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Ann Thorac Surg 2003;76:194-201
© 2003 The Society of Thoracic Surgeons

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Original article: general thoracic

Prognostic value of cytokeratin-positive cells in the bone marrow and lymph nodes of patients with resected nonsmall cell lung cancer: a multicenter prospective study

^a Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
^b Department of Surgery I, Kanazawa University School of Medicine, Kanazawa, Japan
^c Department of Surgery, Tokyo Medical University, Tokyo, Japan
^d Department of Clinical Epidemiology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Kitakyushu, Japan

Accepted for publication January 22, 2003.

^* Address reprint requests to Dr Yasumoto, Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
e-mail: k-yasumo{at}med.uoeh-u.ac.jp

	Abstract

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

 
BACKGROUND: This study was designed to prospectively substantiate the prognostic value of cytokeratin-positive (CK⁺) cells in the bone marrow (BM) and regional lymph nodes (LNs) in resected nonsmall cell lung cancer (NSCLC) patients from a large population within a multicenter study.

METHODS: The study population consisted of 351 patients with stages I to IIIA NSCLC from 15 Japanese institutes. BM aspirates were stained immunocytochemically with the anti-cytokeratin antibody, CK2. The hilar and mediastinal LNs of 216 patients with stage I NSCLC were stained immunohistochemically with the anti-CK antibody, AE1/AE3.

RESULTS: CK⁺ cells were detected in 112 patients (31.9%) of the 351 BM aspirate patients. The frequency of CK⁺ cells showed no differences among pathologic stages. The patients with CK⁺ cells in the BM had a tendency to have shorter survival periods than those without CK⁺ cells (p = 0.076). Although the presence of CK⁺ cells in the BM of patients with stage I did not allow the prediction of overall survival, it reduced the overall survival significantly in patients with stages II to IIIA. CK⁺ cells in the LNs were detected in 34 of 216 patients (15.7%) with stage I. The patients with CK⁺ cells in the LNs had a poor prognosis by both univariate (p = 0.004) and multivariate analyses (p = 0.018).

CONCLUSIONS: The presence of CK⁺ cells in the BM was related to a poor prognosis for patients with stages II to IIIA NSCLC; however, it did not predict the prognosis of patients with stage I. For stage I NSCLC, the detection of CK⁺ cells in the LNs implied a poor prognosis for the patients.

	Introduction

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Lung cancer is the leading cause of cancerous death in North America, and it became the most common cause of cancerous death among Japanese in 1998. Lung cancer is also an aggressive carcinoma with a poor outcome. The TNM staging system of lung cancer [1] is widely used as a guide for predicting prognosis. However, about 30% of patients with pathologic stage I nonsmall cell lung cancer (NSCLC) have a recurrence, despite complete surgical resection [2]. This suggests that occult micrometastatic tumor cells, which are not detected by current clinical staging examinations and conventional histopathologic methods such as hematoxylin-eosin staining, have already spread to the distant mesenchymal organs (hematogenous systemic metastasis) or the regional lymph nodes (lymphatic loco-regional metastasis) at the time of surgical intervention. Therefore, for the accurate prediction of a patient’s prognosis and the design of postoperative therapeutic approaches it is necessary to assess the occult micrometastasis.

Many studies have detected micrometastatic tumor cells in bone marrow (BM) [3–6], lymph nodes (LNs) [7–11], and peripheral blood [12, 13] by either immunohistochemical (IHC) staining or genetic methods, such as a reverse transcriptase polymerase chain reaction with cytokeratin (CK) as a micrometastasis marker, and the prognostic significance of micrometastasis detection has been reported. In our previous study [3] we suggested the positive impact of cytokeratin-positive (CK⁺) cells in the BM on survival of patients with NSCLC. However, our previous study was based on a small population (39 NSCLC patients) with a relatively short follow-up period (median, 140 days). The present study was prospectively designed to substantiate the prognostic value of CK⁺ cells, occult micrometastatic tumor cells, in the BM in resected stages I to IIIA NSCLC patients from a large population within a multicenter study. In addition, the prognostic significance of the CK⁺ cells in the hilar and mediastinal LNs of patients with stage I disease was investigated.

