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  1. Biologic Targeting in RTBill McBrideDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles,

  2. Objectives • Understand the relationship between molecular carcinogenesis and radiation response • Know how to target receptor tyrosine kinases • Know what agents are in clinical trials and the molecules they target • Know the clinical trial data on receptor targeting in combination with RT • Understand why some trials fail • Know what other pathways have and are being targeted, why, and how

  3. Cancer Biology Tumor Oncogenes Tumor Suppressor Genes Tumor Microenvironment Vasculature Hypoxia Metabolism Immunity Radiobiology Intrinsic Radiosensitivity Tumor Cell Proliferation Tumor Cell Death/Survival DNA Repair An increased therapeutic ratio can be achieved only by exploiting some difference between normal and malignant tissues • Differences between cancer and normal cells are known • Where is the link between cancer biology and radiobiology?

  4. Features of Carcinogenesis Mutations with gain in function (oncogenes) conspire with those conferring loss of function (tumor suppressor genes) to affect molecular signaling pathways and cause • Unabated, self-sufficient growth factor signaling (overexpression of ligands or receptors) • Loss of response to anti-proliferative signals • Evasion of cell death programs • Increase in replicative potential (telomeres) • Promotion of tissue invasion and metastasis • Sustained angiogenesis • DNA repair abnormalities and genomic instability After: Hanahan D, Weinberg RA, Cell 57-70, 2000. Overall decrease in cell loss factor and tumors grow

  5. Cell Lines Vary in Intrinsic Radiosensitivity S.F. 2Gy in vitro LYMPHOMA NEUROBLASTOMA MYELOMA SMALL CELL LUNG CANCER MEDULLOBLASTOMA BREAST CA SCC PANCREATIC CA COLORECTAL CA NON-SMALL CELL CA MELANOMA OSTEOSARCOMA GLIOBLASTOMA HYPERNEPHROMA 0.2 (0.08 - 0.37) 0.43 (0.14 - 0.75) 0.52 (0.2 - 0.86) Correlates with histological type and in vivo curability

  6. 0 2 4 6 8 10 Dose (Gy) 10 0 2 4 6 8 1 ras + myc S.F. bcr-abl v-fes c-myc Ha-ras Rat -1/v-mos wt-ras Rat -1 0.1 Surviving fraction 0.01 Dose (Gy) 1 c- myc S.F. v-abl 0.1 Rat -1 c-myc +v-abl myc 0.01 v-abl+ dn-myc Dose (Gy) Oncogenes and Radiation Resistancy (from McKenna et al, 1990) • Cancer is associated with deregulation of the same signaling pathways as determine intrinsic cellular radiosensitivity • This is through activation of signal transduction pathways that alter intrinsic radioresistancy • Transformation is not required

  7. Can you Predict How Mutations will Affect Response to RT? • Activation of cell cycle progression and survival pathways generally increases radioresistance • Activation of pro-apoptotic/cell cycle arrest pathways generally radiosensitize • The deregulated signaling pathways to which the cancer becomes “addicted” will provide the best targets for modifying radioresistance • McBride, W.H. and G.J. Dougherty,Nature Medicine, 1995. 1(11): 1215-1217

  8. A Large Number of Small Molecule Kinase Inhibitors have been Developed(FDA-approved) Imatinib (Gleevec) Bcr-Abl, c-kit, PDGFR-a CML, GIST 80%-CML, 54% GIST Gefitinib (Iressa) EGFR NSLC 10% respond Erlotinib (Tarceva) EGFR NSLC, mesothelioma Median survival 6.7 months Bortezomib (Velcade) Proteasome Multiple myeloma 1 year 23% of patients Sorafenib (Nexavar) c-Raf, BRA, Kit, EGFR,mRCC PFS longer FLT-3, VEGFR, PDGFR-β Sunitinib (Sutent) Multiple RTKs, VEGF, PDGF GIST and mRCC GIST: 25.5% MRCC: 36.5% Dasatinib (Sprycel) BCR-ABL, SRC family, CML and Ph+ ALL In trials c-KIT, EPHA-2, PDGFR-β

  9. A Large Number of Monoclonal Antibodies that inhibit Signaling Pathways are Available(FDA-approved) Bevacizumab (Avastin) VEGF CRC 5 months prolonged survival Alemtuzumab (Campath) CD52 B-cell CLL 9.5 months 30% patients Cetuximab (Erbitux) EGFR (HER-1) CRC, pancreatic Ca increased response HNSCC, NSLC Trastuzumab (Herceptin) HER2 Breast cancer 25 months for 26% Tositumomab (Bexxar) CD20 NHL 57% to 71% respond Rituximab (Rituxan) CD20 NHL, CLL,MM, HCL 3 months in 45% of patients Ibritumomab tiuxetan (Zevalin) CD20 NHL 80% respond Gemtuzumab (Mylotarg) CD33 AML 6 months 30% of patients Panitumumab(Vectibix) EGFR mCRC PFS 96 days Bevacizumab, cetuximab, and panitumumab each cost about $100,000/ patient.yr

