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ERCC1 Expression as a Prognostic Marker in N2(1) Nonsmall-Cell Lung Cancer Patients Treated With Platinum-based Neoadjuvant Concurrent Chemoradiotherapy In Gyu Hwang, MD1 Myung Ju Ahn, MD1 Byeong Bae Park, MD1 Yong Chan Ahn, MD, PhD2 Joungho Han, MD3 Seungkoo Lee, MD4 Jhingook Kim, MD5 Young Mog Shim, MD5 Jin Seok Ahn, MD1 Keunchil Park, MD1 1
Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 2
Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 3
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea. 4 Department of Pathology, Kangwon National University Hospital, Kangwon National University School of Medicine, Chunchon, Kangwon, Republic of Korea. 5
Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea.
BACKGROUND. Excision repair cross-complementation Group 1 (ERCC1) overexpression is associated with resistance to cisplatin-based chemotherapy in patients with nonsmall-cell lung cancer (NSCLC). A preliminary study also suggested that ERCC1 expression is associated with radioresistance in lung cancer cells. The aim of this study was to evaluate the clinical implications of ERCC1 expression in stage IIIA N2-positive NSCLC patients treated with platinum-based neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery.
METHODS. Sixty-eight patients with mediastinoscopy-proven N2-positive NSCLC were enrolled between August 1997 and September 2003. ERCC1 expression was assessed by immunohistochemistry from pretreatment mediastinoscopic biopsy specimens.
RESULTS. ERCC1 expression was positive in 31 of 68 specimens (46%). Among 14 patients who obtained pathologic complete response, 6 were positive for ERCC1 expression and 8 were negative (P 5 .818). On univariate analysis, with median follow-up of 61.8 months (range, 34.3-108.8 months), progression-free survival was 15.9 months for ERCC1-positive and 29.5 months for ERCC1-negative patients (P 5 .062), and there was a statistically significant difference in overall survival between ERCC1-negative tumors and ERCC1-positive tumors (89.2 vs 26.0 months, P 5 .014). On multivariate analysis, ERCC1 negativity (P 5 .041) and achieving mediastinal nodal clearance (downstage to pathological N0 or N1) after neoadjuvant CCRT followed by surgery (P 5 .005) were significant independent prognostic factors for the prolongation of survival.
CONCLUSIONS. These results suggest that N2-positive NSCLC patients with ERCC1 negative tumors show a survival benefit from neoadjuvant CCRT with a platinum-containing regimen. Cancer 2008;113:1379–86. 2008 American Cancer Society.
In Gyu Hwang’s current address: Division of Hematology-Oncology, Department of Internal Medicine, Chung-Ang University Medical Center, Chung-Ang University, School of Medicine, Seoul, Korea. Byeong Bae Park’s current address: Division of Hematology-Oncology, Department of Internal Medicine, Hanyang University College of Medicine, Seoul, Korea. Address for reprints: Myung-Ju Ahn, MD, PhD, Division of Hemato-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwondong, Gangnam-gu, Seoul 135-710 Korea; Fax: (011) 82-2-3410-0041; E-mail:
[email protected] Received December 27, 2007; revision received April 12, 2008; accepted April 15, 2008.
KEYWORDS: ERCC1, nonsmall-cell lung cancer, neoaduvant concurrent chemoradiotherapy, prognosis.
N
onsmall-cell lung cancer (NSCLC) is the commonest cause of cancer deaths worldwide.1 Although surgical resection plays a major role in curative treatments, less than 20% of NSCLC patients present with operable stage disease. In locally advanced stage IIIA disease (pT1-3N2M0), the 5-year cumulative survival rate after resection is 23%.2 Recent data show that survival with locally advanced NSCLC can be improved by the addition of chemotherapy to radiotherapy and/or surgery.3-5 The cytotoxic effects of platinumbased anticancer drugs are attributed to the formation of bulky
ª 2008 American Cancer Society DOI 10.1002/cncr.23693 Published online 12 July 2008 in Wiley InterScience (www.interscience.wiley.com).
