Determinants of Tumor Cure

1. Tumor Responses to RTBill McBrideDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles, Ca.wmcbride@mednet.ucla.edu

2. Determinants of Tumor Cure • Size of the clonogenic pool (stem cells) • Intrinsic radiosensitivity • S.F. 2Gy (pro-apoptotic tendency?) • Repair • T1/2 (HR, NHEJ, SLDR, PLDR, fast and slow repair?) • Rate of repopulation/regeneration during therapy • Tpot (L/I., Ki67?) • Reoxygenation (extent of hypoxia) • PO2 (dependence on tissue type, vascularity?) • Redistribution • Growth fraction (dependence on cell type, growth factors?)

3. Determinants of Tumor Cure (continued) Heterogeneity: • Biological • Number of clonogenic “stem cells” • Intrinsic radiosensitivity • Proliferative potential • Tumor microenvironment • Hypoxia • Metabolism • Host cell infiltrates • Interstitial pressure • Genetic • Oncogenes • Tumor suppressor genes • Single Nucleotide Polymorphisms (SNPs)? • Physical • Dose heterogeneity • Geographic miss

4. TD50 Assay 1. Inject varying numbers of tumor cells into mice 2. Determine the number of cells that are needed to form tumors in 50% of mice. To grow, tumors must have arisen in that specific strain of mice, or the mice must be immune deficient. Even then, not all tumors will grow, and most need an inoculum size of at least 104 cells Concept: Only cancer “stem” cells will grow 100 50 0 Percent of mice with tumors 10 102 103 104 105 106 107 Size of tumor inoculum

5. Renewing stem cell Non-stem cell stem cell Tumor cure Tumor regeneration from stem cell pool The cancer stem cell hypothesis suggests that there are a small number of clonogenic stem cells in a tumor and that, if they are therapy-resistant, they are responsible for recurrences, and accelerated tumor repopulation during therapy.

6. MCF-7 Breast Cancer Stem Cells are Radioresistant and are enriched Following Irradiation “Stem” cells At least some human tumors have a clonogenic subpopulation with stem-like characteristics that can be grown in cytokines as spheres and that are radioresistant and are selected for by fractionated irradiation. Phillips et al J Natl Cancer Inst 98:1777, 2006

7. TCD50 Assay 1. Inject mice with enough cells to form a tumor 2. Irradiate when 6mm diam 3. Determine the dose of radiation that is needed to cure 50% of mice. 100 50 0 Threshold-sigmoid curve that goes from 10% to 90% cure over about 10Gy in a clinical fractionation scheme (which is hard to do in mice). Percent of mice with tumors 0 10 20 30 40 50 60 70 80 Gy

8. Tumor Control Probability • In order to cure a tumor, the last surviving clonogen must be killed, and even then it is a probability function of dose. • TCP = e-x = e-(m. SF) or e-m.e-(ad+bD2) or e -(m. e -(D/D0)) • Where x is the number of surviving clonogenic stem cells, • m is the initial number of clonogens • If there is an average of 1 cell surviving TCP=37%

9. Tumor Control Probability • The slope represents extent of heterogeneity in tumor response • The normalized dose response gradient (g) measures the change in TCP in % points for a 1% increase in dose • Often 1-3%

10. 100 80 9 TCP (%) N=10 SF =0.7 2 60 SF =0.6 2 SF =0.5 2 40 SF =0.4 2 SF =0.3 2 20 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 DOSE (Gy) Heterogeneity in Radiosensitivity Rafi Suwinski

11. Heterogeneity in Clonogen Number 100 SF = 0.5 9 80 N=10 2Gy TCP (%) 10 N=10 60 11 N=10 40 Average 20 0 0 10 20 30 40 50 60 70 80 90 Rafi Suwinski DOSE (Gy)

12. 100 =0.5 SF2Gy 80 3 5 8 N=10 N=10 N=10 n=10 60 PERCENT REDUCTION IN METASTASES RISK 40 N =10-108 20 0 0 10 20 30 40 50 60 70 DOSE (Gy) Heterogeneity in tumor volume Micrometastatic Disease

