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© 2002 American Society for Clinical Oncology Prognostic Impact of Micrometastatic Tumor Cells in the Lymph Nodes and Bone Marrow of Patients With Completely Resected Stage I Non–Small-Cell Lung CancerByFrom the Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. Address reprint requests to Toshihiro Osaki, MD, Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan; email: t-osaki{at}med.uoeh-u.ac.jp
PURPOSE: This study was designed to substantiate the prognostic impact of occult micrometastatic tumor cells in the lymph nodes (LNs) and bone marrow (BM) in stage I non–small-cell lung cancer (NSCLC) patients using cytokeratin (CK) as a micrometastatic marker and the relationship between the micrometastases in the LNs and BM. PATIENTS AND METHODS: A total of 2,432 hilar and mediastinal LNs were removed during surgery from 115 patients with completely resected stage I NSCLC. The LNs were analyzed for micrometastasis using immunohistochemistry with the biclonal anti-CK antibody AE1/AE3. BM aspirates from 115 patients were immunocytochemically stained with the monoclonal anti-CK antibody CK2. RESULTS: CK-positive (CK+) cells were detected in 42 (1.7%) of 2,432 LNs, in 32 (27.8%) of 115 patients, and in 32 (27.8%) of 115 BM aspirates. There was no relationship between the frequencies of CK+ cells in the LNs and in the BM. The patients with CK+ cells in the LNs had a poor prognosis by both univariate (P = .008) and multivariate analyses (P = .01), whereas the presence of CK+ cells in the BM did not allow prediction of survival (P = .32). The prognostic impact of LNs micrometastasis was independent even after adjusting for the status of BM micrometastasis. CONCLUSION: The detection of lymph nodal micrometastatic tumor cells provides an accurate assessment of tumor staging and has powerful prognostic implications for completely resected stage I NSCLC patients.
LUNG CANCER IS the leading cause of cancer death in North America, and it became the most common cause of cancer death among the Japanese in 1998. Lung cancer is also an aggressive carcinoma with a poor outcome. The tumor-node-metastasis staging system of lung cancer1 is widely used as a guide for predicting the prognosis. The presence of lymph nodal metastases along with the tumor and metastasis status represents the most accurate factor currently available for the prediction of a prognosis in patients who undergo complete surgical resection. However, approximately 30% of patients with pathologic stage I non–small-cell lung cancer (NSCLC) have a recurrence of the tumor and die despite complete surgical resection.2,3 This suggests that occult micrometastatic tumor cells, which are not detected by current clinical staging examinations and conventional histopathologic methods such as hematoxylin-eosin (HE) staining, have already spread to the regional lymph nodes (LNs; lymphatic locoregional metastasis) or the distant mesenchymal organs (hematogenous systemic metastasis) at the time of surgical intervention. Therefore, it may be necessary to assess the LNs and bone marrow (BM) micrometastases to accurately predict a patient’s prognosis. Many attempts have been made to detect micrometastatic tumor cells in LNs,4-9 BM,10-13 and peripheral blood14,15 by either immunohistochemical (IHC) staining or genetic methods such as a reverse transcriptase polymerase chain reaction with cytokeratin (CK) as a micrometastasis marker, and micrometastases of the LNs and BM are well appreciated in prediction of prognosis. However, there are few studies describing the simultaneous detection of micrometastases in both the BM and LNs of patients with NSCLC and their prognostic implications. This study was designed to detect occult micrometastatic tumor cells in the pathologic negative (pN0) LNs and BM of completely resected stage I NSCLC patients by CK IHC staining and to evaluate the relationship between the micrometastases in the LNs and BM and their prognostic potential.
Patients, LNs, and BM Materials, and Follow-Up Among a total of 405 consecutive patients with NSCLC who underwent radical surgery at the Department of Surgery II, University of Occupational and Environmental Health, Kitakyushu, Japan, from September 1993 to April 2000, 194 patients had stage I disease identified by routine histopathologic examination. Of these 194 patients, we were able to obtain adequate BM samples and paraffin-embedded pN0 LNs from 115 patients. The pN0 was used throughout this study to denote LNs that were negative for metastasis by routine light microscopic assessment. All patients had undergone standard lobectomy or pneumonectomy with the dissection of the hilar and mediastinal LNs (systematic nodal dissection). The tumor stage was classified according to the "Revisions in the International System for Staging Lung Cancer" (1997).1 There were 74 men and 41 women, with a mean age of 67.7 years (range, 49 to 85 years). The histologic types included 78 adenocarcinomas, 29 squamous cell carcinomas, six large-cell carcinomas, and two other types. Sixty-two patients had stage IA (T1N0M0) and 53 had stage IB (T2N0M0) carcinomas. All patients were informed about the study and gave written consent before BM aspiration and surgery. After the primary operation, the patients were examined every month within the first year and thereafter at 3-month intervals as a rule. The evaluations included physical examination, chest roentgenography, analysis of blood chemistry, and carcinoembryonic antigen assay. Chest, abdominal, and brain computed tomography scans and a bone scintiscan were performed every 6 months within the third year and each year thereafter. If any symptoms or signs of recurrence appeared in these examinations, further frequent evaluations to detect the recurrent site were performed. Survival data were updated in April 2001. The median observation period was 35.8 months (range, 0.1 to 90.6 months), with no patients lost to the follow-up. Recurrences were classified as locoregional (inside the ipsilateral thorax) or distant (outside the ipsilateral thorax).
