Japanese Journal of Clinical Oncology 30:478-486 (2000)
© 2000 Foundation for Promotion of Cancer Research
Useful Prognostic Panel Markers to Express the Biological Tumor Status in Resected Lung Adenocarcinomas
1Thoracic Service of Marília Medical School, Marília, 2Pulmonary Division and 3Department of Pathology, São Paulo University, General Hospital, São Paulo and 4São Paulo Federal University, São Paulo, Brazil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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Background: Tumor stage and its histological subtype remain the most important predictors of clinical behavior in current pulmonary practice of lung cancer. However, many investigators agree that these parameters are not sufficient to predict which tumor will recur, even after radical curative surgery. Therefore, it is necessary to evaluate the significance of other morphological, biological and molecular parameters beyond TNM classification.
Methods: Pathological specimens were collected from 45 patients after resection for stage IA (five), stage IB (10), stage IIB (10), stage IIIA (14) and stage IV (six) lung adenocarcinomas. A panel of two morphological (proportion of stroma within the tumor and degree of tumor differentiation), two biological [DNA ploidy and argyrophilic nucleolar organizer region (AgNOR)] and three molecular (immunohistochemical expression of Ki-67, p53 and bcl-2) markers was chosen for analysis of the primary tumor. Life Tables for Survival were used to analyze the individual impact of each variable on survival. Cox proportional hazards model analysis was used to construct an independent tumor status model for cancer recurrence and death. Chi-squared analyses were used to determine the statistically significant relationship among all the variables present in the study.
Results: Multivariate analysis demonstrated statistically significant risk for the following markers: AgNOR, p53 and bcl-2, controlled for stages and surgical resection.
Conclusions: The immunohistochemical expression of p53 and bcl-2 oncogenes and the expression of AgNOR cell proliferation index are critical values in the progression of lung adenocarcinomas. They can express the biological tumor status and indicate a more accurate prognosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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Tumor stage and its histological subtype remain the most important predictors of clinical behavior in current pulmonary practice of lung cancer (1,2). However, many investigators agree that these parameters are not sufficient to predict which tumor will recur, even after radical curative surgery. Therefore, it is necessary to evaluate the significance of other morphological, biological and molecular parameters beyond TNM classification. There are many studies that report new prognostic variables but most of them fail the test of rigorous statistical analysis within the current staging system. In this context, morphological, biological and molecular markers have been studied in recent years in our patients with lung carcinoma (3). These markers have accurately predicted the outcome in several groups of patients with different stages of small cell (4) and squamous cell lung cancer (5). The results encourage expanding prognostic marker investigation to other series of tumors including lung adenocarcinomas. This paper reports the results of a study using a panel of prognostic markers: two morphological (proportion of stroma within the tumor and degree of tumor differentiation), two biological [DNA ploidy and argyrophilic nucleolar organizer region (AgNOR)] and three molecular (immunohistochemical expression of Ki-67, p53 and bcl-2) controlled for stages and surgical resections in order to assess their applicability in patients with resected lung adenocarcinomas.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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From January 1988, until December 1998, 107 patients with diagnosis of non-small cell lung cancer who underwent complete resection were evaluated at Marília Medical School Thoracic Service. Primary lung adenocarcinoma variants were the most frequent cell type in this group with 49 patients (45.79%). Inclusion criteria for entry into the study group included pathological staging by the International System of Staging for Lung Cancer (1), complete resection, no adjuvant therapy, at least 30 day survival time and pathological available material. During this period, 45 patients, 13 females (28.8%) and 32 males (71.1%), with median age 61.71 ± 9.19 years (range 39–91 years) satisfied these criteria and were studied. In 28 patients (62.22%) pulmonary resections were performed by standard longitudinal lateral posterior thoracotomy (6) and in 17 patients (37.77%) pulmonary resections were performed by posterolateral thoracotomy. The following resections were performed: five wedge resections (11.11%), two segmentectomies (4.4%), 27 lobectomies (60%), two bilobectomies (4.4%) and nine pneumonectomies (20%). Mediastinal lymph node resections were performed in all the patients. After the surgery tumors were staged according to the TNM stage system (1): IA (five), IB (10), IIB (10), IIIA (14) and stage IV (six). The mean follow-up time for all patients was 27.6 ± 21.52 months (range 4–90 months). The clinical data for each patient are given in Table 1.
