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© 2002 American Society for Clinical Oncology Cooperative Role of Telomerase Activity and p16 Expression in the Prognosis of Non–Small-Cell Lung CancerByFrom the Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense; and Servicios de Cirugía II, Medicina Preventiva y Oncología, Hospital Clínico San Carlos, Madrid, Spain. Address reprint requests to Manuel Benito, PhD, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Madrid, Spain 28040; email: benito{at}eucmax.sim.ucm.es
PURPOSE: Telomerase activity and p16 expression can be considered two of the most important molecular markers implicated in tumorigenesis. Our main aim was to study the cooperative role of both molecular alterations in the prognosis of patients surgically resected for non–small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We have determined telomerase activity and p16 expression in a series of 98 prospectively collected NSCLC specimens obtained from patients who had undergone surgery without other treatment. Telomerase activity was investigated by a telomeric repeat amplification protocol enzyme-linked immunosorbent assay–based procedure, and p16 expression was examined by Western blot. Associations with survival were evaluated. RESULTS: Positive results for telomerase activity were found in 82% of the cases, and this variable correlated with poor differentiation and recurrence of tumors. Lack of p16 expression was observed in 61% of tumors, and a significant association with tumor recurrence was also observed. By univariate analysis, both negative telomerase activity and p16-positive expression were significantly correlated with a better prognosis. Moreover, statistics for equality of survival distributions for telomerase, adjusted for p16, indicated a positive interaction between both parameters. For telomerase-positive tumors, p16 expression emerged as a significant independent protective variable, as indicated by Cox multivariate analysis (relative risk [RR], 0.214; P = .014). This protective effect was maintained only for stage I and II tumors (RR, 0.108; P = .046). CONCLUSION: These results suggest that the combined telomerase activity and p16 expression analyses may be of prognostic importance in NSCLC, especially for patients affected by stage I and II tumors.
THE EXPRESSION OF telomerase, the enzyme that synthesizes telomeric DNA de novo, is suppressed in most normal human somatic cells but is reactivated during tumorigenesis. This reactivation seems to arrest the normal loss of telomeric DNA incurred as somatic cells divide. Because continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may be an important step in the acquisition of cell immortalization, which occurs during tumor progression.1,2 Recently, it has been shown that telomerase activity is not sufficient for immortalization of human keratinocytes or mammary epithelial cells. However, the downregulation of p16 expression in combination with telomerase activity is able to immortalize epithelial cells efficiently.3 The loss of cell-cycle inhibition by negative regulators, such as p16, frequently occurs in certain primary malignant neoplasms, and p16 is considered a major target in carcinogenesis. Its mechanism of action involves binding to and inactivating the D-cyclin-dependent kinase 4 (or 6) complex and, thus, renders the retinoblastoma protein inactive. This effect blocks the transcription of important cell-cycle regulatory proteins and results in cell-cycle arrest.4-7 Lack of expression of p16 has been associated with different abnormalities that occur in the gene, including homozygous deletion, methylation of the promoter, and point mutations.7 Non–small-cell lung cancer (NSCLC) represents one of the most frequent fatal malignancies in the world. Among men in Spain, lung cancer is the leading form of cancer, and squamous cell carcinoma (SCC) is the main histologic subtype. Spanish women have one of the lowest average incidences of the European cancer registers.8,9 Although much is known about the causes, clinical features, and molecular pathogenesis of NSCLC, there is no molecular marker that has major clinical prognostic predictive value. Both telomerase activity and p16 expression have been separately considered to be of importance in relationship to the development and to predict clinical outcome in NSCLC. Thus, it has been reported that telomerase activity may be useful both as a diagnostic marker to detect the existence of immortal lung cancer cells in clinical materials and as a target for therapeutic intervention.10 On the other hand, alterations in the molecular machinery that controls the transition from G1 to S phase might represent central events that lead to NSCLC generation. To this respect, alterations in the p16/retinoblastoma (Rb) pathway have been considered in tumors from different origins,7 including NSCLC.11 According to these reports, p16 abnormalities are a frequent event in this pathology, and data suggest that the reduction or loss of p16 expression correlates with a worse patient outcome.12 Therefore, according to results published previously, telomerase activity and p16 expression can be considered two of the most important molecular markers implicated in the development of NSCLC. However, there are no reports that examine abnormalities that affect both parameters in a cohort of patients affected by this pathology. In this context, our main aim was to study the prognostic value of telomerase activation in combination with p16 expression in 98 patients surgically resected for NSCLC. Data reported here could expand the ability of clinicians to better predict clinical outcome and lead to better therapeutic strategies that can improve the clinical course of NSCLC.
