Catalytic Subunit of Human Telomerase Reverse Transcriptase Is an Independent Predictor of Survival in Patients Undergoing Curative Resectio
http://www.100md.com
▲還散笫雖悝◎
the Departments of Medicine, Biostatistics, and Surgery, Institut Gustave Roussy, Villejuif
Centre de Chirurgie Viscerale et de Transplantation, Hopital de Hautepierre, Strasbourg
Commisariat 角 l'Energie Atomique, Fontenay-aux-roses, France
the Departments of Surgical Oncology, Gastrointestinal Medical Oncology, and Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX
ABSTRACT
PURPOSE: To determine the role of the catalytic subunit of human telomerase reverse transcriptase (hTERT) in predicting survival after resection of hepatic colorectal metastases (CRM).
PATIENTS AND METHODS: Two hundred one patients who underwent curative resection of hepatic CRM between 1990 and 2000 were identified from a multicenter database. The CRM were analyzed for hTERT nucleolar expression by standard immunohistochemical techniques. hTERT expression and known clinicopathologic factors of survival were examined.
RESULTS: With a median follow-up of 80 months, 152 patients (75.6%) had died; the 5-year overall survival was 30.7%. On univariate analysis, number of metastases greater than two (P = .0005), extrahepatic disease (P = .0054), disease-free interval less than 12 months (P = .006), carcinoembryonic antigen level greater than 200 ng/mL (P = .0071), and positive hTERT nucleolar staining (P < .0001) were associated with decreased survival. On multivariate analysis, three factors independently predicted survival: number of metastases (relative risk [RR] = 1.74; P = .0011); disease-free interval (RR = 1.70; P = .0035); and positive hTERT nucleolar staining (RR = 2.03; P < .0001). Patients with none or one of these factors had a 5-year survival rate of 48%, whereas those with two or three of these factors had a 5-year survival of 15% (P < .0001).
CONCLUSION: hTERT nucleolar expression is associated with worse survival after resection of hepatic CRM. hTERT expression in conjunction with number of hepatic metastases and disease-free interval may permit more accurate prediction of survival after resection of hepatic CRM.
INTRODUCTION
Colorectal cancer is the fourth most common type of cancer in the West and the second leading cause of cancer-related deaths in the United States.1 Approximately 35% to 55% of patients with colorectal cancer develop hepatic metastases during the course of their disease.2 Although surgical resection and adjuvant chemotherapy are potentially curative in such cases, local or distant recurrences develop in many patients. Because of this, only 30% to 58% of patients who undergo a curative liver resection with complete extirpation of liver metastases are alive at 5 years.3-5 There has been considerable interest, therefore, in identifying parameters for predicting which patients have more aggressive lesions and are more likely to experience recurrence after resection, and which patients may benefit most from surgery.
Traditionally, primary tumor stage, preoperative carcinoembyronic antigen (CEA) level, time from primary tumor treatment to diagnosis of hepatic metastases (disease-free interval), hepatic tumor size, number of hepatic metastases, and presence of extrahepatic disease have been reported to be independent predictors of survival after resection.3,6 Fong et al6 have developed a clinical prognostic score based on a number of these clinicopathologic factors.
More recently, specific molecular markers have been linked with clinical outcome for patients with colorectal cancer.7-11 Proliferation markers such as Ki-67 labeling index,7,11,12 p53 expression,13 tritiated thymidine uptake,14 and thymidylate synthase expression9 have been shown to be independent predictors of recurrence and survival. In a previous study,11 our group reported that nucleolar staining for human telomerase reverse transcriptase (hTERT) was a better predictor of survival than the Ki-67 labeling index in patients with resected hepatic colorectal metastases.
Telomerase is a ribonucleoprotein enzyme that specifically maintains telomeres〞noncoding tandemly repeated DNA sequences (5'-TTAGGG-3') present at the ends of chromosomes.15-17 Telomerase has two core functional components: the catalytic subunit of hTERT and a telomerase RNA template. Whereas hTERT is expressed only during cellular proliferation, the telomerase RNA template is expressed constitutively. In most human somatic cells, telomerase expression is repressed and telomere length is progressively shortened with each cell division, eventually resulting in chromosomal instability16,18 and cellular senescence.19-21 In contrast, most human tumors express telomerase, resulting in stabilized telomere length and cell immortalization.22-24 Activation of telomerase in cancer cells, therefore, may represent an important step in tumorigenesis.25 In fact, telomerase activity has been observed in a wide variety of epithelial cancers, including stomach,26 lung,27 renal,28 and hepatocellular carcinoma.29 Increased hTERT expression has also been noted in primary colon carcinoma, whereas it has not been noted in colonic adenomas or normal colonic tissue.30,31
Because hTERT activity is associated with tumorigenesis, we sought to determine whether hTERT nucleolar expression is associated with prognosis following curative resection of hepatic colorectal metastases. Specifically, the goal of the current study was to determine the relative prognostic importance of standard clinicopathologic characteristics versus hTERT expression in predicting overall survival (OS) in patients with hepatic colorectal metastases.
