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© 2001 American Society for Clinical Oncology Molecular Staging of Early Colon Cancer on the Basis of Sentinel Node Analysis: A Multicenter Phase II TrialFrom the Departments of Molecular Oncology, Surgical Oncology and Pathology, John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica, CA; Michigan State University, McLaren Regional Medical Center, Flint, MI; Century City Hospital, Los Angeles, CA; and Department of Biomathematics, University California Los Angeles School of Medicine, Los Angeles, CA. Address reprint requests to Anton J. Bilchik, MD, PhD, FACS, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404; email: bilchika{at}jwci.org
PURPOSE: Approximately 30% of patients with American Joint Committee on Cancer stage I or II colorectal cancer (CRC) develop systemic disease. We hypothesized that multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of sentinel lymph nodes (SNs) draining a primary CRC could detect micrometastases not detected by conventional histopathologic analysis. PATIENTS AND METHODS: In a multi-institutional study, 40 patients with primary CRC underwent dye-directed lymphatic mapping at the time of colon resection. Each dye-stained SN was tagged, and the tumor and regional nodes were resected en bloc. All lymph nodes were examined by conventional hematoxylin and eosin (HE) staining. In addition, each SN was cut into multiple sections for cytokeratin immunohistochemical (CK-IHC) staining and for RT-PCR and electrochemiluminescent detection of three markers: ß-chain human chorionic gonadotropin, hepatocyte growth factor receptor, and universal melanoma-associated antigen. Whenever possible, RT-PCR assay was also performed on primary tumor tissue. The detection sensitivity of individual markers was 10-3 to 10-4 µg of RNA and one to five tumor cells in 107 lymphocytes of healthy donors. RESULTS: One to three SNs were identified in each patient. An average of 15 nodes were removed from each CRC specimen. No nonsentinel (untagged) node contained evidence of tumor if all tagged (sentinel) nodes in the same specimen were histopathology tumor-negative. HE staining of SNs identified tumor in 10 patients (25%), and CK-IHC of SNs identified occult micrometastases in four patients (10%) whose SNs were negative by HE. Of the remaining 26 patients with no evidence of SN involvement by HE or CK-IHC, 12 (46%) had positive RT-PCR results. The number of markers expressed in each SN correlated (P < .04) with the T stage of the primary tumor. There was 79% concordance in marker expression for the respective pairs (n = 38) of primary tumor and histopathologically positive SNs, and 86% (12 of 14) concordance between RT-PCR positive and histopathologically positive SNs. CONCLUSION: Identification and focused examination of the SN is a novel method of staging CRC. CK-IHC and RT-PCR identified occult micrometastases in 53% of patients whose SNs were negative by conventional staging techniques. These ultrasensitive assays of the SN can identify patients who may be at high risk for recurrence of CRC and therefore are more likely to benefit from systemic adjuvant therapy.