  10. Where is Molecular Targeting Going? • Most molecular targeting agents are likely to be more cytostatic than cytotoxic, and are unlikely to be curative on their own. • To cure cancer, you need to kill all the cancer cells !! • It makes sense to use molecular targeted therapy to enhance tumor response to RT (and vice-versa)!

  11. Robert et al., J. Clin Oncol 19:3234-3243, 2001 13 CR and 2PR of 15 evaluable advanced HNSCC cases receiving Cetuximab plus RT • Volume 354:567-578 February 9, 2006 Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and Neck James A. Bonner, M.D., Paul M. Harari, M.D., Jordi Giralt, M.D., Nozar Azarnia, Ph.D., Dong M. Shin, M.D., Roger B. Cohen, M.D., Christopher U. Jones, M.D., Ranjan Sur, M.D., Ph.D., David Raben, M.D., Jacek Jassem, M.D., Ph.D., Roger Ove, M.D., Ph.D., Merrill S. Kies, M.D., Jose Baselga, M.D., Hagop Youssoufian, M.D., Nadia Amellal, M.D., Eric K. Rowinsky, M.D., and K. Kian Ang, M.D., Ph.D. The median duration of locoregional control was 24.4 months among patients treated with cetuximab plus radiotherapy and 14.9 months among those given radiotherapy alone (hazard ratio for locoregional progression or death, 0.68; P=0.005). With a median follow-up of 54.0 months, the median duration of overall survival was 49.0 months among patients treated with combined therapy and 29.3 months among those treated with radiotherapy alone (hazard ratio for death, 0.74; P=0.03). Radiotherapy plus cetuximab significantly prolonged progression-free survival (hazard ratio for disease progression or death, 0.70; P=0.006). With the exception of acneiform rash and infusion reactions, the incidence of grade 3 or greater toxic effects, including mucositis, did not differ significantly between the two groups.

  12. P PTEN P P P P P P P binds phosphotyrosine residues SH2 SH3 binds proline-rich sequences binds lipid ligands (products of PI-3K) PH EGFR (ErbB-1) Also ErbB-2, 3, 4 EGF/TGF-a The Pathways ATP Glucose Amino acids GLUT1 PIP2 PIP3 P P sos Akt Glucose Glucose-6-P Glycolysis SH3 PH PKA PI-3K Grb2 P SH2 P GDP sos SH2 x PIP2 PIP3 LKB1 Ras GTP P110 a, b, d Raf-1 AMPK p110g MEK ERK1 ERK2 Src MAPK/ERK signaling BAD NF-kB FKHD GSK3 MDM2 mTOR Multiple downstream targets p27 FasL p53 cell death/survival cell cycle arrest/progression DNA repair/misrepair cell metabolism

  13. EGFR Targeting Agents in the Clinic • Monoclonal antibodies • Cetuximab (Erbitux) chimeric IgG1 • Panitumumab humanized IgG2a • Matuzumab (discontinued) • Trastuzumab (Herceptin directed to Her2/neu; ErbB-2) • Small molecule TK inhibitors • Gefitinib (Iressa) • Tarceva (Erlotinib) • PKI-166 (ErbB-1 and -2) • Lapatinib (ErbB-1 and -2) • EKB-569 (ErbB-1, -2, -4) • CI-1033 (ErbB-1, -2, -4) • Antisense oligonucleotides • Dominant negative truncated receptor gene therapy

  14. EGFR Targeting: Rationale • Many tumors overexpress or activate EGFR and it generally correlates with outcome • Increased gene copy • Gene Mutation • Domain deletion (EGFRviii in GBM and other cancers) • EGFR is a strong independent prognostic determinant for overall and disease-free survival as well as a strong predictor for locoregional relapse, but not distant metastasis, for patients receiving definitive RT (Ang et al., Cancer Res, 2002) • EGFR activation causes radioresistance. • Blocking EGFR activation radiosensitizes • RT induces EGFR phosphorylation and tumor cell proliferation • Sadly, there is little correlation between EGFR expression and response to EGFR inhibitors….and there are no good in vitro models of cellular response to EGFR blockade