PATIENTS AND METHODS
Patients
We reviewed all the records of 201 patients who underwent an R0 resection (negative microscopic surgical margins) of hepatic colorectal metastases between 1990 and 2000 at three major hepatobiliary centers: Institut Gustave Roussy, Villejuif, France (n = 80); Hautepierre Hospital, Strasbourg, France (n = 55); and The University of Texas M.D. Anderson Cancer Center, Houston, TX (n = 66). Each center's institutional review board approved this study. The 66 patients from M.D. Anderson Cancer Center had previously been included in an analysis comparing Ki-67 and hTERT.11 All patients had complete follow-up data available, and no patient was lost to follow-up. Samples obtained during hepatic resection were available for each patient. Patients who underwent an R1 (positive microscopic surgical margins) or R2 (positive gross surgical margins) hepatic resection were not included in the current study.
The following data were collected for each patient: demographics, nodal status of the primary colorectal tumor, disease-free interval (time from primary tumor treatment to diagnosis of hepatic metastases), number of hepatic metastases, size of largest hepatic metastasis, CEA level, presence of extrahepatic disease, disease status at last follow-up, date of last follow-up, and date of death. Data were recorded as follows: clinical features, present or absent; age, less than 60 years versus 60 years; CEA level, equal or less than 200 ng/mL versus > 200 ng/mL; and tumor number, two or less versus greater than two. A clinical prognostic score was derived for each patient on the basis of the Fong scoring system (Table 1).6 hTERT expression was determined using immunohistochemical staining of paraffin sections.
Immunohistochemical Staining for hTERT
Five-micron-thick sections of hepatic resection specimens were deparaffinized through a series of xylene baths, and the samples were rehydrated in graded alcohols. To enhance epitope exposure, slides were pretreated at 98∼C with 10-mmol/L citrate buffer at pH 6 for 90 minutes. Sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 20 minutes to block the endogenous peroxidase activity, and then incubated in 2.5% blocking serum to reduce nonspecific binding. Following this, the samples were incubated at 37∼C with primary monoclonal mouse hTERT 44F12 antibody (Novocastra, Newcastle on Tyne, UK) at a dilution of 1:50 on paraffin sections for 90 minutes. The sections were processed with standard avidin-biotin immunochemistry according to the manufacturer's recommendations (Vector Laboratories, Burlingame, CA). The final chromogen used was diaminobenzidine, and the nuclear counterstain used was hematoxylin. As a negative control, the staining procedure was performed with the primary antibody omitted. The cell line NCI-H460 (American Type Culture Collection, Manassas, VA) was used as an external positive control. The number of neoplastic cells with nucleolar hTERT staining in representative high-power fields of the tumor was divided by the total number of neoplastic cells in those fields, and the result was expressed as a percentage. Since nucleolar hTERT is associated with telomere function,32,33 only cells with nucleolar staining were included in the numerator. hTERT staining was classified as negative (< 50% of tumor cells positive at the nucleolar level) or positive ( 50% of tumor cells positive at the nucleolar level) as previously described.11
Statistical Methods
The primary end point was OS. OS was calculated from the date of liver resection to the date of death. The goal was to evaluate the independent prognostic potential of hTERT nucleolar expression. This was achieved by subjecting hTERT nucleolar expression, along with the other collected clinicopathologic characteristics, to univariate analysis. The results of the univariate analysis were used to determine the variables that were included in the subsequent multivariate analysis.
Median follow-up was computed using inverted Kaplan-Meier method, while survival probabilities were estimated using the standard Kaplan-Meier method. Univariate relationships between OS and each of the clinicopathologic factors and hTERT were evaluated using the log-rank test. Cox proportional hazards models were used for multivariate analysis with forward stepwise selection of the variables. All analyses were performed using SAS software version 8 (SAS Institute, Cary, NC). All tests were two sided and were considered significant at P = .05.
