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TS expression predicts postoperative recurrence in adenocarcinoma of the lung
Lung Cancer 72 (2011) 360–364
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
TS expression predicts postoperative recurrence in adenocarcinoma of the lung Hidehiko Shimokawa, Hidetaka Uramoto ∗ , Takamitsu Onitsuka, Teruo Iwata, Makoto Nakagawa, Kenji Ono, Takeshi Hanagiri Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
a r t i c l e
i n f o
Article history: Received 11 May 2010 Received in revised form 19 July 2010 Accepted 8 August 2010 Keywords: TS DHFR Recurrence Lung cancer Adenocarcinoma Survival
a b s t r a c t Background: Not all patients with lung cancer require postoperative adjuvant chemotherapy after a complete resection. However, no useful markers for either selecting appropriate candidates or for predicting clinical recurrence exist. Methods: Tumor specimens were collected from 183 consecutive patients who underwent a complete resection for lung adenocarcinoma from 2003 to 2007 in our department. We analyzed the thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expressions in the primary lung adenocarcinoma by immunohistochemisty. Results: The strong expression of TS and DHFR was identified in 39 (21.3%) and 120 (65.6%) patients, respectively. The strong TS expression was identified in 11 (39.3%) of 28 patients and 28 (18.1%) of 155 patients in patients with and without recurrence, respectively (p = 0.012). The strong DHFR expression was also identified in 23 (82.1%) and 97 (62.6%) of the patients with and without recurrence, respectively (p = 0.045). Logistic regression models indicated the strong TS expression to be an independent factor for tumor recurrence. The strong TS and DHFR expression was associated with a poorer disease-free survival (DFS) according to the survival analysis. A multivariate analysis demonstrated the strong TS expression to be independently associated with an increased risk for poor DFS. Conclusions: The strong TS expression may be a useful marker for predicting postoperative recurrence in patients with lung adenocarcinoma following surgery. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is the leading cause of cancer-related deaths in the majority of world wide countries [1]. Non-small cell lung cancers (NSCLC) account for approximately 80% of all lung cancers, and the proportion of adenocarcinoma is notably increasing [2]. Although surgery is the only mainstay of treatment to obtain a cure for this disease, approximately 30% of all patients with pathological stage I NSCLCs have tumor recurrence and eventually succumb to the cancer, despite a complete surgical resection [3]. Recently, the benefits of adjuvant chemotherapy have been confirmed [4]. However, the 5-year survival rate of patients with resected NSCLCs is reported to be as high as 60% without adjuvant chemotherapy, thus suggesting that not all patients require chemotherapy after a complete resection [4]. Therefore, it is necessary to identify the patients who might benefit the most from postoperative adjuvant chemotherapy to not only precisely select the patients who require additional treatment,
but also to prevent the occurrence of adverse events in patients who do not require treatment [5]. As a result, it is important to evaluate the biological and molecular characteristics of NSCLC to identify the factors related to recurrence following surgery. However, no useful markers for predicting clinical recurrence exist at present. TS is an essential enzyme for de novo DNA synthesis and the TS expression status has been shown to correlate with the proliferative activity of cancer cells [6]. Previous clinical studies have also demonstrated that high TS expression is associated with a poor prognosis in a variety of malignant tumors [7–9], but the exact clinical reason of the poor prognosis remains unclear. DHFR is also a key enzyme in nucleotide synthesis and DNA replication. Furthermore, TS and DHFR are target enzymes that have been shown to be involved in purine and pyrimidine synthesis for antifolate inhibitors [10]. Therefore, we hypothesized that TS and DHFR may also be useful predictive indicators of tumor recurrence in patients following lung resection. 2. Materials and methods
Abbreviations: TS, thymidylate synthase; DHFR, dihydrofolate reductase; DFS, disease free survival; NSCLC, non-small cell lung cancer; IHC, immunohistochemical; UFT, tegafur–uracil; PS, proportional score; IS, intensity score; OR, odds ratio; 95% CI, 95% confidence interval. ∗ Corresponding author. Tel.: +81 93 691 7442; fax: +81 93 692 4004. E-mail address: [email protected] (H. Uramoto). 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.08.024
2.1. Patients, clinical features, and follow-up The institutional review board approved this study and informed consent for the use of the tumor specimens was obtained
H. Shimokawa et al. / Lung Cancer 72 (2011) 360–364
either from all the patients or their legal guardians. Tumor samples were obtained from 296 patients with primary lung adenocarcinoma who had undergone a surgical resection between 2003 and 2007 in our department. Nine of these patients were stage IV and 25 underwent an incomplete resection. The tumor samples from 79 patients were too small to evaluate by immunohistochemical (IHC) staining for TS and DHFR status. As a result, 113 patients were excluded from further analysis. Therefore, 183 tumor specimens were evaluated. The patients were followed-up every month within the first postoperative year and at approximately 2- to 4-month intervals thereafter. The evaluations included a physical examination, chest roentgenography, an analysis of blood chemistry, and measurements of tumor markers. Chest and abdominal computed tomography, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years after surgery. Additional examinations were performed if any symptoms or signs of recurrence were detected. A follow-up was conducted in all patients. The median follow-up period was 34.2 months. Twentyseven (15.3%) patients had received adjuvant chemotherapy as follows: carboplatin plus paclitaxel (n = 18), carboplatin plus gemcitabine (n = 7), and tegafur–uracil (UFT) (n = 2). At the last follow-up, 144 patients were alive and free of cancer, while 11 patients had died of other causes without evidence of cancer, 12 patients were alive with recurrent cancer, and 16 patients had died of cancer. In total, 28 (15.3%) of the 183 patients demonstrated disease recurrence after surgery.