  15. Preclinical Data: Cetuximab potentiates RT against tumor growth in mice Control C225 x1 C225 x3 18 Gy 1 4 18 Gy + C225 x1 1 2 1 0 18 Gy + C225 x3 Tumor Size (mm) 8 6 4 0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 Days Clin Cancer Res 6: 701, 2000 (Huang and Harari, 2000) Int J Radiat Oncol Biol Phys 51: 474, 2001

  16. Mechanisms of Action of EGFR Inhibition with RT • Blocks ligand binding (MoAb) • Prevents EGFR phosphorylation and downstream signaling • Decreases DNA repair after irradiation • EGFR nuclear translocation • Binds to and blocks DNA-Pk? • Increases radiation-induced apoptosis • Cell cycle effects • Decreases tumor cell proliferation • accelerated repopulation? • Improves reoxygenation • Blocks angiogenesis • Inhibit VEGF and radiosensitizes vasculature? • Inhibition of tumor cell invasion and metastasis • Kills tumor cells through antibody-dependent cellular cytotoxicity (MoAb)

  17. There are lots of clinical trials ongoing, planned, or closed like these two More toxicity with IMRT than non-IMRT

  18. PI3K Inhibitors in the Clinic Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy. Nathan T. Ihle and Garth Powis Molecular Cancer Therapeutics 8, 1, January 1, 2009 There are currently 9 PI3K inhibitors in Phase I/II trials and 10 mTOR inhibitors

  19. But not all trials are successful! Machiels, JP et al Phase I/II study of preoperative cetuximab, capecitabine (Xeloda), and external beam radiotherapy in patients with rectal cancer Ann Oncol, 18:738-744, 2007. Only 5% pathological CR Rodel, C. et al., Multicenter phase II trial of chemoradiation with oxiplatin for rectal cancer. J. Clin Oncol 25:110-117, 2007. Only 9% pathological CR

  20. Why may they fail? • Need for Biomarkers to identify those who are going to respond • Acneiform rash predicts better than most markers that have been investigated! One week after Cetuximab and RT - courtesy K. Haustermans

  21. EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib (Iressa) TherapySCIENCE VOL 304 4 JUNE 2004 J. Guillermo Paez,1,2* Pasi A. Janne,1,2* Jeffrey C. Lee,1,3* Sean Tracy,1 Heidi Greulich,1,2 Stacey Gabriel,4 Paula Herman,1 Frederic J. Kaye,5 Neal Lindeman,6 Titus J. Boggon,1,3 Katsuhiko Naoki,1 Hidefumi Sasaki,7 Yoshitaka Fujii,7 Michael J. Eck,1,3 William R. Sellers,1,2,4  Bruce E. Johnson,1,2  Matthew Meyerson1,3,4  Receptor tyrosine kinase genes were sequenced in non-small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15 of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.

  22. P PTEN P P P P P P P binds phosphotyrosine residues SH2 SH3 binds proline-rich sequences binds lipid ligands (products of PI-3K) PH Why may they Fail? EGFR (ErbB-1) Also ErbB-2, 3, 4 EGF/TGF-a Bypass Mutations ATP Mutant EGFR Glucose Amino acids Mutant PI-3K PTEN loss Mutant AKT GLUT1 PIP2 PIP3 P P sos Glucose Glucose-6-P Glycolysis SH3 PH Akt Mutant Ras PKA PI-3K Grb2 P SH2 P GDP sos SH2 x PIP2 PIP3 LKB1 Ras GTP P110 a, b, d Raf-1 AMPK p110g MEK ERK1 ERK2 Src MAPK/ERK signaling Mutant mdm2 BAD NF-kB FKHD GSK3 MDM2 mTOR Multiple downstream targets Mutant Src p27 FasL p53 Mutant p53 cell death/survival cell cycle arrest/progression DNA repair/misrepair cell metabolism

  23. Radiological Response mt KRAS + pan wt KRAS + pan PFS mutant KRAS Pantumumab PFS wt KRAS Pantumumab mt KRAS + BSC wt KRAS + BSC Panitumumab in mCRC Overall Survival Amado, R. G. et al. J Clin Oncol; 26:1626-1634 2008

  24. Why may they fail? Poor targets? • Superior targets are probably • Binding domain of a kinase to which the tumor is “addicted” • Imatinib (Gleevac) is very effective in CML and GIST. Dasatinib binds even better and is even more effective as an up-front therapy. • A mutation, rather than an overexpressed normal protein. • Don’t forget the pharmacokinetics/pharmacodynamics

