FIBROBLAST GROWTH FACTOR 2 A PREDICTOR OF OUTCOME FOR PATIENTS IRRADIATED FOR STAGE II-III NON SMALL-CELL LUNG CANCER

Transcription

1 doi: /j.ijrobp Int. J. Radiation Oncology Biol. Phys., Vol. 82, No. 1, pp , 2012 Copyright Ó 2012 Elsevier Inc. Printed in the USA. All rights reserved /$ - see front matter CLINICAL INVESTIGATION Thoracic Cancer FIBROBLAST GROWTH FACTOR 2 A PREDICTOR OF OUTCOME FOR PATIENTS IRRADIATED FOR STAGE II-III NON SMALL-CELL LUNG CANCER DIRK RADES, M.D.,* CORNELIA SETTER, M.D.,* OLAV DAHL, M.D., PH.D., yz STEVEN E. SCHILD, M.D., x AND FRANK NOACK, M.D. { *Department of Radiation Oncology and { Institute of Pathology, University of Lubeck, Lubeck, Germany; y Section of Oncology, Institute of Medicine, University of Bergen, Bergen, Norway; z Department of Oncology, Haukeland University Hospital, Bergen, Norway; and x Department of Radiation Oncology, Mayo Clinic Scottsdale, Scottsdale, Arizona Purpose: The prognostic value of the tumor cell expression of the fibroblast growth factor 2 (FGF-2) in patients with non small-cell lung cancer (NSCLC) is unclear. The present study investigated the effect of tumor cell expression of FGF-2 on the outcome of 60 patients irradiated for Stage II-III NSCLC. Methods and Materials: The effect of FGF-2 expression and 13 additional factors on locoregional control (LRC), metastasis-free survival (MFS), and overall survival (OS) were retrospectively evaluated. These additional factors included age, gender, Karnofsky performance status, histologic type, histologic grade, T and N category, American Joint Committee on Cancer stage, surgery, chemotherapy, pack-years, smoking during radiotherapy, and hemoglobin during radiotherapy. Locoregional failure was identified by endoscopy or computed tomography. Univariate analyses were performed with the Kaplan-Meier method and the Wilcoxon test and multivariate analyses with the Cox proportional hazard model. Results: On univariate analysis, improved LRC was associated with surgery (p =.017), greater hemoglobin levels (p =.036), and FGF-2 negativity (p <.001). On multivariate analysis of LRC, surgery (relative risk [RR], 2.44; p =.037), and FGF-2 expression (RR, 5.06; p <.001) maintained significance. On univariate analysis, improved MFS was associated with squamous cell carcinoma (p =.020), greater hemoglobin levels (p =.007), and FGF-2 negativity (p =.001). On multivariate analysis of MFS, the hemoglobin levels (RR, 2.65; p =.019) and FGF-2 expression (RR, 3.05; p =.004) were significant. On univariate analysis, improved OS was associated with a lower N category (p =.048), greater hemoglobin levels (p <.001), and FGF-2 negativity (p <.001). On multivariate analysis of OS, greater hemoglobin levels (RR, 4.62; p =.002) and FGF-2 expression (RR, 3.25; p =.002) maintained significance. Conclusions: Tumor cell expression of FGF-2 appeared to be an independent negative predictor of LRC, MFS, and OS. Ó 2012 Elsevier Inc. Non small-cell lung cancer, radiotherapy, fibroblast growth factor 2, FGF-2 expression, prognostic factors, treatment outcomes. INTRODUCTION Lung cancer is the leading cause of cancer-related death worldwide (1). About 80% of all lung cancers are non small-lung cancer (NSCLC). Thus, the improvement of treatment strategies for NSCLC is of great interest for both patients and treating physicians. Angiogenesis, the formation of new blood vessels, plays a major role in tumor progression (2). The anti-angiogenic antibody bevacizumab and the small molecule erlotinib, an inhibitor of the epidermal growth factor receptors, have been approved for the treatment of advanced NSCLC (3). The fibroblast growth factor (FGF) family is also involved in angiogenesis, wound healing, and embryonic development (4). In particular, the basic fibroblast growth factor, b-fgf, also known as FGF-2, has been demonstrated to stimulate angiogenesis. However, the prognostic effect of FGF-2 in patients with NSCLC is unclear. A few retrospective studies have suggested that tumor cell expression of FGF-2 is associated with poor survival (5 9). In contrast, other retrospective studies did not find such an association (10, 11). The most recent analysis that suggested a negative association between tumor cell expression of FGF-2 and survival included mostly (63%) patients with Stage I disease who had undergone surgery for NSCLC (8). Only 18% of the patients of that analysis had undergone postoperative radiotherapy (RT). In the present analysis, the tumors were more advanced, with 77% of the patients having Stage III disease. Furthermore, this is the first study to investigate the potential Reprint requests to: Dirk Rades, M.D., Department of Radiation Oncology, University of Lubeck, Ratzeburger Allee 160, Luebeck Germany. Tel: (0049) ; Fax: (0049) ; Rades.Dirk@gmx.net 442 Conflict of interest: none. Received May 19, 2010, and in revised form Aug 19, Accepted for publication Aug 23, 2010.