Patients and Tumor Samples Ninety-eight non–small-cell lung tumors and their corresponding control tissue samples were obtained from patients who underwent radical surgery between 1994 and 1999 at San Carlos Hospital in Madrid. None of these patients had received neoadjuvant therapy before surgical resection. Informed consent was obtained from patients before investigation. Six patients were female, and 92 were male, with an average age of 63.85 ± 9.33 years (range, 40 to 83 years). The median follow-up period for patients was 37.6 months (range, 11 to 79 months). During surgery, tissue samples were obtained from tumoral specimens and from macroscopically normal lung parenchyma (at least 10 cm from the distal margin of the tumor, when possible). All tissue samples were snap-frozen in liquid nitrogen immediately after surgery and stored at -80°C. Cryostat-sectioned, hematoxylin-eosin–stained samples from each tumor block were examined microscopically by two independent pathologists to confirm the presence of more than 80% tumor cells. Paired normal tissues from the same patient, used as controls, were also microscopically confirmed. Cancers were staged pathologically using the tumor-node-metastasis system.13 Forty-two patients had stage I tumors; eight had stage II; 37 had stage IIIA; nine had stage IIIB, and two patients had stage IV tumors. Therefore, 87 patients who had stage I, II, or IIIA tumors were subjected to curative surgery, whereas only a biopsy was taken from patients who suffered from more extensive disease. According to World Health Organization criteria, 56 tumors were SCC; 38 were adenocarcinomas (AC); and four were large-cell undifferentiated carcinomas. The histologic classification of tumors was established according to previous criteria.14 Thus, 26 tumors were classified as well differentiated; 46, moderately; and 26, poorly differentiated.
Analysis of Telomerase Activity We used an extract of the telomerase-positive embryonic kidney cell line 293 as a positive control. The sensitivity of the procedure was sufficient to detect telomerase activity in an extract that contained 10 cells of the telomerase-positive cell line used as control. Negative controls were prepared in each case by treating cell extracts with DNase-free RNase. This treatment destroys telomerase activity because telomerase essentially requires the integrity of its internal RNA component as a template for the addition of the telomeric repeat sequences to the telomerase-specific primer. Several authors have described occasional biopsies that contain inhibitors of Taq polymerase, and extracts that contain such inhibitors become amplifiable once the original extracts are diluted.16-18 In our study, to avoid the effect of Taq polymerase inhibitors, we stimulated the activity of telomerase by serial dilutions of the extracts. Thus, for each of the samples, we evaluated telomerase activity directly in the cell extract (which contained 6 µg of total proteins) as well as in serial dilutions (1:10, 1:100, and 1:1000) established from the original extracts. Moreover, in all cases, we included a negative control. This protocol was applied both to tumor samples and the corresponding control tissues.