RESULTS
Patient Characteristics
The clinical features of the 201 patients included in the study are presented in Table 2. There were 107 men and 94 women, for a male-female ratio of 1.1:1. Eighty patients had a primary colorectal tumor associated with positive regional lymph nodes. Most patients had a disease-free interval of less than 12 months. Most patients had fewer than three hepatic metastases (n = 127; 63.2%), and most metastases were smaller than 5 cm (n = 141; 70.1%). Extrahepatic disease was present in 33 patients. Patients were equally distributed between a Fong score of 2 or less and a Fong score of 3 or more.
hTERT nucleolar staining was restricted to the metastatic colon cancer cells; no such staining was observed in the adjacent liver tissue. In the majority of cases, hTERT staining was limited to the nucleolar structures; in a few cases, diffuse nucleoplasmic staining with nucleolar reinforcement was observed. Representative examples of hTERT nucleolar staining are shown in Figure 1. Eighty-six patients had positive hTERT nucleolar staining. The differences in staining rates between the three study centers were not significant (P = .23).
Univariate Survival Analysis
At a median follow-up of 80.0 months (range, 75.1 to 84.5 months), 152 patients (75.6%) had died. The median survival time after hepatic resection was 37.3 months, and the 5-year actuarial survival rate was 30.7%. The median survival for patients from each of the surgical centers was similar: Institut Gustave Roussy, 40.7 months; Hopital de Hautepierre, 35.6 months; M.D. Anderson Cancer Center, 35.6 months (P = .23; Fig 2).
On univariate analysis, number of metastases greater than two, extrahepatic disease, disease-free interval less than 12 months, CEA level greater than 200 ng/mL, Fong score of 3 or greater, and positive hTERT nucleolar staining were associated with shortened OS after hepatic resection (Table 3). Sex, age, and nodal status of the primary tumor were not associated with OS.
Patients with positive nucleolar hTERT staining had shortened survival: median survival was 46 months for hTERT-negative patients versus 23 months for hTERT-positive patients (P < .0001; Fig 3). When the prognostic significance of hTERT nucleolar staining was analyzed according to surgical center, this factor remained a significant predictor of shortened OS at both the Institut Gustave Roussy and M.D. Anderson Cancer Center (P = .04 and P = .02, respectively). At Hopital de Hautepierre, the institution with the fewest patients, positive hTERT nucleolar staining showed a trend toward significance in relation to OS (P = .07; Fig 4).
Multivariate Survival Analysis
On multivariate analysis, number of hepatic metastases greater than two (hazard ratio [HR] = 1.74; 95% CI, 1.25 to 2.42; P = .0011), disease-free interval less than 12 months (HR = 1.70; 95% CI, 1.20 to 2.42; P = .0035), and positive hTERT nucleolar staining (HR = 2.03; 95% CI, 1.46 to 2.82; P = .0001) remained independent predictors of poor survival, and patients with positive nucleolar hTERT staining had the highest relative risk of death.
Using these three independent prognostic variables, a clinical score was devised in an attempt to stratify patients with regard to prognosis. Patients with no factor or one factor had a longer median survival (53 months) compared with patients who had two or three factors (23 months; P < .0001). The 5-year actuarial survival rate was 48% for patients with no factor or one factor and 15% for patients with two or three factors (P < .0001; Fig 5).
DISCUSSION
An aggressive approach to resection of hepatic metastases has been advocated by many major centers.6,34 With the improvements seen in the morbidity and mortality associated with hepatic resection, even patients with advanced local disease (multiple, bilobar, large metastases) are candidates for hepatic resection.34,35 Despite hepatic resections performed with curative intent, only 20% to 30% of these patients have a disease-free survival time of at least 5 years.36-38 Furthermore, many recurrences after hepatic resection occur locally within the liver itself early after resection.10,38 Currently, there are few criteria for predicting which patients have more aggressive disease and are, therefore, more likely to experience recurrence and shortened survival. In the study reported here, we found that hTERT nucleolar expression was an independent predictor of OS after curative resection of hepatic colorectal metastases.
Many prior studies have examined prognostic factors for tumor recurrence after hepatic resection.3,39 There has not been complete agreement, however, concerning what clinicopathologic factors are associated with a better prognosis. The two factors that appear to have the most prognostic importance are the disease-free interval and the status of the hepatic resection margin.40,41 Other, more controversial factors include number of metastases, preoperative CEA values, primary tumor stage, and type of liver resection performed.41-43 In the current study, we found that number of hepatic metastases and disease-free interval were independent predictors of survival.
In an attempt to derive more prognostic information, some investigators have combined multiple clinicopathologic factors to formulate prognostic scoring systems.3,6 In the current study, one such scoring system〞the Fong scoring system〞was indeed found to be predictive of survival on univariate analysis. Smith et al have shown, however, that patients with similar scores may have different long-term outcomes.11 In the current study, three of the five criteria of the Fong scoring system (node-positive primary tumor, CEA level > 200 ng/mL, and largest hepatic tumor > 5 cm) were not found to be significant predictors of OS on multivariate analysis when hTERT was introduced into the regression model. Conventional clinicopathologic factors, therefore, may be inadequate. As such, more recently, there has been increased interest in identifying biologic markers that may help better define patients at risk for recurrence after hepatic resection for colorectal metastases.