2.2. IHC staining in paraffin-embedded tumor samples IHC staining was conducted using serial sections from the same paraffin-embedded blocks by previously described methods [11,12]. Briefly, all tissue specimens were formalin-fixed and processed similarly, according to the standard histology practices. A 3 m-thick formalin-fixed, paraffin-embedded tissue section was prepared from each specimen. All specimens were stained with hematoxylin–eosin for the histological diagnosis. The sections were briefly immersed in citrate buffer [0.01 mol/L citric acid (pH 6.0)] and then were incubated for two 10 min at 121 ◦ C in a high-pressure sterilization oven for antigen retrieval. They were then incubated with the TS (TS106, Santa Cruz Biothechnology, CA) diluted at 1:25, and DHFR (ab82171, Abcam, Cambridge, MA) diluted at 1:50, respectively, in phosphate-buffered saline 60 min
361
in a room-temperature. Thereafter, IHC staining was performed by the labeled polymer method (Histofine Simple Stain MAX-PO kit, Nichirei, Tokyo, Japan) according to the manufacturer’s instructions [12,13]. The positive and the negative controls were processed by the HeLa cells and the exclusion of the primary antibody, respectively. 2.3. Evaluation of the stained specimens Following the IHC detection of protein expression in each specimen, the percentage of immunoreactive tumor cells in five 400× fields selected randomly on one slide was recorded, and then the final value of positive tumor cells was determined as the average of the positive immunostained cells. To evaluate any correlations with the clinicopathological characteristics, these protein expression scores were divided into positive or negative groups. The stained specimens for cytoplasm of cancer cells were then categorized into eight degrees according to previous report [14]. Initially, six degrees of the proportional score (PS) for the positive staining cells were assigned according to the frequency of positive tumor cells (0, none; 1, <1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5, >2/3). Thereafter, four degrees for the intensity score (IS) were assigned according to the intensity of the staining (0, none; 1, weak; 2, intermediate; and 3, strong). The PS and the IS were then added to each other to obtain a total score, which ranged from 0 to 8. According to the total score, the TS and DHFR of the tumor was categorized as a weak expression when the score was 0–4 and a strong expression when the score was 5–8. The slides were independently examined by two of the investigators (H.S. and T.O.) who were blinded to the clinicopathological data. When a discrepancy was found between the two investigators, a consensus was reached via their simultaneous examination using a double-headed microscope. 2.4. Statistical analyses Statistical significance was evaluated using the chi-square test or Fisher’s exact test. The A multivariate logistic regression was used to evaluate independent associations. Kaplan-Meier method was used to estimate the probability of survival, and survival differences were analyzed by the log-rank test. A multivariate analysis was then performed according to Cox’s proportional hazards model. The odds ratio (OR) and 95% confidence interval (95% CI)
Table 1 Relationships between the molecular parameters and clinicopathological characteristics. Variables
Gender Age (y) Pathologic stage T status N status Smoking Differentiationc CEAc a b c
Category
Male Female <69 ≥69 I II–III T1 T2–4 Negative Positive C/Ea Nb Well No-well <2.5 ≥2.5
C/E: current/ever. N: never smoker. Unclassified patients were excluded.
No. of patients n = 183
102 81 80 103 148 35 128 55 152 31 113 70 93 74 122 58
TS expression
DHFR expression
Strong (%) 39 (21.3)
Weak 144
Strong (%) 120 (65.6)
Weak 63
23 (22.5) 16 (19.8) 25 (31.3) 14 (13.6) 28 (18.9) 11 (31.4) 26 (20.3) 13 (23.6) 30 (19.7) 9 (29.0) 29 (25.7) 10 (14.3) 10 (1.1) 26 (35.1) 29 (23.8) 10 (17.2)
79 65 55 89 120 24 102 42 122 22 84 60 83 48 93 48
89 (87.3) 51 (63.0) 47 (58.8) 73 (70.9) 93 (62.8) 27 (77.1) 79 (61.7) 41 (74.5) 96 (63.2) 24 (77.4) 76 (67.3) 44 (62.9) 59 (63.4) 51 (68.9) 78 (63.9) 40 (69.0)
33 30 33 30 55 8 49 14 56 7 37 26 34 23 44 18
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H. Shimokawa et al. / Lung Cancer 72 (2011) 360–364 Table 3 Univariate analysis of the factors contributing to the recurrence.