COLORECTAL CARCINOMA (CRC) is the most common gastrointestinal malignancy and the second leading cause of cancer-related deaths in the United States. More than 130,000 new cases are expected this year.1 The 5-year survival for American Joint Committee on Cancer stage I colorectal cancer is 90% but decreases to 75% and 50% for stages II and III patients, respectively. The presence of lymph node metastases is therefore an extremely important prognostic factor. Alarmingly, however, one third of patients whose regional lymph nodes are tumor-free by conventional histopathologic methods will develop systemic disease. Although the etiology of recurrence is likely multifactorial, these statistics suggest that conventional methods of assessing the lymph nodes fail to detect micrometastatic tumor deposits. Several retrospective studies have reported nodal micrometastases in CRC specimens examined by cytokeratin immunohistochemistry (CK-IHC), multilevel sectioning, monoclonal antibody staining, and reverse transcriptase-polymerase chain reaction assay (RT-PCR).2-9 However, these ultrastaging techniques are too costly and time-consuming for use on the 15 or more lymph nodes in the average CRC specimen. Sentinel node (SN) mapping, first developed and popularized by Morton et al10 in melanoma, can identify the initial lymph node to receive metastatic cells from a primary tumor that drains via the lymphatics. Giuliano et al11,12 and Kelemen et al13 later applied the technique to breast and thyroid carcinomas. More recently, we have applied SN mapping to a variety of solid neoplasms, including CRC.14-16 Because the SN is the regional lymph node most likely to contain early metastases, a focused examination of this node should provide a more efficient and cost-effective approach for detecting occult regional-node metastases in patients with primary CRC. In this study, we hypothesized that examination of the SN by CK-IHC and a novel multimarker RT-PCR assay would improve the detection of occult micrometastases in the nodal basins that drain CRC. We previously reported a multimarker RT-PCR assay to detect micrometastases in blood and tissues of patients with breast cancer, melanoma, and pancreatic cancer.17-19 The multimarker system eliminates some of the problems associated with single-marker detection techniques, such as tumor cell heterogeneity, clonal selection during tumor metastasis, and variable expression of individual genes within tumor cells. In the present study, three tumor mRNA markers were evaluated: beta-chain human chorionic gonadotropin (ß-hCG), hepatocyte growth factor receptor (c-Met), and universal melanoma-associated antigen-A family (uMAGE). ß-hCG is overexpressed in many gastrointestinal malignancies, including CRC.19-21 c-Met is an oncogene encoding the receptor for hepatocyte growth factor, a ligand that regulates cell functions.22,23 c-Met is frequently overexpressed in CRC and has been associated with tumor progression.23,24 The MAGE-A gene family, which consists of 12 different genes on the X chromosome that have high nucleotide sequence homologies, is expressed in various tumors (including carcinomas) but not in normal tissue except male germline cells.25,26 Multiple members of the MAGE-A gene family are expressed in CRC.25,27,28 We developed a universal primer set that covers the common areas of several MAGE-A gene families so that multiple members (MAGE-A-1,3,6,12) could be detected in a single reaction.29 To test our hypothesis in this study, serial sections of SNs were assessed by CK-IHC, and paired primary CRCs and SNs were assessed by RT-PCR and electrochemiluminescent (ECL) analysis. Expression of mRNA tumor markers ß-hCG, c-Met, and uMAGE were also assessed in CRC cell lines, healthy donor peripheral blood lymphocytes (PBLs), and benign lymph nodes.
Patient Population In 1998, the John Wayne Cancer Institute (Santa Monica, CA), McLaren Regional Medical Center (Flint, MI), and Century City Hospital (Los Angeles, CA) began a multicenter trial of SN mapping in patients with CRC. All patients enrolled onto this study had early stage primary CRC with no evidence of distant metastases.
SN Mapping and Colectomy
Processing of CRC Specimen Each tagged SN was excised, measured, and bisected longitudinally (bivalved). An 8-µm imprint slide was prepared from the cut surface and stained with Diff-Quik I & II (Dade Int, Miami, FL) for the pathologist intraoperative diagnosis. The remainder of the lymph node was frozen. After minimal trimming of the frozen tissue block, an 8-µm section was prepared on the cryostat. Six to eight further sections, representing approximately 72 µm, were cut, layered on a sterile glass slide, and stored at -80°C until they could be processed for RNA. For RT-PCR analysis, we also determined the necessary amount of RNA that could be consistently isolated from a minimum number of frozen sections of SN. The next 8-µm section of tissue was cut for examination by Diff-Quik staining. The remainder of the bisected node was then placed in 10% formalin and embedded in paraffin. Four-micrometer sections of each paraffin-embedded tissue block were cut at least two different levels separated by approximately 200 µm and then examined by hematoxylin and eosin (HE) and CK-IHC, as previously described.15,16 CK-IHC was performed with MAK-6 antibody cocktail (Ciba-Corning, Alameda, CA) and an automated immunostainer (Ventana ES 320, Ventana Medical Systems, Tucson, AZ). A CK-IHC stain was considered positive if it demonstrated strongly positive cell clusters or individual cells that had anatomic and cytologic features of tumor cells. Whenever possible, tissue from the primary tumor was paired with SN tissue for RT-PCR analysis. All tissue specimens were obtained in accordance with protocols approved by the institutional review boards of each participating center. Tissues used in the RT-PCR analysis were collected, dissected, and processed under defined sterile conditions to prevent RNA contamination. Tissue specimens were either immediately processed or cryopreserved to avoid RNA degradation.