Previous studies have shown that proliferation markers such as Ki-67,7,11,12 tritiated thymidine uptake,14 p53 expression,44 absence of epithelial growth factor,13 and thymidylate synthase9 are predictive of survival. Other studies, however, have failed to show a correlation between many of these markers and overall prognosis.13,45 Similarly, studies of other biologic markers〞such as ploidy, p53 and K-ras mutations, and expression of p53, Bcl-2, c-myc, Her-2/neu, epithelial growth factor receptor, H-ras, p53, nm23, and dcc〞have yielded inconsistent results with regard to prognosis (Table 4). 8,9,12,14,45-47 Because of this, our group chose to focus on hTERT expression as a possible novel prognostic factor in patients undergoing curative resection of hepatic colorectal metastases.
Telomerase is expressed in most human tumors, resulting in stabilized telomere length and cell immortalization. Yan et al48 showed that hTERT is concentrated in the nucleolus and that functional assembly of telomerase takes place in the nucleolus. Telomerase activity can be studied by a variety of techniques, including telomeric repeat amplification, hTERT mRNA expression by reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. Immunohistochemical staining is routinely performed in pathology laboratories, making it an ideal tool for the screening of clinical tissue. Immunohistochemical hTERT staining is most intense in the nucleoli, and nucleolar immunostaining corresponds to telomerase activity and hTERT mRNA expression.27,32,33,48 Our data demonstrate that immunohistochemical evaluation of hTERT protein in paraffin-embedded and formalin-fixed resection specimens from patients with hepatic colorectal metastases can identify specific subsets of patients with distinctly different outcomes.
In a previous study,11 positive hTERT nucleolar staining was found on univariate analysis to be a better predictor of survival than the Ki-67 labeling index in patients with resected colorectal liver metastases. The study we describe here represents a multicenter, multivariate validation of the prognostic importance of hTERT expression. The current study indicated that hTERT expression was indeed independently associated with shorter survival after curative resection of hepatic colorectal carcinoma metastases. In fact, on both univariate and multivariate analysis, hTERT nucleolar staining was the most powerful predictor of survival. Patients who had hTERT-positive colorectal metastases had a more than two-fold increase in the risk of death compared with patients with hTERT-negative tumors (HR = 2.03; 95% CI, 1.46 to 2.82; P = .0001). The hazard ratio for hTERT was higher than that reported in previous large multivariate analyses of clinical predictors of prognosis.6 Patients with hTERT-positive colorectal metastases had a median survival (23 months) half that experienced by patients with hTERT-negative colorectal metastases (46 months; P < .0001). These findings are consistent with those of other investigators who have shown the importance of hTERT status in the primary colon tumor.49,50 For example, Shoji et al49 reported that in sporadic colon carcinomas, primary tumor telomerase activity was associated with depth of invasion, venous invasion, and the incidence of liver metastasis. Primary colon carcinoma hTERT status has also been shown to correlate with OS.50 The current study is important as it proves that hTERT status within the resected colorectal metastasis itself provides additional important prognostic information with regard to long-term patient outcome.
In the present study, a clinical scoring system was devised in an attempt to identify which patients with resected colorectal metastases benefited the most from surgical resection. Three factors〞number of liver metastases greater than two, disease-free interval greater than 12 months, and positive hTERT nucleolar staining〞were utilized. The clinical scoring system was able to categorize patients into two distinct prognostic groups: patients with no or one factor present, and patients with two or three factors present (Fig 4). In fact, patients with two or three factors present had only a 15% 5-year survival rate. These data strongly suggest that hTERT expression in conjunction with number of hepatic metastases and disease-free interval may provide for a more accurate prognostic model for assessing survival after hepatic resection for colorectal metastases.
In conclusion, the current study validates the independent prognostic value of hTERT expression in predicting OS for patients with surgically resected hepatic colorectal metastases. The hTERT biomarker in conjunction with disease-free interval and number of hepatic tumors may allow for the establishment of a novel prognostic model based on clinical and molecular information. Such a prognostic model may help in clinical decision making regarding adjuvant therapy and design of adjuvant therapy trials. Future studies will need to include hTERT as a prognostic factor to confirm the prognostic power of this biologic marker.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by a fellowship grant from the Foundation Bettencourt (J.D.).
