Ann Thorac Surg 2001;71:1765-1771
© 2001 The Society of Thoracic Surgeons
Original article: general thoracic
Prognostic implication of Ki-67 immunostaining in treating subclinical pleural cancer found at thoracotomy in lung cancer patients
Mitsutoshi Shiba, MDa,
Kimitaka Kakizawa, MDa,
Hiromasa Kohno, MDa,
Kiyoshi Shibuya, MDa,
Hisami Yamakawa, MDa,
Kenzo Hiroshima, MDb,
Takehiko Fujisawa, MDa
a Department of Surgery, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, Chiba, Japan
b Department of Pathology, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, Chiba, Japan
Accepted for publication February 13, 2001.
Address reprint requests to Dr Fujisawa, Department of Surgery, Institute of Pulmonary Cancer Research, Chiba University School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260-8670, Japan
e-mail: msiba{at}hotmail.com
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Abstract
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Background. Therapeutic principles for managing subclinical pleural cancer found unexpectedly during intraoperative examination are unclear. We analyzed prognostic factors including the tumor proliferative marker Ki-67 in these circumstances.
Methods. The cases of 65 surgically treated patients with lung cancer and subclinical T4 pleural cancer, microscopic in 25 and macroscopic in 40, were reviewed.
Results. The overall 5-year survival rate of patients undergoing lobectomy was 14.3%. For patients with T4 N0 disease, the 5-year survival rate was 46.7%. In patients with a low Ki-67 labeling index, the 5-year survival rate was 28.6%. The Ki-67 labeling index was a significant (p < 0.05) indicator of survival. Multivariate analysis demonstrated Ki-67 labeling index, lymph node involvement, and tumor differentiation to be the most influential prognostic factors for postoperative survival (p < 0.01).
Conclusions. In the treatment of lung cancer patients with subclinical pleural cancer found at thoracotomy, tumor resection is not necessarily contraindicated. Resection appears to be beneficial in patients with no nodal involvement or a low tumor Ki-67 labeling index. This index is a good therapeutic indicator for lung cancer patients.
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Introduction
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When preoperative chest roentgenograms and chest computed tomograms show no evidence of pleural effusion in a patient with lung cancer but effusion is found at thoracotomy, we define this finding as subclinical pleural effusion. We discovered subclinical pleural effusion in 39% (320 patients) of 831 patients undergoing operation for lung cancer including 49 patients (15.3%) who had cancer cells in the pleural fluid. The prognostic implication of such unexpected T4 cancers is unclear, and therapeutic guidelines are lacking.
Immunostaining with the antibody that reacts with the Ki-67 nuclear antigen associated with cell proliferation and found throughout the cell cycle (G1, S, G2, and M phases) has been studied and found to be absent in resting (G0) cells [1, 2]. Such immunohistochemical staining provides a reliable method for evaluating tumor growth fraction in many malignant tumors including those in the lung [3], and it appears to be a useful prognostic marker of non–small cell lung cancer, especially in the early stage [4–7]. Therefore, we correlated outcome with Ki-67 antigen expression in resected tumor tissue and analyzed the effectiveness of the Ki-67 labeling index as a prognostic indicator in the presence of subclinical pleural cancer.
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Material and methods
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From 1985 to 1995, 923 patients with primary lung cancer had pulmonary resection at the Institute of Pulmonary Cancer Research, Chiba University. There were 613 men and 310 women with a mean age of 62.1 years. The following types of cancer were found: 511 adenocarcinomas, 323 squamous cell carcinomas, 28 large cell carcinomas, 23 small cell carcinomas, and 38 miscellaneous types. TNM stage was classified according to the system of the Union Internationale Contre Cancer [8]. The distribution of pathological extent was as follows: T1 N0 M0, 237; T2 N0 M0, 172; T1 N1 M0, 34; T2 N1 M0, 65; T3 N0 M0, 62; T3 N1–2 M0, 68; T1–2 N2 M0, 107; T1–3 N3 M0, 20; T4 N1–3 M0, 105; and T1–4 N1–3 M1, 35. The stage could not be accurately determined for 18 patients in this retrospective study. No patient in this series underwent induction therapy.
Subclinical pleural cancer was found in 65 patients at the time of thoracotomy. Lobectomy with lymph node dissection was done in 55 patients who had complete resection of primary lesions. Exploratory thoracotomy or partial lung resection was done in 10 patients in the hope of improving the quality of life. Partial pleurectomy was added to lobectomy in 30 patients with macroscopic pleural cancer. After resection, the pleural cavity was washed with saline solution and exposed to mitomycin-C at the time of closing.
Patients were followed up every month during the first postoperative year, every 2 to 3 months for 2 to 5 years postoperatively, and every 6 months thereafter. Contact with the patient by telephone or postal card determined the need of additional follow-up and his or her survival status. The apparent causes of death were confirmed by telephone contact with the primary physicians.