CRC Cell Lines and Control Specimens for RT-PCR Analysis Colon cancer cell lines HT-29 and SW480 were obtained from ATCC (Rockville, MD). JAR choriocarcinoma cell line was obtained from ATCC. CRC cell lines JWCI-0044 (A), JWCI-0044 (B), JWCI-0361, JWCI-0427, JWCI-0485, JWCI-1100, and JWCI-1203 were established and characterized at the John Wayne Cancer Institute. All nine cell lines were cultured in RPMI 1640 media supplemented with 10% heat-inactivated fetal calf serum (Gemini, Calabasas, CA), penicillin, and streptomycin. Cells were washed with sterile physiologic phosphate-buffered saline (PBS). Total RNA was extracted from cells when cultures reached 70% to 80% confluence.
RNA Isolation Tissue was finely minced in TRI Reagent while on ice. Tissue mincing and RNA extraction were performed as previously described.18,19 All RNA extractions were performed in a designated sterile laminar flow hood using RNase-free lab ware. RNA was quantitated and assessed for purity by ultraviolet spectrophotometry. RNA was then treated with RQ1 RNase-Free DNase (Promega, Madison, WI) to degrade any genomic DNA carried over from the extraction. Expression of porphobilinogen deaminase mRNA (housekeeping gene mRNA standard control) was verified by RT-PCR/ECL in all RNA samples used in the study.19 Tissue processing, RNA extraction, RT-PCR assay set up, and post–RT-PCR product analysis were performed in separate designated rooms and facilities to prevent nucleic acid and cDNA product carryover contamination.
Synthesis of Primers and Probes Oligonucleotide probes were specifically designed as internal probes spanning at least one intron region. Oligonucleotide probes were synthesized and labeled with tris (2,2-bipyridine) ruthenium (II) chelate (Midland Certified Reagent Company). The probe sequences synthesized were as follows: porphobilinogen deaminase, 5';-tris (2,2-bipyridine) ruthenium (II) chelate-GTATGCGAGCAAGCTGGCTCTTGCGG-3';; ß-hCG, 5';-tris (2,2-bipyridine) ruthenium (II) chelate-GCAGAGTGCACATTGACAGCT-3';; c-Met, 5';-tris (2,2-bipyridine) ruthenium (II) chelate -ACTTCATATAAGGGGTCTGGGC-3';; and uMAGE, 5';-tris (2,2-bipyridine) ruthenium (II) chelate-TGAGCAGAGGAGTCAGCACTG-3';.
RT-PCR Assay
ECL Analysis
The average age of the 40 patients accrued for this study was 70.1 years (± 11.5 years); 17 (43%) were men. Of the 40 primary tumors, 13 were in the right colon, nine were in the sigmoid colon, seven in the rectum, six in the cecum, three in the left colon, and two in the transverse colon. Lymphatic mapping identified one to three SNs in all patients. Most commonly, a blue-stained lymphatic channel running from the tumor into the mesentery of the bowel was identified within 30 seconds after injection. Lymphatic mapping lengthened the procedure by an average of 5 to 10 minutes. The average number of SNs identified was two; the average number of nodes in each CRC specimen was 15 (range, two to 28). In three cases, an SN was mapped outside the margins of a conventionally planned mesenteric resection. In two of these cases, lymphatic channel from a proximal ascending colon cancer drained aberrantly to an SN to the left of the middle colic vessel ( Fig 2).