Jean-Charles Soria and Jean-Nicolas Vauthey contributed equally to this work. This work is original. The analysis reported here has not previously been published in full or in part.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Centre de Chirurgie Viscerale et de Transplantation, Hopital de Hautepierre, Strasbourg
Commisariat 角 l'Energie Atomique, Fontenay-aux-roses, France
the Departments of Surgical Oncology, Gastrointestinal Medical Oncology, and Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX
ABSTRACT
PURPOSE: To determine the role of the catalytic subunit of human telomerase reverse transcriptase (hTERT) in predicting survival after resection of hepatic colorectal metastases (CRM).
PATIENTS AND METHODS: Two hundred one patients who underwent curative resection of hepatic CRM between 1990 and 2000 were identified from a multicenter database. The CRM were analyzed for hTERT nucleolar expression by standard immunohistochemical techniques. hTERT expression and known clinicopathologic factors of survival were examined.
RESULTS: With a median follow-up of 80 months, 152 patients (75.6%) had died; the 5-year overall survival was 30.7%. On univariate analysis, number of metastases greater than two (P = .0005), extrahepatic disease (P = .0054), disease-free interval less than 12 months (P = .006), carcinoembryonic antigen level greater than 200 ng/mL (P = .0071), and positive hTERT nucleolar staining (P < .0001) were associated with decreased survival. On multivariate analysis, three factors independently predicted survival: number of metastases (relative risk [RR] = 1.74; P = .0011); disease-free interval (RR = 1.70; P = .0035); and positive hTERT nucleolar staining (RR = 2.03; P < .0001). Patients with none or one of these factors had a 5-year survival rate of 48%, whereas those with two or three of these factors had a 5-year survival of 15% (P < .0001).
CONCLUSION: hTERT nucleolar expression is associated with worse survival after resection of hepatic CRM. hTERT expression in conjunction with number of hepatic metastases and disease-free interval may permit more accurate prediction of survival after resection of hepatic CRM.
INTRODUCTION
Colorectal cancer is the fourth most common type of cancer in the West and the second leading cause of cancer-related deaths in the United States.1 Approximately 35% to 55% of patients with colorectal cancer develop hepatic metastases during the course of their disease.2 Although surgical resection and adjuvant chemotherapy are potentially curative in such cases, local or distant recurrences develop in many patients. Because of this, only 30% to 58% of patients who undergo a curative liver resection with complete extirpation of liver metastases are alive at 5 years.3-5 There has been considerable interest, therefore, in identifying parameters for predicting which patients have more aggressive lesions and are more likely to experience recurrence after resection, and which patients may benefit most from surgery.
Traditionally, primary tumor stage, preoperative carcinoembyronic antigen (CEA) level, time from primary tumor treatment to diagnosis of hepatic metastases (disease-free interval), hepatic tumor size, number of hepatic metastases, and presence of extrahepatic disease have been reported to be independent predictors of survival after resection.3,6 Fong et al6 have developed a clinical prognostic score based on a number of these clinicopathologic factors.
More recently, specific molecular markers have been linked with clinical outcome for patients with colorectal cancer.7-11 Proliferation markers such as Ki-67 labeling index,7,11,12 p53 expression,13 tritiated thymidine uptake,14 and thymidylate synthase expression9 have been shown to be independent predictors of recurrence and survival. In a previous study,11 our group reported that nucleolar staining for human telomerase reverse transcriptase (hTERT) was a better predictor of survival than the Ki-67 labeling index in patients with resected hepatic colorectal metastases.
Telomerase is a ribonucleoprotein enzyme that specifically maintains telomeres〞noncoding tandemly repeated DNA sequences (5'-TTAGGG-3') present at the ends of chromosomes.15-17 Telomerase has two core functional components: the catalytic subunit of hTERT and a telomerase RNA template. Whereas hTERT is expressed only during cellular proliferation, the telomerase RNA template is expressed constitutively. In most human somatic cells, telomerase expression is repressed and telomere length is progressively shortened with each cell division, eventually resulting in chromosomal instability16,18 and cellular senescence.19-21 In contrast, most human tumors express telomerase, resulting in stabilized telomere length and cell immortalization.22-24 Activation of telomerase in cancer cells, therefore, may represent an important step in tumorigenesis.25 In fact, telomerase activity has been observed in a wide variety of epithelial cancers, including stomach,26 lung,27 renal,28 and hepatocellular carcinoma.29 Increased hTERT expression has also been noted in primary colon carcinoma, whereas it has not been noted in colonic adenomas or normal colonic tissue.30,31
Because hTERT activity is associated with tumorigenesis, we sought to determine whether hTERT nucleolar expression is associated with prognosis following curative resection of hepatic colorectal metastases. Specifically, the goal of the current study was to determine the relative prognostic importance of standard clinicopathologic characteristics versus hTERT expression in predicting overall survival (OS) in patients with hepatic colorectal metastases.
