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Prognostic Value of Genetically Diagnosed Lymph Node Micrometastasis in Non-Small Cell Lung Carcinoma Cases¹

Laboratory of Cancer Diagnosis and Therapy, Saitama Cancer Center Research Institute, Saitama 362-0806 [T. H., Y. T., M. H., S. H., E. T.]; Department of Pathology, Saitama Cancer Center Hospital, Saitama 362-0806 [Y. K., K. Ni., E. T.]; Department of Pathology, Cancer Institute, Tokyo 170-0012 [Y. I.]; Department of Chest Surgery, Cancer Institute Hospital, Tokyo 170-0012 [S. T., S. O., K. Na.]; and Department of Thoracic and Cardiovascular Surgery, Niigata University School of Medicine, Niigata 951-8122 [T. H., J. H.], Japan

	ABSTRACT

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The predictive value of lymph node micrometastasis, detected by immunohistochemical or genetic methods, is well appreciated in terms of prognosis. However, a major problem is high false-positive rates, because most methods focus on cytokeratin, which is a component not only of carcinoma but also normal epithelial and nonepithelial cells. Mutant allele-specific amplification (MASA) can detect DNAs derived from cancer cells itself, reportedly with high sensitivity. It was, therefore, used with nested-PCR using p53 or K-ras mutation for analysis of lymph node micrometastasis in non-small cell lung carcinoma (NSCLC) patients in the present study, in comparison with the immunohistochemical method using an anti-cytokeratin reagent for the same samples. Lymph nodes from 31 NSCLC patients with p53 and K-ras mutated tumors (30 and 1, respectively) staged as pathological (p)-T_1–4 N_0–1 and M₀ were examined. Genetic and immunohistochemical methods demonstrated positive reactions in 34 (15%) and 61 (27%) of 229 lymph nodes, respectively (9 cases, 29%, and 24 cases, 77%). The concordance with the two methods was 77%, but 13 (39%) of 34 genetically positive lymph nodes could not be detected by immunohistochemistry (IHC). Of 22 cases with p-N₀ disease, 6 (27%) were genetically positive in hilar and/or mediastinal lymph nodes, and 4 (67%) of them died after cancer relapse. In contrast, none of the patients without micrometastasis died of cancer (P < 0.001, log rank analysis). Of the same p-N₀ patients, 17 (77%) were positive by IHC, and 4 (24%) of them died of cancer, whereas 5 negative patients did not suffer cancer relapse. Survival did not significantly differ between cases positive and negative (P = 0.246) by IHC. According to the g-N (N factor restaged by a genetic method), patients with g-N₁ and g-N₂ disease had a shorter survival than those with g-N₀ disease (P = 0.042 and P < 0.001, respectively). However, no significant difference was observed with grading by IHC. Thus, detection of micrometastasis in regional lymph nodes with the MASA method, in other words with a carcinoma-specific marker, is of greater prognostic significance for early stage NSCLC patients than immunohistochemical results. This approach should facilitate selection of patients for whom postoperative adjuvant chemotherapy should be performed.

	INTRODUCTION

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Numbers of operable NSCLC³ cases are increasing because of improved detection of the disease with sophisticated diagnostic techniques. However, even after complete or potentially curative surgery, the long-time survival rate remains unsatisfactory (1) . This is related to the fact that disseminated tumor cells, undetectable by routine diagnostic methods, may be present at the time of surgery. If they could be detected, prediction of relapse or prognosis would be facilitated, allowing additional appropriate treatment to be performed for high-risk patients. Micrometastasis to regional lymph nodes, not detected by routine histology, can now be identified by immunohistochemical or genetic methods, and this is reported to be useful for assessment of prognosis (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . High false-positive rates are a major problem, but the MASA genetic method has high sensitivity and specificity for carcinomas; one tumor cell containing genetic changes was detected in a background of thousands of normal cells (13, 14, 15, 16, 17) . It has established prognostic value for colon cancer (2 , 13) , but data for lung cancer are limited. One reason is that it is difficult to collect enough cases of early stage carcinomas having genetic alteration for MASA analysis. The other is that amplification of the mutated DNA sequence in lymph nodes presented practical difficulties in the past.

In the present study, we therefore collected many NSCLCs with mutations and analyzed regional lymph node micrometastasis by a modified improved MASA method and assessed its prognostic potential. For comparison, the immunohistochemical method using a monoclonal anti-CK reagent was also performed for the same specimens.

