Prediction of response to chemoradiation in rectal cancer by a gene polymorphism in the epidermal growth factor receptor promoter region

Int. J. Radiation Oncology Biol. Phys., Vol. 66, No. 2, pp. 500 –504, 2006 Copyright © 2006 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/06/$–see front matter

doi:10.1016/j.ijrobp.2006.03.030

CLINICAL INVESTIGATION

Rectum

PREDICTION OF RESPONSE TO CHEMORADIATION IN RECTAL CANCER BY A GENE POLYMORPHISM IN THE EPIDERMAL GROWTH FACTOR RECEPTOR PROMOTER REGION KAREN-LISE GARM SPINDLER, M.D.,* JENS NEDERBY NIELSEN, PH.D.,† JAN LINDEBJERG, M.D.,‡ IVAN BRANDSLUND, D.M.SC.,† AND ANDERS JAKOBSEN, D.M.SC.* *Department of Oncology, †Department of Clinical Biochemistry, ‡Department of Pathology, Danish Colorectal Cancer Group South, Vejle Hospital, University of Southern Denmark, Vejle, Denmark Purpose: Epidermal growth factor receptor (EGFR) has been associated with radioresistance in solid tumors. Recently a polymorphism in the Sp1 recognition site of the EGFR promoter region was identified. The present study investigated the predictive value of this polymorphism for the outcome of chemoradiation in locally advanced rectal cancer. Methods and Materials: The study included 77 patients with locally advanced T3 rectal tumors. Treatment consisted of preoperative radiation therapy at a total tumor dose of 65 Gy and concomitant chemotherapy with Uftoral. Blood samples from 63 patients were evaluated for Sp1 -216 G/T polymorphism by polymerase chain reaction analysis. Forty-eight primary tumor biopsies were available for EGFR immunostaining. Patients underwent surgery 8 weeks after treatment. Pathologic response evaluation was performed according to the tumor regression grade (TRG) system. Results: Forty-nine percent had major response (TRG1–2) and 51% moderate response (TRG 3– 4) to chemoradiation. The rates of major response were 34% (10/29) in GG homozygote patients compared with 65% (22/34) in patients with T containing variants (p ⴝ 0.023). Fifty-eight percent of biopsies were positive for EGFR expression (28/48). The major response rates with regard to EGFR immunostaining were not significantly different. EGFR-positive tumors were found in 83% of the GG homozygote patients compared with 38% of patients with TT or GT variants (p ⴝ 0.008). Conclusions: There was a significant correlation between EGFR Sp1 -216 G/T polymorphism and treatment response to chemoradiation in locally advanced rectal cancer. Further investigations of a second set of patient and other treatment schedules are warranted. © 2006 Elsevier Inc. Epidermal growth factor receptor, Sp1 -216 polymorphism, Predictive marker, Rectal cancer, Chemoradiation.

Advances in surgical methods, especially total mesorectal excision and development of preoperative radiation therapy, have led to major improvements in the treatment of rectal cancer. The risk of local relapse has been reduced by approximately 50% but the overall survival was only improved by 4% (1). Preoperative radiation therapy of rectal cancer is now considered standard treatment in many European countries. A large Dutch study evaluated the effect of short-course preoperative radiation therapy and found that patients with T3 tumors and a close circumferential margin had a high risk of local recurrence and no significant effect of short-course treatment (2). Consequently these patients should be offered a

longer course of radiation treatment, preferably in combination with chemotherapy, as shown in recent randomized trials (3, 4). A number of studies have clearly indicated that downsizing of the tumor and consequently R0 resection is essential to prognosis (5). Furthermore, a recent trial showed that long-course chemoradiation with endorectal brachytherapy has promising potential in terms of a high rate of complete pathologic remission and a low toxicity (6). However, additional reliable preoperative selection criteria are needed to identify subgroups of patients who will or will not benefit from extensive long-course chemoradiation. The epidermal growth factor receptor (EGFR) is a transmembrane receptor important to tumor growth and

Note—An online CME test for this article can be taken at www.astro.org under Education and Meetings. Reprint requests to: Karen-Lise Garm Spindler, M.D., Department of Oncology, Vejle Hospital, Kabbeltoft 25, DK-7100 Vejle, Denmark. Tel: (⫹45) 79-406833/79-406000; Fax: (⫹45) 79-406907; E-mail: [email protected]

The study was supported by grants from Vejle County. Acknowledgments—We thank Lone Frischknecht and Birgit Roed Sørensen for technical assistance and Kirsten Birgitte Hjermitslev for proofreading. Received Feb 3, 2006, and in revised form March 24, 2006. Accepted for publication March 28, 2006.