Alkaline Phosphatase Activity
Evaluation of p16 Expression by Western Blot
Clinical Correlations
This study attempted to determine the effect of telomerase activity and p16 expression on the prognosis of patients who had undergone potentially curative surgery for NSCLC. Telomerase activity was measured in tumor and nontumor tissues using a telomerase semiquantitative PCR-ELISA assay, the results of which correlated with the appearance of a telomerase-mediated 6 nucleotide-ladder, as we have previously published.15 Thus, telomerase activity was investigated in 85 tumors because, in 13 cases, a partial RNA degradation was observed previous to the activity assay. Because RNA integrity is an essential parameter for telomerase activity evaluation, these samples were excluded. Thus, telomerase-positive tumors were classified into three groups according to activity levels: low, intermediate, and high activity. Twenty cases (23.5%) showed low activity levels, and intermediate or high levels were detected in 23 and 27 tumors (27.1% and 31.8%), respectively. In 15 cases (17.6%), negative telomerase values were obtained. Therefore, positive results for enzyme activity were found in 70 (82%) of the 85 NSCLCs. Moreover, weak levels of telomerase activity were found in 21 (24.7%) of the 85 normal tissues analyzed. Our data indicated that poor differentiation of tumors was significantly associated with positive telomerase activity (P = .015). In addition, telomerase activity showed a borderline correlation with recurrence of tumors (P = .071). This variable did not show an association with other clinicopathologic features included in this study (Table 1). Tumor recurrence and telomerase activity were clearly associated in tumors staged within the groups I to IIIA that corresponded to patients who had undergone potentially curative surgery and who did not die in the postoperative period (n = 69; P = .017). In this case, we have found tumor recurrence in 21 (36.2%) of the 58 tumors that showed telomerase activity (eight tumors with low levels, two patients with intermediate activity, and 11 tumors with high enzyme activity). No cases of tumor recurrence were observed in the 11 telomerase-negative tumors. For tumors staged as I to IIIA, a significant correlation was also found between poor differentiation of tumors and positive telomerase activity (P = .007), with no differences between different activity levels.
p16 expression was estimated by densitometry of immunoblots (Fig 1). After beta-actin normalization, all nontumor tissues showed low or undetectable levels of protein expression (range, 0 to 0.26 arbitrary densitometric units), whereas tumor tissues expressed p16 at different levels. Thus, tumors were divided into three groups according to protein expression in relation to p16 levels in normal tissues: tumors with low, intermediate, and high p16 expression. Low expression was defined as patients with p16 levels similar to or lower than those in the corresponding control tissues. The group of intermediate-p16 expression was established for tumors that overexpressed p16 from 1.1-fold to three-fold in relationship to control tissues. Finally, tumor samples with more than threefold levels of p16 with respect to normal tissues were included in the high-expression group. According to our data, 60 tumor samples (61.2%) showed low p16 levels, 17 (17.3%) showed intermediate levels, and a distinctly higher expression was found in 21 samples (21.4%). All correlations included here have been performed on the basis of p16-negative expression for tumors of the first group and p16-positive expression for tumors with intermediate or high p16 levels. Thus, the percentage of tumors with positive expression of p16 was 38.7% (38 of 98).
Expression of p16 was not associated with age or sex of patients nor with stage, histology, or differentiation of tumors. However, a significant correlation was detected between lack of p16 expression and tumor recurrence (P = .030) (Table 2). When we considered the group of tumors included in I to IIIA stage that corresponded to patients who survived into the postoperative period (n = 72), we detected a strong association between tumor recurrence and lack of p16 expression (P = .001). In fact, only three of the 22 patients who experienced recurrence showed significant p16 expression, whereas in 29 (58%) of 50 cases without recurrence, intermediate or high p16 expression levels were detected. p16-positive expression was significantly correlated with intermediate or high telomerase activity levels (Table 3).
Next, we calculated the DFS from the date of surgery until the time of tumor recurrence. Analyses of these survival data only included patients who underwent curative surgery. In relation to telomerase activity, a significant difference on survival was found between negative and positive cases (log-rank P = .04). The 3-year survival probability was 100% in patients with telomerase-negative tumors and 57.7% in the group of patients who showed telomerase-positive tumors because 21 cases of tumor recurrence were detected within the 58 patients included in this group (Fig 2). When we considered different telomerase-positive levels, no significant differences in the relationship to survival probability were detected.
The lack of p16 expression was also correlated with an adverse outcome in patients affected by NSCLC (log-rank P = .004). The 3-year survival probability was 46% in patients without p16 expression, compared with 88.2% in the group of patients with a significant value for p16 expression. In fact in the first group, 29 of the 50 patients showed tumor recurrence, whereas only three recurrences within the 22 cases were detected in the second group (Fig 3).
By univariate analysis, stage IIIA tumors were associated with a significant survival reduction. Moreover, both negative telomerase activity and p16-positive expression were correlated with a better prognosis for patients (Table 4). Relative risk (RR) was significantly lower in patients whose tumors did not show telomerase expression (RR, 0.036; P = .009) or positive p16 expression (RR, 0.205; P = .003).
