1

Paul A. Bunn, Jr, MDProfessor, Director of University of Colorado Cancer Center James Dudley Chair in Cancer ResearchDenver, Colorado

Paul M. Harari, MDJack Fowler ProfessorDepartment of Human OncologyUniversity of Wisconsin Medical SchoolMadison, Wisconsin

Hak Choy, MDNancy B. and Jake L. Hamon Distinguished Chair in Therapeutic Oncology ResearchProfessor, University of Texas Southwestern Medical Center at DallasDallas, Texas

Michael S. O’Reilly, MDAssistant Professor, Radiation Oncology and Cancer BiologyRadiation Treatment CenterThe University of Texas M. D. Anderson Cancer Center

Combining TargetedTherapies WithRadiation Therapy

Head and

Neck:

Supported by an educational grant from Bristol-Myers Squibb Companyand ImClone Systems Incorporated.

Program Description Clinical trials have established that the epidermal growth factor receptor (EGFR) and the vascularendothelial growth factor (VEGF) are valid targets for cancer therapies. Novel agents that targetother molecular entities are showing promising activity in early studies. Since most of these tar-geted therapies do not have much clinical activity as single agents, it is critical to develop activecombinations with other modalities, such as chemotherapy and radiation therapy. Some combi-nations with traditional chemotherapy have already shown effectiveness, others have not.Combinations with radiations are not as developed.

This monograph is based on a symposium that examined the state of combined treatment with targeted agents and radiation therapy for the treatment of patients with solid tumors, especially focusing on head and neck cancer. Topics discussed included: the preclinical and clini-cal rationale for combining these modalities, the current state of clinical development of thiscombined modality treatment, and issues that will have to be addressed to make further progressin this arena.

Learning Objectives• Compare the early clinical development of anti-EGFR tyrosine kinase inhibitors and

monoclonal antibodies combined with radiation therapy for the treatment of solid tumors

• Evaluate the rationale for combining anti-EGFR targeted agents with radiation therapy for the treatment of patients with head and neck cancer or other solid tumors

• Outline the rationale for combining angiogenesis inhibitors with radiation therapy for the treatment of head and neck cancer or other solid tumors

• Break down the preclinical rationale for combining agents that target NF-�B and the protea-some, the mammalian target of rapamycin, or cyclin-dependent kinases with radiation therapy for the treatment of head and neck cancer or other solid tumors

• Summarize challenges faced in the clinical development of combination treatment withtargeted agents and radiation therapy

Target AudienceThis activity is intended for medical and radiation oncologists and other health care profession-als involved in the care of patients with head and neck cancer.

Date of Release: December 1, 2006Date of Expiration: December 31, 2007

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Head and Neck: Combining Targeted Therapies With Radiation Therapy

FacultyPaul A. Bunn, Jr, MD

Professor, Director of University of Colorado Cancer Center James Dudley Chair in Cancer ResearchDenver, Colorado

Paul M. Harari, MDJack Fowler ProfessorDepartment of Human OncologyUniversity of Wisconsin Medical SchoolMadison, Wisconsin

Hak Choy, MDNancy B. and Jake L. Hamon Distinguished Chair in Therapeutic Oncology Research Professor, University of Texas Southwestern Medical Center at DallasDallas, Texas

Michael S. O’Reilly, MDAssistant Professor, Radiation Oncology and Cancer BiologyRadiation Treatment CenterThe University of Texas M. D. Anderson Cancer Center

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AccreditationThis activity has been planned and implemented in accordance with the Essential Areas andPolicies of the Accreditation Council for Continuing Medical Education (ACCME). The AmericanAcademy of CME (Academy) is accredited by the ACCME to provide continuing medical educa-tion for physicians.

CreditThe Academy designates this educational activity for a maximum of 1.5 hours AMA PRA Category1 Credits™. Physicians should only claim credit commensurate with the extent of their participa-tion in the activity.

Faculty DisclosuresPaul A. Bunn, Jr, MDDr Bunn is a consultant to AstraZeneca, Bristol-Myers Squibb Company, ImClone SystemsIncorporated, Genentech, Hoffman La Roche, and OSI Pharmaceuticals. Dr Bunn discussesnon–FDA approved or investigational uses of erlotinib, gefitinib, and cetuximab.

Hak Choy, MDDr Choy is on the advisory boards of Bristol-Myers Squibb Company, Eli Lilly and Company,Genentech, ImClone Systems Incorporated, Novartis, and Sanofi-Aventis. Dr Choy does not dis-cuss any non–FDA approved or investigational uses of any products.

Paul M. Harari, MDDr Harari has research agreements with, has received honoraria as a speaker from, and is on theadvisory boards of the following: Amgen, AstraZeneca, Bristol-Myers Squibb Company, Genentech,ImClone Systems Incorporated. Dr Harari discusses non–FDA approved or investigational uses ofcetuximab, erlotinib, bevacizumab, gefitinib, tirapazamine, temozolomide, and A12.

Michael S. O’Reilly, MDDr O’Reilly has received research funding from AstraZeneca. Dr. O’Reilly does not discuss anynon–FDA approved or investigational uses of any products.

4

IntroductionAmple evidence from preclinical and clinical studies has validated targeting the epidermalgrowth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) for the treatment ofvarious solid tumors. Anti-EGFR and anti-VEGF agents already in the clinic include orally admin-istered small molecule tyrosine kinase inhibitors (eg, erlotinib and gefitinib) and intravenousmonoclonal antibodies (eg, bevacizumab, cetuximab). In addition to other agents that targetEGFR and VEGF (and its receptors), other molecular entities that appear to be good targets forcancer treatment include: NF-�B and the proteasome, the mammalian target of rapamycin, andcyclin-dependent kinases. These novel targeted agents offer great potential, but, except for a fewcases, their use is still being optimized. Given the generally low activity of even the most clinical-ly advanced targeted agents as monotherapy, it has become evident that these agents need to becombined with chemotherapy or radiation to increase therapeutic benefit for patients withadvanced malignancies. The focus of the present monograph is to examine how far we havegone and how much farther we still have to go toward effectively combining targeted therapieswith radiation to improve the treatment of patients with head and neck cancer.

This monograph highlights the proceedings from a satellite symposium held June 23, 2006, duringthe June 22-25. Radiation Therapy Oncology Group (RTOG) meeting held at the Fairmont RoyalYork, Toronto, Canada. During this symposium four experts in the field discussed the early clinicaldevelopment of EGFR inhibitors; the preclinical rationale and supporting clinical data for combin-ing EGFR inhibitors, anti-VEGF inhibitors, and other molecularly targeted therapies with radiationtherapy; and the challenges associated with testing, optimizing, and validating these treatments.

5

Combining EGFR Inhibitors With Radiation: Early Clinical DevelopmentPaul A. Bunn, Jr, MD

The epidermal growth factor receptor (EGFR), which is involved in proliferation of normalepithelium, becomes overexpressed in preneoplastic processes, and during progression of malig-nant tumors in squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lungcancer (NSCLC). In fact, EGFR is highly expressed in most, but not all, SCCHN and lung cancers.Evidence suggests that increased EGFR gene copy number may be associated with poor prognosisof patients with SCCHN and potentially lung cancer.1,2 In the presence of tobacco and other car-cinogens, the epithelium thickens and nuclei become larger and atypical. All the cells in thisexpanded epithelium express EGFR. During the preneoplastic and neoplastic processes, bloodvessels are recruited into the epithelium through the process of angiogenesis (see Dr O’Reilly’smanuscript for further discussion of this topic).

EGFR Structure and FunctionEGFR is a transmembrane-spanning tyrosine kinase receptor with extracellular and intercellulardomains. The extracellular domain is recognized by two ligands, transforming growth factor(TGF)-alpha and EGF. Ligand binding to EGFR causes receptor dimerization, which leads to inter-nal conformational changes that allow phosphorylation of the intercellular portion of the recep-tor. Phosphorylation then triggers a cascade of intracellular signals causing cellular proliferation,angiogenesis, and inhibition of apoptosis. The EGFR family (known as the HER/ErbB family) oftransmembrane receptor tyrosine kinases includes HER1 or EGFR, HER2, HER3, and HER4, andtheir various ligands. In the presence of ligand, homodimerization and heterodimerization arepossible.3 The EGFR gene is large and is located on chromosome 7. Tumors may harbor anincreased EGFR gene copy number, gene amplification, and/or mutations in the EGFR gene.

Anti-EGFR AgentsAnti-EGFR agents in clinical development include the orally administered small molecule tyro-sine kinase inhibitors (TKIs) and intravenously administered monoclonal antibodies. TKIs such aserlotinib and gefitinib recognize the intercellular portion of EGFR. The monoclonal antibodycetuximab binds to the extracellular portion of this receptor. In head and neck cancer and lungcancer these agents caused objective responses in a minority of patients and stable disease (SD),usually with symptomatic benefit, in a larger group of patients.4-7

Tyrosine Kinase InhibitorsEarly in the development of these agents, particularly in lung cancer, it became apparent thatcertain clinical features were associated with response. Women, never smokers, and patients withadenocarcinoma had higher responses with gefitinib and erlotinib treatment compared withmen, smokers, and those with other NSCLC histologies, respectively (Iressa package insert).8,9 Themost common EGFR inhibitor class side effects include dose-related acneiform rash and diarrhea.Interstitial lung disease (ILD) has been reported in about 1% of patients treated with erlotinib,with as high as a 2% incidence in Japanese patients treated in the post-marketing experience.8

ILD had lethal consequences in about one third of affected patients.