	MATERIALS AND METHODS

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Patients, Clinicopathological Data, and Follow-Up.
Fresh frozen samples from 151 NSCLCs (113 adenocarcinomas and 38 squamous cell carcinomas), which were resected consecutively from 1989 to 1993 at Cancer Institute Hospital, Tokyo, Japan, were screened for p53 mutations in exons 4–8 and 10 using single-strand conformation polymorphism, followed by sequencing, and for K-ras mutations of codons 12, 13, and 61 using the MASA method, as described previously (18, 19, 20) . All patients analyzed had undergone a complete or potentially curative resection with lobectomy or pneumonectomy, combined with pulmonary hilar and mediastinal lymph node dissection. Histopathological classification and differentiation of the tumors were determined according to the 1981 WHO classification of lung tumors (21) . The stage of the disease was based on the TNM staging system of the International Union against Cancer (UICC; Ref. 22 ). Of the 68 p53 and 12 K-ras mutated cases, 35 cases with p53 mutations and one case with a K-ras mutation were staged as pathological (p)-T_1–4 N₀ and N₁ M₀. Of those cases, 5 cases with p53 mutation were excluded because the PCR products with the MASA method for the positive controls could not be consistently detected. Finally, 31 cases staged as p-T_1–4 N₀ and N₁ M₀ with enough follow-up were eligible for analysis (Table 1) . Location and type of mutations for each patient are shown in Table 2 . None had received chemotherapy or radiotherapy before surgery, but 13 (41.9%) underwent postoperative adjuvant therapy. The patients comprised 24 men and 7 women with a mean age at surgery of 60 years (range, 26–76 years). Twenty-two had adenocarcinomas, and 9 had squamous cell carcinomas, with 8 well, 16 moderate, and 7 poorly differentiated lesions. Two hundred and twenty-nine lymph nodes (median, 8; range, 4–9 for each case) from the 31 patients were eligible for comparison of the micrometastasis positivity between MASA and immunohistochemical methods and for evaluation of prognostic value (Table 3) . One hundred and seventy lymph nodes of 22 p-N₀ (no lymph node metastasis by conventional histological examination) cases were examined in both pulmonary hilar and mediastinal regions. For the remaining nine p-N₁ patients, 59 lymph nodes from mediastinal regions were examined by both methods. Survival duration was calculated from the date of operation until the date of the last follow-up (censored) or the date of death. The end point was cancer-related death, and deaths attributable to other causes were treated by censoring. Complete follow-up information was available on all 31 patients, with a median follow-up period of 73 months (range, 8–108). Within 5 years after surgery, six patients died with distant or intrapulmonary metastasis, and the others, with the exception of 1 patient who died of another cause (case no. 8), were free of disease (Table 3) .

Nested-PCR for Genetic Analysis Using the MASA Method.
DNAs used for nested-PCR were extracted from histologically negative lymph nodes. As positive controls, DNAs were extracted from primary tumors fixed in formalin and embedded in paraffin for each patient, and diluted tumor DNAs (1:10, 1:100, and 1:1000) with DNAs extracted from lymph nodes of other cases were used. Also, lymph node DNAs extracted from other cases were used as negative controls. For nested PCR, two sets of primers for each patient were prepared. For the first-round PCR amplification, primers located outside of the mutational site were synthesized to amplify the products within 150 bp, because genomic DNAs from tissues fixed in formalin and embedded in paraffin were in fragments. Mutant allele-specific synthesized primers of 15–22 bp with 3'-ends corresponding to each p53 or K-ras variant were used for the second round of PCR (MASA method; Table 2 ; Refs. 13 and 16 ).

Genomic DNAs from lymph nodes were subjected to first PCR amplification with the set of outer primers (0.5 µM) in 25-µl mixtures consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 0.2 mM deoxynucleotide triphosphates (dATP, dTTP, dGTP, and dCTP), and 0.625 unit of AmpliTaq GOLD (Perkin-Elmer Corp., Foster City, CA), with denaturing at 95°C for 9 min, annealing and extension at 52–66°C for 30 s in each cycle, using a GeneAmp PCR System 9700 (Perkin-Elmer Corp.). Because of varied qualities of genomic DNAs from lymph nodes, PCR were performed for 45–50 cycles to obtain equivalent amplified products by saturation. After purification for removal of the primers and deoxynucleotide triphosphates, the amplified products were diluted 50-fold in 10 mM Tris (pH 8)-1 mM EDTA buffer, and 1 µl of aliquots of diluted products was subjected to a second round of PCR using nested primers (mutant allele-specific primers) under the same conditions, except for the temperature of annealing and extension (64–76°C) and the figures of PCR cycles (35–41 cycles), which differed with each set of primers. Primer sequences and PCR conditions of each primer set are shown in Table 2 . The PCR products were then subjected to 3% agarose gel electrophoresis and visualized by ethidium bromide staining. All specimens were analyzed at least twice to confirm the results. Furthermore, in the first two cases analyzed, we examined the sequences of the final MASA products and confirmed their identity as those targeted.