2 Effect of FGF-2 in patients irradiated for NSCLC d D. RADES et al. 443 prognostic effect of tumor cell expression of FGF-2 in NSCLC that included only patients who had undergone RT. In addition to FGF-2, 13 additional potential prognostic factors were investigated with respect to locoregional control (LRC), metastasis-free survival (MFS), and overall survival (OS). An understanding of the prognostic factors would help physicians evaluate the underlying tumor biology and identify potential targets for therapy. Prognostic factors could also help guide the physician in selecting appropriate therapy for an individual patient. Furthermore, prognostic factors are important for proper stratification when designing clinical trials. METHODS AND MATERIALS The tumor cell expression levels of FGF-2 were evaluated in tumor samples obtained before RT from 60 patients who had undergone RT for NSCLC at the University of Lubeck between January 2000 and December These patients were a subgroup with tissue samples available and successful staining for FGF-2 from our preceding study of the prognostic factors in NSCLC (12). All the patients were smokers at the diagnosis of NSCLC. The patient characteristics are summarized in Table 1. Immunohistochemistry The resected NSCLC tissues were fixed in 10% buffered formalin (ph 7.0) and embedded in paraffin. The formalin-fixed, paraffinembedded tumor samples were used for the preparation of a tumor tissue microarray block. The tissue microarray block was constructed using a Manual Tissue Arrayer 1 (Beecher Instruments, Silver Spring, MD) with a 1.0-mm diameter core biopsy needle. Subsequently, 4-mm-thick serial sections were prepared from the TMA block, deparaffinized in xylene, and rehydrated in graded alcohols. Antigen retrieval was performed in 0.01 mmol/l sodium citrate buffer (ph 6.0) for 5 min in a microwave. Endogenous peroxidase was blocked with 0.3% hydrogen peroxidase for 5 min. Next, the sections were incubated with a polyclonal antibody (rabbit, AB 1458, 1/1,000 dilution, Millipore, Billerica, MA). The sections were washed with Tris-buffered saline containing 0.1% Tween 20 (ph 7.0), and subsequent reaction was performed with the biotin-free horseradish peroxidase enzyme-labeled polymer of the Powervision system (ImmunoLogic, Duiven, The Netherlands). Diaminobenzidine complex was used as the chromogen. The sections were counterstained with hematoxylin. The tumors were considered to express FGF-2 if $10% of the tumor cells were positive. Treatment Radiotherapy was performed after computed tomography-based three-dimensional treatment planning with a linear accelerator and 6 18-MV photons. The target volume included the primary tumor and the locoregional lymph nodes with a 2-cm margin. The total dose given as the equivalent dose in 2-Gy fractions (EQD2) was Gy and was determined by the treatment schedule favored at our institution during different periods and on the extent of resection performed in patients undergoing surgery. The EQD2 was calculated using the equation: EQD2 = D [(d + a/b)/(2 Gy + a/b)], where D is the total dose, d is the dose per fraction, a is the linear component of cell killing, b is the quadratic component of cell killing, and the a/b ratio is the dose at which both components are equal (13, 14). The a/b ratio for tumor cell kill was assumed to be 10 Gy. The EQD2 was Gy after an R2 resection Table 1. Patient characteristics Characteristic Patients (n) Age (y) #65 32 (53) >65 28 (47) Gender Female 14 (23) Male 46 (77) Karnofsky performance score #70 21 (35) >70 39 (65) Histologic type SCC 31 (52) Adenocarcinoma 25 (42) Large cell carcinoma 4 (7) Histologic grade (35) 3 39 (65) T category (63) 4 22 (37) N category (38) (62) AJCC stage Stage II 14 (23) Stage III 46 (77) Surgery No 28 (47) Yes 32 (53) Chemotherapy No 27 (45) Yes 33 (55) Smoking pack-years #50 29 (48) >50 21 (35) Unknown 10 (17) Smoking during RT No 40 (67) Yes 20 (33) Hemoglobin level during RT (g/dl) <12 37 (62) $12 23 (38) FGF-2 expression Negative 34 (57) Positive 26 (43) Abbreviations: SCC = squamous cell carcinoma; AJCC = American Joint Committee on Cancer; RT = radiotherapy; FGF-2 = fibroblast growth factor 2. Data in parentheses are percentages. (macroscopically residual tumor) or for definitive treatment, Gy after an R1 resection (microscopically residual tumor), and Gy after an R0 resection (no residual tumor). The distribution of the resection status/dose-fractionation schedules was not significantly different in patients with FGF-2 positive or FGF-2 negative tumors (p =.59, chi-square test). Chemotherapy consisted of two to four cycles of a cisplatin-based regimen. Two cycles were administered concurrently with RT. Potential prognostic factors A total of 14 potential prognostic factors were retrospectively evaluated with respect to LRC, MFS, and OS. These factors included age (<65 vs. $65 years, median age, 65 years), gender,

