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© 2002 American Society for Clinical Oncology Comparative Detection of Lymph Node Micrometastases of Stage II Colorectal Cancer by Reverse Transcriptase Polymerase Chain Reaction and ImmunohistochemistryByFrom the Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University; and Department of Pathology, School of Allied Health Science, Faculty of Medicine, Osaka University, Osaka, Japan. Address reprint requests to Hirofumi Yamamoto, MD, PhD, Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan; email: kobunyam{at}surg2.med.osaka-u.ac.jp
PURPOSE: Inconsistent conclusions have been drawn about the clinical significance of micrometastases in lymph nodes (LNs) of node-negative colorectal cancer (CRC) patients. We performed a comparative study of detection of micrometastases using immunohistochemistry (IHC) by anti-cytokeratin antibody and carcinoembryonic antigen (CEA)-specific reverse-transcriptase polymerase chain reaction (RT-PCR) in the same patients, in an attempt to move closer to their clinical application. PATIENTS AND METHODS: Sixty-four CRC patients, with RNA of good quality available from paraffin-embedded LN specimens, were selected from 84 stage II patients who underwent curative surgery between 1988 and 1996. We investigated associations between the presence of micrometastases by each method and prognosis. RESULTS: Micrometastases were detected in 19 (29.6%) of 64 patients by RT-PCR and in 35 (54.7%) of 64 patients by IHC. By RT-PCR analysis, patients exhibiting a positive band for CEA mRNA had a significantly worse prognosis than those who were RT-PCR–negative, with respect to both disease-free and overall survival (P = .027 and .015, respectively). By IHC analysis, the presence of micrometastasis did not predict patient outcome in terms of either disease-free or overall survival. Infiltrating pattern of tumor growth characteristic was significantly associated with shorter disease-free survival among various clinical or pathologic factors. By multivariate Cox regression analysis, micrometastasis detected by RT-PCR and the Crohn’s-like lymphoid reaction were both independent prognostic factors. CONCLUSION: Micrometastases detected by RT-PCR, but not IHC, may be of clinical value in identifying patients who may be at high risk for recurrence of CRC and who are therefore likely to benefit from systemic adjuvant therapy.
METASTASIS TO regional lymph nodes (LNs) is an important prognostic factor and is used for clinical decision-making regarding the selection of the most appropriate cancer treatment.1-5 Histologic metastasis in regional LNs is clinically identified through examination of a few slices of hematoxylin and eosin (H&E)-stained sections. Studies have shown that occult cancer metastasis (also designated as micrometastasis) exists in the LNs of colorectal cancer patients.6 Micrometastases are thought to exist in the LNs of patients with various types of human malignancies.7-12 This type of metastasis is so minimal that it can be overlooked in H&E sections.7 Accordingly, immunohistochemistry (IHC)11,13-21 and/or molecular genetic techniques7,10,11,22-24 have been used to detect micrometastases. Immunohistochemical studies, mainly targeting cytokeratin (CK), a specific marker of epithelial cells, have shown that micrometastasis to regional LNs is an important prognostic factor in N0 gastric cancer.25-27 However, the value of micrometastasis for prognostication in node-negative colorectal cancer remains controversial. In Dukes’ stage A and B tumors, many immunohistochemical studies have shown no relationship between the existence of micrometastasis and unfavorable prognosis,13,16-19 although a few investigators have reported a significant correlation.14,20,21 Using molecular genetics techniques, only two groups of investigators have so far suggested a positive correlation between the presence of micrometastases and poor prognosis in patients with node-negative colorectal cancer (CRC).22,24 The first study targeted alterations of DNA within regional LNs, that is, mutations of the K-ras and p53 genes.22 Liefers et al24 reported that micrometastasis to LNs was a sensitive marker for poor prognosis in 26 patients with stage II node-negative colorectal cancers, by reverse-transcriptase polymerase chain reaction (RT-PCR) using carcinoembryonic antigen (CEA) as a genetic marker. To confirm and extend their results, many prospective studies are currently underway in larger population samples, but firm conclusions are still several years away. To assess the possible clinical application of micrometastasis detection, the previous discrepant findings between genetic diagnosis and IHC need to be resolved. Because most of the previous studies used only one method, we examined in the present study the presence of micrometastases in pericolic LNs by means of both RT-PCR and IHC in the same N0 CRC patients, who underwent curative surgery and whose clinical outcomes were already known. For this purpose, we used a novel technique that was recently developed in our laboratories, which accomplished efficient extraction of RNA from archived formalin-fixed LN specimens.28,29 Our data provide an important insight into the possible clinical application of micrometastasis detection, in advance of the results of the prospective studies still underway.