Clinical Development of TKIsIn previously treated patients with advanced NSCLC, a randomized phase III trial (NationalCancer Institute of Canada Clinical Trials Group Study BR.21) showed a median overall survival

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of 6.7 months for erlotinib compared with 4.7 months for placebo (hazard ratio [HR], 0.73; P <.001), indicating a 27% reduction in the hazard ratio of death.9 In this trial, while all subsets ofpatients benefited from erlotinib treatment, the best survival advantage occurred in never-smok-ers. These data have been overinterpreted by some, leading to exclusion of certain subgroups insome studies despite evidence for survival benefit in all subsets.

Following these trials comparing TKI monotherapy with placebo, the anti-EGFR agents weretaken directly to phase III clinical trials in combinations with chemotherapy. Four large random-ized trials showed no benefit when gefitinib10,11 or erlotinib12,13 was added to standard chemother-apy in unselected patients with NSCLC. In the ISEL phase III trial, in unselected patients withadvanced refractory NSCLC, gefitinib also had no statistically significant median overall survival(gefitinib 5.6 months versus placebo 5.1 months) and 1-year survival (gefitinib 27% versus place-bo 22%) advantage compared with placebo (HR, 0.89; P < .11).14 The lack of patient selection forEGFR expression and negative interaction with chemotherapy likely contributed to the poorresults achieved by the TKIs in these randomized trials. To draw a parallel from breast cancer, onecan imagine that tamoxifen would also have produced poor results if it had been tested inpatients with breast cancer unselected for expression of the estrogen receptor. Thus, one of theproblems faced in the clinical development of anti-EGFR agents may be that of targeted agentsnot being studied with due consideration to the target. Another contributing factor to the poorperformance of TKIs in combination with chemotherapy for NSCLC may be a negative interac-tion between the mechanism of action of the anti-EGFR TKIs and standard chemotherapy.Indirect evidence supporting this hypothesis comes from the INTACT 2 trial, where the time toprogression curves for chemotherapy and chemotherapy + gefitinib are identical in the first 6months, but seem to separate at later times when treatment with gefitinib continued afterchemotherapy stopped (Figure 1).11 Results of in vitro experiments suggest a possible explanationfor the poor effect of combining TKIs with chemotherapy. Anti-EGFR TKIs inhibit cells in the G1

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Population: intention-to-treat.

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500 mg4.67

-1.6338(p=0.10)

250 mg5.32

-1.5833(p=0.11)

Placebo5.06

Group 500 mg/dayGroup 250 mg/dayPlacebo

Figure 1. Time to progression in INTACT-2 trial: curves appear to separate after chemotherapy stopped, while gefi-tinib treatment continued. (From Herbst et al,11. permission needed)

phase of cell cycle, whereas chemotherapy, such as taxanes, kills cells in G2. A drug that kills atG2 would not be expected to have an effect on cells already arrested. In cell culture experiments,erlotinib abrogated the G2/M blockade effect of paclitaxel in H322 cells when given simultane-ously.15

Early Studies of Anti-EGFR Inhibitors Combined With Radiation TherapyAs was seen in clinical trials of anti-EGFR inhibitors used as monotherapy or in combinationwith chemotherapy, early studies of combinations with radiation therapy were done in unselect-ed patients. In the SWOG 0023 trial, patients first treated with concurrent chemotherapy + radia-tion, followed by consolidation therapy with docetaxel, were randomized to maintenance thera-py with placebo or gefitinib.16 Many patients dropped off the trial after the consolidation phase(perhaps partly because of the high dose of docetaxel used), and only 255 of the originallyaccrued 620 patients were eligible for randomization to maintenance therapy with gefitinib orplacebo. Interim results of this trial presented at ASCO 2005 showed that the placebo group hadslightly better overall survival and time to tumor progression compared with the gefitinib groupand that it was unlikely that gefitinib would be superior if the trial continued (Figure 2). Thus,the trial closed early indicating that giving maintenance gefitinib to unselected patients afterchemotherapy-radiation is not indicated (see Dr Harari’s manuscript for discussion of combininganti-EGFR agents with radiation therapy).

Selection of Patients for Anti-EGFR Therapy With TKIsSelection of patients to undergo therapy with EGFR TKIs could be based on clinical or biologiccriteria, or a combination of the two. Clinical selection criteria being investigated include gender,histology, smoking status, and ethnicity. Biologic criteria that are being studied by various groupsinclude EGFR protein expression assessed by immunohistochemistry (IHC), EGFR gene high copynumber or amplification measured by fluorescent in situ hybridization (FISH), EGFR and K-Rasmutations determined by DNA sequencing, and pAKT activation status assessed by IHC. AKT is aserine/theronine kinase that affects cell survival, proliferation, growth, and other important cel-

8

0%

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N124131

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1929

The P-value for testing the alternative hypothesis of a 33% improvement with gefitinib was P = 0.0015.

P =0.092-sided stratified

log-rank

Figure 2. SWOG 0023 survival from randomization to gefitinib versus placebo. .(From Kelly et al,16 will need permission)

lular processes by regulating intracellular signaling pathways located downstream of EGFR.17 Amultivariate analysis of clinical and biologic sub-group data from two phase II trials with nearly200 patients showed that smoking status, performance status, EGFR gene copy number, andamount of EGFR protein expression were independent prognostic factors.18 In this study that hasbeen submitted for publication, patients who had NSCLC tumors that were EGFR-positive byboth IHC and FISH analysis had a longer median survival (21 months) compared with patientswho were EGFR-positive with only one assay (median survival 11 months), and with those whowere EGFR-negative with both assays (median survival 6 months).

My colleagues and I also performed an analysis of data from an Italian study cohort of 105patients treated with gefitinib 250 mg/day (Expanded Access Protocol for second- or third-lineNSCLC) and 100 patients treated with 500 mg/day gefitinib as first-line or second-line treatmentfor bronchealveolar carcinoma (BAC).19 In this study, patients with tumors containing EGFR exon19 mutations had a higher probability of response to gefitinib and a trend toward longer medialsurvival compared with patients with exon 21 mutations or wild type EGFR. Prospective random-ized studies are needed to validate whether EGFR exon 19 mutations truly have a predictive valuefor response to gefitinib treatment.

Retrospective analyses of data from phase III trials with gefitinib and erlotinib suggest the poten-tial predictive utility of some of these biomarkers, particularly EGFR FISH status (Figure 3).20,21

FISH-positive status seems to be associated with improved survival with gefitinib treatment com-pared with placebo in the ISEL trial and with erlotinib treatment in the BR.21 trial. 20,21 In thesetwo studies, the HR for gefitinib was 0.61 and for erlotinib 0.44 in FISH-positive patients com-pared with placebo. These data suggest that FISH-positive status for EGFR gene high copy numberor amplification may be a good selection factor for treatment with these TKIs. Cappuzzo and col-

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Log-rank: p=0.008HR=0.44 (0.23, 0.82)

Log-rank: p=0.59HR=0.85 (0.48, 1.51)

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Cox: p=0.07HR=0.61 (0.36, 1.04)

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Figure 3. Retrospective analysis of data from randomized studies with gefitinib and erlotinib suggests that FISH may beuseful in predicting benefit of EGFR-TKIs. (From Hirsch et al,20, Tsao et al21, will need permission)

leagues came to the same conclusion after their retrospective analysis of tumor samples from 102patients with NSCLC treated with gefitinib.22 In multivariate analysis, high EGFR gene copy num-ber determined by FISH analysis was associated with better survival with a statistically significantHR of 0.44 (95% CI, 0.23 to 0.82).

Preliminary results of the prospective ONCOBELL trial, reported by Cappuzzo et al at ASCO2006, also indicate that EGFR FISH analysis may be a useful predictor of response to gefitinib inpatients with NSCLC.23 This study found that the presence of EGFR exon 19/21 mutations wasassociated with increased response to this TKI; whereas the presence of EGFR exon 20 and HER2mutations appeared linked to gefitinib resistance. In Japan, Inoue and colleagues used the pres-ence of mutations of EGFR exons 18 to 23 to prospectively select patients with previouslyuntreated advanced NSCLC for treatment with gefitinib (250 mg/d).24 From a group of 75patients, they found that 25 (33%) had EGFR mutations, which appeared more frequently inwomen (P < .01) and never or light smokers (P < .001). Sixteen of the patients with EGFR muta-tions received gefitinib and had an overall response rate of 75% (95% CI, 54% to 96%) and amedian progression-free survival time of 9.7 months (95% CI, 7.4 to 9.9 months). Initial resultsfrom another study indicate that EGFR protein expression by IHC may be a weaker predictivefactor than FISH.25

Ongoing Studies With Patient Selection for Anti-EGFR TKI TreatmentOngoing studies with some of the TKIs are focusing on patients with EGFR+ disease (Figure 4).For example, a phase II trial is comparing erlotinib monotherapy with carboplatin/paclitaxelalternating with erlotinib in patients with untreated NSCLC that is EGFR FISH+ and/or IHC+(OSI Protocol #724-203). The industry-supported Randomized Double-Blind Trial in AdjuvantNSCLC With Tarceva (RADIANT) began recruitment in September of 2006. In this trial, patientswith stage IB-IIIA disease that is FISH+ and/or IHC+ after surgery and chemotherapy (unless stageIB) are being randomized 2:1 to erlotinib or placebo. Another promising trial of this type was theCanadian NCIC BR.19 adjuvant trial of gefitinib monotherapy for resected NSCLC.Unfortunately, this trial closed early after a preceding trial in patients with stage IV NSCLCresulted in a negative outcome for the investigational agent.

