Tumor Responses to RT Bill McBride Dept
Tumor Responses to RT Bill McBride Dept. Radiation Oncology David Geffen School Medicine UCLA, Los Angeles, Ca. Radiation Biology is study of the effects of radiation on living things.For the most part, this course deals with the effects of radiation doses of the magnitude of those used in radiation therapy. 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?) 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 TD50 Assay Concept: Only cancer stem cells will grow
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 Percent of mice with tumors Size of tumor inoculum 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. MCF-7 Breast Cancer Stem Cells are Radioresistant and are enriched Following Irradiation
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 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 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 Gy 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% 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% Heterogeneity in Radiosensitivity
DOSE (Gy) 20 40 60 80 100 10 30 50 70 90 110 120 130 N=10 9 SF 2 =0.3 =0.4 =0.5 =0.6 =0.7 TCP (%) Rafi Suwinski Heterogeneity in Clonogen Number
100 SF =0.5 9 80 N=10 2Gy TCP (%) 10 60 N=10 11 40 N=10 Average 20 10 20 30 40 50 60 70 80 90 Rafi Suwinski DOSE (Gy) Micrometastatic Disease
DOSE (Gy) PERCENT REDUCTION IN METASTASES RISK 20 40 60 80 100 10 30 50 70 n=10 5 N =10-108 SF2Gy =0.5 N=10 8 3 Heterogeneity in tumor volume 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 = Tpot where 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 Tumor Kinetics Human SCC 36 hrs Tc Cell cycle time 0.25
6 days 60 days 0.9 Tc Cell cycle time G.F Growth fraction Tpot Pot. doubling time TdActual 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 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 Tumors can regenerate at the same time as they regress!
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 Control 15 Gy 25 Gy 35 Gy The regrowth rate of surviving clonogens varies with the surviving fraction - Lewis Lung Carcinoma (Stephens and Steel) 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. Accelerated Tumor Repopulation
Total Dose (2 Gy equiv.) Treatment Duration local control no local control 70 55 40 T2 T3 T2 T3 local control no local control 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 even1.6 1.8 Gy/day around week 6-7. 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 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 History 1909 Schwarz - radium dose on human skin Gray, Mottram, Flanders - oxygen effects in biology 1955 Thomlinson & Gray - tumor cords Powers & Tolmach - survival curves in vivo Churchill Davidson - HBO in patients 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 Chronic Hypoxia Within areas of need, oxygen is released from red blood cells and enterstumor tissue by diffusion. It is metabolized by respiring cells.As a result,at distances greater than about 100 m from the nearest blood vesselinsufficient 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 V BLOOD VESSEL Necrosis Hypoxia Proliferation 100 m HIGH LOW Proliferation, O2, pH, cell viability 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 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 and in the higher range at higher doses For neutrons, O.E.R is about 1.6 S.F. O.E.R.= 2.67 1.0 0.1 0.01 oxic hypoxic 1.0 1.5 2.0 2.5 3.0 O.E.R. 760 200 50 40 30 20 10 Partial Pressure of Oxygen (mm Hg) at 37o C air % oxygen Dose (Gy) RBE and OER as a function of LET
1 2 3 4 2 4 6 8 RBE (for cell kill) OER Fast Neutrons Alpha Particles RBE Co-60 gamma rays Diagnostic X-rays OER 0.1 1000 100 10 1 At the optimal LET of around 100 keV/m, the average separation is around 2 nm, which is the distance between DNA strands. Linear Energy Transfer (LET in keV/m) OER is the inverse of RBE because it depends on the indirect action of ionizing radiation 35 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) 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) 1 Hypoxic Fraction -1 10 -2 10 HYPOXIC S.F. 10 -3 -4 14 Days 10 -5 10 AIR -6 OXIC 10 Colony assay 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 Eppendorf Polarographic Fine Needle pO2 Probe
Slide 10 I then measured the oxygen tension of these tumors in vivo .I was quite fortunate in that Dr. Lavey had recently acquired one of the few eppendorph Po histographs which are necessary for this type of measurement. The eppendorf has a very thin 300um probe which is smaller than a 27 gauge needle.The needle is carefully inserted into the tumorof the unanesthetized restrained mouse.The probe sequentially moves through the tumor in steps of 0.7mm and several tracks were taken of eachtumor to insure a good data set since tumors are not homogenous. Insulating glass Gold Wire 12 m Membrane 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 Eppendorf Polarographic Probe
50% 40% 40% 30% NFSA NFSA NFSA TNF 30% NFSA IL7 20% 20% Slide 12 The results of the po histography are intriguing. As you can see from the graphs, the tnf transfected lines appear to be more oxic than the nfsa control. In the interleukin 7 lines the data issuggestive of the same result, although the difference is not as dramatic.Note the inflexion points at the group.Below this point, the nfsa lines areeither the same or significantly more hypoxic.Above this point in the tnf line, all the measures are significantly more oxic.In the il7 group, the trend is similar, with the group being significantly more oxic than the nfsa. These results wereintriguing and we are currently repeating the experiments to obtain a larger data set to insure the validity of our results. 10% 10% 0% 0%