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platinum-DNA adducts and interstrand cross-links. The nucleotide excision repair pathway has a central role in DNA repair and is associated with resistance to platinum-based chemotherapy.6-9 Excision repair cross-complementation Group 1 (ERCC1) plays a key role in nucleotide excision repair and removes platinum-induced DNA adducts.10-12 Several clinical studies have shown that ERCC1 expression is associated with resistance to platinum-based chemotherapy and poor prognoses in several tumors, including NSCLC.13-17 A preliminary study also suggested that ERCC1 expression is associated with radioresistance in lung cancer cells.18 This study was conducted to evaluate the association between ERCC1 expression and clinical benefits such as pathologic complete responses, mediastinal lymph node clearance, progression-free survival (PFS), and overall survival (OS) in stage IIIA N2-positive NSCLC patients treated with platinum-based neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery.
MATERIALS AND METHODS Patients and Treatment Eighty-three patients with mediastinoscopic biopsyproven N2-positive NSCLC were treated with CCRT followed by surgery between August 1997 and December 2003. All specimens were collected by mediastinoscopic lymph node biopsy before any therapy. Sixty-eight tissue samples from the 83 patients were adequate for ERCC1 expression analysis. All the patients had histologically or cytologically confirmed NSCLC and mediastinoscopic biopsy-proven stage IIIA, N2 disease. Their Eastern Cooperative Oncology Group performance status was 0 to 1. Patients selected for the study had adequate bone marrow, liver, and renal functions. None of them had undergone any previous oncological treatment. Neoadjuvant CCRT included chemotherapy and concurrent thoracic radiotherapy. The chemotherapy regimens consisted of weekly paclitaxel (50 mg/m2 per week intravenously [iv]) or docetaxel (20 mg/m2 per week iv) plus cisplatin (25 mg/m2 per week iv) or carboplatin (AUC 1.5/week iv) for 5 weeks (from March 2001 to September 2003) and cisplatin (60-100 mg/m2 iv, Day 1) plus oral etoposide (50 mg/m2 per os, Day 1-14) every 4 weeks (from September 1997 to March 2001). The radiation dose was 45 grays (Gy) over 5 weeks (1.8 Gy/fraction per day, 5 fractions/ week) using 10-MV x-rays. Surgical resection was planned at around 4 weeks after the completion of neoadjuvant CCRT (Fig. 1). Postoperative radiation therapy (18 Gy in 10 fractions) was administered in the event of persistent histologically positive N2
FIGURE 1. The scheme of treatments is shown. CCRT indicates concurrent chemoradiotherapy; RT, radiotherapy; wks, weeks; cGY, centigrays; d, day; IV, intravenously; D1, Day 1; PO, per os; AUC, area under the curve.
lymph nodes, positive multi-N1 lymph nodes, or a positive resection margin at the time of surgery.
Assessments The pretreatment evaluation included the patient’s history, physical examination, performance status, chest x-ray, complete blood count, blood chemistry, computed tomography (CT) scan of the chest, whole-body bone scan, and fiberoptic bronchoscopy. Surgical staging was performed by 1 of 2 experienced thoracic surgeons (Y.M.S. and J.K., 19 and 14 years of experience, respectively). During mediastinoscopy, the surgeons routinely examined the American Thoracic Society (ATS) lymph node map areas of 2R, 4R, 2L, 4L, and 7; during thoracotomy, they dissected all visible and palpable lymph nodes accessible in the mediastinum according to our routine surgical protocol. All encountered lymph nodes were removed from 10R, 9, 8, 7, 4R, 3, and 2R ATS lymph node map areas in tumors of the right lung and from areas 10L, 9, 8, 7, 6, 5, and 4L of the left lung. When necessary, especially when imaging results suggested possible nodal metastasis in nodal stations other than routine lymph node dissection, Group 1 (highest mediastinal) or 2L (tumors located in the left lung) nodes were also evaluated during mediastinoscopy or thoracotomy. Surgeons labeled dissected lymph nodes by numbering the nodes according to the lymph node map definition for lung cancer staging proposed by Mountain and Dresler.19 Subsequently, a pathologist assessed nodes numbered in surgical fields and recorded the presence or absence of tumor in these nodes. The pathologist also described tumors (ie, histopathologic types, surrounding organ involvement, necrosis, fibrosis, tumor volume, and distance from resection margin). Restaging procedures with
ERCC1 as a Prognostic Marker in NSCLC/Hwang et al
complete clinical, radiological evaluations including CT scan of the chest were performed after CCRT; clinical responses to CCRT were assessed according to the World Health Organization criteria. Pathological responses (complete pathologic response, mediastinal lymph node clearance) were evaluated after surgery and the completion of CCRT. Complete pathologic response was defined as 95% necrosis and fibrosis, and mediastinal lymph node clearance as down-staged to pN0-1 after CCRT.20 Patients who underwent surgery were evaluated by chest CT every 3 months for 2 years after surgery, and then every 6 months thereafter. Approval was obtained from the institutional review boards, according to legal requirements.