13. Tumor Growth and Regression The kinetics of tumor growth and regression depend upon • Cell cycle • Growth fraction (G.F.) • G.F. is the proportion of proliferating cells • G.F. = P / (P + Q) where P = proliferating cells and Q = non-proliferating cells (quiescent/senescent/differentiated cells) • Cell loss factor • Cell Loss Factor measures loss of cells from a tissue • If = 0, Td = Tpotwhere Td is the actual volume doubling time and Tpot is potential volume doubling time • = 1 - Tpot / Td • if G.F. = 1 then Tpot = Tc • Under steady state conditions, constant cell number is maintained by the balance between cell proliferation and cell loss i.e.  = 1.0. In tumors (and embryos)  < 1.0

14. Tumor Kinetics Human SCC 36 hrs 0.25 6 days 60 days 0.9 Tc Cell cycle time G.F. Growth fraction Tpot Pot. doubling time Td Actual doubling time Cell loss factor (36hr x 4) (1-6/60) Rate of tumor growth and rate of tumor regression after therapy are determined largely by the cell loss factor, that varies greatly from tumor to tumor

15. Tumor Growth and Regression • Slow growing tumors may regress rapidly • Slow regression is not an indication of treatment failure • Rapidly growing tumors would be expected to regress and regrow rapidly • In general, the rate of tumor regression after Tx is not prognostic

16. Tumor Regeneration 20Gy X-rays Relative tumor volume Control Irradiated Tumors can regenerate at the same time as they regress! Growth delay Surviving clonogens measured in vitro Time Rat rhabdomyosarcoma Hermans and Barendsen, 1969

17. Control 15 Gy 25 Gy 35 Gy The regrowth rate of surviving clonogens varies with the surviving fraction - Lewis Lung Carcinoma (Stephens and Steel)

18. EVIDENCE FOR ACCELERATED REPOPULATION IN TUMORS • After RT, tumors recur faster than than would be expected from the original growth rate • Split-course RT often gives poor results • Protraction of treatment time often gives poor results • Accelerated treatment is sometimes of benefit.

19. T2 T3 T2 T3 local control 70 55 40 local control Total Dose (2 Gy equiv.) no local control no local control Treatment Duration Accelerated Tumor Repopulation Withers et al, 1988 Maciejewski et al., 1989 • T2 and T3 SCC head and neck (excluding nasopharynx and vocal cord). TCD50 values are consistent with onset of repopulation at 4 weeks followed by accelerated repopulation with a 3-4 day doubling time, implying a loss in dose of about 0.6 Gy/dy • If the red line is correct, onset may be about day 21 and repopulation may not be constant. It may increase from 0.6 Gy/dy around week 3-4 to even 1.6 – 1.8 Gy/day around week 6-7.

20. Tpot in a Large Multicenter HNSC Trial 476 patients (Begg et al 1999) • It was thought that shortening treatment time by accelerated hyperfractionation and that this might be predicted by Tpot , but a large multicenter trial was unable to confirm this • But note that Tpot in HNSCC was 3-5dys for most patients, confirming the potential for very rapid growth

21. Sources of Heterogeneity • Biological Dose • Number of clonogenic “stem cells” • Intrinsic radiosensitivity • Proliferative potential • Tumor microenvironment • Hypoxia • Metabolism • Physical Dose • Need to know the importance of dose-volume constraints

22. History • 1909 • Schwarz - radium dose on human skin • 1930-1950 • Gray, Mottram, Flanders - oxygen effects in biology • 1955 • Thomlinson & Gray - tumor cords • 1960-1965 • Powers & Tolmach - survival curves in vivo • Churchill Davidson - HBO in patients

23. Hypoxia in Tumors • Chronic hypoxia is a result largely of • Limited O2 diffusion due to • oxygen consumption (”diffusion limited hypoxia”) • irregular vascular geometry • Acute/transient/intermittent hypoxia is a result largely of • Chaotic vasculature and interstitial pressure • vascular stasis • flow instabilities