HE staining of all nodal tissue and CK-IHC staining of SN tissue showed that the tumor status of the SN was concordant with the pathology of the lymph node basin in all cases ( Table 1). Ten patients (25%) had a positive SN by routine HE analysis. Four other patients (10%) had a positive SN by CK-IHC ( Fig 3). Therefore, there were 26 patients whose nodes were negative by histopathologic analysis. The SN was the only positive node in seven of the 40 cases; in four of these cases, including one of the patients with aberrant lymphatic drainage (Fig 2), the SN was negative by HE but positive by CK-IHC analysis.
RT-PCR Analysis of CRC Cell Lines Optimal RT-PCR assay conditions were established for ß-hCG, c-Met, and uMAGE mRNA marker detection in CRC cell lines. RT-PCR assay conditions were standardized and uniform throughout the studies. All nine established CRC cell lines showed variable levels of expression of ß-hCG, c-Met, and uMAGE mRNAs. There were five (56%) cell lines positive for ß-hCG mRNA, four (44%) positive for c-Met mRNA, and six (67%) positive for uMAGE mRNA expression ( Table 2). Each CRC cell line expressed at least one of the three mRNA markers.
RT-PCR Assay Sensitivity and Specificity The sensitivity of mRNA marker detection by the RT-PCR assay was validated by serial dilution analysis of total RNA from tumor cells and by an in vitro tumor cell dilution assay using normal donor PBL. The sensitivity limit for ß-hCG, c-Met, and uMAGE mRNA detection was determined by performing RT-PCR on serial dilutions of 1 µg of JAR cell line total RNA. JAR, a choriocarcinoma cell line, was used as a standard for the assay, because it expresses all three tumor mRNA markers consistently in different cell passages. The detection sensitivity limits of ß-hCG, c-Met, and uMAGE were 10-1 ng, 1 ng, and 10-1 ng, respectively ( Figs 4 to 6). An in vitro model in which cultured tumor cells were serially diluted was used to determine the sensitivity of the assay. JAR cells were serially diluted in approximately 107 PBLs from healthy donors. Using this in vitro model, the RT-PCR assay consistently detected one to five tumor cells among 107 normal cells using ß-hCG, c-Met, and uMAGE as tumor detection markers (data not shown). This in vitro model estimates the potential detection sensitivity of the assay for occult tumor cells among lymphoid cells.
PBLs from 25 healthy donors served as negative RT-PCR controls for the tumor mRNA markers. No ß-hCG, c-Met, or uMAGE mRNA expression was detected in RNA from healthy donor PBLs. Lymph nodes from five patients with benign conditions were also negative for all markers. These results confirmed assay specificity for these mRNA tumor markers in CRC tissue.
RT-PCR Analysis of CRC Primary Tumors Thirty-three (87%) of 38 primary tumor specimens were RT-PCR positive ( Table 3). The total number of tumor specimens expressing zero, one, two, or three mRNA markers was five, 13, seven, and 13, respectively. For individual markers, the expression was 68% for ß-hCG (n = 26), 63% for c-Met (n = 24), and 42% for uMAGE (n = 16). There was a correlation between the number of expressed markers and the T stage (Spearman correlation coefficient = 0.510; P = .001): 11% of T1 tumors, 44% of T2 tumors, and 75% of T3/T4 tumors expressed two or three markers, and all three markers were expressed in more than one half (55%) of T3/T4 specimens.
Individual markers showed variations in overlap of expression. Within the group of 26 patients whose tumors tested positive for ß-hCG mRNA, 19 were also positive for c-Met mRNA and 14 for uMAGE mRNA. Of the 24 patients whose tumors were positive for c-Met, 13 were also positive for uMAGE mRNA. ß-hCG and c-Met expression had a significant correlation with uMAGE (kappa coefficient; P < .02 and P < .04, respectively). c-Met expression (Wilcoxon rank-sum test; P = .006) and uMAGE expression (P = .016) were significantly greater in patients with higher T stages.