3 444 I. J. Radiation Oncology d Biology d Physics Volume 82, Number 1, 2012 Table 2. Univariate analysis of locoregional control LRC rate (%) Variable At 1 y At 2 y p Fig. 1. Effect of tumor cell expression of fibroblast growth factor-2 on locoregional control. Karnofsky performance score (#70 vs. >70), histologic type (squamous cell carcinoma vs. adenocarcinoma vs. large cell carcinoma), histologic grade (Grade 1-2 vs. Grade 3), T category (T1-T3 vs. T4), N category (N0-N1 vs. N2-N3), American Joint Committee on Cancer stage (Stage II vs. Stage III), surgery (no vs. yes), chemotherapy (no vs. yes), smoking during RT (no vs. yes), pack-years (#50 vs. >50 years, defined as the average number of cigarette packs smoked daily multiplied by the number of years smoked), most weekly hemoglobin levels during RT (<12 vs. $12 g/dl), and FGF-2 expression of tumor cells (no vs. yes). Five to seven hemoglobin levels were obtained during the RT course. The cutoff of 12 g/dl was chosen according to the data of Vaupel et al. (15). Statistical analysis The LRC, MFS, and OS rates were calculated using the Kaplan- Meier method (16). Differences between the Kaplan-Meier curves were calculated using the Wilcoxon test (univariate analysis). The results were considered significant at p <.05. The potential prognostic factors considered significant or of borderline significance (p #.06) on univariate analysis were also evaluated in a multivariate analysis performed using the Cox proportional hazard model. The intervals to locoregional failure, distant failure, and death were referenced from the end of RT. Locoregional failure was identified by endoscopy or serial computed tomography scans performed every 3 6 months. The patients were followed up until death or for a median of 24 months (range, 13 64) for those alive at the last evaluation. RESULTS Locoregional failure occurred in 24 of the 60 patients. In the entire cohort, the LRC rate at 1 and 2 years was 73% and 60%, respectively. On univariate analysis, improved LRC was significantly associated with surgery (p =.017), greater hemoglobin levels during RT (p =.036), and the absence of FGF-2 expression in tumor cells (p <.001, Fig. 1). The results of the univariate analysis of LRC are summarized in Table 2. On multivariate analysis of LRC, surgery (relative risk [RR], 2.44; 95% confidence interval [CI], ; p =.037) and the absence of FGF-2 expression (RR, 5.06; 95% CI, ; p <.001) maintained significance, but the hemoglobin levels during RT did not (RR, 1.88; 95% CI, ; p =.22). Age (y).18 # > Gender.32 Female Male Karnofsky performance score.33 # > Histologic type.45 SCC Adenocarcinoma Large cell carcinoma 75 Not available Histologic grade T category N category AJCC stage.31 Stage II Stage III Surgery.017 No Yes Chemotherapy.46 No Yes Smoking pack-years.75 # > Smoking during RT.15 No Yes Hemoglobin levels during RT (g/dl).036 < $ FGF-2 expression <.001 No Yes Abbreviation: LRC = locoregional control; other abbreviations as in Table 1. Distant metastases developed in 29 patients. In the entire cohort, the MFS rate at 1 and 2 years was 61% and 49%, respectively. On univariate analysis, improved MFS was significantly associated with squamous cell carcinoma (p =.020), greater hemoglobin levels during RT (p =.007), and absence of FGF-2 expression in tumor cells (p =.001, Fig. 2). The results of the univariate analysis of MFS are summarized in Table 3. On multivariate analysis of MFS, the hemoglobin levels during RT (RR, 2.65; 95% CI, ; p =.019) and the absence of FGF-2 expression (RR, 3.05; 95% CI, ; p =.004) maintained significance; however, the histologic type did not (RR, 1.05; 95% CI, ; p =.18).