INTRODUCTION

500

EGFR Sp1 -216 polymorphism and response to chemoradiation

development. EGFR is located on most epithelial cells and is overexpressed in various solid tumors (7). Binding of ligand to receptor activates a complex signal transduction pathway inside the cell that increases deoxyribonucleic acid (DNA) replication and stimulates cellular growth and proliferation (8, 9). Overexpression of EGFR occurs in 25– 82% of colorectal cancers and has been associated with shorter survival and increased risk of metastasis (10). EGFR has become a molecular target for anticancer therapy in colorectal cancer (11). There might be biologic differences between colon and rectal cancers, and the role of EGFR in rectal cancer needs elucidation. Preclinical and clinical studies demonstrate an association between positive EGFR expression, radiation resistance, and local relapse in patients treated with radiation therapy (12, 13), but only very few studies have described the impact of EGFR expression on the outcome of preoperatively combined chemoradiation in locally advanced rectal cancer (14 –16). Traditionally the EGFRs have been evaluated by immunohistochemistry (IHC) of tumor tissue (17). Tumor heterogeneity and lack of standardization of methodology are considered major obstacles in the detection of EGFR by IHC. Alternative methods for EGFR expression analysis and underlying mechanisms for EGFR regulation are being actively investigated. Regulation of EGFR expression is a complex and not fully understood mechanism. The promoter region of a gene is essential for gene activation and transcription. Binding of so-called Sp1 proteins in this promoter region is regarded as essential to EGFR gene transcription. Recently it has been shown that a single nucleotide polymorphism in the Sp1 binding site of the regulatory EGFR promoter region is common and associated with higher Sp1 binding affinity and affects gene expression in cell lines (18). Replacement of G by T at position -216 increased the promoter activity by 30%. Cell lines with G/G genotype had lower gene expression level than the G/T and T/T genotypes. It was suggested that this Sp1 -216 G/T polymorphism may contribute to the variability of EGFR expression and response to EGFR targeted therapies in cancer. Sp1 -216 G/T polymorphism can be detected by polymerase chain reaction in blood samples, which has obvious advantages compared with EGFR evaluation on limited amounts of available tumor tissue. So far there are no reports on the role of these Sp1 -216 G/T polymorphisms in the clinical setting. The aim of the present study was to investigate the predictive value of EGFR Sp1 -216 G/T polymorphism in patients with locally advanced rectal cancer receiving long-term chemoradiation with pathologic response as primary endpoint. Secondary endpoint was to evaluate prediction of response by traditional IHC of EGFR and to elucidate a possible relation between the two potential markers.

● K.-L. G. SPINDLER et al.

501

Table 1. Patient characteristics Number Age median (y) Gender Male Female Circumferential margin (mm), mean (range) Classification T3N0M0 T3N1M0 T3N2M0 Tumor size, mean (cm) (range) Distance from anal verge (cm) 0–5 5–10 Histopathology adenocarcinomas Available for response evaluation Major response Moderate response Available for Sp1 -216 G/T polymorphism evaluation GG GT TT Available for EGFR immunostaining Positive Negative

77 64 53 (69%) 24 (31%) 1.4 (0–5) 16 (22%) 44 (62%) 11 (16%) 4.9 (1.3–8.0) 33 (48%) 36 (52%) 77 74 36 (49%) 38 (51%) 63 29 (46%) 23 (37%) 11 (17%) 48 28 (58%) 20 (42%)

Abbreviation: EGFR ⫽ epidermal growth factor receptor.

METHODS AND MATERIALS Patient population and treatment protocol Seventy-seven patients were included in the study from 2003 until 2005 at Vejle Hospital, Denmark. Diagnostic biopsies from histopathologically confirmed adenocarcinomas of the rectum were collected along with corresponding blood samples. Only patients with T3 tumors less than 5 mm from the mesorectal fascia by magnetic resonance imaging were included. Patient characteristics are summarized in Table 1. Preoperative radiation was given as an external total tumor dose of 60 Gy combined with an intracavitary boost of a 5 Gy single fraction to the tumor bed. Concurrent chemotherapy consisted of oral Uftoral 300/mg/m2 daily and leucovorin 22.5 mg/daily 5 days a week. The operation was performed 8 weeks posttreatment. Treatment details are given elsewhere (5). The study was approved by the Regional Ethics Committee of Vejle and Funen Counties according to Danish law.

