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Eur J Cardiothorac Surg 2001;20:481-488
© 2001 Elsevier Science NL

Bone marrow micrometastasis might not be a short-term predictor of survival in early stages non-small cell lung carcinoma

^a Department of Cardio-vascular and Thoracic Surgery, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Avenue Hippocrate 10, B-1200 Brussels, Belgium
^b Department of Pathology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium
^c Biostatistics and Epidemiology Unit, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium

Received 21 November 2000; received in revised form 25 May 2001; accepted 26 May 2001.

Corresponding author. Tel.: +32-2-7646107; fax: +32-2-7648960
e-mail: poncelet{at}chir.ucl.ac.be

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objective: To determine the presence of occult micrometastasis (OM) in a selected population of surgically resectable patients presenting with non-small cell lung carcinoma (NSCLC) and to evaluate its prognostic value on relapses and survival. Methods: From February 1996 to December 1999, 99 patients undergoing surgical treatment for NSCLC were prospectively investigated for the presence of occult bone marrow micrometastasis. Tumor cells were detected with monoclonal primary antibodies directed against low molecular weight cytokeratins. Results: Median follow-up time was 14.3 months (range 0.2–45.6 months). Overall prevalence of OM was 22.2% (22 out of 99). The presence of OM was not correlated to pathology, T status, or N status. In survival analysis, the only independent predictors of overall survival were N0 status and Stage I (P=0.016 and 0.004, respectively), while T1 was a predictor of disease-free survival (P=0.044). Metastasis and loco-regional recurrence were observed at follow-up in 18.2 (four out of 22) and 9% (two out of 22) of patients OM(+) and in 14.3 (11 out of 77) and 7.8% (six out of 77) of patients OM(-), respectively (P=not significant). OM was a predictor neither of overall survival nor of disease-free survival (P=0.52 and 0.97, respectively). In Stage I patients, 1-year overall survival and 1-year disease-free survival were 89 and 98% for OM(-) patients and 88 and 90% for OM(+) patients, respectively (P=0.57 and P=0.75). Conclusions: OM was present in >20% of surgically treated NSCLC patients and did not correlate to pathological variables. In contrast to previous published data, in this study the presence of OM had no influence on overall or disease-free survival.

Key Words: Bone marrow • Micrometastasis • Lung carcinoma • Prognosis • Survival

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Over the last two decades, in both Stage I and Stage II disease, surgical resection has been accepted as the standard treatment of non-small cell lung carcinoma (NSCLC) patients [1,2]. In 1995, in a meta-analysis of NSCLC patients stratified according to the first revision of Mountain's classification [3], Nesbitt et al. [4] found that Stage I (T1-2N0M0) patients had a 5-year survival rate of 64.6% (3987 patients), whereas Stage II (T1-2N1M0) patients had a 5-year survival rate of only 41.2% (1384 patients). In 1997, Dobashi et al. [5] reported a 5-year survival rate of 58% in a series of 31 patients with pN0 status and R0 resection. Thus, despite the development of technologies allowing for a more accurate staging (computerized tomography (CT) scan, magnetic resonance imaging (MRI), nuclear scanning), and various preoperative surgical diagnostic procedures, no major breakthrough has been made in improving long-term survival. Several reasons can be put forward to account for this low survival rate. One could be the insufficient sensitivity of current histologic techniques for node pathological examination [5,6]; another, the incomplete mediastinal lymph node sampling during surgery [7]. Finally, one explanation could be the early dissemination of metastatic cells that would remain undiagnosed at the time of disease evaluation. In 1993, Pantel et al. [8] reported their results on a group of 82 patients evaluated for the presence of bone marrow micrometastasis by immunohistology using monoclonal antibodies (mAb) directed against cytokeratin polypeptide CK-18. More recently, the same authors presented the long-term follow-up results of a larger study population [9,10]. The presence of two or more micrometastatic cells in the bone marrow of patients with negative nodes (PN0) was a strong predictor of recurrence and had a statistically significant impact on disease-free [9] and overall survival [10]. In patients with pN1–N2, no impact either on recurrences or on survival was observed.