Immunohistochemical Staining.
Paraffin-embedded tissue sections 4 µm thick were immunostained with the monoclonal anti-CK reagent, CAM5.2 (Becton Dickinson, San Jose, CA), using an immunoperoxidase method. This primary mouse monoclonal antibody is specific for CKs 8 and 18 of epithelial cells and is positive in both adenocarcinomas and squamous cell carcinomas of the lung (24) . After exhaustion of endogenous peroxidase with hydrogen peroxide, deparaffinized sections were incubated with 0.1% trypsin for 45 min at room temperature to expose antigen sites. Possible nonspecific background staining was blocked with 10% normal goat serum for 10 min at room temperature. Then the sections were incubated with a primary antibody at room temperature for 1 h, and the antibody reaction was developed with secondary antibodies using ENVISION polymer reagent (peroxidase-labeled polymers conjugated to goat antimouse and antirabbit immunoglobulins; Dako, Glostrup, Denmark). Next, 0.02% diaminobenzidine was applied as the chromogen, and the sections were counterstained with hematoxylin. For positive controls, tissue sections of both primary tumors and metastatic lymph nodes detected by conventional histopathology were included in each staining batch. Negative control sections for IHC were stained with the primary antibodies omitted.

Statistical Analysis.
To assess any correlations between the presence of micrometastasis and clinicopathological data, Fisher’s exact probability test, Mann-Whitney’ U test, and Student’s t test were used, with P < 0.05 indicating significance. Survival curves were created by the Kaplan-Meier method, and the statistical significance of differences was calculated by the log rank test.

	RESULTS

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Histologically, no lymph node metastases were reconfirmed, even with retrospective examination of the H&E slides after obtaining genetic and IHC results.

Lymph Node Micrometastasis Detected by the MASA Method.
Representative results of the MASA method are shown in Fig. 1 , and a summary of the results is presented in Tables 1 and 3 . The genetic method revealed 34 (14.8%) of 229 lymph nodes and 9 (29.0%) of 31 cases to be positive. Among 22 patients with p-T_1–4 N₀ M₀ disease, 24 (14.1%) MASA-positive lymph nodes of a total of 170 in the hilar and/or mediastinal regions were detected in 6 (27.3%) patients; micrometastases in two patients were limited to pulmonary hilar lymph nodes and 4 to mediastinal regions. Among nine cases with p-T_1–4 N₁ M₀ disease, 10 (16.9%) of 59 lymph nodes in the mediastinal region were MASA positive in 3 (33.3%) patients.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Results of nested PCR for detection of point mutations in the p53 gene. PCR products were electrophoresed in a 3% agarose gel containing 0.5 µg/ml ethidium bromide. A, negative result for case 19. B, positive result (Lanes 2–7) for case 3. Top, first-round PCR; bottom, second-round PCR (MASA method). Lane M, size marker; Lane T, primary tumor (positive control); Lanes N1–2, lymph nodes of different cases (negative controls); Lanes 1–9, lymph nodes (examined samples); Lane 1/10–1/1000, tumor DNAs diluted (1:10, 1:100, and 1:1000) with DNAs extracted from lymph nodes of other cases (positive controls); Lane DW, distilled water (negative control).

View larger version (149K): [in this window] [in a new window]   Fig. 2. An example of a CAM5.2-positive cell by immunohistochemical staining in a lymph node. An oval cell is diffusely positive by CAM5.2 in the cytoplasm. In a serial lymph node section stained with H&E, the cell was histologically interpretable as a macrophage or a histiocyte rather than a carcinoma cell. Several macrophages phagocytizing dust particles are also apparent. x400.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Kaplan-Meier survival curves with respect to disease-specific survival. A, cases with and without micrometastasis detected by the genetic method in pathological-N0 disease. A worse prognosis was observed in patients with micrometastasis (P < 0.001, log rank analysis). B, N0 and N1 disease decided by conventional histology (p-N), without any significant intergroup difference (P = 0.856). C, N0–2 disease restaged by the genetic method (g-N). A worse prognosis was observed in patients with g-N1 and g-N2 than those with g-N0 (P = 0.042 and P < 0.001, respectively). D, N0–2 disease restaged by IHC (IHC-N). No difference in survival is apparent between patients with IHC-N0 and IHC-N1 or IHC-N2 (P = 0.289 or P = 0.239, respectively).