On the other hand, statistics for equality of survival distributions for telomerase adjusted for p16 had significant results with the Breslow test (P = .03). For negative p16 expression, survival differences were significant (P = .04), whereas when p16 expression was considered positive, we did not find significant differences between the two survival curves (P = .65) (Fig 4). These results indicated a positive interaction between the two parameters evaluated in this study, telomerase activity and p16 expression.
To identify which independent factors jointly had a significant influence on survival, Cox multivariate analysis was performed. For this study, variables with a P value lower than .15 in the univariate analysis were selected. However, it was not mathematically possible to establish the Cox multivariate study, including telomerase activity, because one subset of events was empty (no cases of recurrence within the group of telomerase-negative tumors), and we would have obtained an undefined value for RR. So, we performed multivariate analysis only with telomerase-positive cases. Thus, our data indicated that, for patients affected by I to IIIA tumors who showed telomerase activity, p16 expression was a significant protective variable independent of tumor stage. RR was approximately four times lower than in the case of tumors with negative p16 expression (RR, 0.214; P = .014) (Table 5). Interestingly, the protective effect of p16 expression was maintained when we only considered the earlier-stage tumors (stage I and II tumors). p16-positive expression can also be considered a protective biomarker for stage I and II tumors with positive telomerase activity (RR = 0.108; P = .046) (Table 5). In fact, Fig 5 shows the correlation between telomerase-positive activity and p16 expression of tumors in association with patient prognosis, including stage I to IIIA tumors versus only including stage I and II tumors. In both cases, a high rate of relapse was detected in patients who had tumors that lacked p16 expression (P = .001 and P = .015, respectively).
Previous studies have demonstrated that detection of telomerase in human tissue samples may be one of the best markers of neoplasia across a broad range of tumor types.20 Although the expression of telomerase does not by itself lead to a tumorigenic phenotype,21 telomere maintenance might cooperate with additional oncogenic mutations to create a malignantly transformed clone.22 Thus, it seems that, in certain primary cell types, senescence can only be bypassed by concomitant activation of telomerase and loss of either pRb or p53 function.23 These results indicated that the Rb pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. Moreover, recent results in human epithelial cell lines indicate that immortalization may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation in p16INK4a, p14ARF, or other genes to become immortal.24 Despite the fact that a great number of studies regarding molecular abnormalities in relationship to cancer have emerged in recent years, there has been no report to specifically investigate telomerase activity in combination with p16 expression in human cancer. So, considering the role of telomerase activity in cellular immortality and that p16 is a major target in carcinogenesis, for which an involvement in cell senescence has been described,7 we have evaluated both parameters in a cohort of NSCLC patients who only underwent surgical resection in this study. Our data indicated that a high proportion of lung tumors (82%) showed positive telomerase activity, which correlated this marker with negative determinants of prognosis, such as poor differentiation or recurrence of tumors. These results were in agreement with previously published studies.25 Moreover, in other reports, high telomerase activity had been frequently detected in primary NSCLC that exhibited high tumor-cell proliferation rates and advanced pathologic stage.19 In contrast to these data and according to our results, no differences regarding clinicopathology of tumors were detected between the distinct telomerase positive levels. In normal tissues, we detected weak levels of telomerase activity in 24.7% of cases. These data could be attributed to the presence of microscopic telomerase-positive tumor cells or the presence of connective and/or lymphocytic cells that may show positive telomerase activity levels. In relation to p16, we found low or undetectable levels of protein expression in all of the control tissues considered and different levels of p16 expression in tumor samples. Taking into account that, in most cases, our nontumor samples were collected at least 10 cm away from the distal margin of the tumors and, therefore, no contamination by tumor cells would be expected, these results may indicate that p16 in tumor samples is upregulated in response to oncogenic factors. In fact, it has been suggested that upregulation of p16 is not a consequence of the entry into senescence but is directly linked to the accumulation of cell doublings.26 Moreover, in tumor samples, we found altered expression to be a rather frequent event in NSCLC because our results revealed a lack or low levels of protein expression in 61% of cases. Inactivation