1 3 3 1 1 3 1 2 2 2 1 2 2 1 2

2 3 2 3 3 2 2 3 2 3 2 2 2 1 2 1 2

C1 C2 C1 C2 C2 C2 C1 C2 C1 C2 C1 C1 C1 C1 C1 C1 C2

JGR, Japanese General Rules. *Gabriel and Dukes’ modification of Dukes’ into C1 and C2 to denote apical lymph node involvement.24

Table VI. Distribution of lymph nodes according to Japanese General Rules classification* Lymph node position Lymph node status K20 and histology negative K20 only positive Histology only positive K20 and histology positive

n1 492 (87%) 37 (7%) 9 (2%) 28 (5%)

n2 334 (87%) 24 (6%) 6 (2%) 18 (5%)

n3 95 (86%) 9 (8%) 1 (1%) 6 (5%)

*Percent lymph node area.

oped in 2 additional Dukes’ C patients. Clinical follow-up of these patients is ongoing. DISCUSSION In this study, anatomic skip metastases were seen in 10 of the 55 tumors (18%) when examined by lymph node mapping and K20 RT-PCR. This is far higher than previously reported in studies using histopathologic evaluation alone.24-26 Of note, the rate of skip lesions detected by histopathologic evaluation alone (1.8%) in this study was comparable with previous studies. The findings of a significant percentage of skip metastases detected by K20 RT-PCR challenge the accepted concept of lymphatic drainage in the colon, which has been assumed to follow a sequential, tiered pattern. Recent lymphoscintigraphic studies in melanoma have cast doubt over the validity of assuming a sequential pattern of spread through the anatomic

regional lymphatic bed. Nonanatomic lymphatic drainage has been observed in up to 10% of melanomas leading to a revision of traditional concepts of the lymphatic drainage of the skin and subcutaneous tissue.36,37 These phenomena of nonanatomic lymphatic drainage could be explained by nonsequential spread through the lymphatic basin (true skip metastases), or nonanatomic lymphatic spread. Six tumors (10.9%) were shown to have evidence of apical lymph node involvement by K20 RT-PCR, 4 of which were histologically node negative. These findings are in keeping with a recent study by Weitz et al20 that used a nested K20 RTPCR assay. Occult lymph node metastases were grouped into those occurring in the paracolic lymph nodes, those along the main vessel, and those in the apical node(s). Weitz et al found 37.5% of histologically lymph node–negative

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patients had evidence of occult metastases in the apical nodes. However, these findings have to be viewed in the context of a much higher incidence of occult lymph node metastases overall. Previous studies have shown that extended lymph node dissection does not alter survival or the incidence of local recurrence.38-40 This study may offer some explanation because occult skip metastases can occur in the apical lymph node group and may occur outside the resected area. The presence of occult metastatic disease and skip metastases highlights the importance of obtaining adequate mesenteric tissue to allow accurate diagnosis of lymph node status. Limited resection with few lymph nodes would result in the understaging of colonic tumors, even when molecular detection methods are used. Ten lymph nodes in this study were positive on routine histopathology, but negative by K20 RTPCR (Table IV). However, no patients were understaged by the use of K20 RT-PCR (Table III). These findings may be attributable to inadequate lymph node sampling, unsuccessful RT-PCR, unsuccessful electrophoresis, or correlation errors between histologic and K20 RT-PCR results. Specimens with poor β-actin expression or a lack of K20 expression in the normal mucosa or tumor were initially subject to repeat PCR. If this was still negative, cDNA synthesis was repeated. Electrophoresis errors are unusual and easily recognized by the lack of bromophenol blue within the gel. Therefore, the most likely causes for nonexpression are either inadequate sampling or a lack of expression. Sampling of lymph nodes involved bisection of the lymph node with half analyzed by RT-PCR and half by histopathologic evaluation. Sampling errors are, therefore, potentially possible and could account for some of the discrepancy between histologic and K20 RT-PCR findings. Determination of lymph node weight and RNA yield demonstrated that the tissue collection and RNA extraction technique was most successful with smaller amounts of tissue. A lack of β-actin expression was associated with the larger lymph nodes. An analysis of large lymph nodes by molecular techniques remains problematic because there is a greater potential for sampling errors in favor of histology if a smaller tissue biopsy specimen is taken and a greater potential for inadequate extraction. An analysis of the locoregional lymphatic spread of colonic cancer using K20 RT-PCR demonstrated that the anatomic distribution of metastases did not always follow the assumed sequential anatomic pattern of spread. Anatomically it is, therefore, not