Immunohistochemistry for ERCC1 Expression Mediastinoscopic biopsy specimens before any therapy were analyzed for ERCC1 expression. Formalinfixed paraffin-embedded tissue blocks were sectioned to 4 lm thickness. The tissue sections were deparaffinized in xylene and then rehydrated in serially graded alcohol. ERCC1 antigen retrieval consisted of heating in 10 mM citrate buffer at pH 6.0 with microwaves (15 minutes, 700 W), and cooling to room temperature for 20 minutes. After washing in Tris-buffered saline (TBS), the slides were preincubated in 5% normal blocking solution (goat serum) for 10 minutes to reduce nonspecific binding. The slides were incubated overnight at room temperature with mouse monoclonal anti-ERCC1 (8F1; Neomarkers, Fremont, Calif) at a 1:200 dilution in a humidified chamber. The primary antibody was observed with an avidinbiotin complex (ABC) system (Dako, Carpinteria, Calif). After further washing in TBS, each biotinylated goat antimouse immunoglobulin G at a dilution of 1:100 was added to the slide, which was incubated for 20 minutes at room temperature. Finally, the sections were washed again in TBS and incubated for 10 minutes in a solution of streptavidin-ABC-horseradish peroxidase at a dilution of 1:100. Color development was achieved using 3,30 -diaminobenzidine tetrahydrochloride (Immunotech, Cedex, France). Finally, the sections were counterstained with Mayer hematoxylin. Negative controls were processed as above without the primary antibody. Evaluation of Immunostaining Immunohistochemical evaluations were performed independently by 2 pathologists (J.H. and S.L.) with no knowledge of the clinical data. ERCC1 immunoreactivity levels in each case were assessed semiquantitatively under a light microscope. Representative areas of the images were acquired at a magnification
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of 3400 for each specimen. Tumor nuclear staining intensity was graded on a scale of 0 to 3 using adjacent nonmalignant cells as a reference (intensity 2). Discordant cases were reviewed. A total of 100 to 1500 positive or negative tumor nuclei per specimen were counted manually. The percentage of positive tumor nuclei was calculated for each specimen, and a proportional score was given (0 if 0%, 0.1 if 1%-9%, 0.5 if 10%-49%, and 1.0 if 50%), as previously described.21,22 Then this proportional score was multiplied by the staining intensity of nuclei to obtain a final semiquantitative H score. ERCC1-positive tumors were defined as more than the median value of all the H scores.
Statistical Analysis Clinical and pathological variables were compared across groups using the v2 or Fisher exact tests for categorical variables. The duration of OS was calculated from the first day of neoadjuvant CCRT until the date of death or the most recent documented followup. PFS was calculated from the first day of neoadjuvant CCRT to the day when disease progression was recognized or the day of the last follow-up visit. Patients who were alive at the last follow-up were censored at that time. Patients who were taken off study or who died before progression were censored at the time that they were taken off study. OS and PFS were estimated with the Kaplan-Meier method. Comparisons between groups were performed using the logrank test for time-to-event variables. P-values less than .05 were considered statistically significant. Multivariate analysis of the independent predictive or prognostic factors for survival was performed using the forward stepwise (likelihood-ratio statistics based on the conditional parameter estimate) method of the Cox proportional hazard regression model with 95% confidence intervals (CIs).
RESULTS Characteristics of the Patients The median age of the patients was 58 years (range, 33-72 years), and 71% were men. Forty-one patients had adenocarcinomas (60%), and 26 had squamouscell carcinomas (38%). The chemotherapy regimens were as follows: 32 patients were treated with oral etoposide plus cisplatin combination chemotherapy (47%), 29 with taxane (28 paclitaxel and 1 docetaxel) plus cisplatin (43%), and 7 with paclitaxel plus carboplatin (10%) (Table 1). Thoracic radiotherapy was administered as planned to all patients (100%). The median duration of radiotherapy was 36 days (range, 32-47 days).
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TABLE 1 Patient Characteristics
Characteristic Sex Men Women Age, y