Cytological and immunohistochemical examination
Collected effusions were mixed with 0.1 mL of heparin sodium and centrifuged for 10 minutes at 3,000 rpm. A smear of the sediment was made and stained quickly. Cytological evaluations were performed during operation by an expert cytopathologist qualified by the Japanese Society of Clinical Cytology.
Representative tumor samples were obtained from paraffin-embedded specimens fixed by neutral formalin immediately after operation. Tumor specimens 3 to 4 µm thick were cut and mounted on silan-coated glass slides. Ki-67 antigen levels were determined using the MIB-1 monoclonal antibody (Immunotec, Marseilles, France). This antibody recognizes native Ki-67 antigen and recombinant fragments of the Ki-67 molecules, and it is applicable for use on formalin-fixed, paraffin-embedded tissues after microwave pretreatment as well as on frozen section [2].
For immunohistochemical staining, slides were stained according to the streptavidin-biotin staining procedures. Briefly, after deparaffinization, specimens were heated in a microwave (100°C, four times) in 0.01 mol/L citrate buffered solution. After cooling gradually to room temperature, slides were incubated for 20 minutes in 0.3% H2O2 diluted in methanol. They were then washed with water, incubated with normal rabbit serum to block nonspecific binding, and incubated with primary antibody diluted ratio 1:100 in buffered solution overnight at 4°C. After incubation with bridging antibody for 30 minutes, slides were incubated with avidin-biotin complex for 30 minutes. Immunostaining was visualized by the use of diamino-benzidine (stained for 8 minutes) followed by counterstaining with hematoxylin.
All slides were evaluated without any information on clinical outcome or other clinicopathological data. Microscopy was performed in a high-power field (10 x 100) by counting more than 1,000 tumor cells in randomly selected representative parts of the tumor. Usually we looked at more than five different areas in each tumor including at least two central and two peripheral portions to obtain a total cell count. As the median value of a positive ratio of tumor cells was 11% in this series, the Ki-67 LI was defined as high if 10% or more of the tumor nuclei were positively stained with the monoclonal antibody. We also referred to other reports [4, 6, 7] when establishing this definition of high LI.
Statistical analysis
To examine factors affecting postoperative survival, the following variables were analyzed: age, sex, histology, differentiation, tumor size, nodal involvement, Ki-67 LI, and operations performed. Each of these eight variables was divided into two categories as follows: age was divided by mean age, ie, less than or equal to 60 years versus more than or equal to 61 years; sex, male versus female; histology, adenocarcinoma versus other types; differentiation, well or moderate versus poor or undifferentiated; tumor size, equal to or less than 3 cm versus greater than 3 cm; nodal involvement; positive versus negative; Ki-67 LI, equal to or greater than 10% versus less than 10%; and operation performed, lobectomy versus partial resection. In addition, T4 cancers were analyzed by whether they were grossly evident (macroscopic pleural cancer) or whether they were found by cytological study only (microscopic pleural cancer). Pleural invasion by the main tumor was categorized as positive (tumor invasion beyond the visceral pleural) or negative (tumor possibly reaching but not extending beyond the pleural surface).
Postoperative survival in the dichotomized categories of the variables was estimated using the method of Kaplan and Meier [9], and survival distributions between the dichotomized categories were compared with log-rank test to determine significant prognostic indicators. Then with these indicators as variables, the Cox proportional hazards regression model [10] was applied to evaluate joint influences of the variables on postoperative survival. In this statistical analysis, we used StatView statistical software. The level of significance was set at 0.05.
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Results
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Patient characteristics
Of the 923 patients with primary lung cancer, 399 were found to have subclinical pleural effusion (around 20 mL) or small pleural nodules and underwent intraoperative cytological study. Subsequently, subclinical malignant effusions without a visible pleural disseminating nodule were found in 25 patients (6.3%, D0 group), and a subclinical visible pleural cancer nodule with or without malignant effusion was identified in 40 patients (10.0%, D1 group).
The characteristics of these 65 patients, who form the study group, are shown in Table 1. Patient age ranged from 29 to 82 years with a mean age of 60.5 years. Preoperative staging by TNM subset of the 65 patients was as follows: T1 N0 M0, 16; T2 N0 M0, 18; T1 N1 M0, 1; T2 N1 M0, 7; T3 N0 M0, 2; T3 N1 M0, 2; T1 N2 M0, 6; T2 N2 M0, 9; and T3 N2 M0, 4. Adenocarcinomas were observed in 58 patients, and 7 patients had other tumor types. Histological grading showed five well-differentiated tumors, 51 moderately differentiated tumors, seven poorly differentiated tumors, and two adenosquamous carcinomas. The following nodal involvement was found: N0 tumors, 11; N1 tumors, 6; N2 tumors, 36; and N3 tumors, 4. All of the N3 nodes were located in the contralateral hilar lymph node station adjacent to subcarinal lymph nodes. On gross visualization thoracotomy, the grade of macroscopic dissemination was relatively mild, and the nodules were located mainly on the visceral pleura and the diaphragmatic aspect of the parietal pleura.