4 Effect of FGF-2 in patients irradiated for NSCLC d D. RADES et al. 445 Table 3. Univariate analysis of metastasis-free survival MFS rate (%) Variable At 1 y At 2 y p Fig. 2. Effect of tumor cell expression of fibroblast growth factor-2 on metastases-free survival. The median survival time for the entire cohort was 25.5 months. The OS rate at 1 and 2 years was 67% and 56%, respectively. On univariate analysis, improved OS was significantly associated with lower N category (p =.048), hemoglobin levels $12 g/dl during RT (p <.001), and absence of FGF-2 expression in tumor cells (p <.001; Fig. 3). The results of the univariate analysis of OS are summarized in Table 4. On multivariate analysis of OS, greater hemoglobin levels during RT (RR, 4.62; 95% CI, ; p =.002) and the absence of FGF-2 expression (RR, 3.25; 95% CI, ; p =.002) maintained significance, but the N category (RR, 1.42; 95% CI, ; p =.12) did not. DISCUSSION Age (y).40 # > Gender.23 Female Male Karnofsky performance score.40 # > Histologic type.020 SCC Adenocarcinoma Large cell carcinoma 75 Not available Histologic grade T category N category AJCC stage.64 Stage II Stage III Surgery.35 No Yes Chemotherapy.92 No Yes Smoking pack-years.94 # > Smoking during RT.20 No Yes Hemoglobin levels during RT (g/dl).007 < $ FGF-2 expression.002 No Yes Abbreviation: MFS = metastasis-free survival; other abbreviations as in Table 1. The prognostic effect of the tumor cell expression of FGF- 2 in NSCLC patients was unclear. The present study is the first to investigate FGF-2 in a cohort of patients who had undergone RT for locally advanced NSCLC. According to our results, the tumor cell expression of FGF-2 was significantly associated with the treatment outcomes as stratified by LRC, MFS, and OS. The results maintained significance on multivariate analysis. Given the limitations of a retrospective study, the tumor cell expression of FGF-2 appeared to be independent prognostic factor with respect to all three investigated endpoints. These results are in accordance with most of the few studies that have investigated the prognostic value of FGF-2 expression in NSCLC patients. However, in contrast to our study, the previous studies included patients who had generally not undergone RT for NSCLC. Furthermore, most patients in the previous studies had had Stage I disease. Our study is the first to include mainly patients (>75%) with Stage III disease. Therefore, our study is unique, which should be taken into account when comparing our results with those from other series. In 1996, Takanami et al. (7) retrospectively investigated 167 specimens that were mostly from patients with Stage I cancer and found that the overall prognosis was significantly poorer for all patients with tumors positive for FGF-2. Shou et al. (6) performed a retrospective study of 119 NSCLC patients (40% with Stage I disease) who had not undergone RT. The 5-year OS rate was 41% for FGF-2 positive tumors and 74% for FGF-2 negative tumors (p =.017). In the retrospective study by Iwasaki et al. (5), 59% of the 71 patients had Stage I disease and only 34% had Stage III disease. The patients with high FGF-2 levels had significantly worse survival than those with low levels (p =.006). Again, the patients did not undergo RT. The most recent study published in 2009 included 335 retrospectively analyzed patients (8). However, 63% of patients had Stage I disease and only 10% had Stage III disease. RT after surgery was administered only to 18% of patients, and definitive RTwas not used at all. On multivariate analysis, a high tumor cell

