University of Colorado Denver



BIOS 6648 Final ProjectGroup 3: Anna Ivashchenko, Jesse Krisher, Kristen WeishaarAdjuvant Sequential Intravesical BCG and Electromotive Mitomycin-C (EMDA/MMC) after Transuretheral Resection in Patients with Primary High Risk Non-Muscle Invasive Transitional Cell Carcinoma of the Bladder. A one-group phase II study.Background and SettingDisease BackgroundBladder cancer, accounting for 5-10% of all malignancies in males in Europe and the USA, with a growing incidence in females, has an annual estimate of 357,000 newly diagnosed cases and 145,000 deaths worldwide [1]. Transitional cell carcinoma (TCC) is the most common type of cancer found in the urinary system, including the kidneys, urinary bladder, ureters, and urethra. Known risk factors for this type of cancer include cigarette smoking, chemical exposures, and increasing age (> 55 years).DiagnosisThe most common clinical sign with bladder cancer is blood in the urine, which is present in about 80-90% of patients at the time of diagnosis. Other common symptoms include painful urination, increased frequency of urination, or feeling the urge to urinate without being able. As these signs can be associated with a number of conditions other than bladder cancer, additional tests are needed to diagnose this disease. The simplest way to obtain a diagnosis is by taking a small piece of the tumor (biopsy) in a procedure known as cystoscopy, which involves introducing a tube equipped with a camera (cystoscope) into the bladder through the urethra. This allows visualization of the lining of the bladder, and instruments can also be introduced to obtain a biopsy. StagingOne of the most important aspects of cancer diagnosis and treatment is a process known as tumor staging, which provides information about the characteristics of the tumor as well as if there has been any spread to other organs (metastasis). While the biopsies obtained with cystoscopy can confirm a diagnosis of TCC, these samples are not sufficient for tumor staging. Classification of bladder cancer is done using the TNM staging system, which stands for “Tumor” (describes the size of the primary tumor and its invasiveness into surrounding tissues), “Node” (describes involvement of nearly lymph nodes), and “Metastasis” (spread of cancer to other organs. For bladder cancer, the “T” classification ranges from least invasive to most invasive. Tis, which signifies a non-invasive or “flat” tumor, is the lowest stage, and then tumor invasion into the bladder wall increases with stages T1-4, with T4 representing a tumor that invades through the bladder wall and into other organs, such as the prostate, uterus, or vagina.TreatmentThe recommended treatment protocol for bladder cancer will depend on the results of the staging tests, particularly the level of invasiveness of the primary tumor. For tumors that do not invade the muscle layer of the bladder (non-muscle invasive bladder cancer, or NMIBC), a procedure known as transurethral resection (TUR) can be used, which involves using a cautery device attached to a cystoscope to “shave off” the portion of the tumor that is visible in the bladder. While this procedure can be successful in the treatment of superficial tumors, it is not a sufficient treatment for tumors that invade into the muscle layer or deeper, as these tumors have a high likelihood of recurrence following TUR, and can also progress to involve surrounding organs. The major challenge with this disease is preventing tumor recurrence and disease progression, and currently there is no 100% effective way to do this. After TUR alone, 5-year-recurrence rates vary from 31% in low-risk to 78% in high-risk patients, while progression rates range from 0.8% to 45% [2]. In an effort to prevent tumor regrowth after TUR, standard treatment involves infusion of chemotherapy or immune-stimulating agents directly into the bladder (known as intravesical instillation). The most common chemotherapy agents used are mitomycin-C (MMC), doxorubicin, and epirubicin, which have been tested in numerous randomized clinical trials [3,4]. A single instillation of MMC or epirubicin immediately after TUR was shown to reduce the recurrence risk by 39%, which led to standardization of this procedure [5]. Immunotherapy for bladder cancer involves the use of BCG (Bacillus Calmette-Guerin), which is a vaccine against tuberculosis. This is the standard treatment for more aggressive, high-risk bladder cancers. While this treatment is associated with more