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possible to define an index lymph node group for sampling that would reliably represent the lymph node status for a given patient. The increased prevalence of occult anatomic skip metastases detected could be caused by a number of factors: (1) nonanatomic early lymphatic spread to a sentinel or primary draining lymph node; (2) nonsequential spread within the lymphatic bed (true skip metastases); (3) differences in tumor biology, with occult metastases perhaps lacking the invasive and adherent properties of larger metastases. Further investigation of the biology of metastases and lymphatic spread may give greater insight into the natural history of colorectal carcinoma and help define the place of sentinel node biopsy in the treatment of colorectal cancer. We thank Peter Herbison (biostatistician, Department of Preventive and Social Medicine, University of Otago) for his help with the analysis of the results; the staff of the Surgery Department (Dunedin Hospital) for procurement of tissue; the staff of the Department of Anatomic Pathology (Dunedin Hospital) for histopathologic analysis; Dr David Markie (Department of Pathology, Dunedin School of Medicine, University of Otago) for MSI data; and Peter Scott (Department of Information Technology) for line art.

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26. Tang R, Wang JY, Chen JS, et al. Survival impact of lymph node metastasis in TNM stage III carcinoma of the colon and rectum. J Am Coll Surg 1995;180:705-12. 27. Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors, and cultured cells. Cell 1982;31:11-24. 28. Moll R, Lowe A, Laufer J, et al. Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol 1992; 140:427-47. 29. Moll R, Zimbelmann R, Goldschmidt MD, et al. The human gene encoding cytokeratin 20 and its expression during fetal development and in gastrointestinal carcinomas. Differentiation 1993;53:75-93. 30. Adams MD, Kerlavage AR, Fleischmann RD, et al. Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 1995;377(6547 Suppl):3-174. 31. Burchill SA, Bradbury MF, Pittman K, et al. Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. Br J Cancer 1995;71:278-81. 32. Futamura M, Takagi Y, Koumura H, et al. Spread of colorectal cancer micrometastases in regional lymph nodes by reverse transcriptase-polymerase chain reactions for carcinoembryonic antigen and cytokeratin 20. J Surg Oncol 1998;68:34-40. 33. Yun K, Merrie AEH, Gunn J, et al. Keratin 20 is a specific marker of submicroscopic lymph node metastases in colorectal cancer: validation by K-ras mutations. J Pathol 2000;191:21-6. 34. General rules for clinical and pathological studies on cancer of the colon, rectum and anus. Japanese Research Society for Cancer of the Colon Rectum and Anus. Tokyo: Kanahera; 1994. p. 14-25. 35. Merrie AEH, Phillips LV, Yun K, et al. Skip lesions in colon cancer: assessment by lymph node mapping. Eur J Nucl Med 1999;26(Suppl S1):S73. 36. Uren RF, Howman-Giles R, Thompson JF, et al. Lymphoscintigraphy to identify sentinel lymph nodes in patients with melanoma. Melanoma Res 1994;4:395-9. 37. Uren RF, Howman-Giles R, Thompson JF. Lymphatic drainage from the skin of the back to retroperitoneal and paravertebral lymph nodes in melanoma patients. Ann Surg Oncol 1998;5:384-7. 38. Pezim ME, Nicholls RJ. Survival after high or low ligation of the inferior mesenteric artery during curative surgery for rectal cancer. Ann Surg 1984;200:729-33. 39. Surtees P, Ritchie JK, Phillips RK. High versus low ligation of the inferior mesenteric artery in rectal cancer. Br J Surg 1990;77:618-21. 40. Corder AP, Karanjia ND, Williams JD, et al. Flush aortic tie versus selective preservation of the ascending left colic artery in low anterior resection for rectal carcinoma. Br J Surg 1992;79:680-2.