5 446 I. J. Radiation Oncology d Biology d Physics Volume 82, Number 1, 2012 Table 4. Univariate analysis of overall survival OS rate (%) Variable At 1 y At 2 y p Fig. 3. Effect of tumor cell expression of fibroblast growth factor-2 on overall survival. expression of FGF-2 was an independent negative prognostic factor for disease-free survival (p =.038). In contrast to these studies, two other retrospective studies did not find a significant association between tumor cell expression of FGF-2 and the patient s prognosis (10, 11). In neither of these two studies did patients undergo RT. Our study is the only study to date that included only patients who had undergone RT. According to our results, the tumor cell expression of FGF-2 appears to be an independent prognostic factor for treatment outcomes. Mechanistically, this is probably related to FGF-2 s role in cellular proliferation. Understanding the prognostic importance of this factor could lead to a better stratification of patients in future trials and identifying a potential target for future therapy. One group has suggested the possibility of using FGF-antisense mrnas in esophageal tumors overexpressing FGF-2 (17). FGF-2 overexpression is a risk factor for esophageal cancer recurrence and reduced survival, which is ameliorated by coexpression of the FGF-2 antisense gene. The retrospective nature of the present study, and the relatively small number of patients should be considered when interpreting the results. However, prospective studies regarding the potential prognostic effect of tumor expression of FGF-2 are not available. In addition to the tumor cell expression of FGF-2, treatment outcomes were associated with the N category, surgery, and hemoglobin levels during RT. The prognostic effect of the tumor stage has been previously described by several investigators (18 24). The prognostic value of the hemoglobin levels during RT has already been described for other tumor types (25 29). Munstedt et al. (25) demonstrated that the hemoglobin levels during RT was significantly associated with both disease-free (p <.001) and overall (p <.001) survival in a retrospective series of 996 patients with endometrial carcinoma. Our group had previously demonstrated hemoglobin levels of $12 g/dl during chemoradiotherapy were associated with significantly better survival (p =.002) and locoregional control (p <.001) than lower hemogloblin levels in a retrospective study of 108 esophageal cancer patients (26). Several investigators have investigated the prognostic Age (y).82 # > Gender.15 Female Male Karnofsky performance score.37 # > Histologic type.17 SCC Adenocarcinoma Large cell carcinoma 75 Not available Histologic grade T category N category AJCC stage.83 Stage II Stage III Surgery.11 No Yes Chemotherapy.49 No Yes Smoking pack-years.78 # > Smoking during RT.11 No Yes Hemoglobin levels during RT (g/dl) <.001 < $ FGF-2 expression <.001 No Yes Abbreviation: OS = overall survival; other abbreviations as in Table 1. value of hemoglobin levels and the importance of tumor oxygenation in head-and-neck cancer patients (27 29). It has been demonstrated that hemoglobin levels <12 g/dl lead to a reduction of tumor oxygenation owing to the reduced oxygen-carrying capacity of the blood. Appropriate tumor oxygenation is important for the optimal effect of RT based on the induction of cytotoxic free radicals leading to damage of tumor DNA. CONCLUSION The pre-rt tumor cell expression of FGF-2 appeared to be an independent negative prognostic factor for treatment

6 Effect of FGF-2 in patients irradiated for NSCLC d D. RADES et al. 447 outcomes in terms of LRC, MFS, and OS in patients who had undergone RT for Stage II-III NSCLC. Prospective studies are warranted to confirm the data of the present retrospective study. REFERENCES 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, CA Cancer J Clin 2007;57: Presta M, Dell Era P, Mitola S, et al. Fibroblast growth factor/ fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 2005;16: Byers LA, Heymach JV. Dual targeting of the vascular endothelial growth factor and epidermal growth factor receptor pathways: Rationale and clinical applications for non smallcell lung cancer. Clin Lung Cancer 2007;8(Suppl. 