Correlation between clinicopathological factors and D grouping was analyzed (see Table 1). Concerning lymph node metastases, 6 of the 11 patients with N0 tumors were in the D0 group, and 19 of the 46 patients with N-positive tumors were in the D0 group. This difference was not significant.
Survival
The estimated postoperative 5-year survival rates for the dichotomized categories of the ten factors examined are shown in Table 2. Mean and median follow-up times for the 65 patients were 2.45 years (893 days) and 2.03 years (740 days), respectively. The survival rates associated with N0 grade (46.7%) and low Ki-67 LI (28.6%) were especially high.
The results of log-rank testing are also shown in Table 2. As we found no significant difference between the D0 and D1 groups (p = 0.4583), significant differences were calculated according to the total number of patients in each category of the clinicopathological factors. Significant differences in survival rates were observed for histology (adenocarcinoma versus other; p = 0.0262) (Fig 1A); tumor size equal to or less than 3 cm versus greater than 3 cm; (p = 0.0055), (Fig 1B); nodal involvement (negative versus positive, p = 0.0110) (Fig 2A), and Ki-67 antigen LI in resected tumor tissue (high versus low, p < 0.0001) (Fig 2B). The Kaplan-Meier survival curve with respect to lymph node metastasis (N factor) and Ki-67 LI revealed the following 5-year survival rates: 75.0% in the N0 and low LI group, 22.2% in the N0 and high LI group, 19.2% in the N+ and low LI group, and 0% in the N+ and high LI group (Fig 2C). The N0 and low LI group had the best 5-year survival. As for tumor recurrence, intrapulmonary dissemination was the most frequent initial site of relapse, especially in the N+ and high LI group.
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Fig 1. Postoperative survival rate of patients with lung cancer with subclinical pleural dissemination stratified by (A) histology (adenocarcinoma versus nonadenocarcinoma) and by (B) tumor size (> 3.0 cm versus  3.0 cm).
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Fig 2. Postoperative survival rate of patients with lung cancer with subclinical pleural dissemination stratified by (A) lymph node metastasis (positive versus negative), by (B) Ki-67 labeling index (LI) (high,  10.0%, versus low, < 10.0%), and by (C) lymph node status and Ki-67 LI (N0 and LI low versus N0 and LI high versus N+ and LI low versus N+ and LI high).
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We obtained p values greater than 0.05 and less than 0.1 from the comparison of survival distributions between the dichotomized categories of sex and differentiation. Although the p value for operation was 0.136, lobectomy resulted in better postoperative survival than did partial resection. In patients who had lobectomy, postoperative 2-, 3-, and 5-year survival rates were 55.9%, 36.6%, and 14.3%, respectively, whereas in patients who underwent partial resection, the survival rates were 60.0% at 2 years, 10.0% at 3 years, and 0% at 5 years. The p values for age and pleural invasion were greater than 0.5.
We also analyzed survival according to stage independent of subclinical metastasis. The 5-year survival rates were as follows: stage I, 46.7%; stage II, 0%; and stage III, 6.8%. A significant difference between stage I and stages II and III was observed (p = 0.0110).
Prognostic factors
Using the six factors with p values of less than 0.1 by log-rank test as variables, the Cox proportional hazards regression model was applied to evaluate joint influences of these factors on survival. For this evaluation, we used 50 patients with no missing data for the six variables. The results are shown in Table 3. The regression coefficients for nodal involvement, Ki-67 LI, and tumor differentiation were significant (p = 0.0021, 0.0002, and 0.0082, respectively). We also analyzed survival by stage independent of the subclinical pleural cancer. The analysis indicated similar results. We drew log cumulative hazard curves for each dichotomized category of the six factors and confirmed the assumptions of proportions of proportional hazards.
To sum up the results of this study, there was no significant difference in survival between patients with microscopic pleural cancer (D0) and macroscopic pleural cancer (D1). Tumor size, histology, lymph node status, and Ki-67 LI were significant prognostic indicators for postoperative survival. The 55 patients who underwent lobectomy had 2-year, 3-year, and 5-year survival rates of 55.9%, 36.6%, and 14.3%, respectively. The 5-year survival rate after lobectomy was 46.7% for patients with T4 N0 disease and 35.7% for patients with a low Ki-67 LI. Results of Cox proportional hazards regression modeling suggested that tumor differentiation, lymph node status, and Ki-67 LI were the most important prognostic indicators.