Table 2 Relationships between the TS/DHFR expression and recurrence. Variables
Cases with recurrence Cases without recurrence $
DHFR expression
Variables
OR
Strong (%)
TS expression Weak
Strong (%)
Weak
11(39.3)$1 28 (18.1)
17 127
23 (82.1)$2 97 (62.6)
5 58
Gender: male Age: ≤69 pT: 2–4 pN: positive TS expression: strong DHFR expression: strong
1.070 0.854 3.322 8.130 2.933 2.747
Statistically significant: p-value = 0.012$1 , 0.045$2 .
were calculated for each variable. Differences were considered to be statistically significant for p-values < 0.05. The data were analyzed using the Stat View software package (Abacus Concepts, Inc., Berkeley, CA). 3. Results 3.1. Detection of the TS and DHFR expression and clinicopathological factors
95% CI 0.475–2.410 0.249–1.376 1.453–7.576 3.311–20.0 1.240–6.944 0.991–7.634
p-Value 0.871 0.218 0.004 <0.001 0.014 0.052
OR: odds ratio, 95% CI: 95% confidence interval.
Table 4 Multivariate analysis of the factors contributing to the recurrence. Variables
OR
95% CI
Gender: male Age: ≤69 pT: 2–4 pN: positive TS expression: strong DHFR expression: strong
1.083 0.552 2.604 6.410 2.695 2.237
0.428–2.747 0.205–1.486 1.029–6.579 2.481–16.677 1.022–7.092 0.735–6.803
p-Value 0.865 0.240 0.043 <0.001 0.048 0.138
All of the patients were Japanese, consisting of 102 males and 81 females in this series, with a mean age of 68.5 years (range 23–88 years). The tumor stage was classified according to the Revisions in the International System for Staging Lung Cancer [15]. According to the pathological stage, 113 patients were at stage IA, 35 at IB, 7 at IIA, 10 at IIB, 15 at IIIA, and 3 at IIIB. TS and DHFR expression was identified in 39 (21.3%) and 120 (65.6%) patients, respectively. No significant association of TS or DHFR expression was identified with the clinical factors (Table 1). The majority of the sites of tumor recurrence were hematogenous metastases. Twenty-five and 6 cases had hematogenous (9: brain, 10: lung, 5: bone, and 1: adrenal metastasis) and locoregional (4: Lymph node metastasis and 2: pleural dissemination) recurrences, respectively. Two, one, and one subject had recurrent tumors in both brain and bone, brain and adrenal, and bone and lymph nodes, respectively. 3.2. Influence of TS and DHFR expression and the clinicopathological factors on recurrence The strong TS expression was identified in 11 (39.3%) of 28 patients and 28 (18.1%) of 155 patients in patients with and without recurrence, respectively (p = 0.012) (Table 2). The strong DHFR expression was also identified in 23 (82.1%) and 97 (62.6%) of the patients with and without recurrence, respectively (p = 0.045). The strong TS and DHFR expression were significantly correlated with postoperative recurrence. The logistic regression models indicated the strong TS expression, but not the DHFR expression, to be an independent predictor for recurrence, as were the pathological T and N statuses (Tables 3 and 4). 3.3. Influence of TS and DHFR expression and clinicopathological factors on DFS The 5-year DFS rate with weak and strong for TS expression was 85.6% and 69.9%, respectively (p = 0.012) (Fig. 1A). The 5-year
Fig. 1. Kaplan–Meier DFS curves stratified by TS (A) and DHFR (B) expression. The heavy and narrow lines show the strong and weak expressions of the TS and DHFR expressions, respectively.
DFS rate with weak and strong for DHFR expression was 91.0% and 78.0%, respectively (p = 0.043) (Fig. 1B). The strong TS and DHFR expression was associated with a poorer DFS according to the univariate survival analysis (p = 0.016) (Table 5). A multivariate survival analysis demonstrated the strong TS expression to be
Table 5 Univariate analysis using a proportional hazard model for the disease-free survival. Variables
Gender Age (y) T status N status TS expression DHFR expression
Characteristics
Hazard ratio
Unfavorable
Favorable
Male ≤69 T2–4 Positive Strong Strong
Female >69 T1 Negative Weak Weak
1.112 0.655 3.236 6.494 2.551 2.611
95% CI
0.526–2.353 0.302–1.420 1.541–6.803 3.077–13.699 1.193–5.464 0.993–6.897
p-Value
0.780 0.284 0.002 <0.001 0.016 0.052
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Table 6 Multivariate analysis using a proportional hazard model for the disease-free survival. Variables
Gender Age (y) T status N status TS expression DHFR expression
Characteristics
Hazard ratio
Unfavorable
Favorable
Male ≤69 T2–4 Positive Strong Strong
Female >69 T1 Negative Weak Weak
1.035 0.622 2.320 4.739 2.222 1.838
95% CI
0.483–2.217 0.281–1.377 1.072–5.00 2.212–10.204 1.027–4.808 0.669–5.051
p-Value
0.930 0.242 0.033 <0.001 0.043 0.238
independently associated with an increased risk for a poor DFS with a marginal p-value (Table 6).
meaning of histological differences [27], and efficacy of adjuvant chemotherapy [28,29] in a prospective trial.