24. V V V Proliferation, O2, pH, cell viability V 100 m HIGH.................LOW BLOOD VESSEL V V V V V V Proliferation V V V V V V V V V Hypoxia Necrosis Chronic Hypoxia • Within areas of need, oxygen is released from red blood cells and enters tumor tissue by diffusion. It is metabolized by respiring cells. As a result, at distances greater than about 100 µm from the nearest blood vessel insufficient oxygen remains to maintain cell viability. • Adjacent to areas of necrosis, one may find a region 1-2 cell layers thick where oxygen tensions are hypoxic. Within a solid tumor mass, mitotic index and viability decrease with distance from the nearest blood vessel (Tomlinson and Gray; Tannock, Cancer Res 30: 2470, 1970) • Hypoxia does NOT correlate with tumor volume

25. Acute Hypoxia • The vascular network that develops in tumors is structurally abnormal • Vessels are dilated, tortuous, elongated, with A-V shunts and blind ends • Pericytes are frequently absent • The basement membrane is thin • Vessels are more permeable giving increased interstitial pressure • The abnormal vasculature results in spatial and temporal heterogeneity in blood flow that in turn produce regions of temporary or acute hypoxia, acidity and nutrient depletion Brown & Giaccia, 1994 Normal Tissue Konerding et al., 1998 Neoplastic tissue

26. 3.0 2.5 2.0 1.0 0.1 0.01 O.E.R. S.F. 1.5 hypoxic oxic O.E.R.= 2.67 air 100% oxygen 1.0 0 10 20 30 40 50 200 760 Partial Pressure of Oxygen (mm Hg) at 37o C 0 2 4 6 8 10 THE OXYGEN EFFECT • Oxygen is a powerful oxidizing agent and therefore acts as a radiosensitizer if it is present at the time of irradiation (within msecs) • The magnitude of the OER is critically dependent upon oxygen tension. The greatest increase occurs between 0-20 mm Hg with further modest increases to air (155 mm Hg) and above (760 mm Hg=100% oxygen). • Its effects are measured as the oxygen enhancement ratio (O.E.R.) • O.E.R. = the ratio of doses needed to obtain a given level of biological effect under anoxic and oxic conditions = D(anox)/D(ox) • For low LET radiation the O.E.R. is 2.5-3.0 and in the higher range at higher doses • For neutrons, O.E.R is about 1.6 Dose (Gy)

27. 8 4 6 3 4 2 2 1 0 0 1 10 100 1000 RBE and OER as a function of LET RBE (for cell kill) OER Fast Neutrons Alpha Particles RBE Co-60 gamma rays Diagnostic X-rays OER 0.1 Linear Energy Transfer (LET in keV/m) OER is the inverse of RBE because it depends on the indirect action of ionizing radiation

28. Demonstrating hypoxic regions/cells within tumors • Differential radiation sensitivity • Eppendorf polarographic electrode • Immunohistochemistry • Misonidazole • Hypoxyprobe™ immunohistochemistry with pimonidazole • HIF-1 and products • PET imaging • 18F-fluoromisonidazole (FMISO-PET) • EF5 - etanidazole • Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM)

29. Tumor Cell Survival : In vivo-in vitro assay • If solid tumors in mice are irradiated with single doses of radiation under hypoxic conditions or in air and an in vitro clonogenic assay performed, normally a dog-leg curve is obtained in air indicating a radioresistant population whose magnitude can be estimated by extrapolation onto the Y axis. After Rockwell and Kalman, 1973 IRRADIATE tumor After 24hrs make cell suspension Plate cells DOSE (Gy) 0 2 4 6 8 10 12 14 16 18 20 22 1 Hypoxic Fraction -1 10 -2 10 HYPOXIC S.F. -3 10 14 Days -4 10 -5 10 AIR -6 OXIC Colony assay 10