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NSCLC StageIB-MIA

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RANDOMIZE*

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NSCLCEGFR FISH+and/or IHC+

Figure 4. Schema of examples of ongoing trials of TKIs in EGFR+ positive patients. (Need permission from OSI)

CetuximabCetuximab is an IgG1 chimeric monoclonal antibody that exclusively recognized EGFR and itsheterodimers (see Harari’s manuscript for further discussion). Binding of cetuximab to EGFR pre-vents binding of EGF, the natural ligand for EGFR, and leads to a cascade of events that includestimulation of receptor internalization and inhibition of receptor dimerization, tyrosine kinasephosphorylation, and signal transduction. Cetuximab binds to EGFR with an affinity (Kd = 2.0 x10-10 M) that is one log higher than the affinity of the natural ligand.

Predictably for monoclonal antibodies, cetuximab has little activity when used as a single agent.In a phase II trial, cetuximab had a 4.5% overall response rate consisting of 3 partial responsesamong 66 patients with recurrent NSCLC; 20 of these patients (30%) experienced stable disease.26

Cetuximab had higher response rates when given in combination with chemotherapy in phase IItrials (Table 1).27-30 As a follow-up to a phase I/II trial of a cetuximab, paclitaxel, carboplatin regi-men,31 Kelly et al27 reported at ASCO 2006 preliminary results of the randomized phase II selec-tion trial SWOG 0342 which evaluated the combination of chemotherapy (paclitaxel + carbo-platin) + cetuximab and chemotherapy followed by cetuximab in previously untreated patientswith advanced NSCLC. The addition of cetuximab improved ORR and median survival, but didnot seem to affect time to tumor progression. The chemotherapy + cetuximab arm achieved theprimary endpoint of median survival > 10 months and will be used in future trials. Preliminaryresults from the phase II randomized LUCAS trial suggested an advantage when cetuximab wasadded to cisplatin + vinorelbine chemotherapy compared with chemotherapy alone.28 The phaseII results suggest that, unlike with the small-molecule TKIs, there does seem to be an advantageto giving cetuximab with standard chemotherapy. A randomized phase III trial (FLEX trial) is inprogress to compare cisplatin + vinorelbine + cetuximab with cisplatin + vinorelbine in patientswith previously untreated advanced or metastatic NSCLC that expresses EGFR by IHC.32

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Table 1. Phase II clinical trials of cetuximab plus chemotherapy in advanced NSCLC

Study Regimen N Previous Overall Median 1-YearChemotherapy Response Survival, Survival,

(Yes/No) Rate, % mo %

Thienelt Carboplatin+ 31 No 29 10.7 45et al31 paclitaxel+

cetuximab

Rosell Cisplatin+ 43 No 35 8.3 32(LUCAS vinorelbine +trial)28 cetuximab

Cisplatin+ 43 No 28 7.0 26vinorelbine

Robert Carboplatin+ 35 29 10.2 45et al29 gemcitabine+

cetuximab

Kim30 Docetaxel+ 47 Yes 22.2 7.8 38cetuximab

NR = not reported.

It is now clear that the EGFR is an important target for both NSCLC and for SCCHN. The small-molecule TKI erlotinib is approved for use in NSCLCs and cetuximab is approved for use in com-bination with radiotherapy in SCCHN. Retrospective studies indicate that biologic features of thetumor, including cell surface EGFR staining for SCCHN and NSCLC, and EGFR FISH and muta-tion status for NSCLC, can be used to select patients most likely to benefit from these EGFRinhibitors. Ongoing prospective trials should help define the best way to select patients and thebest way to integrate these EGFR therapies with radiotherapy and with chemotherapy.

Reference List

1. Chung CH, Ely K, McGavran L, et al. Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas. J Clin Oncol. 2006;24:4170-4176.

2. Hirsch FR, Bunn PA Jr. Epidermal growth factor receptor inhibitors in lung cancer: smaller or larger molecules, selected or unselected populations? J Clin Oncol. 2005;23:9044-9047.

3. Pinkas-Kramarski R, Soussan L, Waterman H, et al. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J. 1996;15:2452-2467.

4. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL1 Trial) [corrected]. J Clin Oncol. 2003;21:2237-2246.

5. Kelly K. The benefits of achieving stable disease in advanced lung cancer. Oncology (Williston Park). 2003;17:957-963.

6. Perez-Soler R. Phase II clinical trial data with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (OSI-774) in non-small-cell lung cancer. Clin Lung Cancer. 2004;6 (suppl) 1:S20-S23.

7. Shepherd FA, Rodrigues PJ, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med. 2005;353:123-132.

8. Iressa Package Insert. AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA. June, 2004.

9. Tarceva Package Insert. OSI Pharmaceuticals, Inc. and Genentech, Inc., Melville, New York, USA. Nov., 2005.

10. Giaccone G, Herbst RS, Manegold C et al. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial--INTACT 1. J Clin Oncol.2004;22:777-784.

11. Herbst RS, Giaccone G, Schiller JH, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial--INTACT 2. J Clin Oncol.2004;22:785-794.

12. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol. 2005;23:5892-5899.

13. Perez-Soler R. The role of erlotinib (Tarceva, OSI 774) in the treatment of non-small cell lung cancer. Clin Cancer Res. 2004;10:4238s-4240s.

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14. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet. 2005;366:1527-1537.

15. Piperdi B, Ling YH, Kroog G, Perez-Soler R. Schedule-dependent interaction between epidermal growth factor inhibitors (EGFRI) and G2/M blocking chemotherapeutic agents (G2/MB) on human NSCLC cell lines in vitro. J Clin Oncol (Meeting Abstracts). 2004;22:7028.

16. Kelly K, Gaspar LE, Chansky K, et al. Low incidence of pneumonitis on SWOG 0023: A preliminary analysis of an ongoing phase III trial of concurrent chemoradiotherapy followed by consolidation docetaxel and gefitinib/placebo maintenance in patients with inoperable stage III non-small cell lung cancer. J Clin Oncol (Meeting Abstracts). 2005;23:7058.

17. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999;13:2905-2927.

18. Hirsch FR, Varella-Garcia M, Bunn PA Jr. Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small cell lung cancer patients treated with EGFR tyrosine kinase inhibitor therapy. Ann Oncol. 2006;Submitted.

19. Hirsch FR, Franklin WA, McCoy J, et al. Predicting clinical benefit from EGFR TKIs: Not all EGFR mutations are equal. J Clin Oncol (Meeting Abstracts). 2006;24:7072.

20. Hirsch F, Franklin WA, McCoy J, et al. Molecular predictors of outcome with gefitinib in a phase III placebo-controlled study in advanced non-small-cell lung cancer. J Clin Oncol. 2006;24:In press.

21. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer - molecular and clinical predictors of outcome. N Engl J Med. 2005;353:133-144.

22. Cappuzzo F, Hirsch FR, Rossi E, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst. 2005;97:643-655.

23. Cappuzzo F, Toschi L, Trisolini R, et al. Clinical and biological effects of gefitinib in EGFR FISH positive/phospho-akt positive or never smoker non-small cell lung cancer (NSCLC): Preliminary results of the ONCOBELL trial. J Clin Oncol (Meeting Abstracts). 2006;24:7023.

24. Inoue A, Suzuki T, Fukuhara T, et al. Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations. J Clin Oncol. 2006;24:3340-3346.

25. Hirsch FR, Varella-Garcia M, Bunn, PA, Jr. et al. Molecular analysis of EGFR gene copy number, EGR expression and Akt activation status in advanced non-small cell lung cancer (NSCLC) treated with gefitinib or placebo (ISEL trial). Clin Cancer Res. 2005;24:9031s.

26. Lilenbaum R, Bonomi P, Ansari R, et al. A phase II trial of cetuximab as therapy for recurrent non-small cell lung cancer (NSCLC): Final results. J Clin Oncol (Meeting Abstracts). 2005;23:7036.

27. Kelly K, Herbst RS, Crowley JJ, et al. Concurrent chemotherapy plus cetuximab or chemotherapy followed by cetuximab in advanced non-small cell lung cancer (NSCLC): A randomized phase II selectional trial SWOG 0342. J Clin Oncol (Meeting Abstracts). 2006;24:7015.

28. Rosell R, Daniel C, Ramlau R, et al. Randomized phase II study of cetuximab in combination with cisplatin (C) and vinorelbine (V) vs. CV alone in the first-line treatment of patients (pts)with epidermal growth factor receptor (EGFR)-expressing advanced non-small-cell lung cancer(NSCLC). J Clin Oncol (Meeting Abstracts). 2004;22:7012.

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29. Robert F, Blumenschein G, Herbst RS, et al. Phase I/IIa study of cetuximab with gemcitabine plus carboplatin in patients with chemotherapy-naive advanced non-small-cell lung cancer.J Clin Oncol. 2005;23:9089-9096.

30. Kim ES, Mauer AM, Tran HT, et al. A phase II study of cetuximab, an epidermal growth factor receptor (EGFR) blocking antibody, in combination with docetaxel in chemotherapy refractory/resistant patients with advanced non-small cell lung cancer: Final report. ASCO Meeting Abstracts. 2003;22:2581.

31. Thienelt CD, Bunn PA, Jr, Hanna N, et al. Multicenter phase I/II study of cetuximab with paclitaxel and carboplatin in untreated patients with stage IV non-small-cell lung cancer.J Clin Oncol. 2005;23:8786-8793.