  25. NF-kB (nuclear factor that acts on B elements) • Central role in inflammation and immunity • Virchow (1863) first proposed that chronic irritation was the cause of cancer • Inflammatory cytokines activate ROS production leading to DNA damage, genomic instability, and cancer • NF-B is activated which transcribes anti-apoptotic factors • inhibitors of apoptosis (IAPs) like survivin, Bcl-XL, etc • NF-B is induced by doses of radiation in the higher dose range • The majority of cancers have high NF-kB levels

  26. Possible Outcomes Angiogenesis Inflammation Radioprotection Invasion Proliferation Survival Transformation Immunity Ligands TNF-a IL-1 a/b IL-6 TGF-b IL-8 IFN-g bFGF VEGF EGF ErbB2 Hypoxia Signal Transduction ras Akt TRAF Raf MEKK1 IKK IkB  ERK JNK p38 NF-B HIF-1 AP1 Elk1/cEBP HRE NF-IL-6 CRE NF-kB Effectors VEGF bFGF TNF-a IL-1 a/b IL-6 TGF-b MMP PPAR g/d Bcl-2 IAP (survivin) COX-2 PLA-A2 Arachidonic PGH2 PGD2 Acid PGE2 PGF2 PGI2 PGJ2 TBX 5,12,15 LOX LTA4 B4 C4

  27. COX Inhibitors • Non-selective COX I and 2 inhibitors • NSAIDs - aspirin, ibuprofen, indomethacin - • Selective COX2 inhibitors • celecoxib, rofecoxib, meloxicam, NS-398, etc Note: Anti-tumor action of celecoxib may not be solely through COX2 inhibition

  28. Tumor Vasculature as a Therapeutic Target • Vascular targeting • Induction of selective and irreversible damage to established tumor-associated blood vessels • normalize the abnormal tumor vasculature, increase tumor oxygenation, and reduce interstitial fluid pressure (IFP) • Acute treatment • Anti-angiogenesis • Preventing the growth of new tumor-associated blood vessels • Chronic treatment Blood vessel Blood vessel

  29. Advantages of Vascular Targeting • Since many thousands of tumor cells depend upon each blood vessel for the delivery of oxygen and nutrients, theoretically even limited damage to tumor vasculature may occlude a vessel and cause “an avalanche of tumor cell death”. • Since cells being targeted are in contact with the blood stream, delivery problems that limit the efficacy of therapies directed toward tumor cells are not an issue • Since endothelial cells are genetically stable and non-transformed, treatment related resistance is less likely to emerge

  30. VEGF • VEGF is a major angiogenic factor • There is a correlation between VEGF expression in tumor tissue and microvessel counts, which generally results in poorer survival *P=0.01. Imoto H, Osaki T, Taga S, et al. J Thorac Cardiovasc Surg. 1998;115:1007-1014.

  31. Bevacizumab (Avastin) • Monoclonal anti-VEGF • First-line treatment for patients with metastatic carcinoma of the colon or rectum in combination with intravenous 5-FU-based chemotherapy. Also, lung and breast cancer trials. • Randomized, phase III trial, with previously untreated metastatic colorectal cancer. • bolus-IFL plus placebo – O.S. 15.6 months, PFS 6.4 months • bolus-IFL plus bevacizumab – O.S. 20.3 months, PFS 10.6 months • 5-FU/LV plus bevacizumab – 18.3 months, PFS 8.8 months • Overall response rate = 39%, and median duration of response = 8.5 months. • Complications - GI perforations and wound dehiscence 2%

  32. As well as being a growth factor for endothelial cells, VEGF is vascular permeability factor • Cancer cells are killed indirectly by damaging tumor blood vessels (anti-vascular effect) • VEGF-targeted agents increase the response of tumors to radiation in preclinical models • They may sensitize tumors to radiation • By transiently normalizing the tumor vasculature, leading to greater tumor oxygenation, and thereby increasing the cytotoxicity of radiation to cancer cells • By increasing the radiosensitivity of tumor-associated endothelial cells.