Patients We selected 84 unspecified CRC patients with stage II tumors (clinically staged according to the tumor-node-metastasis classification of the International Union Against Cancer30), who underwent curative surgery in our surgery department between 1988 and 1996. Sixty-four patients with well-preserved RNA were further investigated for the presence of micrometastases. These patients (n = 64) included 40 men (62.5%) and 24 women (37.5%), with a mean age of 60.2 ± 10.3 years (± SD; range, 31 to 80 years). Thirty-five tumors were located in the colon and 29 in the rectum, ranging in size from 0.8 to 12.0 cm in diameter (mean, 4.8 ± 2.0 cm). The majority of tumors were well-differentiated adenocarcinomas (n = 40), followed by moderately differentiated adenocarcinomas (n = 23) and mucinous adenocarcinomas (n = 1). None of the patients had preoperative chemotherapy or irradiation. The mean follow-up period was 79.5 months (range, 6.6 to 134.1 months).
Tissue Specimens
RNA Extraction From Pericolic LN Specimens and RT-PCR Two primer sets for PCR amplification of CEA were designed according to the results of our earlier studies.11,29 Primer sequences were as follows:
Previously published glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR primers were used to exclude the possible degradation of RNA in each reaction.31 The sizes of the amplicons for CEA-1, CEA-2, and GAPDH were 366, 160, and 181 bp, respectively. PCR amplification was performed in a 25-µL reaction mixture containing 2 µL of cDNA, 1x PCR buffer, 1.5 mmol/L MgCl2, 0.8 mmol/L deoxynucleotide triphosphatase, 0.2 µmol/L each primer, and 1 unit of Taq DNA polymerase (AmpliTaq Gold; Roche Molecular Systems, Inc, Pleasanton, CA). PCR was set up using the following protocols: one cycle of denaturing at 95°C for 12 minutes, and then (1) for CEA-1, 35 cycles of 94°C for 30 seconds, 69°C for 30 seconds, and 72°C for 60 seconds; (2) for CEA-2, 35 cycles of 95°C for 1 minute and 72°C for 1.5 minutes; and (3) for GAPDH, 18 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds. All PCRs were finished with a final extension at 72°C for 10 minutes. By the above-mentioned method, the minimal detection limit for CEA mRNA was the level derived from one MCF-7 cell among 105 lymphocytes.29
H&E Staining and Immunohistochemistry
Assessment of the Primary Tumor and Occult Cancer Cells Detection of one or more CK-positive cancer cells in at least one LN was regarded as positive for micrometastasis in a patient. Only cells that were diagnosed as definite carcinoma cells on the basis of morphologic features were considered micrometastasis.
Statistical Analysis
Optimal PCR Conditions We initially determined the optimal conditions under which CEA cDNAs prepared from 30 control LNs of noncancer patients were never amplified. After 35 cycles of PCR, none of these control samples expressed CEA transcripts, but at 40 cycles, bands for CEA mRNA appeared in two (6.7%) of 30 LNs, with either CEA primer set 1 or set 2. These experiments were reproducible. On the basis of these results, PCR was performed for 35 cycles throughout the subsequent experiments. CEA mRNAs were expressed in all histologically positive LNs (n = 30). A band of GAPDH mRNA was exclusively noted in the above-mentioned LNs (30 control LNs and 30 histologically positive LNs).
Examination of Preserved mRNA From Paraffin-Embedded Blocks
Detection of CEA mRNA in LNs
Immunohistochemistry When the primary tumors (n = 64) were immunostained with anti-CEA antibody or anti-CK antibody, all tumors expressed both CEA and CK proteins. In addition, 30 histologically positive LNs exclusively expressed both proteins (data not shown). For histologically negative LNs (n = 350), five serial sections were stained with anti-CK antibody and searched for occult cancer cells under a light microscope. Intense immunoreactivity for CK was found in the cytoplasm of occult carcinoma cells (Fig 2). Most occult carcinoma cells were observed singly, but occasionally they formed small clusters. They were located in the subcapsular sinus or paracortical sinus. Among 350 histologically negative LNs, 70 (20.0%) contained micrometastatic carcinoma cells by screening of five consecutive stained sections. In total, 35 (54.7%) of 64 patients harbored micrometastases. When only the first slide of each LN was analyzed, CK-positive LNs and patients were reduced to 8.9% and 28.1%, respectively.
Assessment of Primary Tumor The conspicuous pattern of the Crohn’s-like lymphoid reaction was found in 29 cases (45.3%), and the inconspicuous pattern was noted in 35 cases (54.7%). Lymphoid aggregates were confined to the colonic wall or to the pericolic adipose tissue (Fig 3A). In analysis of tumor growth characteristic, the infiltrating type, characterized by deep and wide infiltration by isolated tumor cells, was found in 28 cases (43.8%) (Fig 3B). The expanding type, whose invasive margin was well circumscribed, was noted in 36 cases (56.2%) (Fig 3C).