PATIENTS AND METHODS
Patients
We reviewed all the records of 201 patients who underwent an R0 resection (negative microscopic surgical margins) of hepatic colorectal metastases between 1990 and 2000 at three major hepatobiliary centers: Institut Gustave Roussy, Villejuif, France (n = 80); Hautepierre Hospital, Strasbourg, France (n = 55); and The University of Texas M.D. Anderson Cancer Center, Houston, TX (n = 66). Each center's institutional review board approved this study. The 66 patients from M.D. Anderson Cancer Center had previously been included in an analysis comparing Ki-67 and hTERT.11 All patients had complete follow-up data available, and no patient was lost to follow-up. Samples obtained during hepatic resection were available for each patient. Patients who underwent an R1 (positive microscopic surgical margins) or R2 (positive gross surgical margins) hepatic resection were not included in the current study.
The following data were collected for each patient: demographics, nodal status of the primary colorectal tumor, disease-free interval (time from primary tumor treatment to diagnosis of hepatic metastases), number of hepatic metastases, size of largest hepatic metastasis, CEA level, presence of extrahepatic disease, disease status at last follow-up, date of last follow-up, and date of death. Data were recorded as follows: clinical features, present or absent; age, less than 60 years versus 60 years; CEA level, equal or less than 200 ng/mL versus > 200 ng/mL; and tumor number, two or less versus greater than two. A clinical prognostic score was derived for each patient on the basis of the Fong scoring system (Table 1).6 hTERT expression was determined using immunohistochemical staining of paraffin sections.
Immunohistochemical Staining for hTERT
Five-micron-thick sections of hepatic resection specimens were deparaffinized through a series of xylene baths, and the samples were rehydrated in graded alcohols. To enhance epitope exposure, slides were pretreated at 98∼C with 10-mmol/L citrate buffer at pH 6 for 90 minutes. Sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 20 minutes to block the endogenous peroxidase activity, and then incubated in 2.5% blocking serum to reduce nonspecific binding. Following this, the samples were incubated at 37∼C with primary monoclonal mouse hTERT 44F12 antibody (Novocastra, Newcastle on Tyne, UK) at a dilution of 1:50 on paraffin sections for 90 minutes. The sections were processed with standard avidin-biotin immunochemistry according to the manufacturer's recommendations (Vector Laboratories, Burlingame, CA). The final chromogen used was diaminobenzidine, and the nuclear counterstain used was hematoxylin. As a negative control, the staining procedure was performed with the primary antibody omitted. The cell line NCI-H460 (American Type Culture Collection, Manassas, VA) was used as an external positive control. The number of neoplastic cells with nucleolar hTERT staining in representative high-power fields of the tumor was divided by the total number of neoplastic cells in those fields, and the result was expressed as a percentage. Since nucleolar hTERT is associated with telomere function,32,33 only cells with nucleolar staining were included in the numerator. hTERT staining was classified as negative (< 50% of tumor cells positive at the nucleolar level) or positive ( 50% of tumor cells positive at the nucleolar level) as previously described.11
Statistical Methods
The primary end point was OS. OS was calculated from the date of liver resection to the date of death. The goal was to evaluate the independent prognostic potential of hTERT nucleolar expression. This was achieved by subjecting hTERT nucleolar expression, along with the other collected clinicopathologic characteristics, to univariate analysis. The results of the univariate analysis were used to determine the variables that were included in the subsequent multivariate analysis.
Median follow-up was computed using inverted Kaplan-Meier method, while survival probabilities were estimated using the standard Kaplan-Meier method. Univariate relationships between OS and each of the clinicopathologic factors and hTERT were evaluated using the log-rank test. Cox proportional hazards models were used for multivariate analysis with forward stepwise selection of the variables. All analyses were performed using SAS software version 8 (SAS Institute, Cary, NC). All tests were two sided and were considered significant at P = .05.