32. Von Pawel J, Park K, Pereira JR, et al. Phase III study comparing cisplatin/vinorelbine plus cetuximab versus cisplatin/vinorelbine as first-line treatment for patients with epidermal growth factor (EGFR)-expressing advanced non-small cell lung cancer (NSCLC) (FLEX). J Clin Oncol (Meeting Abstracts). 2006;24:7109.

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Patients with SCCHN with extensive disease often require narcotic analgesics to control pain andnutritional supplementation to successfully complete therapy. It is a challenge for clinicians tonurture these patients through several months of therapy. There is a clear need for the develop-ment of effective biologic therapies that are less toxic and will, hopefully, complement and sup-plement the advances that are being made in surgery and radiation therapy.

Traditional cytotoxic agents commonly have an effect on DNA synthesis, transcription, transla-tion, or cell division. They are nonselective to the cancer cell compartment and thus generallycause a wide spectrum of side effects (eg, mucositis, hair loss, GI effects, and immunosuppres-sion) as collateral damage. The premise for targeted therapies, such as EGFR inhibitors, is thatthese drugs, which are designed to be more selective in blocking tumor growth, will do so withless toxicity to normal cells. Examples of molecular targets currently being investigated include:

• Growth factors and their receptors (the most advanced area and the focus of this monograph)

• Signal transduction pathways

• Tumor-associated antigens/markers

• Proteasome

• Cell-survival pathways

• Extracellular matrix/angiogenic pathways

15

Figure 1. Simplified schematic of EGFR signaling pathways, their effect on cellular functions, and the effect of anti-EGFRtreatment by 2 classes of agents: mAbs and TKIs. ECM = extracellular matrix; mAb = monoclonal antibody; TKI = tyro-sine kinase inhibitor. (From Harari, Huang, et al,1 with permission)

Combining EGFR Inhibitors With Radiation in Head and Neck CancerPaul M. Harari, MD

Introduction

The EGFR signaling pathway is a very active area for development of targeted agents because ofits central role in regulating cellular functions that affect tumor growth and metastasis (Figure1).1 Agents that target extracellular portions of EGFR include the commercially available mono-clonal antibodies cetuximab and panitumumab (which was approved in September 2006) andthe investigational monoclonal antibody matuzumab. TKIs are small-molecule orally adminis-tered anti-EGFR inhibitors that target the intracellular domain of EGFR. In the past few years,two anti-EGFR agents received US Food and Drug Administration (FDA) approval for oncologyindications: gefitinib in 2003 and erlotinib in 2004. Many more new agents are moving throughinvestigational drug development pipelines. It is important for all involved in clinical care andtrial design to ensure that these agents undergo careful, logical, and judicious clinical testing.

Preclinical Rationale for Combining EGFR Inhibitors With Radiation Therapy The rationale for combining EGFR inhibitors with radiation therapy is based on experimentalfindings in various in vitro and in vivo preclinical models. One of the elements that may con-tribute to the favorable interaction of EGFR inhibitors with radiation therapy is that these 2modalities induce cell cycle arrest at different checkpoints: G1 for the EGFR inhibitors and G2 forradiation. Several studies with different EGFR inhibitors support this notion. For example, Huangand colleagues reported that cetuximab treatment modulated proliferation, apoptosis, andradiosensitivity in squamous cell carcinoma (SCC) cell lines derived from patients with head andneck cancer.2 In these in vitro experiments, cetuximab treatment—which caused an accumula-tion of cells in the G1 phase of the cell cycle and apoptosis of SCC cells—enhanced SCCradiosensitivity and amplified their radiation-induced apoptosis. A subsequent study in SCC

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Figure 2. Antitumor activity of cetuximab in combination with radiation in SCC xenografts. Tumor cells were injectedinto athymic mice. After 23 days (when tumors reached a certain size) cetuximab treatment (1 injection/week for 4weeks) was initiated. Radiation treatment consisted of a single 8-Gy fraction, administered 24 h after each injection ofcetuximab. Values are mean tumor size ± SE (n = 8 mice/group). (From Huang et al,3 with permission)

tumor xenografts showed that these in vitro observations, on the combined effects of anti-EGFRtreatment with cetuximab and radiation, translated nicely to an in vivo human tumor model.3 Inthese human SCC tumor xenografts the combination of cetuximab and radiation therapy inducedcomplete regression of tumors over a 100-day follow-up period (Figure 2). The mechanism of cetux-imab modulation of the radiation response in these models appears to involve inhibition of dam-age repair, cell cycle kinetics, and tumor angiogenesis. Additional in vitro and in vivo experimentsfrom several laboratories have produced further evidence that the anti-metastatic effect of cetux-imab treatment on human SCC may involve disruption of tumor-induced vascularization.4

Similarly, Chinnaiyan et al found that combination treatment with erlotinib and radiation shift-ed cell cycle distribution toward the G1 phase and enhanced radiation therapy—induced apopto-sis in cell lines derived from patients with NSCLC, prostate cancer, and SCCHN, and in humanxenograft tumor models (Figure 3).5 Using a human SCCHN tumor xenograft model, Huang andcolleagues showed that the combination of oral gefitinib and focal radiation treatment resultedin significant tumor regression and delay of regrowth.6 Treatment with each modality alonecaused only partial and transient tumor regression. Gefitinib treatment also reduced cell survivalafter radiation in clonogenic assays of SCCHN cells. Additional experiments published in thesame paper suggested that the antitumor activity of this combination may be attributed to bothinhibition of cellular proliferation (through cell cycle arrest and enhanced radiation-inducedapoptosis) and inhibition of tumor angiogenesis.

In summary, numerous preclinical studies (only a fraction of which could be summarized in thisarticle) have shown that EGFR blockade by various anti-EGFR agents given in combination withradiation has antiproliferative effects at multiple cell cycle checkpoints. The combined effect ofradiation-induced blockade at G2 and anti-EGFR-induced blockade at G1 leads to more successfultumor apoptosis induction, inhibition of DNA damage repair, anti-angiogenic effects, and anti-

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control erlotinib XRT erlotinib/XRT

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Figure 3. Impact of treatment with erlotinib, radiation therapy, and the combination of the 2 modalities on cell cycle S-phase distribution of the human head and neck SCC cell line UM-SSC6. Columns are mean values of duplicate samples. (Modified from Chinnaiyan et al,5 with permission)

metastatic effects. The next key question is how well these impressive in vitro and in vivo obser-vations translate to the clinic.

Clinical Experience With Anti-EGFR Therapy Combined With Radiation TherapyThis strong preclinical rationale for combining anti-EGFR therapy with radiation therapy stimu-lated the clinical development of combining cetuximab with radiation therapy. A phase I trial inpatients with advanced SCCHN showed that the regimen was well tolerated and had measurableefficacy in all assessable patients.7 The activity of cetuximab in combination with cisplatin hadalso been demonstrated in phase II trials involving patients with recurrent or refractorySCCHN.8,9

A phase III trial subsequently established that the combination of cetuximab and radiation thera-py is a good strategy for these patients.10 This international study enrolled 424 patients with stageIII or IV, nonmetastatic measurable SCCHN suitable for definitive radiotherapy. Patients wereexcluded from this study if they had received chemotherapy in the previous 3 years; if they hadundergone surgery for SCCHN; or if they had been treated with radiation therapy for SCCHN.Confirmation of EGFR+ status was not an entry criterion, but tumor samples were obtained forits determination by IHC. Patients—stratified by Karnofsky performance status scores, presence orabsence of positive regional nodes, tumor stage, and radiotherapy fractionation—were random-ized to radiation therapy or radiation therapy plus weekly cetuximab. The primary endpoint ofthis study was independently assessed duration of locoregional control, defined as the absence ofprogression of locoregional disease at scheduled follow-up visits.

As expected for an EGFR inhibitor, the most common cetuximab-associated associated adverseevent was an acneiform rash, with most cases being grade 1 or 2 (Table 1). While cetuximab hadskin-related effects, it did not impact mucosal tissues, as evidenced by a similar frequency ofmucositis in the 2 treatment groups. Infusion reactions were more common in the cetuximab-

18

Table 1. Common adverse events reported in the pivotal phase III trial of radiation therapy ± cetuximab (Data fromBonner et al10)

Percentage of PatientsRadiation Therapy Radiation Therapy +

(n = 212) Cetuximab (n = 208)Adverse Event All Gr Gr 3/4 All Gr Gr 3/4Mucositis 94 52 93 56Acneiform rash 10 1 87* 17*Xerostomia 71 3 72 5Dysphagia 63 30 65 26Nausea 37 2 49† 2Fever 13 1 26‡ 1Headache 8 < 1 19‡ <1Infusion reaction 2 0 15* 3**Chills 5 0 11†† 0Anemia 13 6 3* 1‡‡

*P < .001, all comparisons done by Fisher’s exact test.

†P = .02. ‡P = .001. **P = .01. ††P = .03. ‡‡P = .006.

treated group, not surprising given that the other group was treated with radiation and no intra-venous antineoplastic agents. The incidences of other grade 3 or higher toxicities were not signif-icantly different between the 2 groups. Ninety percent of patients in the cetuximab groupreceived all planned doses (median number of doses, 8).