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Comment
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When patients are known to have T4 cancer, thoracotomy is usually not indicated [8, 11, 12]. However, because preoperative evaluation is not always accurate, cytological results positive for malignant cells can be encountered unexpectedly at thoracotomy. Little is known about the surgical outcomes of lung cancer patients with subclinical pleural dissemination. Our data showed that the survival of patients with N0 disease with pleural invasion was significantly better than that of patients with N1 or N2 disease with pleural invasion. This finding suggests that a tumor designated N0 with pleural dissemination has a relatively mild biological nature and that it could include the early stage of the pleural cancer and a low Ki-67 LI. Thus, T4 N0 patients with subclinical pleural cancer can benefit from surgical treatment.
The choices of treatment of pleural cancer include partial pleurectomy, partial pulmonary resection, lobectomy, and pleuropneumonectomy [13–18]. We chose lobectomy instead of pleuropneumonectomy in the hope of giving our patients the best quality of life possible. On the basis of our results, lobectomy provided a better surgical outcome than partial resection. As the number of patients having partial resection was small in our study, a large prospective study will be necessary to evaluate lobectomy as a standard method of operation for subclinical pleural cancer.
We found no difference in outcome between patients with microscopic and macroscopic pleural cancer. This surprising result suggests that we ought to treat these two groups as one clinical entity. Our surgical outcome, including these two groups, is in general agreement with the results in previous reports of surgical treatment for pleural cancer [13–18]. In three relatively recent studies [16–18] as well as this one involving more than 30 patients having resection for lung cancer with pleural invasion, median survival ranged from 24 to 29 months and the 5-year survival rate, from 14.3% to 24.6%. Although, a difference in survival was not shown for type of operation or adjuvant therapies (local or general), a difference was clearly demonstrated for pathological factors that reflect the biological nature of lung cancer, namely N status and Ki-67 LI.
By univariate and multivariate analysis, we showed Ki-67 LI, lymph node status, and tumor differentiation to be the most significant prognostic indicators among the clinicopathologic factors examined. To our knowledge, this is the first report demonstrating that Ki-67 antigen expression is an independent prognostic factor in lung cancer patients with malignant pleural dissemination. Several investigators [4–7] have studied the role of Ki-67 expression in predicting the surgical outcome of patients with lung cancer. Scagliotti and colleagues [6] reported that in patients with resectable non–small cell lung cancer, disease-free survival was significantly lower for those with a higher Ki-67 labeling score (> 25% positive cells). Harpole and associates [7] also found that a higher Ki-67 proliferation index (> 5%) was a significant predictor of early recurrence and cancer death by univariate analysis but not by multivariate analysis for patients with stage I non–small cell lung cancer. Tungekar and coauthors [4] demonstrated an initial difference in survival between three ranges of Ki-67 LI (0%–10%, 11%–40%, and > 40%), but the difference largely disappeared after 5 years.
In this series, we counted tumor cells under microscopy and set a threshold of 10% to divide Ki-67 LI into two categories. Some investigators [5] have reported the use of image cytometry or flow cytometry to evaluate tumor growth fraction with Ki-67 immunostaining. Compared with our conventional method, these methods seem more objective because a larger number of cells can be counted. On the other hand, when image analysis by cytometry is used, observer discretion may unduly influence outcome and cut off lines because of field selection and variability in staining intensity. Also, it has been suggested that if flow cytometry is used, pretreatment of the tumor is necessary to prevent simultaneous counting of tumor cells and interstitial normal cells.
In the future, a more convenient and objective method to count labeled cells will be needed, especially for rapid assessment of the Ki-67 LI during operation. Such a method will be required for a large, prospective study to evaluate the effectiveness of Ki-67 LI as a postoperative prognostic factor in subclinical malignant pleural disease. To date, significant correlation of Ki-67 labeling score between preoperative biopsy specimens and postoperative surgically resected specimens has been reported [19]. Although the marker index was determined on the resected specimen in our series, our method can be introduced for the preoperative evaluation of patients because our data indicated that the Ki-67 LI was a strong prognostic indicator even for patients with T4 cancer. We believe that the Ki-67 LI will help both to develop a therapeutic strategy for use during operation and eventually to select patients for postoperative adjuvant therapy. With rapid histopathological diagnosis during operation, including proliferative antigen staining [20], it should become possible to calculate the LI of the Ki-67 antigen immediately during operation.
In conclusion, our data suggest that lung cancer patients with subclinical malignant pleural effusion and with either no nodal involvement or a low Ki-67 LI, namely the N0 and low LI group, the N0 and high LI group, and the N+ and low LI group, should receive surgical treatment.
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Acknowledgments
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We thank Dr Yoshihide Tsuchiya for statistical assistance. This investigation was supported in part by Grant-In-Aid for Scientific Research C-08671514 from the Ministry of Education, Science, Culture and Sports of Japan.
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Abstract
Introduction