30. Tumor Hypoxia • If murine tumors are irradiated with varying sized single doses of radiation under clamped (hypoxic) and normal conditions and the % of tumors controlled plotted, the TCP curve is shifted to the right by hypoxia and the O.E.R. can be calculated. Moulder and Rockwell, 1984

31. Eppendorf Polarographic Fine Needle pO2 Probe Membrane Insulating glass Gold Wire 12 m Probe Casing 300 m • A 700 mV polarizing voltage is applied against the Ag/AgCl anode. The measured current is proportional to the local oxygen tension • No longer sold, but other versions are possible

32. Eppendorf Polarographic Probe 50% 40% 40% 30% NFSA NFSA 30% NFSA TNF NFSA IL7 20% 20% 10% 10% 0% 0% <6 <12 <18 <24 <30 <36 <42 <48 <6 <12 <18 <24 <30 <36 <42 <48 mmHg

33. Bioreductive Drugs • Misonidazole forms adducts in hypoxic cells in vitro and in vivo with thiol groups in proteins, peptides and amino acids. Hypoxia (pO2 < 10 mmHg) is required for binding. • FMISO-PET is one of 2 commonly used PET tracers (the other being Cu-ATSM), but it accumulates slowly. Other imidazoles are under study. • EF5 is a fluorinated derivative of etanidazole • Pimonidazole is generally injected in vivo and the adducts stained using antibodies. • Intracellular Cu-ATSMis a non-nitroimidazole that has been shown to be bioreduced and trapped in hypoxic cells and is used for PET. Pimonidazole staining of human CRC tumor

34. Hypoxia and proliferation in a solid tumor blood vessels proliferating cells (IdUrd +) necrosis Biopsy of head/neck squamous cell carcinoma Hypoxia (pimonidazole +) From: Albert Van der Kogel

35. Pimonidazole (green) and vascular staining (red) in human head and neck tumor Chronic Acute From Bussink et al., 2001

36. Hypoxia-induced gene expression • Transcription factors • AP-1, NF-kB, SP-1 activation • which can mediate radioresistancy • p53 induction • which can cause apoptosis with hypoxia-driven p53 mutant selection and increasing genetic instability • HIF-1 and products eg VEGF, CA IX, OPN etc • HIF-1alpha is a target for prolyl hydroxylation by HIF prolyl-hydroxylase, targeting it for rapid degradation in normoxic conditions. Under hypoxia, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a cosubstrate, stabilizing HIF-1α. This upregulates several genes to promote survival in low-oxygen conditions, including glycolytic enzymes and VEGF, which promotes angiogenesis. • In general these surrogate markers do not correlate well with hypoxia, probably because more than hypoxia stabilizes HIF-1

37. Contribution of hypoxia to tumor progression • Enhances resistance to radiation and chemotherapy because of classic oxygen effect • Induces expression of genes that • confer resistance to radiation and other pro-apoptotic insults • triggers genetic instability • cause angiogenesis and potentiate metastasis From Giaccia, 1999

38. Regulation of hypoxia-induced gene expression HER2 IGFR EGFR Src LY294002 PI3K HIF-1 synthesis a PTEN AKT FIH-1 FRAP Rapamycin HIF-1 b Angiogenesis VEGF HIF-1 a Target gene HIF-1 mRNA a IGF-2 Proliferation protein expression HYPOXIA Glucose Metabolism Prolyl hydroxylation transporters VHL Ubiquitination p53 HIF-1 degradation a

39. Inflammatory Cytokines Redox regulation apoptosis/ necrosis IL-1a, IL-8 Heme oxygenase 1, metallothionein, diaphorase, GSH, BNip3 (BCl2 family) pH regulation HIF-1 carbonic Anhydrases CA9 VEGF VEGFR EPO EGF EGFR PDGF-B IGF-1 IGF-2 Glucose transporters Glut1,3 Glycolytic enzymes ALDA, PGK1, PKM, PFKL, LDHA angiogenesis energy metabolism proliferation