	Patients and methods

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Patients, BM materials, and follow-up
This prospective study was performed by the Cooperative Project No. 24 Group of The Japanese Foundation for Multidisciplinary Treatment of Cancer. The inclusion criteria for this study included histologically proven stages I to IIIA NSCLC, aged less than 80 years, and performance status less than 2. Exclusion criteria included a history of malignancy and prior chemotherapy or radiotherapy. Between February 1997 and September 1998, 365 patients with stages I to IIIA NSCLC, who underwent radical surgery at one of the 15 participating Japanese institutes, were enrolled in this study. Fourteen patients were excluded because of protocol violations: 8 were stage IIIB identified by postoperative histopathologic examination; 3 had a history of malignancy; 1 had a nonmalignant tumor identified by postoperative histopathologic examination; and 2 had a new double primary cancer within the first year after surgery. As a result, 351 patients with stages I to IIIA NSCLC were finally enrolled in this study. The median number of enrolled patients of each institute was 22 (range, 2 to 55 patients). The protocol was approved by an independent ethics committee, and all patients were informed about the study and gave written consent before BM aspiration and surgery. No postoperative adjuvant therapies were performed for patients with stage I NSCLC. There were no restrictions on postoperative adjuvant therapies for patients with stages II to IIIA NSCLC.

The tumor stage was classified according to the Revisions in the International System for Staging Lung Cancer [1]. After the primary operation the patients were examined at 3-month intervals for 3 years, and usually thereafter at 6-month intervals. The evaluations included physical examination, chest roentgenograms, analysis of blood chemistry, and carcinoembryonic antigen and squamous cell carcinoma-related antigen assays. Chest, abdominal, and brain computed tomographic scans and a bone scintiscan were performed every 6 months for 3 years, 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 December 2002. The median observation period was 4.02 years (range, 0.08 to 5.34 years). We defined a loco-regional recurrence as a clinical or radiologic manifested disease in the mediastinum, the supraclavicular node, or the bronchial stump, or a malignant pleural or pericardial effusion, and a distant recurrence as disease in the ipsilateral lung or outside the ipsilateral thorax.

Ck staining of BM
Heparinized BM aspirate (5 mL) was collected from one side of the upper iliac crest through a needle while the patient was under general anesthesia in the operating room before surgery. The fraction of mononuclear cells (MNCs) from each sample was obtained by Ficoll-Hypaque (Dainippon Pharmaceutical Co, Ltd, Osaka, Japan) density-gradient centrifugation at 1,500 rpm for 30 minutes. The mean yield of MNCs was 6.5 x 10⁶ from each sample. An aliquot of 1 x 10⁶ MNCs was cytocentrifuged at 1,300 rpm for 5 minutes onto 5 glass slides (2 x 10⁵ cells per slide). After overnight air drying and 4% paraformaldehyde-acetone fixation, the MNCs (10⁶ cells) were stained immunocytochemically and analyzed to detect the CK⁺ cells. Using the method of Pantel and colleagues [4] with slight modifications, the mAb CK2 (IgG [Boehringer, Mannheim, Germany]), which recognizes the CK component no. 18 used (2.5 µg/mL) as the primary antibody, and the antibody reaction was developed using the alkaline phosphatase antiphosphatase technique (DAKO, Tokyo, Japan) [14]. Endogenous phosphatase was inhibited by a preincubation with levamisole. The presence of CK⁺ cells, which stain bright red in the cytoplasm, were accepted as evidence of micrometastatic tumor cells, even if only a single CK⁺ cell was detected. The suitability of the mAb CK2 for NSCLC cell screening of BM aspirates has been evaluated in detail by Pantel and colleagues [4]. When we examined and checked the BM specimens, we referred to samples of typical CK positive or negative cells that we had prepared. Patients with discrepant evaluations were finally evaluated by two authors (KY and TO).