side effects, recurrence rates are lower than after intravesical chemotherapy [6]. In patients whose tumors recur after BCG treatment, the final treatment option is surgery to remove the bladder (cystectomy).While MMC does seem to reduce the risk of tumor recurrence and possibly progression, failure rates are still high. Treatment failure may be due to invasion of tumor into the bladder wall deeper than MMC can be absorbed through the bladder lining, or due to higher resistance to chemotherapy in more aggressive tumors.Electromotive Drug AdministrationElectromotive drug administration (EMDA) is a novel treatment mechanism that can allow for greater MMC accumulation in bladder tissues, which could potentially overcome some of the problems with standard MMC intravesical treatment. Intravesical EMDA is administered by a battery powered generator (Physionizer? 30, manufactured by Physion?, Medolla, Italy) that delivers a controlled electric current. The current passes between two electrodes: the active electrode, which is placed inside the bladder using a specifically designed catheter, and the ground electrodes, which are placed on the skin of the lower abdomen. The electrical current draws the MMC into the cells of the bladder lining, allowing for greater penetration and accumulation of MMC in the bladder tissues.RationaleLargely, there is excellent survival using the standard of care; however, there is no known method for recurrence prevention. As specified above, the patient’s risk status is a key component in the 5-year-recurrence rates and progression rates [2]. MMC appears to reduce the risk of tumor recurrence and possible progression of NMIBC but failures do exist and may be due to either a stage T1 tumor at a depth where MMC cannot reach through passive transport or high-grade cancer cells with reduced sensitivity to chemotherapy. By ensuring increased MMC accumulation in the bladder tissues through MMC-EMDA delivery, the risk of local recurrence may be decreased.Previous in vitro studies have demonstrated that MMC-EMDA delivery rates had a 4- to 7-fold increase over passive diffusion (PD), implying it may improve clinical delivery rates. Few in vivo data exist, however those data appear confirmatory. In a small phase II study [7], marker lesion disappeared at about the same rates between MMC (n=13) and EMDA/MMC (n=15) instillations while recurrence rates were 27% lower in patients treated with EMDA/MMC compared to standard of care. Low recurrence rates of high-risk NMIBC were also reported in an observational study [8]. It was shown that within high-risk NMIBC patients, 56% of patients were recurrent-free after 14 months. Response rates among PD/MMC, EMDA/MMC and BCG were compared in a prospective randomized study [9]. In follow-ups at both 3 and 6 months, EMDA/MMC response rates were significantly better than PD/MMC, however equivalent to BCG after a median follow-up of 82 months. EMDA increased toxic side effects in the bladder but was substantially less than with BCG, a relatively dramatic treatment option [10]. A larger prospective randomized study showed reduced recurrence rate and longer disease-free period in patients receiving EMDA/MMC [11].Overall, in vivo studies suggest an improved response in patients treated with EMDA/MMC. In our trial, we want to show that EMDA/MMC exceeds the standard of care and show marked improvement in recurrence rates. For this reason, we have chosen a superiority study design. Because sufficient evidence from previous randomized control trials exists, use of a single-arm design will be able to discriminate between superiority and inferiority of EMDA/MMC treatment by comparison with the results of these prior studies. Additionally, single-arm trial design was chosen since the epidemiology is already established, an implicit comparison can be made between treatment and controls in previous trials, and will be sufficient for Investigational New Drug (IND) approval from the U.S. Food and Drug Administration. In summary, although EMDA/MMC may be a milestone for future therapeutic concepts, confirmation by other investigators is needed.Synopsis of Proposed StudyStudy PopulationThis study is designed for patients that have been diagnosed with transitional cell carcinoma of the bladder. Specific eligibility criteria are as follows:After a restaging TURBT patients with histologically proven primary high grade (grade3) and/or pT1 transitional cell carcinoma of the bladder, with or without pTis and pTis alone are