Response evaluation Tumor response to chemoradiation was graded by histopathologic examination according to the tumor regression grade (TRG) system as previously described (5). Evaluation was performed by a trained pathologist (J.L.). The grading was based on histopathologic evaluation of the amount of fibrosis and residual carcinoma cells. TRG 1 was defined as complete regression with no residual carcinoma cells left. TRG 2 tumors had a few residual carcinoma cells but mostly fibrosis. TRG 3 tumors contained some residual carcinoma cells but still ⬎50% fibrosis. TRG 4 tumors were dominated by carcinoma cells but had some degree of fibrosis (less than 50%). Tumor regression Grade 5 showed no histopathologic regression. Major response was defined by TRG 1 and 2. TRG 3 and 4 tumors were considered to have a moderate response.

502

I. J. Radiation Oncology

● Biology ● Physics

Table 2. Response to preoperative chemoradiation according to EGFR Sp1 -216 G/T polymorphism and EGFR expression

Marker

Major response

Moderate response

Total

Major response rate (%)

Sp1 variant G/G Sp1 variant G/T - T/T EGFR-positive EGFR-negative

10 22 15 10

19 12 13 10

29 34 28 20

34%* 65%* 54% 50%

Abbreviation: EGFR ⫽ epidermal growth factor receptor. * p ⫽ 0.023.

Immunohistochemistry Rectal tumor biopsies were immersed in 4% buffered neutral formalin and fixed for 24 h. Paraffin embedding was performed according to standard procedure. Sections of 4 ␮m were mounted on coated slides and allowed to dry for 30 min at 60°C and overnight at 37°C. All sections were stained within 24 h of embedding. The slides were deparaffinized in ESTISOL 220 (Esti Chem, Køge, Denmark) and rehydrated in graded alcohol solutions. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Proteolytic antigen retrieval was performed using 0.1% protease at room temperature for 20 min. Slides were incubated in primary mouse anti-EGFR Mab (clone H-11, DAKO, Glostrup, Denmark) for 30 min at room temperature. Visualization of the reaction was performed using ENVISION ⫹ DAB (DAKOCytomation) followed by counterstaining with hematoxylin. Staining was performed manually.

Evaluation of EGFR expression Evaluation was independently performed by two investigators (K.G.S. and J.L.). The inter- and intraobserver reproducibility of assessments of the IHC staining was tested by calculating Cohen’s kappa (K values ⬎0.87). EGFR positivity was defined according to DAKO guidelines; any membrane staining above background level was considered positive. Tumors were graded with regard to intensity and amount of membrane staining. A score of staining intensity was assigned as follows: 1⫹ ⫽ weak, 2⫹ ⫽ moderate, and 3⫹ ⫽ strong membrane staining. The tumor was defined positive if ⱖ1% of the cells had membranous staining for EGFR according to the DAKO guidelines. A tumor with less than 1% positive cells was considered EGFRnegative. The score was defined according to the percentage of positively stained tumor cells as follows: 0 ⫽ less than 1%, 1 ⫽ 1–10%, 2 ⫽ 10 –25%, 3 ⫽ 25–50%, 4 ⫽ ⬎50%.

Sp1 -216 G/T EGFR gene polymorphism analysis Genomic DNA was isolated from whole blood as previously described (19). Polymerase chain reaction analysis of Sp1 gene polymorphism in the EGFR promoter was performed using the ABI PRISM 7900 HT Sequence Detection System. EGFR-Sp1 forward primer was CGT CCG GGC AGC CC. EGFR-Sp1 reverse primer was GGC GCT CAC ACC GTG C. EGFR-Sp1G probe was 6FAM-AGC AGC CTC CGC C. EGFR-Sp1T probe was VICAGC AGC CTC CTC C. The results were verified by sequencing on an ABI 3100 Sequence Detection System. Forward sequencing primer was GGT TCT CCT CCC TCC TCC TCG CA. Reverse sequencing primer was TTG TGG CGT TGG CGG CGA.