Detection of CK⁺ cells in the LNs of stage i patients
Of the 228 patients with stage I, we were able to obtain adequate paraffin-embedded pathologic negative (pN0) LNs from 216 patients at 13 of 15 Japanese institutes. We searched for CK⁺ cells in the LNs using the following CK IHC staining methods. Five 4-µm slices, representing every other slice from 10 slices of each paraffin-embedded LN section were attached to glass slides. The slides were stained with the primary antibody against the CK by using a labeled streptavidin-biotin method (DAKO LSAB kit [Dako Corp, Carpinteria, CA]). The primary antibody was the mouse biclonal antibody (AE1/AE3 [Progen Biotechnik Gmbh, Heidelberg, Germany]) to the CKs, which recognizes most of the type I (acidic type) and the type II (basic type) CKs. The staining procedures were as follows: (1) deparaffinization with xylene and ethanol, (2) incubation with the primary antibody (dilution: AE1/AE3, 1:200), (3) incubation with the secondary antibody (either biotinylated goat antimouse IgG or goat antirabbit IgG), (4) developing with peroxidase-labeled streptavidin and diaminobenzidine-H₂O₂, and (5) counterstaining with hematoxylin. The presence of CK⁺ cells within the whole body section of the LNs was accepted as evidence of micrometastatic tumor cells, even if only a single CK⁺ cell was detected. The CK⁺ cells were further confirmed as being cytologically atypical cells (enlarged nuclear size and apparent increased nuclear to cytoplasmic ratio) before finally being designated as micrometastasis.

Statistical analysis
The associations between the clinicopathologic characteristics and the status of CK⁺ cells were analyzed by the use of a contingency table. Statistical significance was evaluated using the ² test. A univariate survival analysis for each prognostic survival variable was estimated according to the Kaplan-Meier method. The terminal event of overall survival analysis was death attributable to cancer or noncancer causes. The statistical significance of the differences in survival distribution among the prognostic groups was evaluated by the log rank test. The Cox proportional hazards model was applied to the multivariate survival analysis. The statistical difference was considered to be significant if the p value was below 0.05. Data were analyzed with the use of the Abacus Concepts, Survival Tools for StatView program (Abacus Concepts, Inc, Berkeley, CA).

	Results

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Patient characteristics
The characteristics of the 351 assessable patients are summarized in Table 1. There were 227 men and 124 women, with a mean age of 63.1 years (range, 25 to 79 years). The histologic types included 227 adenocarcinomas, 94 squamous cell carcinomas, 15 large cell carcinomas, 12 adenosquamous carcinomas, and three other types (two carcinosarcomas, and one unclassified carcinoma). Of the patients, 135 had stage IA carcinomas, 93 had stage IB, 13 had stage IIA, 37 had stage IIB (22 patients, T2N1; 15, T3N0), and 73 had stage IIIA (51 patients, T1 to 2N2; 11, T3N1; and 11, T3N2). Standard lobectomy or pneumonectomy with the dissection of the hilar and mediastinal LNs (systematic nodal dissection) had been performed on 345 patients. Five patients had undergone lobectomy without the systematic nodal dissection. One patient had undergone a wedge resection. Complete resections were performed in 349 patients, whereas incomplete resections were performed in 2 patients.

Influence of CK⁺ cells in the BM on recurrence and survival
As of the last follow-up in December 2002, 257 patients were alive, 15 had died of other causes without evidence of cancer, and 79 had died of cancer. A total of 116 patients (33.0%) had recurrences during this period (75 patients had a distant recurrence, 17 had a loco-regional recurrence, 23 had both types of recurrences, and 1 patient’s recurrence site was unknown. The overall incidence of recurrence and the recurrence site were not influenced by the status of CK⁺ cells in the BM. The patients with CK⁺ cells in the BM had a tendency to have shorter survival periods than those without CK⁺ cells (p = 0.076) (Fig 1A). No significant difference was seen in the disease-free survival analysis (p = 0.47) (Fig 1B). The survivals were not influenced by the number of CK⁺ cells in the BM sample. Among the patients with stage I, the overall and disease-free survivals did not significantly differ between patients with and without CK⁺ cells (p = 0.64 and 0.43, respectively) (Fig 2A, 2B). Among the patients with stages II to IIIA, the overall survival of patients with CK⁺ cells was significantly shorter than the survival of those without CK⁺ cells (p = 0.047) (Fig 3A), whereas a border-line significance was seen in the disease-free survival (p = 0.087) (Fig 3B). A Cox’s multivariate survival analysis among the patients with stages II to IIIA showed that the status of CK⁺ cells in the BM was a significant independent predictor of a poorer prognosis after adjusting for the N status (p = 0.048; relative risk of death = 1.70) (Table 2).