2):S79 S Berger W, Setinek U, Mohr T, et al. Evidence for a role of FGF- 2 and FGF receptors in the proliferation of non-small lung cancer cells. Int J Cancer 1999;83: Iwasaki A, Kuwahara M, Yoshinaga Y, et al. Basic fibroblast growth factor (bfgf) and vascular endothelial growth factor (VEGF) levels as prognostic indicators in NSCLC. Eur J Cardiothorac Surg 2004;25: Shou Y, Hirano T, Gong Y, et al. Influence of angiogenetic factors and matrix metalloproteinases upon tumour progression in non-small cell lung cancer. Br J Cancer 2001;85: Takanami I, Tanaka F, Hashizume T, et al. The basic fibroblast growth factor and its receptor in pulmonary adenocarcinomas: An investigation of their expression as prognostic markers. Eur J Cancer 1996;32A: Donnem T, Al-Shibli K, Al-Saad S, et al. Prognostic impact of fibroblast growth factor 2 in non-small cell lung cancer: Coexpression with VEGFR-3 and PDGF-B predicts poor survival. J Thorac Oncol 2009;4: Bremnes RM, Camps C, Sirera R. Angiogenesis in non-small cell lung cancer: The prognostic impact of neoangiogenesis and the cytokines VEGF and bfgf in tumours and blood. Lung Cancer 2006;51: Kojima H, Shijubo N, Abe S. Thymidine phosphorylase and vascular endothelial growth factor in patients with stage I lung adenocarcinoma. Cancer 2002;94: Volm M, Koomagi R, Mattern J, et al. Prognostic value of basic fibroblast growth factor and its receptor (FGFR-1) in patients with non-small cell lung carcinomas. Eur J Cancer 1997;33: Rades D, Setter C, Schild SE, et al. Effect of smoking during radiotherapy, respiratory insufficiency, and hemoglobin levels on outcome in patients irradiated for non small-cell lung cancer. Int J Radiat Oncol Biol Phys 2008;71: Barendsen GW. Dose fractionation, dose rate and iso-effect relationships for normal tissue responses. Int J Radiat Oncol Biol Phys 1982;8: Joiner MC, van der Kogel AJ. The linear-quadratic approach to fractionation and calculation of isoeffect relationships. In: Steel GG, editor. Basic Clinical Radiobiology. 2nd ed. New York: Oxford University Press; pp Vaupel P, Thews O, Mayer A, et al. Oxygenation status of gynecologic tumors: What is the optimal hemoglobin level? Strahlenther Onkol 2002;178: Kaplan EL, Meier P. Non parametric estimation from incomplete observations. J Am Stat Assoc 1958;53: Barclay C, Li AW, Geldenhuys L, et al. Basic fibroblast growth factor (FGF-2) overexpression is a risk factor for esophageal cancer recurrence and reduced survival, which is ameliorated by coexpression of the FGF-2 antisense gene. Clin Cancer Res 2005;11: Caglayan B, Fidan A, Salepci B, et al. Effects of prognostic factors and treatment on survival in advanced non-small cell lung cancer. Tuberk Toraks 2004;52: Hsu HC, Wang CJ, Huang EY, et al. Post-operative adjuvant thoracic radiotherapy for patients with completely resected non-small cell lung cancer with nodal involvement: Outcome and prognostic factors. Br J Radiol 2004;77: Jaen Olasolo J, Alonso Redondo E, Leon Jiminez A, et al. Nonsmall lung cancer: Survival after radiotherapy and prognostic factors. Arch Bronconeumol 2003;39: Jeremic B, Milicic B, Dagovic A, et al. Pretreatment prognostic factors in patients with early stage (I/II) non small-cell lung cancer treated with hyperfractionated radiation therapy alone. Int J Radiat Oncol Biol Phys 2006;65: Jeremic B, Milicic B, Acimonic L, et al. Concurrent hyperfractionated radiotherapy and low-dose daily carboplatin/paclitaxel in patients with early-stage (I/II) non small-cell lung cancer: Long-term results of a phase II study. J Clin Oncol 2005;23: Jeremic B, Casas F, Wang L, et al. Radiation therapy for early stage (I/II) non-small cell lung cancer. Front Radiat Ther Oncol 2010;42: Hoyer M. Improved accuracy and outcome in radiotherapy of lung cancer. Radiother Oncol 2008;87: Munstedt K, Volzing M, Von Georgi R. Hemoglobin levels during radiotherapy and their influence on local control and survival of patients with endometrial carcinoma. Oncol Rep 2004;11: Rades D, Lang S, Schild SE, et al. Prognostic value of the hemoglobin levels during concurrent radio-chemotherapy in the treatment of esophageal cancer. Clin Oncol 2006;18: Becker A, Stadler P, Lavay RS, et al. Severe anemia is associated with poor tumor oxygenation in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys 2000;46: Rudat V, Stadler P, Becker A, et al. Predictive value of the tumor oxygenation by means of po 2 histography in patients with advanced head and neck cancer. Strahlenther Onkol 2001;177: Rudat V, Vanselow B, Wollensack P, et al. Repeatability and prognostic impact of the pretreatment po 2 histography in patients with advanced head and neck cancer. Radiother Oncol 2000;57:31 37.