RESULTS
Patient Characteristics
The clinical features of the 201 patients included in the study are presented in Table 2. There were 107 men and 94 women, for a male-female ratio of 1.1:1. Eighty patients had a primary colorectal tumor associated with positive regional lymph nodes. Most patients had a disease-free interval of less than 12 months. Most patients had fewer than three hepatic metastases (n = 127; 63.2%), and most metastases were smaller than 5 cm (n = 141; 70.1%). Extrahepatic disease was present in 33 patients. Patients were equally distributed between a Fong score of 2 or less and a Fong score of 3 or more.
hTERT nucleolar staining was restricted to the metastatic colon cancer cells; no such staining was observed in the adjacent liver tissue. In the majority of cases, hTERT staining was limited to the nucleolar structures; in a few cases, diffuse nucleoplasmic staining with nucleolar reinforcement was observed. Representative examples of hTERT nucleolar staining are shown in Figure 1. Eighty-six patients had positive hTERT nucleolar staining. The differences in staining rates between the three study centers were not significant (P = .23).
Univariate Survival Analysis
At a median follow-up of 80.0 months (range, 75.1 to 84.5 months), 152 patients (75.6%) had died. The median survival time after hepatic resection was 37.3 months, and the 5-year actuarial survival rate was 30.7%. The median survival for patients from each of the surgical centers was similar: Institut Gustave Roussy, 40.7 months; Hopital de Hautepierre, 35.6 months; M.D. Anderson Cancer Center, 35.6 months (P = .23; Fig 2).
On univariate analysis, number of metastases greater than two, extrahepatic disease, disease-free interval less than 12 months, CEA level greater than 200 ng/mL, Fong score of 3 or greater, and positive hTERT nucleolar staining were associated with shortened OS after hepatic resection (Table 3). Sex, age, and nodal status of the primary tumor were not associated with OS.
Patients with positive nucleolar hTERT staining had shortened survival: median survival was 46 months for hTERT-negative patients versus 23 months for hTERT-positive patients (P < .0001; Fig 3). When the prognostic significance of hTERT nucleolar staining was analyzed according to surgical center, this factor remained a significant predictor of shortened OS at both the Institut Gustave Roussy and M.D. Anderson Cancer Center (P = .04 and P = .02, respectively). At Hopital de Hautepierre, the institution with the fewest patients, positive hTERT nucleolar staining showed a trend toward significance in relation to OS (P = .07; Fig 4).
Multivariate Survival Analysis
On multivariate analysis, number of hepatic metastases greater than two (hazard ratio [HR] = 1.74; 95% CI, 1.25 to 2.42; P = .0011), disease-free interval less than 12 months (HR = 1.70; 95% CI, 1.20 to 2.42; P = .0035), and positive hTERT nucleolar staining (HR = 2.03; 95% CI, 1.46 to 2.82; P = .0001) remained independent predictors of poor survival, and patients with positive nucleolar hTERT staining had the highest relative risk of death.
Using these three independent prognostic variables, a clinical score was devised in an attempt to stratify patients with regard to prognosis. Patients with no factor or one factor had a longer median survival (53 months) compared with patients who had two or three factors (23 months; P < .0001). The 5-year actuarial survival rate was 48% for patients with no factor or one factor and 15% for patients with two or three factors (P < .0001; Fig 5).
DISCUSSION
An aggressive approach to resection of hepatic metastases has been advocated by many major centers.6,34 With the improvements seen in the morbidity and mortality associated with hepatic resection, even patients with advanced local disease (multiple, bilobar, large metastases) are candidates for hepatic resection.34,35 Despite hepatic resections performed with curative intent, only 20% to 30% of these patients have a disease-free survival time of at least 5 years.36-38 Furthermore, many recurrences after hepatic resection occur locally within the liver itself early after resection.10,38 Currently, there are few criteria for predicting which patients have more aggressive disease and are, therefore, more likely to experience recurrence and shortened survival. In the study reported here, we found that hTERT nucleolar expression was an independent predictor of OS after curative resection of hepatic colorectal metastases.
Many prior studies have examined prognostic factors for tumor recurrence after hepatic resection.3,39 There has not been complete agreement, however, concerning what clinicopathologic factors are associated with a better prognosis. The two factors that appear to have the most prognostic importance are the disease-free interval and the status of the hepatic resection margin.40,41 Other, more controversial factors include number of metastases, preoperative CEA values, primary tumor stage, and type of liver resection performed.41-43 In the current study, we found that number of hepatic metastases and disease-free interval were independent predictors of survival.
In an attempt to derive more prognostic information, some investigators have combined multiple clinicopathologic factors to formulate prognostic scoring systems.3,6 In the current study, one such scoring system〞the Fong scoring system〞was indeed found to be predictive of survival on univariate analysis. Smith et al have shown, however, that patients with similar scores may have different long-term outcomes.11 In the current study, three of the five criteria of the Fong scoring system (node-positive primary tumor, CEA level > 200 ng/mL, and largest hepatic tumor > 5 cm) were not found to be significant predictors of OS on multivariate analysis when hTERT was introduced into the regression model. Conventional clinicopathologic factors, therefore, may be inadequate. As such, more recently, there has been increased interest in identifying biologic markers that may help better define patients at risk for recurrence after hepatic resection for colorectal metastases.