View larger version (12K): [in this window] [in a new window]   Fig 1. (A) Survival of stage I to IIIA nonsmall cell lung cancer patients with or without cytokeratin-positive (CK+) cells in the bone marrow (p = 0.076). (B) Disease-free survival of stage I to IIIA nonsmall cell lung cancer patients with or without CK+ cells in the bone marrow (p = 0.47).

View larger version (12K): [in this window] [in a new window]   Fig 2. (A) Survival of stage I nonsmall cell lung cancer patients with or without cytokeratin-positive (CK+) cells in the bone marrow (p = 0.64). (B) Disease-free survival of stage I nonsmall cell lung cancer patients with or without CK+ cells in the bone marrow (p = 0.43).

View larger version (12K): [in this window] [in a new window]   Fig 3. (A) Survival of stage II to IIIA nonsmall cell lung cancer patients with or without cytokeratin-positive (CK+) cells in the bone marrow (p = 0.047). (B) Disease-free survival of stage II to IIIA nonsmall cell lung cancer patients with or without CK+ cells in the bone marrow (p = 0.087).

View larger version (12K): [in this window] [in a new window]   Fig 4. (A) Survival of stage I nonsmall cell lung cancer patients with or without cytokeratin-positive (CK+) cells in the lymph nodes (p = 0.004). (B) Disease-free survival of stage I nonsmall cell lung cancer patients with or without CK+ cells in the lymph nodes (p = 0.028).

	Comment

Top Abstract Introduction Patients and methods Results Comment Acknowledgments References

Our hypothesis was that the presence of CK⁺ cells in the BM is significantly associated with poor prognosis in patients with resected NSCLC, especially in stage I patients. If so, then the detection of CK⁺ cells in the BM may allow us to identify the patients at high risk of recurrence in advance, and such patients at stage I may be suitable candidates for postoperative chemotherapy. In this study, the presence of CK⁺ cells in the BM reduced the overall survival significantly in patients with stages II to IIIA, whereas it did not allow the prediction of overall survival in patients with stage I. This finding means that different implications of CK⁺ cells of the BM may exist between patients with stage I (early stage) and patients with stages II to IIIA (advanced stages). In patients with stage I, the presence of an isolated CK⁺ cell in the BM does not always directly reflect the hematogenous systemic metastasis. Most CK⁺ cells in the BM may be in a nonproliferating or dormant state. Pantel and colleagues [16] found that a considerable number of tumor cells in the BM are nonproliferating cells and that bone marrow is a reservoir for these tumor cells. On the contrary, in patients who had stages II to IIIA with tumors that had already progressed locally and metastasized to the regional LNs, the presence of an isolated CK⁺ cell in the BM may provide additional information by reflecting the early phase of hematogenous systemic tumor cell dissemination. The second finding in this study was that no significant difference was seen in the frequency of CK⁺ cells in the BM between patients, in terms of gender, age, histologic type, lymph node involvement (pathologic N factor), tumor extension (pathologic T factor), or pathologic stage. In particular, CK⁺ cells were detected with nearly equal frequencies among stages I, II, and III.

Recently we reported the simultaneous detection of CK⁺ cells in both the BM and LNs of 115 patients with stage I NSCLC from one of our member institutions [15]. This previous study, consisting of patients from a single institution, indicated that stage I patients with CK⁺ cells in the LNs had a poor prognosis by both univariate and multivariate analyses, whereas the presence of CK⁺ cells in the BM did not allow prediction of survival. In the present multicenter study, we investigated LN micrometastasis in a much larger number of patients with stage I NSCLC (216 patients), and we tried to confirm our previous implications. There was no significant relationship between the status of CK⁺ cells in the LNs and in the BM. The detection of CK⁺ cells in the LNs had a prognostic implication for stage I NSCLC patients. The prognostic impact of the status of CK⁺ cells in the LNs in stage I patients was independent, even after adjusting for the status of CK⁺ cells in the BM. Lymph nodal micrometastasis, as well as overt lymph nodal metastasis, reflects not only lymphogenous spread but also the early phase of hematogenous systemic tumor cell dissemination, because the main pattern of recurrence was distant metastases.