The duration of locoregional control, the primary endpoint, was significantly more favorableamong patients treated with the combined modality treatment (Figure 4). The median durationof locoregional control was 24.4 months among patients in the cetuximab plus radiation therapygroup compared with 14.9 months among those treated solely with radiation therapy (HR forlocoregional progression or death, 0.68; P = .005). After a median follow-up of 54 months, themedian duration of overall survival was 49.0 months for patients in the combined modalitygroup and 29.3 months for patients in the radiation therapy–only group (HR for death, 0.74; P =.03). The addition of cetuximab to radiation therapy was associated with a 10% gain in 3-yearsurvival benefit (cetuximab plus radiation therapy, 55% vs radiation therapy, 45%). The minimaladditive toxicity and significant improvement in locoregional control and overall survival seenwith the addition of cetuximab to radiation therapy in this study of patients with SCCHN led tothe addition of this indication to the cetuximab labeling. With this FDA approval, cetuximabbecame the first new drug approved for SCCHN in 45 years. Hopefully, more well-designed andtested targeted therapies will follow.

Economic Challenges to Developing and Implementing New TherapiesThe estimated cost of bringing a new cancer drug to market in the US is on the order of$500,000. Sources of US cancer research funding have shifted from primarily federal support inthe 1970s to primarily industry support in the current era.

The high cost of finding new drugs and bringing them to the market is now reflected in highdrug prices for targeted therapies. This price tag on progress poses serious financial strain onpatients, caregivers, and as public and private heath insurance providers. For example, the intro-duction of new treatment regimens for colorectal cancer has shown the capacity to prolong the

19

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life of selected patients by several months; however, the treatment is generally palliative innature. The annual cost to treat all eligible patients with metastatic CRC in the US with regimensincorporating targeted therapies is now estimated to be approximately $1.2 billion.11 It is clearthat integration of targeted therapies in the care of patients with cancer has raised a host of com-plex pharmacoeconomic issues that still remain to be unraveled. Clinicians face the challenge ofsorting through the available data to make the most judicious applications of these medicationsfor improving patient survival and quality of life. Clinicians who treat patients with SCCHN nowhave a promising new therapy option to consider with the knowledge of a 10% gain in 3-yearsurvival seen with the addition of cetuximab to radiation. This finding seems likely to translateinto more patients cured of their SCCHN cancer since the vast majority of H&N recurrences aremanifested within 24 months following treatment.

Summary and ConclusionMolecular oncology is bringing more cancer-specific therapies with reduced toxicity profiles tocancer patients. As more patients are being treated with EGFR inhibitors there is the vexing issueof how to best treat skin reactions—which oncologists are not yet fully accustomed to do. It willbe valuable to develop algorithms to improve clinician and patient handling of this side effect.Resistance to anti-EGFR therapies limits the impact of this promising class of agents, as evi-denced by the fact that anti-EGFR agents have clinical activity in a minority of patients whenused as monotherapy (response rates 10% to 20%). The future will provide systematic dissectionsof the biological and molecular mechanisms of intrinsic (ie, agent ineffective at start of therapy)and acquired resistance (ie, loss of efficacy with time after initial response to therapy) to molecu-lar targeting agents such as imatinib, trastuzumab, bevacizumab, cetuximab, gefitinib, anderlotinib. The identification of specific molecular mechanisms of resistance to anti-EGFR agentsshould suggest new strategies to enhance activity.

As we transition novel targeted therapeutics to the clinic, another challenge is how to best inte-grate them with other treatment modalities. For example, when considering the potential appli-cations of targeted agents as radiation sensitizers, questions that need to be considered include:

• Should they be administered before, during, or after radiation therapy?

• Could they substitute targeted agent for chemotherapy?

• Should they be part of multiagent chemoradiation regimens, or multitargeted therapieswith radiation?

Reference List

1. Harari PM, Huang SM. Searching for reliable epidermal growth factor receptor response predictors. Clin Cancer Res. 2004;10:428-432.

2. Huang SM, Bock JM, Harari PM. Epidermal growth factor receptor blockade with C225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck. Cancer Res. 1999;59:1935-1940.

3. 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.

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4. Huang SM, Li J, Harari PM. Molecular inhibition of angiogenesis and metastatic potential in human squamous cell carcinomas after epidermal growth factor receptor blockade. Mol Cancer Ther. 2002;1:507-514.

5. Chinnaiyan P, Huang S, Vallabhaneni G, et al. Mechanisms of enhanced radiation responsefollowing epidermal growth factor receptor signaling inhibition by erlotinib (Tarceva). Cancer Res. 2005;65:3328-3335.

6. Huang SM, Li J, Armstrong EA, Harari PM. Modulation of radiation response and tumor-induced angiogenesis after epidermal growth factor receptor inhibition by ZD1839 (Iressa). Cancer Res. 2002;62:4300-4306.

7. Robert F, Ezekiel MP, Spencer SA, et al. Phase I study of anti--epidermal growth factor receptor antibody cetuximab in combination with radiation therapy in patients with advanced head and neck cancer. J Clin Oncol. 2001;19:3234-3243.

8. Baselga J, Trigo JM, Bourhis J, et al. Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinum-based chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck. J Clin Oncol. 2005;23:5568-5577.

9. Herbst RS, Arquette M, Shin DM, et al. Phase II multicenter study of the epidermal growth factor receptor antibody cetuximab and cisplatin for recurrent and refractory squamous cellcarcinoma of the head and neck. J Clin Oncol. 2005;23:5578-5587.

10. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006;354:567-578.

11. Schrag D. The price tag on progress—chemotherapy for colorectal cancer. N Engl J Med. 2004;351:317-319.

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Combining Targeted Therapies With Radiation Therapy

Combining Angiogenesis Inhibitors With RadiationMichael S. O’Reilly, MD

IntroductionThe outcome for patients with advanced cancer is poor even with aggressive therapy. Angiogenesis inhibitors offer great potential, but it is unclear how to optimize their use. Given their generally low activity as monotherapy, antiangiogenic agents need to be combinedwith radiation and other modalities for the treatment of advanced malignancies

For example, studies with bevacizumab in colon cancer1 and lung cancer2 showed that marginalimprovements in overall survival and progression-free survival were seen only when this anti–vas-cular endothelial growth factor (VEGF) monoclonal antibody was combined with chemotherapy.

Rationale for Combining Radiation Therapy With Anti-angiogenic Agents At least in preclinical studies, p53 (a tumor suppressor gene) status may affect tumor response toantiangiogenic therapy. Experiments by Yu and colleagues demonstrated that tumor cells with aninactivated p53 gene have a reduced rate of apoptosis under hypoxic conditions, exactly the con-ditions that would be expected to stimulate angiogenesis.3 These data suggest that resistance mayarise if therapy targets a single proangiogenic molecule, even when given in combination withchemotherapy. To overcome this resistance, a third modality such as radiation therapy might bea sensible addition to combined treatment with an antiangiogenic agent and standardchemotherapy.

Teicher and colleagues were the first to show that antiangiogenic therapy could be used effective-ly in combination with radiation therapy in a preclinical tumor model using Lewis lung tumor

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Figure 1. Increased productionof VEGF by tumor cells afterradiation therapy. A dose-dependent increase in VEGFsecretion is seen for all doses ofradiation (P < 0.05). (FromGorski et al. Cancer Res.1999:59:3374-3378, Figure 1B,requires permission)5

xenografts.4 Prior to these and other studies it was assumed that antiangiogenic agents wouldinduce hypoxia, which would make radiation less effective. Radiation does damage tumor vascu-lature; however, tumors revascularize rapidly partly because of an increased production of pro-angiogenic factors by the tumor cells. After radiation therapy, tumors increase production ofVEGF (Figure 1).5 This effect can be blocked by use of antiangiogetic agents thus improving, atleast in preclinical models, the response to radiation therapy.

Surrogate Markers to Monitor Therapeutic ActivityA problem associated with treating patients with angiogenesis inhibitors and other biologicaltherapeutics (alone or in combination with other modalities) is that they often do not work rightaway, making it difficult to determine the effect of treatment. This necessitates the need to devel-op invasive or noninvasive surrogate markers to predict response. Strategies that have been pur-sued include:

• Analyses of tumor biopsies before, during, and after therapy by immunohistochemistry

• Angiogenesis profiles using bodily fluids through assays that measure levels of angiogenic factors in blood or urine

• Proteomic and genomic analysis before, during, and after therapy

• Functional imaging of tumors and surrounding tissues before, during, and after therapy

Among the many challenges to developing these surrogates was the fact that a clinically validat-ed antiangiogenic agent is required to validate the efficacy of these surrogate markers. This chal-lenge is being overcome with the approval of bevacizumab and other targeted agents.

One approach we are purusing at M. D. Anderson Cancer Center is to use invasive strategies tovalidate noninvasive strategies such as functional imaging. In our preclinical experiments, theanti-angiogenic agent endostatin showed a marked improvement in inhibition of tumor growthwhen given in combination with radiation therapy (O’Reilly et al, unpublished data). We testedthe use of an angiogenesis index (ie, a ratio of endothelial cell proliferation and apoptosis) as asurrogate predictor of response to endostatin, radiation therapy, and the combination of the 2 inxenograft models. Our initial results indicate that this index might predict response before theconventional response on tumor growth is seen. More work is needed to test and eventually vali-date the usefulness of this potential predictive marker.

Willett et al from Massachusetts General Hospital have studied bevacizumab and neoadjuvantchemoradiation in patients with rectal cancer using imaging with positron emission tomography(PET) with [18F]-2-fluoro-deoxy-D-glucose (FDG) uptake.6,7 They examined microvessel density,number of mature vessels, tumor interstitial fluid pressure, and other parameters—none of themas of yet shown to be good surrogates for response (Figure 2). An interesting result was thattumor blood flow and blood volume changed as early as day 12. Additional studies are needed todetermine if this change in tumor blood flow and blood volume is a useful predictor of eventualresponse.