RT-PCR Analysis of the SN
Table 4 shows the number of markers detected in the SN of all 40 patients. Among the markers, only c-MET expression correlated with uMAGE expression (kappa coefficient; P < .02). In the 14 patients whose SN metastasis was identified by HE and/or CK-IHC, only 12 (86%) had RT-PCR evidence of SN metastasis. In the 10 SNs positive by HE, nine (90%) were RT-PCR positive. Of the four SNs positive by CK-IHC, three (75%) were RT-PCR positive. The two false-negative results may have been a result of sampling error; tumor cells may have been in the sections for histopathology and not in the RT-PCR sections. Alternatively, there may not have been sufficient marker mRNA in the RT-PCR sections, or the tumor cells may not have expressed any of the markers. Overall, CK-IHC and RT-PCR identified occult micrometastasis in 53% (16 of 30) of patients whose SNs were negative by HE.
Of the 26 patients with histopathologically negative SNs, 11 had T1 tumors, five had T2 tumors, and nine had T3 tumors. Twelve (46%) of the 26 patients had positive RT-PCR results; these positive RT-PCR findings were associated with four T1 lesions, two T2 lesions, and six T3 lesions. Expression of two or more markers was found in four patients with T1 lesions, two with T2 lesions, and six with T3 lesions, thereby upstaging these patients with histopathologically negative SNs. T3 patients had the greatest number of patients upstaged (67%), followed by T2 (40%). There was a significant correlation of the number of markers detected in SN (Spearman correlation coefficient, 0.33; P < .04) with higher T stages. For individual marker expression in histopathologically negative SNs, there was a significant correlation with ß-hCG detection and higher T stage (Wilcoxon rank-sum test; P = .02). The concordance for paired tumor and SN marker expression was 66% for ß-hCG, 68% for uMAGE, and 53% for c-Met. UMAGE mRNA expression demonstrated the most significant (P < .04) concordance in paired specimens. As expected, there was an overall lower detection of mRNA markers in the paired SN.
The high incidence of distant metastasis of CRC in patients whose nodes are negative for tumor may reflect analysis of insufficient numbers or sections of lymph nodes.5-7,15 Because multiple sectioning and IHC staining cannot be routinely used to examine all lymph nodes in a CRC specimen, we focused on the first regional node(s) to receive lymphatic drainage from a primary tumor. In melanoma and breast cancer, lymphatic mapping and excision of the SN is used to determine the tumor status of the entire nodal basin and avoid complete lymph node dissection in node-negative patients. The application of the SN technique in CRC is different because all regional lymph nodes are routinely removed en bloc with the primary tumor. However, as in melanoma and breast cancer, examination of the SN allows the pathologist to focus on the regional node(s) most likely to contain tumor cells and thus improve tumor cell detection and accuracy of staging. In this multicenter study, lymphatic mapping of the SN draining a CRC was logistically feasible, accurate, fast, and inexpensive. The technique is technically simpler than that performed for melanoma or breast cancer because the tumor, the lymphatic channels, and the SN are directly visible. The findings of this multicenter study verify those of Saha et al,15 whose series demonstrated a 99% rate of SN identification, a 96% rate of accuracy for the SN as an indicator of regional lymph node status, and a 17% rate of upstaging CRC. Moreover, in the current study, unexpected lymphatic drainage was demonstrated in three patients, altering the operative approach. In this study SN and lymphatic mapping was successful in all patients, but we have previously demonstrated16 that false negatives can occur when the SN is replaced by tumor. The tumor occludes the lymphatic vessels resulting in drainage to another (nonsentinel) node.16 Because these nodes are large and firm, it is unlikely that SN and lymphatic mapping will be of value in this group of patients. Almost one half (46%) of the 26 patients with histopathologically negative SNs had positive RT-PCR results. Of the three mRNA markers, ß-hCG was the most frequently expressed in both tumor and nodal tissue, followed by c-Met and then by