Previous studies have shown that proliferation markers such as Ki-67,7,11,12 tritiated thymidine uptake,14 p53 expression,44 absence of epithelial growth factor,13 and thymidylate synthase9 are predictive of survival. Other studies, however, have failed to show a correlation between many of these markers and overall prognosis.13,45 Similarly, studies of other biologic markers〞such as ploidy, p53 and K-ras mutations, and expression of p53, Bcl-2, c-myc, Her-2/neu, epithelial growth factor receptor, H-ras, p53, nm23, and dcc〞have yielded inconsistent results with regard to prognosis (Table 4). 8,9,12,14,45-47 Because of this, our group chose to focus on hTERT expression as a possible novel prognostic factor in patients undergoing curative resection of hepatic colorectal metastases.
Telomerase is expressed in most human tumors, resulting in stabilized telomere length and cell immortalization. Yan et al48 showed that hTERT is concentrated in the nucleolus and that functional assembly of telomerase takes place in the nucleolus. Telomerase activity can be studied by a variety of techniques, including telomeric repeat amplification, hTERT mRNA expression by reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. Immunohistochemical staining is routinely performed in pathology laboratories, making it an ideal tool for the screening of clinical tissue. Immunohistochemical hTERT staining is most intense in the nucleoli, and nucleolar immunostaining corresponds to telomerase activity and hTERT mRNA expression.27,32,33,48 Our data demonstrate that immunohistochemical evaluation of hTERT protein in paraffin-embedded and formalin-fixed resection specimens from patients with hepatic colorectal metastases can identify specific subsets of patients with distinctly different outcomes.
In a previous study,11 positive hTERT nucleolar staining was found on univariate analysis to be a better predictor of survival than the Ki-67 labeling index in patients with resected colorectal liver metastases. The study we describe here represents a multicenter, multivariate validation of the prognostic importance of hTERT expression. The current study indicated that hTERT expression was indeed independently associated with shorter survival after curative resection of hepatic colorectal carcinoma metastases. In fact, on both univariate and multivariate analysis, hTERT nucleolar staining was the most powerful predictor of survival. Patients who had hTERT-positive colorectal metastases had a more than two-fold increase in the risk of death compared with patients with hTERT-negative tumors (HR = 2.03; 95% CI, 1.46 to 2.82; P = .0001). The hazard ratio for hTERT was higher than that reported in previous large multivariate analyses of clinical predictors of prognosis.6 Patients with hTERT-positive colorectal metastases had a median survival (23 months) half that experienced by patients with hTERT-negative colorectal metastases (46 months; P < .0001). These findings are consistent with those of other investigators who have shown the importance of hTERT status in the primary colon tumor.49,50 For example, Shoji et al49 reported that in sporadic colon carcinomas, primary tumor telomerase activity was associated with depth of invasion, venous invasion, and the incidence of liver metastasis. Primary colon carcinoma hTERT status has also been shown to correlate with OS.50 The current study is important as it proves that hTERT status within the resected colorectal metastasis itself provides additional important prognostic information with regard to long-term patient outcome.
In the present study, a clinical scoring system was devised in an attempt to identify which patients with resected colorectal metastases benefited the most from surgical resection. Three factors〞number of liver metastases greater than two, disease-free interval greater than 12 months, and positive hTERT nucleolar staining〞were utilized. The clinical scoring system was able to categorize patients into two distinct prognostic groups: patients with no or one factor present, and patients with two or three factors present (Fig 4). In fact, patients with two or three factors present had only a 15% 5-year survival rate. These data strongly suggest that hTERT expression in conjunction with number of hepatic metastases and disease-free interval may provide for a more accurate prognostic model for assessing survival after hepatic resection for colorectal metastases.
In conclusion, the current study validates the independent prognostic value of hTERT expression in predicting OS for patients with surgically resected hepatic colorectal metastases. The hTERT biomarker in conjunction with disease-free interval and number of hepatic tumors may allow for the establishment of a novel prognostic model based on clinical and molecular information. Such a prognostic model may help in clinical decision making regarding adjuvant therapy and design of adjuvant therapy trials. Future studies will need to include hTERT as a prognostic factor to confirm the prognostic power of this biologic marker.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by a fellowship grant from the Foundation Bettencourt (J.D.).
Jean-Charles Soria and Jean-Nicolas Vauthey contributed equally to this work. This work is original. The analysis reported here has not previously been published in full or in part.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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