regarded as being at high risk for tumour recurrence and progression Patients may enroll in this study if they are thought to have no residual disease after TURBTAge 18 years or over Adequate bone-marrow reserve (ie, white-blood-cell count 4000 x 106 cells/L and platelet count 120 x 109/L) ECOG performance status between 0 and 2 Patients who are known PPD positive will be screened for active tuberculosis prior to starting treatment with BCG. Specific exclusion criteria are as follows:Known allergy to BCG or MMCPrior systemic infection with BCG Prior or concomitant urothelial tumours of the upper urinary tract or urethra Previous muscle-invasive (ie, stage T2 or higher) transitional-cell carcinoma of the bladder Bladder capacity of less than 200 mlUntreated urinary-tract infection Severe systemic infection (ie, sepsis) Known HIV-positivity; therapy with immunosuppressive agents Urethral strictures that would prevent endoscopic procedures and repeated catheterisation Upper urinary tract disease (eg, vesicoureteral reflux or urinary-tract stones) that would make multiple transurethral procedures a risk Previous radiotherapy to the pelvis Other concurrent chemotherapy Treatment with radiotherapy-response or biological-response modifiersHistory of tuberculosisOther malignant diseases within 5 years of trial registration (except for adequately treated basal-cell or squamous cell skin cancer, in situ cervical cancer and any other cancer from which patients has been disease-free for 3 years)Pregnancy or nursing Psychological, familial, sociological, or geographical factors that would preclude study participation Patients with implantable or wearable electrical devices will be excluded from this study. Patients with active tuberculosisStudy TreatmentPatients can begin treatment with the study intervention about 2-8 weeks after their TURBT resection.Prior to enrollment, all patients must sign a consent form and will have a Complete Blood Count, Basic Metabolic Panel and a Urinalysis performed.BCG instillation will occur at weeks 1, 2, 4, 5, 7, and 8 of the study. In this procedure, BCG are suspended in 50 ml bacteriostatic-free 0.9% NaCl solution. After the bladder is emptied, the suspension is infused intravesically through a 14 Fr Foley catheter and retained in the bladder for 60-90 minutes. The bladder is then emptied and the catheter removed. MMC instillation will occur at weeks 3, 6 and 9. Prior to drug administration, a Foley catheter is inserted and the bladder is carefully drained. 40 mg of MMC is dissolved in 100 ml NaCl 0.9% solution are instilled intravesically through the electrode catheter by gravity and retained in the bladder for 30 minutes. Concurrent with MMC treatment, a pulsed electric current of 23 mA is given externally for 30 minutes. Intravesical EMDA is administered by a battery powered generator (Physionizer? 30, manufactured by Physion?, Medolla, Italy) that delivers a controlled electric current of 0-30mA / 0-55V DC. The current passes between two electrodes: the active intravesical electrode, which is integrated into a specifically designed transurethral catheter, and the dispersive ground electrodes, which are placed on the skin of the lower abdomen (Figure 1). Once treatment is complete, the bladder is emptied and the catheter removed. Patients are assigned one course of treatment per week for 6 weeks with sequential BCG and EMDA/MMC. Two BCG instillations and one EMDA/MMC instillation constitute one cycle for a total of two cycles.3259455573405Figure 1. Illustrated depiction of administration of intravesical EDMA using the Physionizer?4000020000Figure 1. Illustrated depiction of administration of intravesical EDMA using the Physionizer?Maintenance treatment of eMMC will be given at month 3, 4, 6, 7, 9 and 10 (after last dose of BCG of the initial treatment). Maintenance of BCG will be given around month 5, 8, and 11. CBC, BMP, urinalysis, and a history/physical will be performed before each EMDA/MMC procedure. Urinalysis and a history/physical will be performed before each BCG.Study MeasurementsA completed blood count (CBC) and basic metabolic panel (BMP) will be performed weekly and at the beginning of each cycle. Prior to dosing with mitomycin Cat week 3, a CBC will be performed and also at weeks 4 and 5 if the CBC has changed. A urinalysis will be performed prior to each intravesicular instillation. Disease response to treatment is assessed by cystoscopy and urinary cytology at approximately 6 weeks after the last cycle of