Volume 66, Number 2, 2006

Statistical methods Fisher exact test was used to compare response rates in the two groups of major responders vs. moderate responders. P-values were considered significant if p ⱕ 0.05. All statistics were carried out using the NCSS statistical software (NCSS Statistical Software, Saugus, MA).

RESULTS The study included 77 patients. All patients had T3 tumors. Additional patient characteristics are shown in Table 1. Seventy-four patients completed chemoradiation (95%) and were assessable for response evaluation after surgery. Forty-nine percent (36/74) had major response and 51% (38/74) moderate response. Hematologic toxicities were minimal and the same applies to nonhematologic toxicities (indicated by no Grade 4 toxicities). Only 1 patient had Grade 3 diarrhea. Response rates according to Sp1 -216 G/T polymorphism and EGFR expression are shown in Table 2. Sixtythree patients were evaluated for Sp1 -216 G/T polymorphism. The remaining 14 did not have available blood samples for analysis. Twenty-nine were homozygote for the GG, and 21 and 11 had GT and TT respectively. The major response rates were 34% (10/29) in GG homozygote patients compared with 65% (22/34) in patients with replacement of G by T (heterozygosity or homozygosity for T). The difference was statistically significant (p ⫽ 0.023). A total of 48 primary tumor biopsies were available for EGFR immunostaining. Twenty-eight biopsies were scored positive for EGFR expression (58%) and 20 (42%) were scored negative. The rates of major responses in the two groups were not significantly different. Choosing a cutoff for EGFR positivity by 10% of tumor cells did not result in any positive relation between the two variables. There was a significantly different distribution of positive and negative EGFR immunostaining according to Sp1 -216 G/T polymorphism. Data are presented in Table 3. EGFRpositive tumors were found in 83% (15/18) of the GG homozygote patients compared with 38% (8/21) of the patients with TT or GT variants ( p ⫽ 0.008). Patients with both GG variant and EGFR-positive immunostaining were found in 40% (6/15) of major responders compared with 58% (14/24) of those with moderate response, indicating a nonsignificant difference between the two groups (p ⬎ 0.05; data not shown).

Table 3. EGFR expression according to Sp1 -216 G/T polymorphism p ⫽ 0.008

EGFR-positive EGFR-negative Total

GG

GT-TT

Total

15 3 18

8 13 21

23 16 39

Abbreviation: EGFR ⫽ epidermal growth factor receptor.

EGFR Sp1 -216 polymorphism and response to chemoradiation

DISCUSSION Locally advanced rectal cancer presents a major therapeutic challenge. The multimodal treatment (chemotherapy, radiation, and operation) implies a rather high risk of serious toxicity, and there is an obvious need for new predictive markers with a perspective of individualized treatment. The possible importance of EGFR in this context needs further elucidation. A number of experimental studies have shown a positive relationship between EGFR expression and tumor resistance to radiation (20 –23). The detailed mechanism by which EGFR signaling leads to radioresistance is not yet fully understood, but radiation seems to activate the EGFR system, which mediates a cytoprotective response (24 –27). Some studies demonstrate significantly enhanced antitumor activity in cell lines treated with a combination of antibody to EGFR and radiation therapy (28). Clinical studies have associated EGFR expression with radioresistance in different solid tumors, including head-and-neck tumors and glioblastomas (12, 29). Only very few studies have addressed the issue in rectal cancer. Giralt et al. found that high EGFR expression indicated poor response and low disease-free interval after preoperative chemoradiation of rectal cancer (15, 16). Furthermore, Azria et al. reported locoregional recurrence rates of 7% for high EGFR expression compared with 20% for low EGFR expression after radiotherapy in rectal cancers (14). The present study found an EGFR expression rate similar to the rate reported by Giralt and colleagues but no correlation between EGFR expression by IHC and tumor response to chemoradiation. These discrepancies might be due to methodological differences. The low number of observations should also be considered. Giralt et al. found an EGFR expression rate of 64% (29/45). One of the 7 patients with complete pathologic response was EGFR-positive. The overall response rate was 34% in the EGFR-positive patients compared with 62% in patients with EGFR-negative tumors. The present study reports an EGFR expression rate of 54%. There was no difference with regard to major response rates between EGFR-negative and EGFR-positive patients (50% and 54%, respectively). The present study includes only patients with T3 tumors, and all patients received full intensive long-term chemoradiation. In the study by Giralt and colleagues, 45 patients received either radiation therapy alone or chemoradiation. Response was defined by complete pathologic response evaluations compared with the present TRG evaluation as described in our study. Finally, it should be noticed that these studies differ with regard to EGFR immunohistochemical methodology and scoring systems. The traditional EGFR evaluation by IHC showed disappointing results in the clinical settings during EGFR targeted treatment. None of the current methods for EGFR evaluation are considered reliable predictors for these new treatment modalities, and the development of

● K.-L. G. SPINDLER et al.