Relationship Between Clinicopathologic Factors and Micrometastases Detected by CEA mRNA Expression or CK Expression Table 1 lists the clinical and pathologic characteristics of the 64 patients stratified by CEA mRNA expression. No significant difference in age, sex, tumor site, histologic grade, lymphatic invasion, venous invasion, tumor size, Crohn’s-like lymphoid reaction, or growth characteristic of the tumor at the invasive margin was noted between the two groups. Table 2 lists the clinical and pathologic features of the patients with and without CK-positive cells. Tumor size alone was significantly larger in the CK-positive group than in the CK-negative group (P = .014). The presence of micrometastases was not related to the other clinicopathologic parameters.
Survival Analyses Among 64 cases, 12 developed disease recurrence within 5 years after surgery. The recurrent patients in stage II are listed in Table 3. Like more advanced stage patients, stage II patients had often hematogenous metastasis (n = 9) rather than local recurrence (n = 3). Two of 64 patients died from diseases other than CRC, and therefore survival analysis was performed on the remaining 62 cases.
Impact of Expression of CEA mRNA on Overall or Disease-Free Survival The overall and disease-free survival curves were drawn as stratified by the results of CEA-specific RT-PCR. The 5-year overall survival rate was significantly worse in CEA-positive patients (n = 19 [78.2%]) than in patients without micrometastases (n = 43 [95.3%]) (P = .015) (Fig 4A). The difference was more evident in the 5-year disease-free survival rates: 61.4% for CEA-positive patients and 88.4% for CEA-negative patients (P = .027) (Fig 4B).
Impact of CK Staining Positivity on Overall or Disease-Free Survival On the basis of the IHC findings of micrometastases, patients were classified into two groups: those now considered to possess micrometastases (n = 34) and those devoid of micrometastases (n = 28). When similar survival analyses were conducted on these two groups, there was no significant difference in either overall or disease-free survival (Fig 5). When only the first slide of each LN was analyzed, the impact of micrometastases on prognosis was not found either (data not shown).
Univariate Analyses Expression of CEA mRNA in LNs, CK-positive staining in LNs, and various clinicopathologic parameters were evaluated for their impact on prognosis of patients with node-negative stage II CRC. CEA mRNA ex-pression in LNs and infiltrative type of the tumor growth characteristic were significantly associated with shorter disease-free survival (P = .027 and .043, respectively). CEA mRNA expression in LNs alone was significantly associated with poor overall survival (P = .015) (Table 4).
Multivariate Analyses Multivariate Cox regression analysis demonstrated that CEA mRNA expression in LNs alone was a significant prognostic factor for disease-free survival (P = .035) (Table 5). For overall survival, CEA mRNA expression in LNs with a hazard ratio of 13.744 and the Crohn’s-like lymphoid reaction with a hazard ratio of 19.010 retained as significant covariates (P = .019 and .039, respectively) (Table 5). However, all other factors were not significant. When several other combinations were analyzed, using five or six covariates, similar results were obtained.
The presence of LN metastases reflects disease progression and implies that the disease is no longer localized. Accordingly, this event is incorporated into various clinical staging systems such as International Union Against Cancer staging, Dukes’ staging, and Astler-Coller staging.30,40,41 It is known that LN metastasis is a sensitive indicator of poor prognosis in patients with colorectal cancer,1-5 but even in node-negative patients, approximately 10% to 20% patients suffer from relapse in less than 5 years.3 To explain the latter phenomenon, micrometastasis has been a focus of research for years. In the present study, we used RNA rather than DNA to investigate the existence of micrometastases. DNA-based methods have the limitation that mutations of K-ras and/or p53 are found in subsets of CRCs, but not in all cases, whereas CEA is expressed exclusively by primary CRC tissues.11,42 We also observed that all primary tumors and histologically positive LNs expressed the CEA protein. Another reason for using this technique is that tumor cells detected by RT-PCR are likely to be viable because mRNA is extremely unstable. In contrast, there is concern that mutated DNA found in regional LNs might represent a fraction of free tumor DNA rather than being derived from viable cancer cells.43,44 We found that 29.7% of patients displayed positive bands for CEA mRNA in their LNs. This rate was lower than those reported by other investigators (range, 52.1% to 100%).10,22-24 This may be attributable to differences in the system of reverse transcription and PCR conditions. The CEA transcript used as a target for amplification of PCR is expressed in both normal epithelial cells and CRC cells and, therefore, it is possible that the detection rate would increase with an increasing number of PCR cycles. Indeed, it has been reported that the nested PCR technique, which can accomplish high sensitivity by two-step amplification of CEA cDNA, resulted in a 100% detection rate in node-negative CRC patients.10 In an effort to avoid false-positive reaction, PCR for CEA transcripts was performed in a single step and amplification cycles were restricted to 35, as CEA mRNA bands appeared after 40 cycles in a few samples of control LNs