Our aim in the present study was two-fold. The first aim was to investigate the prevalence of bone marrow micrometastasis according to TNM and stage of disease in our institutional series of resected NSCLC patients. The second was to assess the prognostic value of bone marrow micrometastasis in early-stage NSCLC patients.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Study population and sample collection
From February 1996 to December 1999, we collected bone marrow samples from 99 successive lung carcinoma patients a priori suitable for pulmonary resection with a curative intent. Patients' tumors were staged according to the latest revisions in the international system for staging lung cancer [11]. There were 22 females and 77 males included in the study. Their clinico-pathological data are summarized in Table 1. Altogether, Stage I and Stage II patients accounted for nearly 80% of our study group, and 65% of our patients were pN0 status according to TNM classification. For each patient, a bone marrow sample of ±8 ml, was aspirated from the homolateral iliac crest bone with a 2-mm diameter cyto-aspiration Klima–Rosegge needle at the end of the surgical pulmonary resection. Samples were obtained from the iliac crest in all cases.

Each immunohistochemical series included a positive control corresponding to a cytocentrifuged scrapping of a mammary carcinoma. Five surgical specimens from the series were also tested to make sure that the primary tumors expressed these cytokeratins. They were all positive. Two bone marrow samples that were not part of the NSCLC series were considered as negative controls as they concerned one case of tuberculosis and one case of a localized fibrous tumor of the pleura.

Fifteen different bone marrow samples included an internal negative control during immunohistochemical staining. On this slide, the cocktail of first antibodies was omitted, whereas the second antibody and the revelation system were the same. They all remained negative.

2.3. Operative strategies and residual (R) status
The surgical procedures consisted of 62 lobectomies (62.6%) (right=32, left=30), six bi-lobectomies (6.1%), 23 pneumonectomies (23.2%) (right=10, left=13).

Extensive parenchymal and chest wall resections were performed in five patients (5.1%) (lobectomies in all cases), right upper sleeve lobectomies in two patients (2.1%), and wedge resection in one patient. This last patient had undergone a right pneumonectomy 4 years earlier.

It is important to notice that four initial lobectomies (4.1%) were perioperatively converted to pneumonectomies because of positive proximal margin findings on frozen sections. One patient with initial chest wall resection and lobectomy underwent completion pneumonectomy on postoperative day 8 for positive margins on final pathological examination even though frozen sections were negatives. Finally, one patient with initial right lower lobectomy underwent a completion right middle lobectomy and omentoplasty on postoperative day 8 for early postoperative bronchopleural fistula. There was no exploratory thoracotomy in this study group.

Complete homolateral mediastinal lymph node dissection was performed in all but one patient and node stations were labeled according to the American Thoracic Society guidelines [12]. There were 1639 lymph nodes available for pathological examination with a mean number of resected lymph nodes per patient of 16.5 (range 0–40). One patient with a clinical Stage Ia adenocarcinoma (T1N0M0) did not have mediastinal nodes sampling and no lymph node was found on the resected specimen.

Resection was classified R0 (macro- and microscopically complete) [13] in 97 patients (97%), R1 (microscopically incomplete) in two patients (2.1%). No patient was classified R2 (macroscopically incomplete). Two out of the 99 patients were classified as pTxNxM0. Indeed, these two patients (one undifferentiated NSCLC and one adenocarcinoma) had an initial clinical staging IIIa and benefited from induction chemotherapy. Their reassessment showed a clinical downstaging and they were proposed for surgical resection. On the surgical specimens, no tumor was observed and nodes were all negative. These two patients are not included in the survival analysis.

2.4. Selection of predictor variables
Predictor variables studied for their potential impact on prognosis were the pathology of tumor, the grade of differentiation, the T status, the N status, the disease's pathological stage, and the presence or absence of occult micrometastasis (OM).

2.5. Data collection and patients' follow-up
All the information necessary for the study was collected from the patient operative reports, the hospitalization charts, and our thoracic surgery databank. Follow-up was completed from September 1999 to March 2000 and was done through contacts with the referring pneumologist, the primary physician, or the patient's family when appropriate. Follow-up was 100% complete (99 out of 99 patients) until closure of the study as of March 1, 2000.

2.6. Statistical analysis
Data were analyzed with the BDMP New System Professional Edition (22) statistica Ref. 22: BDMP New System Professional Edition (registered trademark of Statistical Solution University of California Press, Berkeley, CA, 1995).

The association of bone marrow micrometastasis with clinico-pathological factors was analyzed using the two-tailed Fisher exact test. Survival from the date of operation was calculated using the Kaplan–Meier survival analysis method. Differences in observed survival between groups were tested for significance using the log-rank test [14].