503

alternative methods is urgently needed. The value of EGFR expression by IHC as a predictive marker for the outcome of chemoradiation in rectal cancer has not yet been finally established. The major obstacles regarding IHC and tumor heterogeneity might be overcome by blood analysis for EGFR polymorphism as presented here. Some regulatory sequences of the EGFR gene are proposed to be located in the 5= region where multiple Sp1 binding sites have been discovered. The most common single nucleotide polymorphism in this 5= region is the -216G/T polymorphism. Liu and colleagues investigated the function of this polymorphism and found that the -216 G/T variant had an impact on EGFR transcription in vivo (18). It was suggested that -216 G/T polymorphisms might contribute to the interindividual variability in EGFR expression and response to EGFR targeted therapies. Analysis of EGFR Sp1 -216 G/T polymorphism may be practically convenient and help clinicians overcome the complex issue of IHC. It has potential as a predictive marker for treatment response in this disease. Furthermore, it might provide information on the regulatory mechanism of EGFR expression in general. This is the first clinical report of Sp1 -216 G/T polymorphism in rectal cancer, and the present study indicates that Sp1 -216 G/T polymorphism is a potential predictive marker for treatment response in rectal cancer during chemoradiation. It appears that GG genotype indicates a high risk of only moderate response whereas patients with T containing variants have a better possibility of obtaining major responses (34% vs. 65% major response rates, respectively.) Furthermore, there was a relation between Sp1 -216 G/T genotyping and EGFR expression by IHC. Eighty-three percent (15/18) of GG polymorphic variants were EGFR-positive compared with 38% (8/21) in the GT-TT group, hypothetically indicating a higher influence on EGFR protein expression by the GG variant. The study by Liu et al. showed a low gene expression level in cell lines with GG variant. The exact regulatory relationships between gene expression level and protein expression level in EGFR are not yet fully understood. However, the possibility of high EGFR protein expression as a result of low gene expression level is described by Seth et al. in prostate cancer cells (30). Data from this study might support our findings of a GG variant relating to positive protein expression. The present study has limitations because it is based on a retrospective analysis and includes a rather low number of patients. Despite these shortcomings, the results are encouraging from a clinical point of view but clearly prospective trials are needed. In conclusion, EGFR Sp1 -216 G/T polymorphism is a potential new marker for treatment response to chemoradiation in locally advanced rectal cancer and calls for further evaluation in clinical settings.