treatment. Additional assessments are then done approximately every 3 months during the first year. Patients with carcinoma in situ undergo cystoscopy and urinary cytology in a similar fashion. If findings on cystoscopy or cytology suggest the presence of recurrent/progressive disease, a surgical biopsy will be performed for further evaluation. Table 1 illustrates the complete visit schedule.Statistical DesignThis trial is designed as a phase II, superiority, single treatment arm, longitudinal (5 year) study. The primary objective of this study is to assess the bladder cancer recurrence-free rate after treatment with intravesical instillation of sequential BCG and EMDA/MMC following TUR. Secondary objectives include: to compare complete response rates at 3 and 6 months in patients with carcinoma in situ; to determine progression-free survival, overall and disease-specific survival in the study group, to be compared with literature data of the current standard of care of BCG alone (Lamm’s protocol); to compare the qualitative and quantitative toxicities of experimental regimens in these patients; and to assess long-term morbidity in the study group, as defined by requirement for fewer TURBTs, courses of traditional intravesical therapies, and surveillance cystoscopies over 5 years (cost-effectiveness), compared with literature data of current standard long-term morbidity of BCG alone.Power AnalysisFor the sample size calculation, a population parameter estimate of recurrence-free rate θ1=0.6 and a desired clinically significant reduction of 0.2 with the EMDA/MMC treatment were assumed.Controlling for both Type I error, alpha=0.05 and Type II error, beta=0.20 (90% Power), a single sample size of 50 subjects would be necessary.76200-6350803275WherePopulation parameter pi = 0.60 (standard regimen)Expected proportion p=0.80 (experimental treatment)u=one-sided percentage point of the normal distribution for power=0.90, u=1.28v=percentage point of the normal distribution for alpha=0.05, v=1.96Alternative methods of sample size calculation are presented in Appendix I. Power and Precision Table is provided in Appendix II.Statistical AnalysesData will be analyzed when data for the visits that occur 12, 24, 36, 48, and 60 months after the last cycle of the study from all patients are complete and accurate. Data analysis will be tailored to the needs of a one-sample phase II activity study, with recurrence rate (rather than time to failure) as the outcome variable. Thus, the analysis will evaluate the difference in risk of recurrence (θ = θ1 – θ0) between the two populations (study treatment [θ1] and standard of care [θ0]).Data for the secondary objectives will be analyzed by the method that is most appropriate for each individual objective. Time to first recurrence in patients with carcinoma in situ will be estimated by the Kaplan-Meier method. Time to progression and overall and disease-specific mortality will also be estimated by the Kaplan-Meier method, with the results to be compared with literature data of the current standard of care treatment (BCG alone). Toxicities of the study treatment will be expressed as incident events, and will be analyzed with standard statistical methods for comparing proportions. Long term morbidity in the study group will be assessed in terms of cumulative incidence. Cost-effectiveness will be calculated in terms of NNT (number needed to treat) and NNH (number needed to harm) figures.Median survival time estimates the time beyond which 50% of the patients are expected to survive in the study population. In diseases with favorable prognosis such as bladder cancer, the survival probability may not reach the value 0.5 across the study period, thus preventing estimate of the median survival time.Patients with carcinoma in situ who do not achieve a complete response will be excluded from the analysis of time to first recurrence, as disease-free status is the condition for being at risk of recurrence. However, all of these patients will be included in time to progression and survival analyses.Study Implementation and ProtocolRecruitment and Retention PlansStudy participants will be recruited from patient populations at the University of Colorado Denver (Aurora, CO) and Rutgers-Cancer Institute of New Jersey (New Brunswick, NJ). Potential participants will be identified by evaluation of all patients undergoing the TURBT procedure at participating hospitals. Eligibility will be further assessed using the defined inclusion/exclusion criteria. If