4. Discussion
Conflicts of interest
Metastasis is the most frequent cause of treatment failure [16] and occult metastases present in definite proportions at the time of surgical intervention [11]. The clinical findings and current tissuebased molecular markers are insufficient for the early identification of individuals at high risk for metastasis, in spite of recent developments in the field of molecular biology [17]. Therefore, novel strategies for the prevention of metastasis are urgently required to improve the prognosis, quality of life, and cost for treating cancer patients. The TS activity is necessary for cancer cell proliferation through the essential DNA synthesis. In fact, TS exhibits an oncogenic activity [18] and the overexpression of TS is an independent factor for a poor prognosis in NSCLC patients [19,20]. However, the precise reason from a clinical standpoint for a poor prognosis still remains unknown and there is no other established biomarker that is correlated to postoperative recurrence clinically. Recent studies reported the possibility that chemokine receptors 7 and angiopoietin-1 are potential markers of for predicting disease-free survival and recurrence in patients with early stage NSCLC [21,22]. In the present study, we evaluated the correlations between the TS and DHFR expressions and recurrence in lung adenocarcinoma. The TS and DHFR expressions were thus found to significantly correlate with recurrence after the operation. Moreover, the strong TS expression was found to be an independent factor for tumor recurrence based on logistic regression models. Therefore, the strong TS expression was associated with a poorer DFS. The strong TS expression was independently associated with an increased risk for a poor DFS. This finding suggests that the TS expression is a suitable biomarker to identify those candidates who would benefit most from adjuvant chemotherapy in adenocarcinoma following a complete resection. Interestingly, a previous study reported that patients with TS negative tumors treated with surgery and UFT had significantly better survival than the patients with TS positive tumors [23]. In this study, two patients received UFT treatment as adjuvant chemotherapy. Each patient showed a positive and negative expression of TS and they were alive and cancer-free. Recently, pemetrexed, which is a new generation antifolate, has been reported to be effective for the treatment of advanced mesothelioma and non-sqamous cell carcinoma of NSCLC [10,24]. In fact, TS is also the main cellular target of not only 5-fluorouracil but also pemetrexed. Therefore, our findings might indicate that TS expression may not only predict tumor recurrence, but it may also have an influence on the efficacy of individual therapy using TS inhibitors in either an adjuvant or intensive setting. In conclusion, the current results indicate that the TS expression may therefore be a useful marker for both predicting postoperative recurrence in patients with lung adenocarcinoma following surgery. Further investigations will therefore be necessary to examine the method of detecting TS expression [25,26], validation, the
We hereby declare that we have no conflicts of interest. Acknowledgements We thank Kanako Sasaki for their valuable technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. References [1] Minna JD, Roth JA, Gazdor AF. Focus on lung cancer. Cancer Cell 2002;1:49–52. [2] Hoffman PC, Mauer AM, Vokes EE. Lung cancer. Lancet 2000;355:479–85. [3] Asamura H, Goya T, Koshiishi Y, Sohara Y, Eguchi K, Mori M, et al. A Japanese lung cancer registry study: prognosis of 13,010 resected lung cancers. J Thorac Oncol 2008;3:46–52. [4] Arriagada R, Auperin A, Burdett S, Higgins JP, Johnson DH, Le Chevalier T, et al. Adjuvant chemotherapy, with or without postoperative radiotherapy, in operable non-small-cell lung cancer: two meta-analyses of individual patient data. Lancet 2010;375:1267–77. [5] Pignon JP, Tribodet H, Scagliotti GV, Douillard JY, Shepherd FA, Stephens RJ, et al. Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group. J Clin Oncol 2008;26:3552–9. [6] Nakagawa T, Otake Y, Yanagihara K, Miyahara R, Ishikawa S, Fukushima M, et al. Expression of thymidylate synthase is correlated with proliferative activity in non-small cell lung cancer (NSCLC). Lung Cancer 2004;43:145–9. [7] Yeh KH, Shun CT, Chen CL, Lin JT, Lee WJ, Lee PH, et al. High expression of thymidylate synthase is associated with the drug resistance of gastric carcinoma to high dose 5-fluorouracil-based systemic chemotherapy. Cancer 1998;82:1626–31. [8] Harpole Jr DH, Moore MB, Herndon II JE, Aloia T, D’Amico TA, Sporn T, et al. The prognostic value of molecular marker analysis in patients treated with trimodality therapy for esophageal cancer. Clin Cancer Res 2001;7:562–9. [9] Edler D, Kressner U, Ragnhammar P, Johnston PG, Magnusson I, Glimelius B, et al. Immunohistochemically detected thymidylate synthase in colorectal cancer: an independent prognostic factor of survival. Clin Cancer Res 2000;6:488–92. [10] Scagliotti G, Hanna N, Fossella F, Sugarman K, Blatter J, Peterson P, et al. The differential efficacy of pemetrexed according to NSCLC histology: a review of two Phase III studies. Oncologist 2009;14:253–63. [11] Yamashita T, Uramoto H, Onitsuka T, Ono K, Baba T, So T, et al. Association between lymphangiogenesis-/micrometastasis- and adhesionrelated molecules in resected stage I NSCLC. Lung Cancer; in press. doi:10.1016/j.lungcan.2010.02.013. [12] Onitsuka