uMAGE. The pathophysiologic role of ß-hCG and its significance in carcinomas are still unknown; it may function as a suppressive factor of immune responses. c-Met may be important as a molecular phenotypic marker for metastasis and as a marker for early detection. The physiologic role of MAGE-A gene expression is also unclear, but both MAGE-A-1 and MAGE-A-3 are immunogenic in humans and potential targets for active-specific immunotherapy. The prognostic significance of nodal micrometastases by either CK-IHC or RT-PCR in CRC remains unclear. In a recent study of 46 patients initially reported as node-negative, re-examination using CK-IHC and carcinoembryonic antigen (CEA)-IHC demonstrated evidence of micrometastases in 12 patients (26%).7 However, the presence of nodal micrometastases did not significantly affect 5-year survival. Similarly, Jeffers et al6 detected CK-IHC micrometastases in 25% of 77 patients whose CRCs were initially staged as Duke’s B. Again, the presence of nodal micrometastases had no significant effect on survival; however, random microsectioning may have missed tumor cells, thereby causing nonsignificant survival differences between the two groups. More recently, Greenson et al2 demonstrated that micrometastatic disease missed by routine HE staining but identified by CK-IHC had an adverse effect on survival. The lack of consensus in the literature in part reflects the absence of standard antibody titers and staining techniques; there are considerable interinstitutional variations in the analysis of CRC lymph nodes by CK-IHC. Although to date no randomized study has demonstrated significance for the detection of micrometastases by CK-IHC, the American College of Surgeons Oncology Group currently is conducting a multicenter trial (Z-0010) to assess the utility of CK-IHC in detecting micrometastasis, of SNs draining primary breast carcinoma. Clinical outcome studies of marker expression in CRC are also limited. Hayashi et al4 demonstrated decreased survival in patients with p53 or K-ras mutations in colonic lymph nodes. In another study of patients whose CRC was staged Duke’s B by conventional techniques, Liefers et al3 reported a 5-year survival rate of 50% for patients whose nodes expressed CEA, versus 91% for those whose nodes did not express CEA. Several other investigators have reported that histologically negative lymph nodes contained evidence of occult metastases by RT-PCR using CK2030 or guanylyl cyclase C31 in qualitative assay systems. However, guanylyl cyclase C, CEA, and cytokeratin are expressed by normal tissues and therefore may introduce false-positive results. Our group and others have questioned their utility for the detection of micrometastatic CRC.32 Our approach has been to use a combination of mRNA markers in a semiquantitative assay to detect occult micrometastases. Focused analysis of multiple sections of the SN by CK-IHC and RT-PCR provides a unique tool for accurate staging of CRC. As demonstrated in our study, lymphatic mapping of the SN also can identify unexpected nodal drainage patterns that alter the margins of surgical resection. Focused examination of SN diagnoses micrometastatic disease missed by conventional techniques. Although the significance of micrometastatic disease is yet to be defined in CRC, it is likely to be an important stratifying factor in choosing those who may benefit from adjuvant chemotherapy.
Supported in part by grant no. T32 CA 09689 from the National Cancer Institute, and funding from the Rogovin-Davidow Foundation, Los Angeles, CA, and the Rod Fasone Memorial Cancer Fund, Indianapolis, IN. We thank Dr J. Heroux (IGEN), V.D. Vu, C.T. Kuo, and the John Wayne Cancer Institute clinic for their support and assistance in this study. We also thank Maria Gonzalez for editorial assistance. The ORIGEN analyzer was provided by IGEN International (Gaithersburg, MD). This study is part of an ongoing joint collaboration with IGEN.
Presented in part at the American Society of Clinical Oncology, New Orleans, LA, March 2000, and the American Gastroenterology Association, San Diego, CA, May 2000.
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ABSTRACT
INTRODUCTION
two–RT-PCR marker expression cutoff was considered a tumor-positive result on the basis of our previous RT-PCR detection studies on blood and SNs.