patients are deemed eligible for the study, enrollment will occur after obtaining the patient’s informed consent. This trial is also listed on (NCT02202044).Retention of enrolled patients is likely to be the most difficult once patients enter the maintenance treatment phase (after study week 9). Study personnel will reach out to each participant at least monthly during this time period, either by phone and/or email. This will allow for continued follow up with the patients as well as updating of contact information if this has changed. Participants will also be encouraged to contact study personnel if any questions or concerns arise. A more individualized form of contact with patients will be achieved by sending annual birthday and holiday cards.Randomization and Blinding ProceduresAs this is a single treatment arm study, no randomization or blinding is needed. If this study was being conducted as a prospective, two-arm study comparing the study treatment to the standard of care, both randomization and blinding would be important to ensure reliability of the results. If the study patients were to be randomized, a randomization tool, such as that at ), could be used to create an unbiased randomization schedule (see attached). All investigators participating in patient care would be blinded to the schedule. Unfortunately, it would be nearly impossible to blind patients and treating physicians to the treatment assignment, as the experimental arm would require additional treatments with the EDMA/MMC that the patients receiving standard of care would not have. However, those involved in response assessment (such as the doctors performing cystoscopy and interpretation of cytology/histopathology samples) could be blinded to the treatment assignment, thus making the outcome data more reliable.Procedures for Minimizing Missing DataData will be entered and managed through REDCap (Research Electronic Data Capture), which is a secure, we-based application designed to support data capture for research studies. The secondary site will be given access to REDCap for data entry. Any entry fields that are not completed will be flagged to notify both the data enterer and the data monitors. Data entry will also be monitored for accuracy by checking realistic limits of entries and comparing at least 20% of the entries with the original documentation. These procedures will be conducted throughout the course of the study.References1. Parkin DM (2008) The global burden of urinary bladder cancer. Scand J Urol Nephrol 26:1-9.2. Babjuk M, Oosterlinck W, Sylvester R, Kaasinen E, B?hle A, Palou-Redorta J (2008) EAU Guidelines on Non-Muscle-Invasive Urothelial Carcinoma of the Bladder. Eur Urol 54:303-314.3. Pawinski A, Sylvester R, Kurth KH, Bouffioux C, van der Meijden A, Parmar MK, Bijnens L (1996) A combined analysis of European Organization for Research and Treatment of Cancer, and Medical Research Council randomized clinical trials for the prophylactic treatment of stage TaT1 bladder cancer. European Organization for Research and Treatment of Cancer Genitourinary Tract Cancer Cooperative Group and the Medical Research Council Working Party on Superficial Bladder Cancer. J Urol 156:1934-1940.4. Lamm DL, Riggs DR, Traynelis CL, Nseyo UO (1995) Apparent failure of current intravesical chemotherapy prophylaxis to influence the long-term course of superficial transitional cell carcinoma of the bladder. J Urol 153:1444-1450.5. Sylvester RJ, Oosterlinck W, van der Meijden AP (2004) A single immediate postoperative instillation of chemotherapy decreases the risk of recurrence in patients with stage Ta T1 bladder cancer: a meta-analysis of published results of randomized clinical trials. J Urol 171:2186-2190.6. B?hle A, Jocham D, Bock PR (2003) Intravesical bacillus Calmette-Guerin versus mitomycin C for superficial bladder cancer: a formal meta-analysis of comparative studies on recurrence and toxicity. J Urol 169:90-95.7. Brausi M, Campo B, Pizzocaro G, Rigatti P, Parma A, Mazza G, Vicini A, Stephen RL (1998) Intravesical electromotive administration of drugs for treatment of superficial bladder cancer: a comparative Phase II study. Urology 5: 506-509. 