Differences were considered significant when the P-value was less than 0.05. For patients presenting recurrences, the number of days was calculated from the date of pulmonary resection to the first documentation of either loco-regional or distant recurrence.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Mean age of the studied population was 64 years (range 45–87) with a 4:1 male to female ratio. The mean follow-up time was 535 days (range 55–1388 days). Overall 30-day mortality was 1% (one patient out of 99).

3.1. Prevalence of bone marrow micrometastasis (Tables 2 and 3).
Out of the 99 patients, 22 (22.1%) were found to have metastatic cells in their bone marrow aspirates. There were no differences in the prevalence when histologic differentiation (squamous cell vs. adenocarcinoma) or grade (poorly, moderately, or well differentiated) was analyzed (see Table 2). There was a trend for a higher frequency with increased tumor size (T2 vs. T1), nodal status (N1 vs. N0), and tumor stage (Stage II vs. I). The small cohort of patients with T4 and/or N2 disease (Stage III disease) with OM (n=3/17) does not allow for a confident statistical analysis. However, none of the differences in the frequency of the variables studied reached statistical significance (Fischer exact test, two-tailed).

3.3. Survival (Figs. 1a,b, 2 and 3a,b ).
One patient died in the early postoperative period from multiple organ failure following intrapericardial right pneumonectomy for a cT4N0M0 tumor. During follow-up, there were ten additional cancer unrelated deaths (four cardiac, one pulmonary embolism, four pulmonary infection, one suicide) and in another patient the cause of death could not be determined. All patients were included for survival analysis. Details of survival according to predictor variables are given in Table 5.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Overall survival according to bone marrow OM status (n=99) is depicted in (a) (no micrometastasis vs. micrometastasis, log-rank P=0.52) and the disease-free survival according to bone marrow OM status is depicted in (b) (no micrometastasis vs. micrometastasis, log-rank P=0.97 (MicroM, micrometastasis; no MicroM, no micrometastasis).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Overall survival and the disease-free survival according to stage status (n=97) (Stage I vs. II, log-rank P=0.07; Stage I vs. II–IV, log-rank P=0.004*; Stage I and II vs. III and IV, log-rank P=0.01*).

View larger version (10K): [in this window] [in a new window]   Fig. 3. Overall survival according to bone marrow OM status in Stage I patients (n=59) is depicted in (a) and the disease-free survival according to bone marrow OM status in Stage I patients is depicted in (b) (Stage I without micrometastasis vs. Stage I with micrometastasis, log-rank P=0.74) (MicroM, micrometastasis; no MicroM, no micrometastasis).

The 1-year disease-free survival was 80±9% (95% CI: 62–97) in patients with OM, not significantly lower than 85±5% (95% CI: 75–95) in patients without OM (entire survival log-rank P=0.97).

We further analyzed survival according to the stage of the disease (Tables 5 and 6). For patients with Stage I disease, the 1-year overall and disease-free survival were 89 and 96%, respectively. Indeed, six patients died during the first year of follow-up, four of cancer unrelated causes (one cardiac, two infections, one pulmonary embolism) and two of cancer related causes (one loco-regional recurrence and one brain metastasis). For patients with Stages II, III, and IV disease, the 1-year overall and disease-free survivals were 78 and 61%, respectively (89 vs. 78%, log-rank P=0.004 and 96 vs. 61%, log-rank P<0001).

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
We have found that 22% of surgically treated patients with NSCLC, mainly Stages I and II, present with bone marrow OM at the time of surgery. Contrary to the current opinion, this had no prognostic value in terms of survival or disease-free survival in the present series. Several points have to be analyzed carefully in order to explain these differences.

Our study has at least one weakness, which is the relatively short follow-up (mean and median time=17.6 and 14.3 months, respectively). The prevalence of bone marrow micrometastatic cells in our study is lower than the one found by Cote et al. (40%, 17 out of 43 patients) [15], Pantel and co-workers (59.7%, 82 out of 139 patients) [9,10], or Oghami et al. (39%, 15 out of 39 patients) [16]. In Pantel et al.'s report, though the stage classification was not available, one might notice that there was a 25.9% prevalence of T3–T4 tumors and a 49.7% prevalence of N1–N2 disease. In addition, our calculated prevalence of 22% is very similar to the one initially reported by their group in their first report on 82 patients [8].

In previous studies, bone marrow sampling was obtained from fragmented ribs [15], one iliac crest [8,16], and iliac crest and ribs (two to four samples) [6,10]. It is of interest to note that in Pantel et al.'s report, the prevalence of micrometastatic cells per sample was close to 30% (124 out of 376 bone marrow aspirates) and that multiple sampling resulted in an overall prevalence of 59.7%.