	DISCUSSION

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In the present study, a conventional MASA method was initially applied to analyze mutations in DNAs extracted from formalin-fixed, paraffin-embedded lymph nodes, but the results were sometimes equivocal. Because the main reason was probably that DNAs extracted from these samples were fragmented and not uniform, we first amplified those DNA fragments that include the mutated region to unify the quality of samples, and then MASA analysis was performed using amplified DNA, in which targeted sequences could be reliably detected. Furthermore, we used several positive and negative controls for confirmation. As positive controls, primary tumor DNAs and diluted DNAs were used. The dilution fold was determined on the basis of initial experiments for several cases in which diluted tumor DNAs at 10-, 100-, 1,000-, and 10,000-fold were amplified with the modified MASA method. The results showed that in all cases a MASA-specific product was constantly observed in the range from 10- to 1,000-fold but not always identified at 10,000-fold. By this method, we demonstrated that 30% of patients with histologically negative lymph nodes indeed had micrometastases, and that these were linked to a poor outcome.

Thus far, for detection of micrometastasis in lymph nodes the immunohistochemical method with anti-CK regent has been used frequently and reported to be effective for prediction of a poor prognosis (3, 4, 5 , 7, 8, 9) . However, our results showed the modified MASA method to be superior to IHC in terms of NSCLC relapse. In regional lymph nodes, macrophages that phagocytize CKs derived from degraded normal lung epithelial cells are presumably present, and these could be misinterpreted and give rise to false-positives. In fact, we could not recognize carcinoma cells histologically in CK-positive lymph nodes. Furthermore, there have been reports that CK expression may be detected in both normal and neoplastic nonepithelial cells (11 , 25 , 26) .

As a genetic approach to detect lymph node micrometastasis or systematic tumor dissemination, reverse transcription-PCR analysis for several markers, including CK19, has been described (10, 11, 12 , 27) . Although having a high sensitivity, this is again plagued with the problem of a high percentage of false-positive results, depending on the number of PCR cycles, because of nonspecificity for cancer cells (11 , 12 , 27) . The modified MASA method applied here is not only highly sensitive but also highly specific for carcinoma cells with particular mutations, so that we could show clearly the correlation between the micrometastasis and postoperative survival in lung carcinoma patients. As to mutated DNAs, they may be present in macrophages if they phagocytize carcinoma cells or carcinoma DNAs (28) , but this would reflect the likelihood of metastasis.

Carcinoma relapse was found in contralateral lungs or other remote organs in our patients with micrometastasis. Two pathways of metastasis are probable; one is that carcinoma cells pass through the regional lymph nodes and reach blood vessels, and the other is that vascular invasion of carcinoma cells occurs at the primary site. In our study, all six patients with relapse had lymph node metastasis histologically and/or genetically, and in four patients of them metastasis extended to mediastinal region. However, none of the patients without such lymph node involvement showed recurrence. Therefore, the main root for systemic metastasis may be via lymphatic pathways, which strengthens the importance of micrometastasis in lymph nodes. However, in two relapsed patients, lymph node metastasis was limited to the hilar region, so that the second route cannot be ruled out.

If lymph node or distant metastasis is within a "micro level," undetectable by routine histological examination, intensive adjuvant therapy may prevent carcinoma recurrence. Therefore, a finding of micrometastasis in lymph nodes is important not only for prediction of prognosis but also for selection of patients for whom postoperative extensive adjuvant chemotherapy may be performed to advantage. Further analysis of a large number of patients with this method, possibly in a prospective fashion, is warranted to confirm our results.

	ACKNOWLEDGMENTS

 
We thank Drs. H. Sugano (Cancer Institute), Y. Nakamura (Institute of Medical Science, University of Tokyo), S. Sugai (Cancer Institute), and T. Kozu (Saitama Cancer Center Research Institute) for helpful advice and discussions. The technical assistance of T. Yoshikawa and Y. Yamaoka is gratefully acknowledged.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan; by a research grant from the Ministry of Health and Welfare of Japan; by the Vehicle Racing Commemorative Foundation; and by the Smoking Research Foundation.

² To whom requests for reprints should be addressed, at Saitama Cancer Center Research Institute, 818 Komuro, Ina, Kitaadachi-gun, Saitama 362-0806, Japan. Phone: 81-48-722-1111; Fax: 81-48-722-1739; E-mail: etuchiya{at}cancer-c.pref.saitama.jp

³ The abbreviations used are: NSCLC, non-small cell lung carcinoma; MASA, mutant allele-specific amplification; TNM, Tumor-Node-Metastasis; IHC, immunohistochemistry; CK, cytokeratin.

Received 3/27/00. Accepted 9/18/00.

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