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Histological Analysis
Tumor tissue samples were obtained by surgical resection of lung adenocarcinoma and fixed in 10% buffered formalin. For each case, one representative slide of the main tumor was selected by light microscope. The tumor sections considered were those with at least 10 high-power fields which represented adenocarcinoma and presented no or little necrosis and minimal preparation artifact. Their respective paraffin-embedded blocks were sectioned at 5 µm and stained with hematoxylin and eosin. These slides were reviewed in a blind and randomized fashion by two independent pathologists, who agreed on the diagnosis of lung adenocarcinoma. The histological classification of each case was done according to the World Health Organization guidelines (7).
p53, bcl-2 and Ki-67 Immunohistochemical Staining
Immunostaining was performed by the streptavidin–biotin method using an LSAB kit, (Dako, Carpinteria, CA) in a 3 µm thick section. The slides were then incubated with the primary antibody: clone 124, 1:400 for bcl-2, clone DO.7, 1:400 for p53 and Ki-67 antigen, 1:1800 for Ki-67.
AgNOR and DNA Staining
AgNOR staining was performed by the modified one-step silver colloid method (8) in 3 µm thick sections and DNA staining by Feulgen’s technique (9) in 5 µm thick sections.
Quantitation of Stroma, p53, bcl-2 and Ki-67
Quantitation was performed by the point counting technique: a 100 point and 50 line coherent system was attached to the eyepiece of a light microscope (10). The stroma proportion was determined in 10 non-coincident microscopic fields in each case, at x25 magnification and covering a 1 mm2/field area. The expression of p53, bcl-2 and Ki-67 was determined in 10 non-coincident microscopic fields in each case at x400 magnification. A total of 1000 points were counted and covered a 62 500 µm2/field area per section. The results were expressed as a percentage obtained by the following relationship: % structure = Ps/Pt, where Ps is the number of points overlying stained tumor cells or stroma and Pt is the number of points overlying tumor tissue. For all morphometric procedures, the error estimate (CE) was kept under 10%.
Quantitation of AgNOR and DNA Ploidy
The quantitatition of AgNOR expression and DNA nuclei content was performed by means of digital image analysis [integrated optical density (IOD)], by using the specific software Bioscan-Optimas (Bioscan, Edmonds, WA) for AgNOR and WING, version 1.1 (Mcom Informatica, São Paulo, Brazil) for DNA ploidy. The quantification method has been described previously (10). For AgNOR, a total of 100 cells per case were analyzed at x1000 magnification and the results were expressed in terms of mean area (µm2) per individual case. For DNA a total of 200 cells per slide were analyzed at x40 magnification with 100 control cells (pneumocytes) and 100 tumor cells. The results were expressed in terms of DNA index (DI) and calculated by the relationship DI = DNA tumors cells/DNA control cells.
Statistical Analysis
Survival Analysis
Survival was defined from the date of surgery to the date of the last follow-up or death from cancer. We considered as a positive event any death caused by cancer recurrence. Death from other causes and live patients were considered as a censored point in the models. The significance level (p) used for entering or removing a covariable from this model was 0.05.
Life Tables for Survival were used to select the isolated variables that showed a statistical significant association with survival (p < 0.05). In this analysis the measured parameter values (p53, bcl-2, Ki-67 and stroma) were arranged in increasing order and gathered in two categories (low and high degree). AgNOR was gathered in five categories. DNA ploidy was gathered in two categories as follows: diploid DNA (DNA index = 1) and non-diploid DNA (DNA index <1 or DNA index >1). The degree of tumor differentiation was gathered in two categories (well and poorly differentiated).
These statistically significant parameters selected by univariate analysis (p < 0.05) were considered in a multivariate analysis by the Cox proportional hazards model.
Chi-squared Analysis
This analysis was used to test relationships among all measured variables present in the study. Fisher’s exact test was used when the frequency of a cell in a 2 x 2 table was <5. In this analysis TNM stages, N status, T status, extension of pulmonary resection, age and AgNOR were gathered in two categories as follows: early TNM stage (IA, IB and IIA), advanced TNM stage (IIIA and IV), lymph nodes negative (N0) and lymph nodes positive (N1 and N2), small tumors (T1 and T2) and large tumors (T3 and T4), small resections (wedge resection, segmentectomy and lobectomy), large resections (bilobectomy and pneumonectomy), young patients ( age <65 years) and old patients (age >65 years), low AgNOR expression (AgNOR <8.70) and high AgNOR expression (AgNOR 
9.05).
All statistical tests were performed by using SPSS v. 6.0 statistical software (11).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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The expression of morphological, biological and molecular markers of each tumor is shown in Table 2.
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Univariate analysis (Table 3) of the relationship between survival and the panel of clinical morphological and biological markers was statistically significant for age, stages, N status, extension of the pulmonary resection performed, degree of tumor differentiation, proportion of stroma within the tumor, expression of p53 (Fig. 1), bcl-2 (Fig. 2) and AgNOR (Fig. 3). There was no statistically significant difference in survival by gender, T status, DNA ploidy and Ki-67 expression. Multivariate analysis (Table 4) controlled by stages and the extension of pulmonary resection demonstrated a statistically significant association between survival and the expression of AgNOR, p53 and bcl-2 controlled by stages and surgical resection. The biological status model obtained for resected lung adenocarcinomas indicates that patients with localized disease, without nodal involvement, who underwent a limited pulmonary resection with low expression of AgNOR and p53 and high expression of bcl-2 will have a better survival.