T, Uramoto H, Nose N, Takenoyama M, Hanagiri T, Sugio K, et al. Acquired resistance to gefitinib: the contribution of mechanisms other than the T790M, MET, and HGF status. Lung Cancer 2010;68:198–203. [13] Onitsuka T, Uramoto H, Ono K, Takenoyama M, Hanagiri T, Oyama T, et al. Comprehensive molecular analyses of lung adenocarcinoma with regard to the epidermal growth factor receptor, K-ras, MET, and hepatocyte growth factor status. J Thorac Oncol 2010;5:591–6. [14] Toi M, Ikeda T, Akiyama F, Kurosumi M, Tsuda H, Sakamoto G, et al. Predictive implications of nucleoside metabolizing enzymes in premenopausal women with node-positive primary breast cancer who were randomly assigned to receive tamoxifen alone or tamoxifen plus tegafur–uracil as adjuvant therapy. Int J Oncol 2007;31:899–906. [15] Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111:1710–7. [16] Christofori G. New signals from the invasive front. Nature 2006;441: 444–50.
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Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
TS expression predicts postoperative recurrence in adenocarcinoma of the lung Hidehiko Shimokawa, Hidetaka Uramoto ∗ , Takamitsu Onitsuka, Teruo Iwata, Makoto Nakagawa, Kenji Ono, Takeshi Hanagiri Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
a r t i c l e
i n f o
Article history: Received 11 May 2010 Received in revised form 19 July 2010 Accepted 8 August 2010 Keywords: TS DHFR Recurrence Lung cancer Adenocarcinoma Survival
a b s t r a c t Background: Not all patients with lung cancer require postoperative adjuvant chemotherapy after a complete resection. However, no useful markers for either selecting appropriate candidates or for predicting clinical recurrence exist. Methods: Tumor specimens were collected from 183 consecutive patients who underwent a complete resection for lung adenocarcinoma from 2003 to 2007 in our department. We analyzed the thymidylate synthase (TS) and dihydrofolate reductase (DHFR) expressions in the primary lung adenocarcinoma by immunohistochemisty. Results: The strong expression of TS and DHFR was identified in 39 (21.3%) and 120 (65.6%) patients, respectively. The strong TS expression was identified in 11 (39.3%) of 28 patients and 28 (18.1%) of 155 patients in patients with and without recurrence, respectively (p = 0.012). The strong DHFR expression was also identified in 23 (82.1%) and 97 (62.6%) of the patients with and without recurrence, respectively (p = 0.045). Logistic regression models indicated the strong TS expression to be an independent factor for tumor recurrence. The strong TS and DHFR expression was associated with a poorer disease-free survival (DFS) according to the survival analysis. A multivariate analysis demonstrated the strong TS expression to be independently associated with an increased risk for poor DFS. Conclusions: The strong TS expression may be a useful marker for predicting postoperative recurrence in patients with lung adenocarcinoma following surgery. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is the leading cause of cancer-related deaths in the majority of world wide countries [1]. Non-small cell lung cancers (NSCLC) account for approximately 80% of all lung cancers, and the proportion of adenocarcinoma is notably increasing [2]. Although surgery is the only mainstay of treatment to obtain a cure for this disease, approximately 30% of all patients with pathological stage I NSCLCs have tumor recurrence and eventually succumb to the cancer, despite a complete surgical resection [3]. Recently, the benefits of adjuvant chemotherapy have been confirmed [4]. However, the 5-year survival rate of patients with resected NSCLCs is reported to be as high as 60% without adjuvant chemotherapy, thus suggesting that not all patients require chemotherapy after a complete resection [4]. Therefore, it is necessary to identify the patients who might benefit the most from postoperative adjuvant chemotherapy to not only precisely select the patients who require additional treatment,
but also to prevent the occurrence of adverse events in patients who do not require treatment [5]. As a result, it is important to evaluate the biological and molecular characteristics of NSCLC to identify the factors related to recurrence following surgery. However, no useful markers for predicting clinical recurrence exist at present. TS is an essential enzyme for de novo DNA synthesis and the TS expression status has been shown to correlate with the proliferative activity of cancer cells [6]. Previous clinical studies have also demonstrated that high TS expression is associated with a poor prognosis in a variety of malignant tumors [7–9], but the exact clinical reason of the poor prognosis remains unclear. DHFR is also a key enzyme in nucleotide synthesis and DNA replication. Furthermore, TS and DHFR are target enzymes that have been shown to be involved in purine and pyrimidine synthesis for antifolate inhibitors [10]. Therefore, we hypothesized that TS and DHFR may also be useful predictive indicators of tumor recurrence in patients following lung resection. 2. Materials and methods
Abbreviations: TS, thymidylate synthase; DHFR, dihydrofolate reductase; DFS, disease free survival; NSCLC, non-small cell lung cancer; IHC, immunohistochemical; UFT, tegafur–uracil; PS, proportional score; IS, intensity score; OR, odds ratio; 95% CI, 95% confidence interval. ∗ Corresponding author. Tel.: +81 93 691 7442; fax: +81 93 692 4004. E-mail address: [email protected] (H. Uramoto). 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.08.024