from noncancer patients. Moreover, the two sets of primers yielded bands in identical samples from the patients tested, which reassured us of the accuracy of the RT-PCR results. However, we should mention a possibility that only a small number of occult cancer cells might be missed under our relatively mild sensitivity of PCR. We found that CEA mRNA expression in LNs was a significant factor for poor prognosis. RT-PCR assay targeting CEA mRNA in stage II colorectal cancer was previously reported by Liefers et al24; however, they examined only 26 patients. Our present study succeeded in examination of survival rates in a larger population (n = 62) by using a novel RNA extraction technique from paraffin blocks.28,29 The increased number of patients facilitated meaningful statistical analysis of the population sample on the basis of appropriate power analysis. When we examined the recurrent sites in stage II CRC, hematogenous metastasis was the most frequent rather than LN recurrence (Table 3). This implies that micrometastasis detected by RT-PCR may serve as an indirect marker to indicate such CRC being from localized disease into expanding disease. Multivariate analyses also revealed that Crohn’s-like lymphoid reaction was an independent prognostic factor for overall survival. This finding suggests that host immune reaction represented by Crohn’s-like lymphoid reaction may appreciably affect patients’ outcome of stage II CRC and disease expansion characterized by micrometastases. Formalin-fixed samples are generally considered to be poor material for molecular biologic applications. However, we previously succeeded in establishing a new method for efficiently extracting RNA from formalin-fixed samples for use in RT-PCR analysis.28,29 The principle of the method is to eliminate the majority of chemical modifications of mRNA caused by formaldehyde by adding a simple heating process before reverse transcription. Approximately 25% of the cases had unsatisfactory RNA quality. This is unlikely to be attributable to the unique extraction method, as we previously obtained well-preserved RNA in 129 of 131 (98%) LN specimens from breast cancer patients. It is probable that the difference could be generated during the sampling procedure before the fixation step. CK has often been used for immunohistochemical detection of micrometastases of colorectal cancer.11,13-21,45 Because our preliminary studies demonstrated that anti-CK antibody yielded more intense cytoplasmic staining than did several anti-CEA antibodies, with less nonspecific signals (data not shown), we used CK antibody rather than CEA antibody to detect occult cancer cells. We found micrometastases in 20.0% of LNs and 54.7% of patients and most of the occult cancer cells were detected as single cells. This rate was relatively high compared with those reported by other investigators. Most previous studies used only one slice for searching for micrometastases, and the detection rate ranged from 0% to 39%.13-20 Here, we used five serial sections per LN because, with one or two slices, definite diagnosis was quite difficult to make. In a separate study, we found that the rate of detection became apparently higher as the slice number increased from one to two to five and we could make convincing diagnosis with five slices.46 Similarly, Yasuda et al21 reported high positive values in 32 of 42 Dukes’ stage B patients (76.2%) with five sections and Sasaki et al45 obtained 100% positivity (19 of 19 Dukes’ stage A and B patients) with 10 sections. These findings suggest that micrometastasis may be so frequent in stage II CRC that discrete detection of the relative abundance of occult cancer cells (many or few) might be more clinically useful, rather than simply their presence or absence. In this regard, IHC is limited for quantification of micrometastases because it is impractical to examine enough slides to correspond to the whole LN. In contrast, the RT-PCR technique has the advantage of allowing examination of a large bulk of LNs, and therefore reproducible quantification is possible. Indeed, we previously succeeded in measurement levels of CEA mRNA in LNs devoid of histologic metastasis of colorectal cancer by a real-time PCR method.47 From a clinical viewpoint, our results are important, especially in prevention of disease recurrence, because the disease-free survival rate was as low as 60%, which was far worse than we had expected. Although patients with stage II colorectal cancer do not usually receive postoperative adjuvant chemotherapy, it is better to administer some therapy to patients at high risk of disease relapse, as there is evidence that fluorouracil-based therapy contributed to better prognoses of patients with colorectal cancer.48-53 Molecular diagnosis may be a powerful tool for identifying patients who are likely to develop disease recurrence. In conclusion, our present data strongly suggest that micrometastases in LNs detected by CEA-specific RT-PCR, but not by IHC, may be useful for predicting high risk for relapse in stage II CRC patients. A controlled randomized study will be necessary to ascertain whether high-risk patients identified by this method can benefit from adjuvant therapy.
Supported by Grant-in-Aid for Cancer Research no. 12213078 from the Ministry of Education, Science, Sports and Culture, Japan, to H.Y.
The first two authors contributed equally to this study.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test or Student’s t test. Values of P < .05 denoted the presence of a statistically significant difference. 