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Chi-squared analysis (Table 5) of the relationship among all variables studied showed that the greatest number of statistically significant relationships was reached by p53 and AgNOR. High expression of p53 and AgNOR was related to parameters which express worse prognoses such as advanced stages, lymph node metastasis and poorly differentiated tumors. A statistically significant association between lymph node metastasis and p53 high expression and bcl-2 low expression was found. Tumors without nodal involvement expressed low p53 and high bcl-2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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The degree of tumor differentiation and the proportion of the stroma framework (desmoplasia) are morphological parameters currently used by pathologists to evaluate lung adenocarcinomas aggressiveness (12,13). In this context, we found a statistically significant relation between poorly differentiated tumors and worse prognosis and also between a high stroma framework proportion and better prognosis (Table 3). However, these results were only confirmed by univariate analysis.
At present the contribution of DNA ploidy to lung cancer prognosis evaluation remains disputable (14,15). We found no difference in survival between diploid and non-diploid tumors and there was no statistically significant relationship between DNA ploidy and TNM stages or other variables present in the study.
AgNOR has been considered to be a simple and practical method to study the proliferative activity in lung adenocarcinoma (16,17). In our research, the relation between AgNOR expression and survival was statistically significant in the univariate analysis (Table 3 and Fig. 3) and in the multivariate analysis (Table 4). The AgNOR high expression in tumor cells showed a statistically significant correlation with lymph node involvement, poorly differentiated tumors and advanced disease (Table 5). There was a profound impact of all these data on the overall survival of patients with lung adenocarcinoma.
In several studies, the Ki-67 proliferation index has been demonstrated to be associated with decreased survival (18,19). In our study we did not find a statistically significant relationship between death risk and Ki-67 expression (Table 3), but there was a statistically significant relationship between high Ki-67 expression and lymph node involvement (Table 5).
The bcl-2 gene is related to apoptosis by its ability to prolong cell survival without increasing cell proliferation (20). Studies of non-small cell lung cancer (21) and lung adenocarcinoma (22) indicated better survival for those patients with tumors which showed a high bcl-2 expression. In our study, patients with a high bcl-2 expression in tumors cells showed statistically better survival than patients with low bcl-2 in tumors cells according to univariate (Table 3) and multivariate analyses (Table 4). There was a statistically significant relationship between a high bcl-2 expression and parameters related to a better prognosis such as absence of nodal involvement and an inverse relationship between the bcl-2 and p53 expression: among 15 tumors with high bcl-2 expression only two had high p53 expression (Table 5).
Mutant p53 is associated with many solid tumors and is the most common gene mutation identified in lung cancer up to now. The mechanism of p53 transformation is controversial and accumulated data show that p53 protein is also related to the apoptosis pathway and is opposed to bcl-2 action. In our study, the impact of p53 expression on survival was statistically significant according to univariate (Table 3) and multivariate analyses (Table 4). A high p53 expression was statistically significant and related to parameters which indicate worse prognosis in the case of young patients, advanced disease, nodal involvement and large resection (Table 5). These results are consistent with previous literature reports (18,19).
A significant correlation between the anatomic tumor stages determined with the TNM staging system and the biological tumor status was found in this study. It is not clear whether with the biological tumor status changes together with the anatomic tumor stage or a tumor with high expression of biological markers such as p53 and AgNOR spreads so fast that it is always in advanced stages by the time of diagnosis.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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The immunohistochemical expression of p53 and bcl-2 oncogenes and the expression of AgNOR cell proliferation index are critical values in the progression of lung adenocarcinomas. They can express the biological tumor status and indicate a more accurate prognosis.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONSAcknowledgmentsREFERENCES |
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We thank Dr Tsuguo Naruke, National Cancer Center, Tokyo, for his scientific support throughout this research. The Brazilian Funding Agencies FAPESP, CAPES, CNPq and LIM-HCFMUSP are thanked for financial support.
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FOOTNOTES |
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+ For reprints and all correspondence: Paulo Eduardo de Oliveira Carvalho, Marília Medical School, José Alfredo de Almeida 395, Marília SP, 17502-220 Brazil. E-mail: peocarva@unimedmarilia.com.br
Abbreviations: AgNOR, argyrophilic nucleolar organizer region; Ki-67, Ki-67 monoclonal antibody; bcl-2, bcl-2 oncogene; p53, p53 mutant tumor suppressor gene; DI, DNA index
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Received April 3, 2000; accepted September 1, 2000.
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