2.1. Patients, clinical features, and follow-up The institutional review board approved this study and informed consent for the use of the tumor specimens was obtained
H. Shimokawa et al. / Lung Cancer 72 (2011) 360–364
either from all the patients or their legal guardians. Tumor samples were obtained from 296 patients with primary lung adenocarcinoma who had undergone a surgical resection between 2003 and 2007 in our department. Nine of these patients were stage IV and 25 underwent an incomplete resection. The tumor samples from 79 patients were too small to evaluate by immunohistochemical (IHC) staining for TS and DHFR status. As a result, 113 patients were excluded from further analysis. Therefore, 183 tumor specimens were evaluated. The patients were followed-up every month within the first postoperative year and at approximately 2- to 4-month intervals thereafter. The evaluations included a physical examination, chest roentgenography, an analysis of blood chemistry, and measurements of tumor markers. Chest and abdominal computed tomography, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years after surgery. Additional examinations were performed if any symptoms or signs of recurrence were detected. A follow-up was conducted in all patients. The median follow-up period was 34.2 months. Twentyseven (15.3%) patients had received adjuvant chemotherapy as follows: carboplatin plus paclitaxel (n = 18), carboplatin plus gemcitabine (n = 7), and tegafur–uracil (UFT) (n = 2). At the last follow-up, 144 patients were alive and free of cancer, while 11 patients had died of other causes without evidence of cancer, 12 patients were alive with recurrent cancer, and 16 patients had died of cancer. In total, 28 (15.3%) of the 183 patients demonstrated disease recurrence after surgery.
2.2. IHC staining in paraffin-embedded tumor samples IHC staining was conducted using serial sections from the same paraffin-embedded blocks by previously described methods [11,12]. Briefly, all tissue specimens were formalin-fixed and processed similarly, according to the standard histology practices. A 3 m-thick formalin-fixed, paraffin-embedded tissue section was prepared from each specimen. All specimens were stained with hematoxylin–eosin for the histological diagnosis. The sections were briefly immersed in citrate buffer [0.01 mol/L citric acid (pH 6.0)] and then were incubated for two 10 min at 121 ◦ C in a high-pressure sterilization oven for antigen retrieval. They were then incubated with the TS (TS106, Santa Cruz Biothechnology, CA) diluted at 1:25, and DHFR (ab82171, Abcam, Cambridge, MA) diluted at 1:50, respectively, in phosphate-buffered saline 60 min
361
in a room-temperature. Thereafter, IHC staining was performed by the labeled polymer method (Histofine Simple Stain MAX-PO kit, Nichirei, Tokyo, Japan) according to the manufacturer’s instructions [12,13]. The positive and the negative controls were processed by the HeLa cells and the exclusion of the primary antibody, respectively. 2.3. Evaluation of the stained specimens Following the IHC detection of protein expression in each specimen, the percentage of immunoreactive tumor cells in five 400× fields selected randomly on one slide was recorded, and then the final value of positive tumor cells was determined as the average of the positive immunostained cells. To evaluate any correlations with the clinicopathological characteristics, these protein expression scores were divided into positive or negative groups. The stained specimens for cytoplasm of cancer cells were then categorized into eight degrees according to previous report [14]. Initially, six degrees of the proportional score (PS) for the positive staining cells were assigned according to the frequency of positive tumor cells (0, none; 1, <1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5, >2/3). Thereafter, four degrees for the intensity score (IS) were assigned according to the intensity of the staining (0, none; 1, weak; 2, intermediate; and 3, strong). The PS and the IS were then added to each other to obtain a total score, which ranged from 0 to 8. According to the total score, the TS and DHFR of the tumor was categorized as a weak expression when the score was 0–4 and a strong expression when the score was 5–8. The slides were independently examined by two of the investigators (H.S. and T.O.) who were blinded to the clinicopathological data. When a discrepancy was found between the two investigators, a consensus was reached via their simultaneous examination using a double-headed microscope. 2.4. Statistical analyses Statistical significance was evaluated using the chi-square test or Fisher’s exact test. The A multivariate logistic regression was used to evaluate independent associations. Kaplan-Meier method was used to estimate the probability of survival, and survival differences were analyzed by the log-rank test. A multivariate analysis was then performed according to Cox’s proportional hazards model. The odds ratio (OR) and 95% confidence interval (95% CI)
Table 1 Relationships between the molecular parameters and clinicopathological characteristics. Variables
Gender Age (y) Pathologic stage T status N status Smoking Differentiationc CEAc a b c
Category
Male Female <69 ≥69 I II–III T1 T2–4 Negative Positive C/Ea Nb Well No-well <2.5 ≥2.5
C/E: current/ever. N: never smoker. Unclassified patients were excluded.