Animal Models and the Road to the ClinicAn important step in incorporating radiation therapy into a multimodality treatment of cancer isthe development of suitable animal models for preclinical studies. In collaboration with Drs Onnand Herbst, our laboratory developed an orthotopic lung cancer model to deliver radiation thera-

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py to the lung or mediastinum, and study it in combination with antiangiogenic agents. In thismodel, lung adenocarcinoma cells are injected directly into the lung of mice through the inter-costal space. The model appears to recapitulate what is seen in patients: a solitary lesion spreadswithin the lung, into the mediastinum, eventually moving into the contralateral lung andspreading more widely, killing the mouse. Using this system, we are able to deliver radiationtherapy to these animals, either hemi-thorax or hemi-thorax plus mediastinal radiation. Myers etal have developed a similar model for the study of SCCHN.8

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Figure 2. Neoadjuvant chemoradiation and bevacizumab treatment in patients with rectal cancer. Panels show vari-ous parameters pretreatment, 12 days after bevacizumab treatment, and after bevacizumab + chemoradiation. (a)Blood flow; (b) Blood volume; (c) Permeability; (d) Microvessel density; (e) Mature vessels; (f) Tumor interstitial pres-sure; (g) FDG uptake; (h) Circulating endothelial cell (EC) progenitors; (i) Viable circulating ECs.6 (From Willett etal. Nat Med. 2004;10:145-147; requires permission)6

We are using this lung adenocarcinoma model to study the effects of ZD6474, a small-molecule oralTKI that targets both VEGFR-2 and EGFR. In our experiments, treatment with ZD6474 alone pro-duced 50% inhibition of tumor growth. Moreover, when we gave ZD6474 with radiation, tumorsshrunk and then became stabilized with an effect superior to the combination of paclitaxel and radi-ation therapy (Shibuya et al, unpublished data). The effect of ZD6474 was sustained control of tumorgrowth, interestingly with recurrence of disease or progression generally occurring in the chest wall.This finding suggests that the chest wall microenvironment might be different than the lung’s, atleast in this animal model. Antiangiogenesis treatment might lead to tumor growth control in onesite, but not affect tumor progression in the other site. This localized resistance to antiangiogenictherapy with ZD6474 appears to be overcome by the addition of radiation therapy. In our experi-ments, ZD6474 plus radiation seemed to primarily block tumor cell proliferation rather than causetumor cell apoptosis. However, when we looked at the effect of this combined-modality treatmenton the vascular endothelium, we observed significant enhancement of endothelial cell apoptosisand, to a lesser degree, tumor cell proliferation, at least compared with controls and the individualtreatment modalities. Thus, our preliminary in vivo data suggest that tumor and vasculature may betargeted differently by antiangiogenic and radiation combined modality therapy (eg, by differentiallyblocking tumor proliferation and inducing apoptosis of vasculature).

We also investigated the production of other proangiogenic molecules (besides VEGF) to test thehypothesis that tumors become resistant to targeted therapy against one proangiogenic factorsuch as VEGF by increasing production of other proangiogenic factors. We found that radiationtherapy increased expression of basic fibroblast growth factor (bFGF), which is known to stimu-late angiogenesis and tumor revascularization. This effect on increased expression of bFGF is off-set by treatment with ZD6474 given in combination with radiation. ZD6474 alone suppressedbFGF expression to a small degree. After chemotherapy and chemoradiation we noted anincrease in bFGF, even though ZD6474 should not have activity, at least in theory, against bFGFproduction. We are still working on understanding the biology behind these experimentalresults. At the least, these experiments show no increased production of one other proangiogenicmolecule after combined-modality therapy with radiation and anti-VEGFR treatment withZD6474. Much work remains to be done to examine the effects of multimodality therapy onother proangiogenic signaling pathways that might engender tumor resistance.

Based on the results of our preclinical studies, we are planning a clinical trial of ZD6474 andradiotherapy for patients who would normally be treated with radiation therapy to the chestalone. Cohort 1 will enroll patients receiving palliative radiation therapy (45 Gy in 15 fractions)to the chest. Cohort 2 will consist of patients with inoperable disease receiving definitive radia-tion therapy (66 Gy in 33 fractions), but no chemotherapy. Cohort 3 will include patients to betreated with radiation therapy alone after induction chemotherapy led to stable or progressivedisease. We plan 3 dose escalation levels of ZD6474 (100, 200, and 300 mg/kg) within each of the3 radiation cohorts, with oral therapy to be started 1 week before radiation therapy. Three to 6patients will be accrued per each dose level of ZD6474 and treatment with this TKI will continueafter radiation therapy until disease progression. The design of this trial is based on the design ofa phase I trial of radiotherapy and the cyclooxygenase-2 enzyme inhibitor celecoxib in patientswith lung cancer (Figure 3).9 The feasibility of this previously published study suggests the useful-ness of this phase I clinical trial design for studying molecularly targeted agents in combinationwith radiation therapy.

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Itasaka and colleagues have developed a metastatic lung cancer to brain model. In this model,spontaneous metastasis developed in the contralateral side of the brain 26 days after intracarotidinjections of tumor cells to one side of the brain (Itasaka et al, unpublished data). In our experi-ments using this model, the triple combination of ZD6474, docetaxel, and radiation was moreeffective than either the single modalities or the various possible doublets (Itasaka et al, unpub-lished data). Studies of microvessel density indicated that, while docetaxel treatment increasedtumor vasculature, ZD6474 treatment given in combination with docetaxel offset this apparentincrease in angiogenesis so that the tumor vasculature acquired a more normal appearance. Thisobservation is in line with Dr Jain’s10 hypothesis that antiangiogenic treatment initially normal-izes tumor vasculature by trimming off aberrant blood vessels, making the tumor more suscepti-ble to infiltration by chemotherapeutic agents. We currently are trying to adapt this mousemodel to deliver radiation to head and neck tumor models, working in collaboration with DrJeffrey Myers (M. D. Anderson Cancer Center), who is developing a model of tongue cancer.

Future Directions for Antiangiogenic and Biological TherapeuticsAmple evidence supports the combination of emerging and existing therapeutic agents and treat-ment modalities such as radiation therapy. As discussed in this article, preclinical evidence andemerging clinical results suggest that the antiangiogenic agents used alone are not very effectivein the treatment of advanced and metastatic cancers. To effectively combine these treatmentmodalities we need to develop and validate noninvasive and invasive surrogates that will helppredict which agents will most likely be efficacious and allow monitoring of therapeutic efficacyduring treatment. To achieve this goal, preclinical models are being optimized to evaluate thera-pies and response. Hopefully, we will be able to apply the preclinical models that we are develop-ing to clinical practice. However, strategies will most likely need to be optimized on a site- and

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Figure 3. Results of a phase I clinical trial of radiation therapy and celecoxib in patients with lung cancer. Combined-modality treatment (radiation therapy after chemotherapy with celecoxib) appeared to improve local progression-freesurvival (LPFS) compared with radiation therapy alone. The design used in this trial is a potentially useful model forstudying molecularly targeted agents in combination with radiation therapy.9

disease-specific basis, and ultimately also on a patient-by-patient basis. The translation of biologi-cally targeted therapies into the clinic is now inevitable, clearly within our grasp. However, quitea bit of work remains to be done to achieve these goals.

Reference List

1. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004;350:2335-2342.

2. Sandler AB, Gray R, Brahmer J, et al. Randomized phase II/III trial of paclitaxel (P) plus carboplatin (C) with or without bevacizumab (NSC #704865) in patients with advanced non-squamous non-small cell lung cancer (NSCLC): An Eastern Cooperative Oncology Group (ECOG) Trial - E4599. J Clin Oncol (Meeting Abstracts). 2005;23:LBA4.

3. Yu JL, Rak JW, Coomber BL, Hicklin DJ, Kerbel RS. Effect of p53 status on tumor response to antiangiogenic therapy. Science. 2002;295:1526-1528.

4. Teicher BA, Holden SA, Ara G, Korbut T, Menon K. Comparison of several antiangiogenic regimens alone and with cytotoxic therapies in the Lewis lung carcinoma. Cancer Chemother Pharmacol. 1996;38:169-177.

5. Gorski DH, Beckett MA, Jaskowiak NT, et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res. 1999;59:3374-3378.

6. Willett CG, Boucher Y, di TE, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med. 2004;10:145-147.

7. Willett CG, Boucher Y, Duda DG, et al. Surrogate markers for antiangiogenic therapy and dose-limiting toxicities for bevacizumab with radiation and chemotherapy: continued experience of a phase I trial in rectal cancer patients. J Clin Oncol. 2005;23:8136-8139.

8. Myers JN, Holsinger FC, Jasser SA, Bekele BN, Fidler IJ. An orthotopic nude mouse model of oral tongue squamous cell carcinoma. Clin Cancer Res. 2002;8:293-298.

9. Liao Z, Komaki R, Milas L, et al. A phase I clinical trial of thoracic radiotherapy and concurrent celecoxib for patients with unfavorable performance status inoperable/unresectable non-small cell lung cancer. Clin Cancer Res. 2005;11:3342-3348.

10. Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science. 2005;307:58-62.

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Combining Other Novel Targeted Therapies With RadiationHak Choy, MD

In addition to the anti-EGFR inhibitors and the angiogenesis inhibitors discussed by my col-leagues in the accompanying manuscripts, there are many other novel targeted therapies thatappear quite amenable to combination with radiation therapy. Agents that target nuclear factor-kappa B (NF-�B) and the proteasome, the mammalian target of rapamycin (mTOR), and cyclin-dependent kinases (CDKs) offer promising opportunities for combination with radiation in thetreatment of head and neck cancer and other solid tumors.

