New Treatment Strategies for Prostate Cancer

New Treatment Strategies for Prostate Cancer Combination Treatment: Cryoablation with Radiation Research • Purpose – Explore the use of adjuvant radiation as a way to cryosensitize and enhance direct killing of human prostate cancer cell lines (PC3) and (DU145) BACKGROUND • Most common noncutaneous cancer & 2nd leading cause of cancer death in men in the US • Prevalence increases with age – 1/3 of men in the 4th decade will have foci of cancer – 3/4 of men in the 8th decade will have foci of cancer BACKGROUND • Lifetime risk that an American will be diagnosed with CaP is 17 % • Only 16%of men diagnosed with CaP ultimately die from it. • Natural hx varies from indolent to highly aggressive. CHALLENGE • Advise treatment in those in whom treatment is necessary. • Due to its prevalence and wide spectrum of aggressiveness different methods of treatment have been developed. • Preferred methods are controversial. TREATMENT OPTIONS • Radical prostatectomy – Perceived as morbid by some • Radiation Therapy – Criticized for its uncertain efficacy CONCERNS • Regarding conventional treatment have stimulated a search for alternative treatments that are effective and associated with limited morbidity. CRYOTHERAPY recognized as a therapeutic option in 1996 • Investigational label was removed • Offers minimally invasive option – – – – low morbidity minimal blood loss short hospital stay high rate of negative post treatment biopsies Cryobiology • Freezing causes cell death via several mechanisms – – – – direct cell injury vascular injury apoptosis Up regulation of the immune system. Direct Cell Death • Mechanical – Rapid formation of intracellular and extracellular ice crystals exerting shear forces on cell membranes and organells • Biochemical – Ph changes – Osmmolarity Direct Cell Death • Complete Cell death is unlikely to occur at temperatures warmer than -40 º C – This is important to note. • Edge of ice ball is 0˚C Vascular Injury • Endothelial Damage • This leads to Platelet Aggregation and micro thrombi formation causing tissue ischemia and cell death Apoptosis • Temperatures of -5º to -15º C causes mitochondrial-mediated apoptosis – up-regulation of pro-apoptotic proteins (such as Bax). Apoptosis is thought to be the primary method of cell death in the peripheral zone. Activation of Immune System • Release of cytokines in serum causing activation of immune system. Morbidities associated with with current cryotherapy • Impotence: >80% – With total ablation • Incontinence: 2% - 10% – Higher if salvage cryosurgery • Obstruction: 3.8% to 23% – Uretheral Sloughing • Usually 3 -8 wks post treatment • Fistula: 0% - 3% – Most commonly seen with previous radiation Isotherms • Isotherms extend radially – -190 degrees C at the probe – and warm to 0 degrees at the ice ball periphery • On ultrasound the freeze zone periphery (ice ball) is seen as a hyper echoic edge, providing a definable destructive freeze margin Isotherms • These isotherms result in two tissue zones: – A central necrotic zone – A peripheral zone of cell damage, but not necrosis. Downloaded from: Campbell-Walsh Urology (on 16 May 2007 03:18 AM) © 2007 Elsevier Isotherms • Uncertain tissue ablation near the ice ball periphery. – It is this area which is felt to be the cause of local recurrence Recurrence Rate • Positive biopsy rate after cryotherapy ranges between 7.7% and 23%. Can We Improve Outcomes • Persistent cancer after cryoablation is felt to represent cell survival in the peripheral zone of the ice ball – where temperatures are warmer than 40˚C. Earlier Research • Previously, we were able to show a synergistic effect on clonogenic inhibition in a human prostate cancer cell line (PC3) – However this synergy was not statistically different than the radiation only treatment group. Hypothesis • That by decreasing the dose of radiation, combined with decreased manipulation (stress) of the cells we would show a statistically significant difference in clonogenic inhibition. Design • Pre plated cells cells were exposed to • Vs. Single cell suspension in prior experiment • -5˚C, • -10˚C, • -20˚C – for 30 minutes and thawed at 37˚C • Or, a combination of the above temperatures with 2 Gy radiation, – non lethal dose Design In combination experiments single dose radiation was given 24 hours prior to cryotherapy same as prior experiment d i u m Medium changed treatment Cells plated for colony forming assay plated for colony forming assay Counted 24 hours 30 minutes: 11 days Materials Cell Culture • Human Prostate cancer cell lines (PC3) and (DU145) was used to test the hypothesis. • Pre plated cells were treated – Prior experiment used • 80 % confluence Cells cultures were treated then plated Materials • Human Prostate cancer cell lines (PC3) and (DU145) were exposed to: – only (PC3) cell line used with prior experiment 1. 2Gy radiation c/t 5 Gy with prior experiment 2. -5 degrees C 3. -10 degrees C 4. -20 degrees C 5. Or a combination of radiation and cryotherapy at above temperatures Data Analysis • Colonies containing 50 or more cells were counted. Surviving fraction (S.F.) was calculated using the plating efficiency. • Each experimental group was repeated three times to obtain mean and standard deviation. RESULTS PC3 cryotreated cells: 5 Gy vs. 2 1.1 1 0.9 0.8 0.7 SF 0.6 0.5 0.4 0.3 0.2 0.1 0 5Gy groups 2Gy Control pc3 -5 C -10 C -20 IR -5 C/ IR -10 C / IR -20 C / IR Dose dependent kill with freezing PC3 VS DU145 1.2 1 0.8 SF 0.6 0.4 0.2 0 0 IR ol C C IR IR nt r -5 -2 0 C/ / -1 / IR PC3 DU145 C 0 -1 co -5 groups Temperature dependant decrease in SF -2 0 C PC3 VS DU145 1.2 1 0.8 SF 0.6 0.4 0.2 0 0 IR ol C C IR IR nt r -5 -2 0 C/ / -1 / IR PC3 DU145 C 0 -1 co -5 groups PC3 CELL MORE CRYOSENSITIVE -2 0 C PC3 VS DU145 1.2 1 0.8 SF 0.6 0.4 0.2 0 PC3 DU145 0 IR ol C C IR IR / C 0 -2 0 C nt r -5 -1 0 -2 / co -5 C groups DU 145 CELLS MORE RADIO - RESISTANT -1 / IR Cryotherapy Enhancement Ratio 20 2 15 CER 10 5 0 Series1 Series2 1 1 2 16 2 PC3 and DU145 2 Series1 3 3 3 0 0 Series2 1 3.8 2.3 SIGNIFICANT INCREASES IN CER FOR PC3 CELLS Conclusion • Adjuvant radiation, 2 Gy, has a statistically significant synergistic effect on PC3 cells when combined with cryoablation • DU145 cell line appears to be cryo resistant at warmer temperatures,this is of unknown significance • Synergy with radiation appears to be present – Currently repeating experiment with DU 145 cell line Current and future studies • Assess cell death and understand the molecular mechanism of this synergistic effect. • Progress to animal studies Future treatment regimens Cryotherapy alone Cryotherapy + 2 Gy x5 2 Gy x5 2 Gy x5 cryotherapy