40. Clinical Relevance of Tumor Hypoxia • Evidence for hypoxia in human tumors • Hyperbaric chambers anecdotally show benefit • normobaric oxygen/carbogen has alsobeen applied and, at times, combined with nicotinamide, a B6vitamin analog thought to counteract the acute hypoxia (ARCON) • Anemia correction has benefit especially in cervix ca • Note that erythropoietin has a deleterious effect in HNSCC due to stimulating tumor growth • Nitroimidazoles - immunohistochemistry and PET • Microelectrode measurements - several studies have correlated hypoxia with poor local response and survival • Nordsmark et al. Radiother Oncol 41, 31, 1996 showed local tumor control correlates with pre-treatment oxygen levels in head and neck ca. • Brizel et al IJROBP 38:285, 1997 showed DFS correlates with hypoxia in T3 and T4 and large node mets from head and neck • Hockel et al Cancer Res 56:4509, 1996 showed hypoxia correlated with local invasion and survival in cases treated with RT or only with surgery

41. Clinical Hypoxia Tumor type Median pO2 fraction <10 mm Hg Breast C. 23-28 26-32 Cervical C. 2-21 21-46 Rectal C. 19-25 - Lung Ca 14 36 Soft tissue sarcomas 18-27 44 Glioblastomas 7 61 Head & Neck C. 19-26 33 H&N lymph nodes 9-25 14-54 Melanoma 10 49 From Vaupel et al., 1998

42. Hypoxia and Local Tumor Control • Local tumor control correlates with pre-treatment oxygen levels in head and neck ca., as measured with an Eppendorf electrode. Tumors were stratified by whether the fraction of pO2 values less than 2.5 mm Hg was above or below the median (15%). 66-68 Gy was given in 33-34 Fx. • Nordsmark et al Radiother Oncol 41, 31, 1996 Small Hypoxic Fraction Large Hypoxic Fraction

43. Tumor Hypoxia and DFS • DFS in cervix ca depends on pO2, irrespective of type of treatment, surgery/RT. Hockel et al, Sem. Radiat. Oncol. 6:30, 1996. • This suggests that hypoxia is linked to tumor aggression

44. From Zeman, 2000 Radiosensitizers

45. Radiosensitizers • Radiosensitizers such as nitroimidazoles can “mimic” oxygen and fix damage • Associated with some toxicity and there were only rarely efforts to determine if the tumors were hypoxic in advance of treatment • However there have been positive trials • DAHANCA 5 trial using nimorazole in treatment of advanced squamous cell carcinoma of the head and neck

46. And a meta-analysis by Jens Overgaard has shown significantly improved survival and loco-regional control Journal of Clinical Oncology, 25: pp. 4066-4074, 2007

47. Hypoxic Cytotoxins • Quinones • Mitomycin C • Nitroaromatics • Benzotriazine di-N-oxides • Tirapazamine • Phase III clinical trials with cisplatin • Phase II with RT • Currently off the market!

48. Tumor Reoxygenation • Since well oxygenated cells are more sensitive than hypoxic cells to ionizing radiation, one might reasonably expect that the hypoxic fraction (i.e. the proportion of hypoxic cells) to increase during the course of radiation therapy • In fact, Putten & Kallman and others demonstrated that the proportion of hypoxic cells present within a tumor varies a lot, but does not increase during a course of fractionated radiation therapy showing REOXYGENATION exists. Multiple mechanisms exist and the variation seems considerable from tumor to tumor.

49. Hypoxic Fraction Days post RT N.B. All single dose!

50. Angiogenesis • Development of new blood vessels from pre-existing capillaries. • Although tumors smaller than approximately 1 mm can receive sufficient oxygen and nutrients by diffusion, continued growth depends upon the development of an adequate blood supply. In the absence of angiogenesis, tumors do not increase in size and remain localized • Angiogenesis also occurs during • wound repair • pregnancy • certain times in the menstrual cycle