504

I. J. Radiation Oncology

● Biology ● Physics

Volume 66, Number 2, 2006

REFERENCES 1. Colorectal Cancer Collaborative Group. Adjuvant radiotherapy for rectal cancer: A systematic overview of 8,507 patients from 22 randomised trials. Lancet 2001;358:1291– 1304. 2. Marijnen CA, Nagtegaal ID, Kapiteijn E, et al. Radiotherapy does not compensate for positive resection margins in rectal cancer patients: Report of a multicenter randomized trial. Int J Radiat Oncol Biol Phys 2003;55:1311–1320. 3. Gerard JP, Bonnetain F, Conroy T, et al. Preoperative (preop) radiotherapy (RT) (⫹/⫺) 5-FU/folinic acid (FA) in T3– 4 rectal cancers: Results of the FFCD 9203 randomized trial [Abstract]. J Clin Oncol 2005;23(Suppl.):247s. 4. Bozzetti F, Andreola S, Baratti D, et al. Preoperative chemoradiation in patients with resectable rectal cancer: Results on tumor response. Ann Surg Oncol 2002;9:444 – 449. 5. Bouzourene H, Bosman FT, Seelentag W, et al. Importance of tumor regression assessment in predicting the outcome in patients with locally advanced rectal carcinoma who are treated with preoperative radiotherapy. Cancer 2002;94:1121– 1130. 6. Jakobsen A, Mortensen JP, Bisgaard C, et al. Preoperative chemoradiation of locally advanced T3 rectal cancer combined with an endorectal boost. Int J Radiat Oncol Biol Phys 2006;64:461– 465. 7. Mendelsohn J. Targeting the epidermal growth factor receptor for cancer therapy. J Clin Oncol 2002;20(18 Suppl.):1S–13S. 8. Marmor MD, Skaria KB, Yarden Y. Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys 2004;58:903–913. 9. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–137. 10. Goldstein NS, Armin M. Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: Implications for a standardized scoring system. Cancer 2001; 92:1331–1346. 11. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004;351:337– 345. 12. Ang KK, Berkey BA, Tu X, et al. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res 2002;62:7350 –7356. 13. Eriksen JG, Steiniche T, Askaa J, et al. The prognostic value of epidermal growth factor receptor is related to tumor differentiation and the overall treatment time of radiotherapy in squamous cell carcinomas of the head and neck. Int J Radiat Oncol Biol Phys 2004;58:561–566. 14. Azria D, Bibeau F, Barbier N, et al. Prognostic impact of epidermal growth factor receptor (EGFR) expression on locoregional recurrence after preoperative radiotherapy in rectal cancer. BMC Cancer 2005;5:62. 15. Giralt J, Eraso A, Armengol M, et al. Epidermal growth factor receptor is a predictor of tumor response in locally advanced rectal cancer patients treated with preoperative radiotherapy. Int J Radiat Oncol Biol Phys 2002;54:1460 –1465.

16. Giralt J, de las Heras M, Cerezo L, et al. The expression of epidermal growth factor receptor results in a worse prognosis for patients with rectal cancer treated with preoperative radiotherapy: A multicenter, retrospective analysis. Radiother Oncol 2005;74:101–108. 17. Spaulding DC, Spaulding BO. Epidermal growth factor receptor expression and measurement in solid tumors. Semin Oncol 2002;29(5 Suppl. 14):45–54. 18. Liu W, Innocenti F, Wu MH, et al. A functional common polymorphism in a Sp1 recognition site of the epidermal growth factor receptor gene promoter. Cancer Res 2005;65: 46 –53. 19. Jakobsen A, Nielsen JN, Gyldenkerne N, et al. Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphism in normal tissue as predictors of fluorouracil sensitivity. J Clin Oncol 2005;23:1365–1369. 20. Gupta AK, McKenna WG, Weber CN, et al. Local recurrence in head and neck cancer: Relationship to radiation resistance and signal transduction. Clin Cancer Res 2002;8:885– 892. 21. Milas L, Mason KA, Ang KK. Epidermal growth factor receptor and its inhibition in radiotherapy: In vivo findings. Int J Radiat Biol 2003;79:539 –545. 22. Milas L, Fan Z, Andratschke NH, et al. Epidermal growth factor receptor and tumor response to radiation: In vivo preclinical studies. Int J Radiat Oncol Biol Phys 2004;58:966 – 971. 23. Nasu S, Ang KK, Fan Z, et al. C225 antiepidermal growth factor receptor antibody enhances tumor radiocurability. Int J Radiat Oncol Biol Phys 2001;51:474 – 477. 24. Schmidt-Ullrich RK, Contessa JN, Lammering G, et al. ERBB receptor tyrosine kinases and cellular radiation responses. Oncogene 2003;22:5855–5865. 25. Contessa JN, Hampton J, Lammering G, et al. Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells. Oncogene 2002;21:4032– 4041. 26. Dent P, Reardon DB, Park JS, et al. Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death. Mol Biol Cell 1999;10:2493–2506. 27. Lammering G, Hewit TH, Hawkins WT, et al. Epidermal growth factor receptor as a genetic therapy target for carcinoma cell radiosensitization. J Natl Cancer Inst 2001;93:921– 929. 28. Huang SM, Harari PM. Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: Inhibition of damage repair, cell cycle kinetics, and tumor angiogenesis. Clin Cancer Res 2000;6:2166 –2174. 29. Barker FG, Simmons ML, Chang SM, et al. EGFR overexpression and radiation response in glioblastoma multiforme. Int J Radiat Oncol Biol Phys 2001;51:410 – 418. 30. Seth D, Shaw K, Jazayeri J, et al. Complex post-transcriptional regulation of EGF-receptor expression by EGF and TGF-alpha in human prostate cancer cells. Br J Cancer 1999; 80:657– 669.