Agents That Target NF-�B and the ProteasomeRationale for Targeting NF-�BAs its name implies, NF-�B is a nuclear transcription factor involved in controlling the expressionof genes that regulate apoptosis, viral replication, tumorigenesis, inflammation, and someautoimmune diseases.1,2 The proteasome is a large proteinase complex that degrades most intra-cellular proteins and, thus plays a key role in regulating cell growth and apoptosis (reviewed inMyung et al3). After protein translation, NF-�B localizes to the cell cytoplasm in an inactive form,because of binding by inhibitory proteins. Following activation by tumor necrosis factor (TNF)-�,these inhibitory proteins become phosphorylated and are degraded by the proteasome, leadingto the liberation and activation of NF-�B. Together, NF-�B and the proteasome are componentsof a signal pathway that is evolutionarily important in mediating programmed gene responses toinjury from various causes (eg, radiation, cytotoxic agents, hypoxia, chemicals, pathogens).

As a mediator of cytokine activity linking inflammation and cancer, NF-�B has been implicatedin tumor development and survival by data from a variety of preclinical experiments (reviewedby Karin4,5). For example, investigators from the National Institutes of Health (NIH, Bethesda,Md) showed that NF-�B is constitutively activated in cell lines derived from human SCCHN andthat it is involved in promoting the expression of the pro-inflammatory and pro-angiogeniccytokine interleukin-8.6,7 Studies conducted by Kato and colleagues demonstrated that radiationinduced activation of NF-�B in SCCHN.8 In these in vitro experiments, resistance to radiationcorrelated with activation of NF-�B and inhibition of NF-�B–sensitized cells to radiation. Morerecent in vitro and in vivo work involving microarray assays showed that NF-�B plays an impor-tant role in directly or indirectly modulating expression of genes linked to major biologicalprocesses (eg, proliferation, apoptosis, adhesion, and angiogenesis) that lead to the developmentof SCCHN.9

BortezomibThe proteasome inhibitor bortezomib (formerly PS-341) was developed in grandpart because ittargets NF-�B, presumably preventing its activation by inhibiting proteasome activity.Bortezomib received FDA approval in 2003 for the treatment of patients with multiple myelomawho had failed previous therapy. In vitro and in vivo experiments demonstrated that bortezomibinhibits activation of NF-�B, cell survival, tumor growth, and angiogenesis in SCCHN.10 Preclin-ical experiments indicated that bortezomib inhibited radiation-induced NF-�B activation .

Similarly, in vitro experiments using a colorectal cancer cell line showed that bortezomib treat-ment suppressed radiation-induced NF-�B activation, increasing the radiosensitivity of the cells.11

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Results of a small pilot study led by Carter Van Waes (National Institute on Deafness and OtherCommunication Disorders, Bethesda, Md) in patients with SCCHN further strengthened thenotion that combined therapy with bortezomib and radiation therapy inhibits activity of NF-�Band expression of genes involved in apoptosis, cell cycle, and angiogenesis.12 Some promisingresponses were observed in individual patients (Figure 1). However, the regimen (bortezomib 0.6or 0.9 mg/m2 given twice weekly plus daily radiation 50 Gy to 70 Gy) used in this trial proved tobe too toxic for patients, with the maximum tolerated dose exceeded at bortezomib 0.6 mg/m2.

An ongoing phase I trial of bortezomib (Protocol 01-C-0104) and concurrent radiation therapy inpatients with recurrent SCCHN also led by Van Waes shows initially promising results seen insome patients (Van Waes et al, personal communication). In this trial, bortezomib is being givenat the same dose levels as in the previous trial by the same group, but with lower doses of radia-tion therapy (1.8 Gy to 60 Gy given daily, 5 days a week). Five of 16 patients had partial respons-es by Response Evaluation Criteria in Solid Tumor (RECIST), 4 had prolonged stable disease, and1 patient had no evidence of disease 1 year after surgeries for residual and recurrent disease. Thisstudy is continuing enrollment and a phase II study is planned.

Agents That Target mTORRationale for targeting mTORRapamycin (sirolimus)—a natural product derived from the soil bacteria Streptomyces hygrosopiusand used in transplantation—has been shown to arrest the cell cycle at the G1 to S phase transi-tion (reviewed in Dutcher13). The serine/threonine kinase mTOR was identified as a critical cellcycle regulator in the process of uncovering the mechanism of action of rapamycin. mTOR is akey kinase that acts downstream of activation of the phosphatidylinositol 3 kinase (PI3K)/Aktpathway (reviewed in Vignot et al14). Rapamycin binds to mTOR’s immunophilin, FK binding

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Figure 1. Responses to bortezomib plus radiation therapy in patients with SCCHN, results from a pilot trial (VanWaes, et al, with permission).12

Tumor Reduction, Pt 1

Tumor Reduction, Pt 4

protein (FKBP12), and this complex interacts with mTOR, inhibiting its function, causing cellcycle arrest and inhibition of cellular proliferation. (Figure 2)

The Akt pathway appears to be constitutively activated in many SCCHN-derived cell lines.15

Preclinical experiments with rapamycin have shown that the Akt-mTOR pathway is a potentialtherapeutic target in SCCHN.16 In these studies, investigators frequently found aberrant accumu-lation of the p70-S6 kinase, an important downstream target of the pathway, in clinical speci-mens from patients with SCCHN and cell lines derived from their tumors. Rapamycin treatmentrapidly decreased accumulation of the phosphorylated form of S6 in cell culture and in SCCHNxenografts, where it also caused tumor regression through inhibition of DNA synthesis and stim-ulation of apoptosis.

Preclinical Activity of mTOR Inhibitors Combined With Radiation TherapyTemsirolimus (CCI-779, an ester of rapamycin); everolimus (RAD001, a hydroxyl ether ofrapamycin); and AP23573 (a non-prodrug analog of rapamycin) are mTOR inhibitors in currentclinical development. Along with rapamycin, these newer inhibitors of mTOR have demonstratedantitumor activity in various preclinical experiments, alone and in combination with radiation.

Eshleman and colleagues from the Mayo Clinic (Rochester, Minn) showed that while rapamycinhad no effect on radiation sensitivity of monolayer cultures of U87 and SKMG-3 malignantglioma cells, it significantly increased the efficacy of fractionated radiation on well-establishedU87 xenografts in nude mice (Figure 3).17 Others demonstrated that rapamycin and everolimushad improved antiangiogenic activity (as determined by blood vessel density) when given incombination with radiation in GL261 glioma tumor xenografts (Shinohara et al. Oncogene.2005;24:5414-5422) (Figure 4).18 Yet-to-be published in vitro experiments indicated that both

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Figure 2. The mTOR axis in intracellular signaling pathways provides multiple therapeutic targets in both cancer cellsand endothelial cells.

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Figure 3. The mTOR inhibitor rapamycin (Rap) enhanced sensitivity to radiation therapy (RT) of established U87malignant glioma human tumor xenografts implanted in nude mice (Eshleman et al,17 with permission). Nude micewith established U87 xenografts were randomized into 4 treatment groups: placebo, radiation only (4 Gy 4),rapamycin only (1 mg/kg), or radiation and rapamycin. The treatment schedule is depicted at the bottom of the figure.

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Figure 4. The combination of RAD-001 and radiation therapy was much more efficient than either treatment alone inreducing the blood vessel density of GL261 tumor xenografts grown in mice (Shinohara et al,18 with permission).

rapamycin and everolimus inhibited growth of human NSCLC cells (F. Khuri, MD, WinshipCancer Institute, Emory University, personal communication).

The combination of rapamycin and docetaxel also augmented effects on decreasing cell survivalof human NSCLC cells (F. Khuri, MD, personal communication).

Investigators at the Mayo Clinic (Rochester, Minn) recently completed a phase I rapamycin dose-escalation trial with concurrent cisplatin-based chemotherapy and radiation trial (NCCTGMC0211) in patients with NSCLC. Results have not yet been presented or published. A phase IItrial will open soon.

Agents That Target CDKsRationale for Targeting CDKsCDKs are key regulators of cell cycle progression (reviewed in Senderowicz and Sausville19). Thereare at least 9 CDKs and they act during different phases of the cell cycle. Some CDKs phosphory-late the retinoblastoma (Rb) protein, a tumor suppressor gene that is actively involved in regulat-ing the G1 phase of the cell cycle. Most human cancers harbor abnormalities in some aspect ofthe Rb pathway caused by over-activation of CDKs. Thus, inhibition of CDKs offers a reasonableapproach for the development of novel antineoplastic therapeutics.

FlavopiridolFlavopiridol (L86-8275), a synthetic flavone compound, is a cyclin-dependent kinase inhibitor (ofCDK1, 2, 4, 6, 7, 9) that causes cell cycle arrest at the G1 and G2 phases of the cell cycle. Studiesby various laboratories indicate that flavopiridol may be useful in combination with standardchemotherapy. My laboratory also demonstrated that docetaxel and flavopiridol enhanced theeffects of radiation in H460 human lung cancer cells in vitro and in vivo.20 In these studies, doc-etaxel plus flavopiridol appeared to act by stimulating apoptosis (Table 1 ) and mediatingchanges in the cell cycle. These results, suggesting the potential of flavopiridol as an enhancer ofradiosensitivity, are corroborated by data from many other laboratories. For example, Raju and co-workers from the M.D. Anderson Cancer Center (Houston, Tex) report-ed that flavopiridol strongly enhanced radiosensitivity of ovarian carcinoma cells in vitro, asassessed by the clonogenic cell survival assay.21 Investigators at Memorial Sloan Kettering CancerCenter (New York, NY), showed that flavopiridol potentiated gamma-irradiation-induced apopto-sis of colon and gastric cancer cells in vitro and in vivo.22 As we had shown in our experiments,these investigators also found that the most effective potentiation of tumor reduction occurredwhen flavopiridol was given after radiation.