Cytokeratin, which forms the intermediate filaments of the cytoskeleton within both normal and malignant epithelial cells [17], 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 [18–20]. However, CKs are not specific to only tumor cells, but are also present in normal epithelial cells, suggesting that a false positive reaction may also be seen within nontumor cells in the LNs [10, 21]. Hashimoto and colleagues [10] reported that the detection of lymph nodal micrometastasis, using a mutant allele-specific amplification method with a carcinoma-specific marker such as 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 with p53-overexpressing tumors, and p53-positive cells were identified in 45.2% of this population [22]. The p53 tumor suppressor gene is mutated in about half of the various types of malignant diseases, including NSCLC [23, 24]. Thus, p53 as a micrometastatic marker is available for only about half of the patients with malignancies. Recently we reported the simultaneous detection of LNs micrometastases by combining the CK and p53 protein IHC stainings [25]. Seventeen of the 49 patients (35%) 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 using the p53 and the CK IHC staining. A combination assay using several micrometa-static markers may increase the diagnostic efficiency for micrometastasis.

Presently, postoperative adjuvant chemotherapy is not a routine standard therapy for completely resected NSCLC patients because of its unreliable results for the improvement of prognosis. Adjuvant chemotherapy may be useful only for patients with occult micrometastasis, because patients with a minimal amount of residual tumor may respond better to chemotherapy. When we consider the application of adjuvant chemotherapy for patients with advanced stage NSCLC (stage II or III), the detection of CK⁺ cells in the BM may be useful to select the patients who will get a survival benefit from adjuvant chemotherapy. On the other hand, for patients with stage I NSCLC who have pathologic negative LNs, the detection of lymph nodal micrometastasis may provide useful clues for the selection of candidates for postoperative adjuvant chemotherapy.

	Acknowledgments

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This study was performed by the Cooperative Project No. 24 Group of The Japanese Foundation for Multidisciplinary Treatment of Cancer (JFMC). Additional study investigators are listed in the Appendix as follows.

	Appendix

 
The following investigators and institutions also participated in this study: Kenji Sugio, Department of Surgery II, Kyushu University, Fukuoka, Japan; Tetsuya Mitsudomi, Department of Chest Surgery, Aichi Cancer Center, Nagoya, Japan; Akira Nagashima, Department of Chest Surgery, Kitakyushu Municipal Medical Center, Kitakyushu, Japan; Tsuguo Furukawa, Department of Surgery, Saga Prefectural Hospital Koseikan, Saga, Japan; Hisashi Nakahashi, Center for Chest Disease, Matsuyama Red Cross Hospital, Matsuyama, Japan; Hideo Sato, Department of Thoracic Surgery, Ishikawa Prefectural Central Hospital, Kanazawa, Japan; Osamu Taira, Department of Thoracic Surgery, Tokyo Medical University Hachioji Medical Center, Hachioji, Japan; Kazuo Yoneyama, Department of Chest Surgery, Tokyo Medical University Kasumigaura Hospital, Ibaraki, Japan; Hisanobu Sakata, Department of Surgery, Nippon Steel Yawata Memorial Hospital, Kitakyushu, Japan; Nobuhiro Koyanagi, Department of Surgery, Iizuka Hospital, Iizuka, Japan; Hideo Manabe, Department of Surgery, Kyushu-kousei-nenkin Hospital, Kitakyushu, Japan; and Yukito Ichinose, Department of Thoracic Oncology, National Kyushu Cancer Center, Fukuoka, Japan.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Yoh Watanabe Harubumi Kato Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yasumoto, K. Articles by Yoshimura, T. Search for Related Content PubMed PubMed Citation Articles by Yasumoto, K. Articles by Yoshimura, T. Related Collections Lung - cancer