CK Staining for LNs
CK Staining for BM The LNs and BM specimens were examined and checked by two of the authors (T.O. and C.D.G.) with no knowledge of the patients’ molecular biologic or clinical data, including the patients’ outcomes.
Statistical Analysis
Detection of CK+ Cells in LNs CK+ cells, either alone or in small clusters, were most often seen in the subcapsular or medullary sinuses and less frequently in the afferent lymphatics of the LN specimens (Fig 1A). The CK+ cells were found in 42 (1.7%) of 2,432 LNs; namely, 32 (27.8%) of 115 patients had CK+ cells in the pN0 LNs. The frequency of CK+ cells in the LNs showed no differences in sex, age, or histologic type; however, the frequency of CK+ cells in the T2 status patients was significantly higher than that in the T1 status patients (P = .03) (Table 1).
Detection of CK+ Cells in BM CK+ cells were easy to identify among the MNCs because the cytoplasms of most of the CK+ cells were uniformly stained bright red (Fig 1B). Clusters of CK+ cells were not detected in this study. The mAb CK2 detected CK+ cells in 32 (27.8%) of 115 BM samples. The frequency of CK+ cells in the BM showed no differences in sex, age, histologic type, or tumor status (Table 1).
Relationship Between Micrometastasis in LNs and the BM
Influence of Micrometastasis on Recurrence and Survival At the last follow-up, 83 patients were alive and free of cancer, 10 patients had died of other causes without evidence of cancer, eight patients were alive with recurrent cancer, and 14 patients had died of cancer. Twenty-two patients (18.8%) had recurrences during this period: 13 distant, two locoregional, and seven patients had both sites of recurrences. The mean duration from the surgery to the recurrence in these 22 patients was 16.9 months (range, 3.5 to 47.6 months). We discriminated these recurrent diseases from second primary malignancies based on the clinical findings, such as the duration from the primary surgery and the number of the second lesion. As shown in Table 3, 11 (34.4%) of the 32 patients with CK+ cells in the LNs had recurrences, whereas 11 (13.3%) of the 83 patients who were free of CK+ cells had recurrences (P = .01). However, the presence of CK+ cells in the BM was not associated with recurrence in patients with stage I NSCLC (P = .95). We also noted that the presence of micrometastasis in the pN0 LNs was predictive of the pattern of recurrence. All 11 patients with CK+ cells in the LNs and recurrent diseases had hematogenous distant recurrences.
The overall 5-year survival of the 115 patients in this study was 70.9%. The Kaplan-Meier survival curves demonstrated that the patients with CK+ cells in the pN0 LNs had significantly shorter survival periods than those without CK+ cells (P = .008) (Fig 2A). Among the patients with stage IA (n = 62) disease, the survival did not significantly differ between patients with and without CK+ cells (P = .40). Among the patients with stage IB (n = 53) disease, the survival of patients with CK+ cells was significantly shorter than the survival of those without CK+ cells (P = .02). The presence of CK+ cells in the BM did not allow the prediction of survival among the patients with stage I (Fig 2B), IA, or IB (P = .32, .45, and .65, respectively) disease. A Cox multivariate survival analysis demonstrated that the LNs micrometastatic status was a significant independent predictor of a poorer prognosis even after adjusting for the tumor status and the BM micrometastatic status in patients with completely resected stage I NSCLC (P = .01; relative risk of death = 2.96) (Table 4).
Restaging of Nodal Status Based on CK Staining Among the patients with pN0 disease identified by a conventional HE histopathologic study, 19 patients (16.5%) were restaged as N1 status (CK-N1) and 13 patients (11.3%) were restaged as N2 status (CK-N2) based on the CK IHC staining. Figure 3A shows the survival curves according to the nodal factor restaged by the CK staining. The survival of patients with CK-N2 was significantly shorter than the survival of those with CK-N0 (P < .001). There were no significant differences between the survivals of the other groups (CK-N0 v CK-N1, P = .29; CK-N1 v CK-N2, P = .08). For comparison with a pathologic nodal (pN) factor, we used a control group of patients with completely resected pathologic T1/2N1M0 (n = 29) and T1/2N2M0 (n = 54) NSCLC assessed by conventional HE staining during the same period. Figure 3B shows the survival curves according to the restaged nodal (CK-N) status and the conventional pN status. There were no differences in the survival between the patients with CK-N1 and pN1 (P = .46) and those with CK-N2 and pN2 (P = .92).