No. of patients n = 183
102 81 80 103 148 35 128 55 152 31 113 70 93 74 122 58
TS expression
DHFR expression
Strong (%) 39 (21.3)
Weak 144
Strong (%) 120 (65.6)
Weak 63
23 (22.5) 16 (19.8) 25 (31.3) 14 (13.6) 28 (18.9) 11 (31.4) 26 (20.3) 13 (23.6) 30 (19.7) 9 (29.0) 29 (25.7) 10 (14.3) 10 (1.1) 26 (35.1) 29 (23.8) 10 (17.2)
79 65 55 89 120 24 102 42 122 22 84 60 83 48 93 48
89 (87.3) 51 (63.0) 47 (58.8) 73 (70.9) 93 (62.8) 27 (77.1) 79 (61.7) 41 (74.5) 96 (63.2) 24 (77.4) 76 (67.3) 44 (62.9) 59 (63.4) 51 (68.9) 78 (63.9) 40 (69.0)
33 30 33 30 55 8 49 14 56 7 37 26 34 23 44 18
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H. Shimokawa et al. / Lung Cancer 72 (2011) 360–364 Table 3 Univariate analysis of the factors contributing to the recurrence.
Table 2 Relationships between the TS/DHFR expression and recurrence. Variables
Cases with recurrence Cases without recurrence $
DHFR expression
Variables
OR
Strong (%)
TS expression Weak
Strong (%)
Weak
11(39.3)$1 28 (18.1)
17 127
23 (82.1)$2 97 (62.6)
5 58
Gender: male Age: ≤69 pT: 2–4 pN: positive TS expression: strong DHFR expression: strong
1.070 0.854 3.322 8.130 2.933 2.747
Statistically significant: p-value = 0.012$1 , 0.045$2 .
were calculated for each variable. Differences were considered to be statistically significant for p-values < 0.05. The data were analyzed using the Stat View software package (Abacus Concepts, Inc., Berkeley, CA). 3. Results 3.1. Detection of the TS and DHFR expression and clinicopathological factors
95% CI 0.475–2.410 0.249–1.376 1.453–7.576 3.311–20.0 1.240–6.944 0.991–7.634
p-Value 0.871 0.218 0.004 <0.001 0.014 0.052
OR: odds ratio, 95% CI: 95% confidence interval.
Table 4 Multivariate analysis of the factors contributing to the recurrence. Variables
OR
95% CI
Gender: male Age: ≤69 pT: 2–4 pN: positive TS expression: strong DHFR expression: strong
1.083 0.552 2.604 6.410 2.695 2.237
0.428–2.747 0.205–1.486 1.029–6.579 2.481–16.677 1.022–7.092 0.735–6.803
p-Value 0.865 0.240 0.043 <0.001 0.048 0.138
All of the patients were Japanese, consisting of 102 males and 81 females in this series, with a mean age of 68.5 years (range 23–88 years). The tumor stage was classified according to the Revisions in the International System for Staging Lung Cancer [15]. According to the pathological stage, 113 patients were at stage IA, 35 at IB, 7 at IIA, 10 at IIB, 15 at IIIA, and 3 at IIIB. TS and DHFR expression was identified in 39 (21.3%) and 120 (65.6%) patients, respectively. No significant association of TS or DHFR expression was identified with the clinical factors (Table 1). The majority of the sites of tumor recurrence were hematogenous metastases. Twenty-five and 6 cases had hematogenous (9: brain, 10: lung, 5: bone, and 1: adrenal metastasis) and locoregional (4: Lymph node metastasis and 2: pleural dissemination) recurrences, respectively. Two, one, and one subject had recurrent tumors in both brain and bone, brain and adrenal, and bone and lymph nodes, respectively. 3.2. Influence of TS and DHFR expression and the clinicopathological factors on recurrence The strong TS expression was identified in 11 (39.3%) of 28 patients and 28 (18.1%) of 155 patients in patients with and without recurrence, respectively (p = 0.012) (Table 2). The strong DHFR expression was also identified in 23 (82.1%) and 97 (62.6%) of the patients with and without recurrence, respectively (p = 0.045). The strong TS and DHFR expression were significantly correlated with postoperative recurrence. The logistic regression models indicated the strong TS expression, but not the DHFR expression, to be an independent predictor for recurrence, as were the pathological T and N statuses (Tables 3 and 4). 3.3. Influence of TS and DHFR expression and clinicopathological factors on DFS The 5-year DFS rate with weak and strong for TS expression was 85.6% and 69.9%, respectively (p = 0.012) (Fig. 1A). The 5-year
Fig. 1. Kaplan–Meier DFS curves stratified by TS (A) and DHFR (B) expression. The heavy and narrow lines show the strong and weak expressions of the TS and DHFR expressions, respectively.