  33. Bevacizumab (Avastin) Feasibility of using bevacizumab with radiation therapy and temozolomide in newly diagnosed high-grade glioma One-year progression-free survival and overall survival rates were 59.3% and 86.7%, respectively. Int J Radiat Oncol Biol Phys. 72:383, 2008. RTOG 0417 A PHASE II STUDY OF BEVACIZUMAB IN COMBINATION WITH DEFINITIVE RADIOTHERAPY AND CISPLATIN CHEMOTHERAPY IN UNTREATED PATIENTS WITH LOCALLY ADVANCED CERVICAL CARCINOMA Phase II: Docetaxel, Cisplatin, Fluorouracil, Bevacizumab, and Radiation Therapy in Treating Patients With Advanced Nasopharyngeal Carcinoma

  34. Targeting Loss of Function Tumor Suppressor Genes Is tougher than targeting oncogenes • Replacement Gene therapy • Requires all tumor cells to be targeted • Currently, no vector has 100% efficiency of gene transfer in vivo • Vectors have associated toxicity that limits dosage • Targets downstream of the mutation • E.g. Akt in PTEN deficient tumors • But it is divorced from the real target

  35. 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 10 20 30 40 50 Radiosensitization by Ad-p53 AdVluc+Irrad. 1.00 AdVp53 control SKOV Tumor Diameter (cm) S.F. 0.10 SKOV/P53 AdVp53 +irr. irrad. irrad. xxx xxx 0.01 4 2 0 Time (days) DOSE (Gy) In Vitro In Vivo

  36. Current approaches to overcoming vector limitations are unlikely to greatly improve the situation • Replicating adenoviruses • Tissue specific/ hypoxia-induced/radiation-induced promoters

  37. Exploiting Low Tumor Oxygenation with Hypoxic Cytotoxins

  38. . O O 2 - 2 * Hypoxia * TPZ Radical * Mechanism of Hypoxic Cytotoxicity of Tirapazamine O O N N N N - + NH N NH N 1 e + H 2 2 Reductase OH O TPZ M. Brown

  39. 0 10 HCR = 300 -1 10 -2 10 Surviving Fraction -3 air 10 hypoxia -4 10 -5 10 Tirapazamine Conc (M) 1 10 100 1000 10000 Tirapazamine is Toxic for Hypoxic Cells in vitro M. Brown

  40. Tirapazamine showed Promise when Combined with XRT or Chemotherapy in Phase I/II Trials Lung Cancer Cervix Cancer Head & Neck Cancer

  41. But failed in several Phase III trials, although several have yet to report • In 2007, Sanofi-Aventis sold Tirapazamine to SRI International • It is now in trials with Cisplatin and RT in Treating Patients With Stage IB, Stage II, Stage III, or Stage IVA Cervical Cancer • Importantly, it seems to work better in patients whose tumors have a high hypoxic fraction, indicating the need for preclinical testing

  42. The pathways that are responsible for cancer are often also the pathways responsible for treatment resistance! Most molecular targeting agents are likely to be more cytostatic than cytotoxic, and are unlikely to be curative on their own. It makes sense to use molecular targeted therapy to enhance tumor response to conventional treatments - these will be largely adjunctive therapies! They are potentially very powerful, but the pathways they target are complex What will be the role of RT in the post-genomic era? There is an urgent need to co-ordinate treatment so as to include all the biological factors that are needed for individualization.

  43. Molecular Profiling • Proteomics • TMA, SELDI, • MALDI, Immuno • Genomics • microarrays, SNP • Epigenomics • Bioinformatics • Pathway usage • Biomarker • identification Prognosis Critical Target Identification Diagnosis • Molecular Imaging • Spread • Metabolism • Hypoxia • Proliferation • Vascularity Treatment Decision Treatment Validation • Molecular Imaging • Spread • Metabolism • Hypoxia • Proliferation • Vascularity Data Analysis Monitoring Biomarker validation Phosphoprofiling Repair Cell cycle Survival Angiogenesis Response Assessment Biostatistics

  44. Questions:Biologic Modulation of Radiation Delivery

  45. Which of the following has shown Phase III efficacy in combination with RT. • Trastuzumab • Gefitinib • Bortezomib • Cetuximab • Avastin

  46. Trastuzumab is a monoclonal antibody that targets • EGFR • Her2neu • NF-kB • TP53

  47. Which of the following has proven to be the best marker for response to EGFR inhibitors • EGFR levels assessed pre-treatment by immunohistochemistry • Acneiform rash during therapy • Inhibition of MAP kinase activity in tumor biopsy during therapy • PTEN status

  48. The response to the EGFR inhibitor Panitumumab in metastatic colorectal cancer correlates with • PTEN loss • EGFR over expression • Ras mutation • TP53 mutation

  49. Bevacizumab is a first-line treatment for patients with • Metastatic carcinoma of the colon or rectum in combination with intravenous 5-FU-based chemotherapy • Glioblastoma in combination with temozolomide and RT • HNSCC with chemoradiation therapy • Multiple myeloma that have failed other forms of therapy

  50. Bortezomib (Velcade) is approved in the US for the treatment of patients with multiple myeloma. It targets • CD20 • EGFR • Proteasomes • VEGF