of the p16 protein was demonstrated in 30% to 50% of NSCLC and in 20% to 40% of lung ACs, with the rate of p16 inactivation lower in AC than that in SCC (40% to 75%).27,28 Our present correlation analyses demonstrated that loss of p16 expression was significantly correlated with tumor recurrence, but no other associations were observed with clinicopathologic features of tumors. These data are in agreement with the findings of Groeger et al,12 who reported a high frequency of aberrant p16 expression in NSCLC but did not correlate this finding with histopathologic parameters. Kratzke et al,29 however, reported a high frequency of p16 aberrant expression in NSCLC to be inversely related to the pathologic stage of the disease. Looking for correlations between telomerase activity and p16 expression in lung carcinomas, we observed that tumors that showed moderate or high levels of telomerase activity were those in the groups with a higher proportion of cases that expressed p16. These results could indicate a possible mechanism to arrest cycling in cells with an extra capacity for proliferation that may result from telomerase reactivation in an earlier stage. Next, we evaluated both telomerase activity and p16 expression in relation to prognosis through DFS data. Very few studies have addressed the relationship between telomerase activity and prognosis in lung cancer, and controversial results have been obtained. Thus, although telomerase activity has been considered one of the most important prognostic factors in patients affected by NSCLC,25 other authors have not found such association.19 According to our results, telomerase activity has emerged as a prognostic marker of importance in NSCLC. In addition, altered p16 expression is an unfavorable prognostic factor for this pathology. Loss of p16 expression correlates with a worse patient outcome. Previous studies examined p16 expression by immunohistochemical techniques and revealed similar data.12 Moreover, taking into account the high proportion of tumors with telomerase activity, we grouped telomerase-positive tumors by function of p16 expression. Thus, our results revealed a positive interaction between these parameters, with p16 expression as a protective variable independent of tumor stage, in patients who had developed tumors that showed positive telomerase activity. Our observations clearly provided in vivo evidence that supports recent proposals that multiple clocks function to limit the proliferative capacity of human cells. Thus, in human keratinocytes that express human telomerase reverse transcriptase, replicative potential was limited by a p16-dependent mechanism. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.30 It is possible that, in NSCLC cells that show telomerase reactivation, p16 is upregulated to control cellular senescence under a mechanism sensitive to telomere length. Loss of this mechanism by the altered regulation of p16 expression could provide an advantage to gain proliferation, with important prognostic implications in patients affected by NSCLC. Therefore, considering that it is now highly likely that telomere maintenance contributes to oncogenesis, p16 expression could reverse immortality in cells that have reactivated telomerase. Alternatively, the lack of p16 expression could contribute to tumor progression and malignancy. These results in NSCLC and, more specifically, in the earlier stages of tumor progression, could be useful for the selection of patients with potentially unfavorable outcomes, to establish adjuvant therapy protocols.
Supported by grant no. 99/1044 from the Ministerio de Sanidad y Consumo, Madrid, Spain, and grant no. 08.1/0017/1998 from the Comunidad Autónoma de Madrid, Madrid, Spain.
The first two authors contributed equally to this work.
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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; telomeric fragments added in the telomerase activity assay > 86 bp); tumors with intermediate levels (OD450nm, 1 to 1.999; telomeric fragments, 62 to 86 bp); and tumors that show low levels of telomerase activity (OD450nm, 0.200 to 0.999; telomeric fragments < 62 bp). 
2 test. A P value of less than .05 was judged to be significant. Distributions of disease-free survival (DFS) were estimated with the Kaplan-Meier method, and comparisons were made with log-rank and Breslow statistics. Results were considered significant for P values less than .05. For the survival analysis, only patients who had undergone potentially curative surgery (patients with tumor-node-metastasis stages I to IIIA tumors) were considered. Also excluded were patients who died in the postoperative period. Univariate and multivariate analysis were performed using the Cox proportional hazards model to identify which independent factors jointly had a significant influence on survival. Adjusted-rate ratios were calculated. The existence of interactions and proportionality assumptions were evaluated. Variables that showed a P value less than .15 in the univariate analysis were selected for the multivariate analysis. The null hypothesis was rejected in each statistical test when P < .05. Analysis was performed using SPSS for Windows (version 10.0; SPSS, Inc, Chicago, IL). 