Although the timing of the bone marrow harvesting could appear to be arguable since it was performed after the pulmonary resection, we feel that it does not jeopardize the validity of positive finding in the cyto-aspirates. Indeed, this important question was addressed by Pantel et al. in a prospective series of 36 patients in whom bone marrow micrometastases were evaluated by needle aspirates performed both before and after the pulmonary resection. No difference in the prevalence of micrometastasis could be found, whether the samples were taken before or after the resection, showing that there was no surgery-induced dissemination [8,9].

The sensitivity of the CAM5.2 and AE1 mAb for the low molecular weight cytokeratin (CK) proteins has been largely demonstrated previously [17,18] and as the other groups, our mAb were tested both on primary epithelial tumor and on benign non-epithelial samples for positive and negative controls.

Both our study and the other reports confirmed a trend for higher prevalence of OM with increased tumor burden. However, when predictor variables such as age, sex, histology, differentiation, T status, N status, or Stage are analyzed, no correlation can be found between micrometastasis prevalence and any of those variables.

Similarly, analysis of the prevalence of loco-regional and distant recurrences did not reveal any significant difference when patients' groups with and without bone marrow micrometastatic cells were compared. It is of some interest that even though bone marrow micrometastatic cells were present in 22 patients, out of the four patients who had metastasis during follow-up, only one developed clinical bone metastasis whereas the other three developed brain metastasis. In the group without bone marrow micrometastatic cells, six out of the 11 patients developed clinical bone metastasis during follow-up. This finding contradicts the previous observations from Pantel et al. who found an increased incidence of bone metastasis in patients with micrometastatis (four out of six) than in patients without micrometastatis (one out of six) [8].

Our study has the largest number of patients with early-stage NSCLC since 65 out of 99 patients (66%) were pN0 and 59 out of 99 patients (60%) were in Stage I. When overall and disease-free survival were analyzed, no difference could be found between patients' groups with and without bone marrow micrometastatic cells, either when all stages or when only early-stage (Stage I) patients were considered.

In our series, there was no difference in disease-free survival between patients with and without micrometastasis. In other words, the presence of OM in early-stage NSCLC did not affect disease-free survival.

This is in contradiction to the results found by Cote et al. (Stage I and II=17 patients), Pantel et al. (Stage I and II=66), and Oghami et al. (Stage I and II=26).

Except for Pantel et al.'s latest report [10], none of the previous studies showed that overall survival was considerably modified by the presence or absence of bone marrow micrometastatic cells either in Stage III or in Stage I or II. However, in their reports, Pantel et al. analyzed only the subgroup of patients who presented with two or more micrometastatic cells per sample whereas for the other subgroup of patients who presented with one micrometastatic cell (about 50% of the overall group), no survival differences were noted. By contrast, we defined micrometastatic patients as those patients who presented with at least one positive bone marrow micrometastatic cell/0.5x10⁶ cells.

In conclusion, our study confirms that more than 20% of early-stage NSCLC patients harbor bone marrow micrometastatic cells at the time of their intended curative pulmonary resection. No correlation could be found between the presence of micrometastatic cells and conventional clinico-pathological variables.

Our study suggest that the presence of micrometastatic cells in a sanctuary site has no major impact on the prognosis and survival of patients with operable NSCLC. Therefore, embarking on adjuvant therapies for early-stage NSCLC based solely on the finding of bone marrow micrometastatic cells seems not justified at this point. Further follow-up of this cohort of patients might strengthen our conclusions.

	Acknowledgments

 
The authors are grateful to Anne Marie Feyens and Esther Haumont for their precious and skilled technical assistance.

	Footnotes

 
Presented at the 8th European Conference on General Thoracic Surgery of the European Society of Thoracic Surgeons, London, UK, November 1–4, 2000.

¹ Members of the ‘Groupe d'Oncologie Thoracique des Cliniques Universitaires Saint-Luc, Université Catholique de Louvain’ and cooperating in the study are E. Coche, Ph. Collard, Y. Humblet, G. Liistro, M. Lonneux, Ph. Noirhomme, Th. Pieters, A. Poncelet, D. Rodenstein, P. Scaillet, and B. Weynand.

	References

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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 Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Poncelet, A.J. Articles by Noirhomme, P.H. Search for Related Content PubMed PubMed Citation Articles by Poncelet, A.J. Articles by Noirhomme, P.H. Related Collections Lung - cancer