Micrometastases to BM and regional LNs can now be identified by IHC or genetic methods using several markers for epithelial cells, such as CK and Ber-EP4, and oncogenes/oncoproteins, such as K-ras and p53. This information is useful for the assessment of the postoperative survival in patients with NSCLC.4-12,20 However, there have been few studies of the simultaneous detection of micrometastases in both the BM and LNs of patients with early-stage NSCLC and their prognostic implications. In this study, CK+ cells in the LNs and the BM of patients with completely resected stage I NSCLC were detected with the same frequency (27.8%), and there was no relationship between micrometastases in the LNs and in the BM. This finding suggests that different regulations or mechanisms may exist in micrometastasis to the LNs and to the BM and that micrometastases to the LNs and the BM may occur independently. The analysis of the tumor relapse pattern in patients with LNs micrometastasis revealed that it was hematogenous. All 11 patients with LNs micrometastasis and recurrent diseases had hematogenous distant recurrences. LN micrometastasis, as well as overt LN metastasis, does not only reflect lymphogenous spread but it may also signal the early phase of hematogenous systemic tumor cell dissemination. Furthermore, the presence of even an isolated CK+ cell in the LNs had a significant impact on survival in patients with early-stage NSCLC, and the survival impact was independent even after adjusting for the BM micrometastatic status and other covariates. In addition, survival differences were observed with restaged nodal status by CK staining, and no survival differences were observed between restaged nodal status and pathologic nodal status (CK-N1 v pN1 and CK-N2 v pN2), suggesting that the detection of LN micrometastasis provides an accurate assessment of tumor staging as well as the pathologic overt LN metastases. On the other hand, the presence of an isolated CK+ cell in the BM was not associated with survival. However, there is a possibility that the presence of CK+ cell in the BM, which may be one of the strong predictable factors for poor survival, had no survival impact because the number of patients included in this study was relatively small for providing the BM micrometastatic status with statistical significance. Concerning this important issue, definitive conclusions await further study maturation. CK, which forms the intermediate filaments of the cytoskeleton within both normal and malignant epithelial cells,21 is widely used as a marker of epithelial cells. The wide distribution of CKs in all epithelial tumors means that antibodies to CK can detect occult metastases from different kinds of cancers.22-25 However, CKs are not only specific to tumor cells but also are present in normal epithelial cells, suggesting that a false-positive reaction may also be seen within nontumor cells in the LNs.8,26 Hashimoto et al8 reported that the detection of LN micrometastasis using a mutant allele-specific amplification method with a carcinoma-specific marker, p53 or K-ras, can reduce the false-positive rates as compared with the methods using anti-CK reagents. We previously reported the use of p53 IHC staining in the detection of occult tumor cells in the LNs of NSCLC patients, and p53-positive cells were identified in 45.2% of this population.20 The p53 tumor suppressor gene is altered in a high proportion of human neoplasms27; however, it is mutated in approximately half of the various types of malignant diseases, including NSCLC.28,29 Thus, p53, as a micrometastatic marker, is available for about half of patients with malignancies. This means that using oncogenes and oncoproteins, such as p53 and K-ras, as micrometastatic markers is disadvantageous for the detection of occult tumor cells. Recently, we reported the simultaneous detection of LNs micrometastases by combining the CK and p53 protein IHC stainings.30 Seventeen (35%) of the 49 patients had micrometastatic tumor cells using the CK IHC staining alone, whereas we were able to identify micrometastasis in the pN0 LNs in 22 patients (45%) by adding the p53 IHC staining to that of the CK. Although performing both IHC stainings increases the cost and the time of the analyses, it may be necessary to increase the diagnostic efficiency for micrometastasis. In conclusion, the detection of LN occult micrometastatic tumor cells provides an accurate assessment of tumor staging and has powerful prognostic implications for completely resected stage I NSCLC patients, especially in stage IB disease. At present, postoperative adjuvant chemotherapy is not a routine standard therapy for completely resected NSCLC patients because of its unreliable results for the improvement of the patients’ prognosis. Adjuvant chemotherapy may be useful for patients with occult micrometastasis in the LNs because patients with a minimal amount of residual tumor may respond better to chemotherapy. On the other hand, numerous trials, including two randomized trials31,32 of induction chemotherapy for stage IIIA NSCLC, have shown that it was feasible and provided higher response rates and a survival advantage. When we consider the application of induction chemotherapy, the detection of micrometastasis in the mediastinal LNs, preoperatively sampled by mediastinoscopy, may also be useful to identify patients with occult N2 disease.
Supported in part by a grant-in-aid no. 09771021 for scientific research from the Ministry of Education, Science and Culture, Japan (to T.O.). We are grateful to Yoshihisa Fujino, MD (Department of Clinical Epidemiology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan), for his statistical advice during the preparation of the article.
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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. A univariate survival analysis for each prognostic variable on overall survival was estimated according to the Kaplan-Meier method.