32

Table 1. Enhancement of radiation effects by combined docetaxel and flavopiridol treatment in lung cancer cellsthrough increased apoptosis (Kim et al,20 with permission). The most effective treatment sequence in this experimentwas docetaxel, then radiation, followed by flavopiridol.

Treatment Dose Enhancement Ratio Apoptosis, %Docetaxel → flavopiridol 1.40 16.5Docetaxel → radiation 1.10 9.4Flavopiridol → radiation 1.18 17.0Radiation → flavopiridol 1.42 25.4Docetaxel → radiation → flavopiridol 1.69 30.9

Sato and colleagues from Japan reported that low-dose flavopiridol enhanced sensitivity to radia-tion of 3 different esophageal carcinoma cell lines.23 Flavopiridol given before or after radiationenhanced response of human esophageal adenocarcinoma (SEG-1) cells in tissue culture anddelayed growth of tumor xenografts generated from implantation of these cells into nude mice.24

Flavopiridol treatment also enhanced tumor-cell radiation response in a GL261 murine gliomatumor model.25 These and other studies have shown the intriguing potential of flavopiridol incombination with radiation therapy and the need for proper sequencing to optimize the efficacyof this combined-modality treatment.

The clinical development of this combination is still at its early stages. An ongoing phase I trialat Memorial Sloan-Kettering Cancer Center (NCT00047307) is examining a dose-escalating regi-men of flavopiridol in combination with radiotherapy (once daily, 5 days per week for 5.5weeks), followed 4 to 5 weeks later by gemcitabine treatment (alone or in combination withother agents, at the discretion of the treating oncologist), in patients with locally advanced,unresectable pancreatic cancer.

Conclusions and Future DirectionsNumerous molecular targeted agents have been developed to treat a wide array of cancers. A fewhave already received FDA approval to be used either alone or in combination. However, respons-es to monotherapy with molecular targeted agents are rare. Data from preclinical studies in vari-ous tumor types support the notion that several molecular targeted agents have radiation-enhancing effects. In fact, clinical trials are ongoing or planned combining targeted agents, suchas bortezomib, rapamycin, and flavopiridol, with radiation for the treatment of NSCLC, head and neck cancer, or other solid tumors. Hopefully, interesting data will become available in thenext few years. This is a promising but still very undeveloped area of clinical research. It remains to be seen how well the preclinical data will translate to the clinic. As those of us who work in oncology know so well: Unfortunately, one dumb tumor cell is still smarter than 10 smart oncologists.

Reference List

1. Maldonado V, Melendez-Zajgla J, Ortega A. Modulation of NF-kappa B, and Bcl-2 in apoptosisinduced by cisplatin in HeLa cells. Mutat Res. 1997;381:67-75.

2. Ghobrial IM, Witzig TE, Adjei AA. Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin. 2005;55:178-194.

3. Myung J, Kim KB, Crews CM. The ubiquitin-proteasome pathway and proteasome inhibitors. Med Res Rev. 2001;21:245-273.

4. Karin M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006;441:431-436.

5. Karin M. NF-kappaB and cancer: mechanisms and targets. Mol Carcinog. 2006;45:355-361.

6. Dong G, Chen Z, Kato T, Van WC. The host environment promotes the constitutive activation of nuclear factor-kappaB and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma. Cancer Res. 1999;59:3495-3504.

7. Ondrey FG, Dong G, Sunwoo J, et al. Constitutive activation of transcription factors NF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines. Mol Carcinog. 1999;26:119-129.

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8. Kato T, Duffey DC, Ondrey FG, et al. Cisplatin and radiation sensitivity in human head and neck squamous carcinomas are independently modulated by glutathione and transcription factor NF-kappaB. Head Neck. 2000;22:748-759.

9. Loercher A, Lee TL, Ricker JL, et al. Nuclear factor-kappaB is an important modulator of the altered gene expression profile and malignant phenotype in squamous cell carcinoma. Cancer Res. 2004;64:6511-6523.

10. Sunwoo JB, Chen Z, Dong G, et al. Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-kappa B, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma. Clin Cancer Res. 2001;7:1419-1428.

11. Russo SM, Tepper JE, Baldwin AS Jr, et al. Enhancement of radiosensitivity by proteasome inhibition: implications for a role of NF-kappaB. Int J Radiat Oncol Biol Phys. 2001;50:183-193.

12. Van WC, Chang AA, Lebowitz PF, et al. Inhibition of nuclear factor-kappaB and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma. Int J Radiat Oncol Biol Phys. 2005;63:1400-1412.

13. Dutcher JP. Mammalian target of rapamycin inhibition. Clin Cancer Res. 2004;10:6382S-6387S.

14. Vignot S, Faivre S, Aguirre D, Raymond E. mTOR-targeted therapy of cancer with rapamycin derivatives. Ann Oncol. 2005;16:525-537.

15. Amornphimoltham P, Sriuranpong V, Patel V et al. Persistent activation of the Akt pathway in head and neck squamous cell carcinoma: a potential target for UCN-01. Clin Cancer Res. 2004;10:4029-4037.

16. Amornphimoltham P, Patel V, Sodhi A, et al. Mammalian target of rapamycin, a molecular target in squamous cell carcinomas of the head and neck. Cancer Res. 2005;65:9953-9961.

17. Eshleman JS, Carlson BL, Mladek AC, Kastner BD, Shide KL, Sarkaria JN. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res. 2002;62:7291-7297.

18. Shinohara ET, Cao C, Niermann K, et al. Enhanced radiation damage of tumor vasculature by mTOR inhibitors. Oncogene. 2005;24:5414-5422.

19. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst. 2000;92:376-387.

20. Kim JC, Saha D, Cao Q, Choy H. Enhancement of radiation effects by combined docetaxel and flavopiridol treatment in lung cancer cells. Radiother Oncol. 2004;71:213-221.

21. Raju U, Nakata E, Mason KA, Ang KK, Milas L. Flavopiridol, a cyclin-dependent kinase inhibitor, enhances radiosensitivity of ovarian carcinoma cells. Cancer Res. 2003;63:3263-3267.

22. Jung C, Motwani M, Kortmansky J, et al. The cyclin-dependent kinase inhibitor flavopiridol potentiates gamma-irradiation-induced apoptosis in colon and gastric cancer cells. Clin Cancer Res. 2003;9:6052-6061.

23. Sato S, Kajiyama Y, Sugano M, Iwanuma Y, Tsurumaru M. Flavopiridol as a radio-sensitizer for esophageal cancer cell lines. Dis Esophagus. 2004;17:338-344.

24. Raju U, Ariga H, Koto M, et al. Improvement of esophageal adenocarcinoma cell and xenograft responses to radiation by targeting cyclin-dependent kinases. Radiother Oncol. 2006.

25. Newcomb EW, Lymberis SC, Lukyanov Y et al. Radiation sensitivity of GL261 murine glioma model and enhanced radiation response by flavopiridol. Cell Cycle. 2006;5:93-99.

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Self-Assessment Questions

1. What are among the most common EGFR inhibitor class side effects?

a. rash and diarrhea

b. vomiting and neutropenia

c. diarrhea and mucositis

d. neutropenia and anemia

2. According to Dr Bunn, retrospective analyses of data from phase III trials with gefitinib and erlotinib suggest the potential predictive utility of which of the following markers?

a. EGFR ICH status

b. EGFR FISH status

c. EGFR exon 16 mutations

d. EGFR intron 19 mutations

3. One of the elements that may contribute to the favorable interaction of EGFR inhibitors withradiation therapy is that these 2 modalities induce cell cycle arrest at different check points. Select the correct check points.

a. G2 for the EGFR inhibitors and G1 for radiation

b. G1 for the EGFR inhibitors and S for radiation

c. S for the EGFR inhibitors and G1 for radiation

d. G1 for the EGFR inhibitors and G2 for radiation

4. Results of a phase III trial that randomized patients with stage III or IV nonmetastatic measurable SCCHN to radiation therapy or radiation therapy plus weekly cetuximab showed that treatment with the EGFR inhibitor significantly improved ___________, the primary endpoint. (Fill in the blank)

a. quality of life

b. duration of locoregional control

c. overall survival

d. progression-free survival

5. Which of the following agents has an U.S FDA approved indication for use in combination with radiation therapy in the treatment of patients with SCCHN?

a. bevacizumab

b. erlotinib

c. gefitinib

d. cetuximab

35

6. According to Dr O’Reilly, which of the following anti-angiogenesis agents is being tested in a clinical trial in combination with radiotherapy for patients who normally would be treated with radiation therapy to the chest alone?

a. ZD6474

b. VEGF-Trap

c. bevacizumab

d. pazopanib

7. Which of the following agents being investigated for use in combination with radiation therapy is a proteasome inhibitor?

a. flavopiridol

b. temsirolimus

c. bortezomib

d. everolimus

36

PROGRAM EVALUATION AND ANSWER SHEETHead & Neck: Combining Targeted Therapies With Radiation Therapy

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