DFS rate with weak and strong for DHFR expression was 91.0% and 78.0%, respectively (p = 0.043) (Fig. 1B). The strong TS and DHFR expression was associated with a poorer DFS according to the univariate survival analysis (p = 0.016) (Table 5). A multivariate survival analysis demonstrated the strong TS expression to be
Table 5 Univariate analysis using a proportional hazard model for the disease-free survival. Variables
Gender Age (y) T status N status TS expression DHFR expression
Characteristics
Hazard ratio
Unfavorable
Favorable
Male ≤69 T2–4 Positive Strong Strong
Female >69 T1 Negative Weak Weak
1.112 0.655 3.236 6.494 2.551 2.611
95% CI
0.526–2.353 0.302–1.420 1.541–6.803 3.077–13.699 1.193–5.464 0.993–6.897
p-Value
0.780 0.284 0.002 <0.001 0.016 0.052
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Table 6 Multivariate analysis using a proportional hazard model for the disease-free survival. Variables
Gender Age (y) T status N status TS expression DHFR expression
Characteristics
Hazard ratio
Unfavorable
Favorable
Male ≤69 T2–4 Positive Strong Strong
Female >69 T1 Negative Weak Weak
1.035 0.622 2.320 4.739 2.222 1.838
95% CI
0.483–2.217 0.281–1.377 1.072–5.00 2.212–10.204 1.027–4.808 0.669–5.051
p-Value
0.930 0.242 0.033 <0.001 0.043 0.238
independently associated with an increased risk for a poor DFS with a marginal p-value (Table 6).
meaning of histological differences [27], and efficacy of adjuvant chemotherapy [28,29] in a prospective trial.
4. Discussion
Conflicts of interest
Metastasis is the most frequent cause of treatment failure [16] and occult metastases present in definite proportions at the time of surgical intervention [11]. The clinical findings and current tissuebased molecular markers are insufficient for the early identification of individuals at high risk for metastasis, in spite of recent developments in the field of molecular biology [17]. Therefore, novel strategies for the prevention of metastasis are urgently required to improve the prognosis, quality of life, and cost for treating cancer patients. The TS activity is necessary for cancer cell proliferation through the essential DNA synthesis. In fact, TS exhibits an oncogenic activity [18] and the overexpression of TS is an independent factor for a poor prognosis in NSCLC patients [19,20]. However, the precise reason from a clinical standpoint for a poor prognosis still remains unknown and there is no other established biomarker that is correlated to postoperative recurrence clinically. Recent studies reported the possibility that chemokine receptors 7 and angiopoietin-1 are potential markers of for predicting disease-free survival and recurrence in patients with early stage NSCLC [21,22]. In the present study, we evaluated the correlations between the TS and DHFR expressions and recurrence in lung adenocarcinoma. The TS and DHFR expressions were thus found to significantly correlate with recurrence after the operation. Moreover, the strong TS expression was found to be an independent factor for tumor recurrence based on logistic regression models. Therefore, the strong TS expression was associated with a poorer DFS. The strong TS expression was independently associated with an increased risk for a poor DFS. This finding suggests that the TS expression is a suitable biomarker to identify those candidates who would benefit most from adjuvant chemotherapy in adenocarcinoma following a complete resection. Interestingly, a previous study reported that patients with TS negative tumors treated with surgery and UFT had significantly better survival than the patients with TS positive tumors [23]. In this study, two patients received UFT treatment as adjuvant chemotherapy. Each patient showed a positive and negative expression of TS and they were alive and cancer-free. Recently, pemetrexed, which is a new generation antifolate, has been reported to be effective for the treatment of advanced mesothelioma and non-sqamous cell carcinoma of NSCLC [10,24]. In fact, TS is also the main cellular target of not only 5-fluorouracil but also pemetrexed. Therefore, our findings might indicate that TS expression may not only predict tumor recurrence, but it may also have an influence on the efficacy of individual therapy using TS inhibitors in either an adjuvant or intensive setting. In conclusion, the current results indicate that the TS expression may therefore be a useful marker for both predicting postoperative recurrence in patients with lung adenocarcinoma following surgery. Further investigations will therefore be necessary to examine the method of detecting TS expression [25,26], validation, the
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