8. Riedl CR, Knoll M, Plas E, Pfluger H (1998) Intravesical electromotive drug administration technique: preliminary results and side effects. J Urol 159:1851-1856.9. Di Stasi SM, Giannantoni A, Stephen RL, Capelli G, Navarra P, Massoud R, Vespasiani G (2003) Intravesical electromotive mitomycin C versus passive transport mitomycin C for high risk superficial bladder cancer: a prospective randomized study. J Urol 170: 777–782.10. Di Stasi SM, Storti L, Giurioli A, Zampa G, Liberati E, Sciarra M, Iorio B, Stephen RL (2008) Carcinoma in situ of the bladder: long-term results of a randomized prospective study comparing intravesical electromotive mitomycin-C, passive diffusion mitomycin-C and bacillus Calmette-Guerin. Eur Urol Suppl 7 (3): 180, abs #439.11. Di Stasi SM, Verri C, Capelli G, Brausi M, Leprini G, Zampa G, Stephen RL (2010) Single preoperative intravesical instillation of electromotive mintomycin-C for primary non-muscle invasive bladder cancer: a randomized trial. J Urol 183 (suppl. 4): 520, abs #1346.Table 1. Study TimelineStudy TimelineScreening(2-8 weeks post TURBT)Pre-cysto-scopyPre-Treat-ment Prior to start of cyclePrior to MMC dosingWeek 1Week2Week4Week5, 7, 8Week 3Week 6, 9Informed consentXMedical history XXXXXXXVital SignsXXXXXXXXUrinalysis, CBC w/ diff, BMPXCBC, BMPXXXXXXXCBCXX**X**UrinalysisXAEs/SAEsXXXXXXXPhysical ExamXXXXXXXDosing* BCG XXXXDosing* MMCXX***Maintenance Dose of eMMC****Maintenance Dose of BCGStudy Timeline6 weeks after last cycle3months after last cycle 6 months after last cycle9 months after last cycle12, 24, 36, 48 and 60 months after last cycleCystoscopyXX XXXVital Signs/Physical ExamXXXXXUrinalysis, CBC w/diff, BMPXAEs/SAEsXXXXXMaintenance tx given after neg. cystoscopyXX*.? Dosing may be delayed up to 2 weeks for cystitis or hematuria.**Only performed if the CBC at week 3 is unchanged***Will occur at months 3, 4, 6, 7. 9 and 10****Will occur at months 5, 8 and 11.Appendix I: Additional Power Analysis MethodologyTwo-Group N Calculation:Functional: True mean improvement in risk of cancer recurrence free rates with BCG (θ0) or BCG with EMDA/MMC (θ1)Contrast: θ = θ1 - θ0 Risk differenceθ0 = 0.60 θ1 = 0.80Hypotheses: Superiority testH0: θ ≤ 0(Inferiority)H1: θ ≥ 0.20(Clinically important benefits)Variance: Under H1Var(θ) = Var(θ1 - θ0 ) = θ01-θ0N+θ11-θ1N Choose: NN = 1.96+1.280.8-0.620.8*0.2+0.6*0.4N = 105 per groupSimple One-Sample N Calculation:θ1 = 0.80Goal: Superiority testIf observed θ1=0.8 then need lower CI limit to be above 0.60.8 - 1.96se = 0.60.8 – 1.96 sqrt 0.8*0.2N = 0.6 N = 1.960.8-0.620.8*0.2N = 15.3716 per group too small for normal approximationInstead, if want lower limit at 0.7 N = 1.960.8-0.720.8*0.2N = 61.47Power Calculation in Current Protocol:θ1 = 0.80N = 5076200-317500-952516129000WhereType I error alpha=0.05Type II error beta=0.20, Power=0.90Population parameter (p) = 0.60 (standard regimen)Expected proportion p=0.80 (experimental treatment)u=one-sided percentage point of the normal distribution for power=0.90, u=1.28v=percentage point of the normal distribution for alpha=0.05, v=1.96NOTE: Similarities to 2-group or 1-group studyIf we use 0.8*0.2 and 0.8*0.2:N = 1.28+1.960.8-0.620.8*0.2 = 1.28 sqrt0.8*0.2+1.98 sqrt0.8*0.20.8-0.62= 42If we use 0.6*0.4 and 0.6*0.4:N = 1.28+1.960.8-0.620.6*0.4 = 1.28 sqrt0.6*0.4+1.98 sqrt0.6*0.40.8-0.62= 63Logic Behind Power Analysis in Protocol:Goal: PowerWant 90% power to reject θ1 ≥ 0.80 if θ1 = 0.60 Type I ErrorWhat does it mean to reject θ1 ≥ 0.80?5%α=0.051.96 se0.80.8 – 1.96 se = 0.8 – 1.96 sqrt 0.8*0.2NYields the Critical Value.What is power when θ1 = 0.60?10%observed 0.61.28 seΒ=900.6 + 1.28 se = 0.6 + 1.28 sqrt 0.8*0.2NWant this cut-off for power to be same as critical value.So, want: 0.6 + 1.28 sqrt 0.8*0.2N = 0.8 – 1.96 sqrt 0.8*0.2NRearranging yields:76200-6350803275NOTE: At N=50, CV=0.698CONCLUSION:The justification in the protocol implies that we primarily want to reject θ1 ≥ 0.80.Appendix II: Power and Precision TableNObservedCI 95% lowCI 95% high150.60.352077430.847923150.650.408620080.89138150.70.468089670.93191150.750.530865340.969135150.80.597572071.002428200.60.385292760.814707200.650.440958860.859041200.70.499159760.90084200.750.560223820.939776200.80.624692270.975308250.60.407960.79204250.650.463027920.836972250.70.520363030.879637250.750.580259020.919741250.80.64320.9568300.60.424692270.775308300.650.479318620.820681300.70.536014630.863985300.750.595048390.904952300.80.656861840.943138350.60.437696580.762303350.650.491979750.80802350.70.548179050.851821350.750.606542690.893457350.80.667479810.93252400.60.448179050.751821400.650.502185590.797814400.70.557984510.842015400.750.615807970.884192400.80.676038720.923961450.60.456861840.743138450.650.510639240.789361450.70.566106510.833893450.750.623482540.876517450.80.683128180.916872500.60.464207220.735793500.650.517790770.782209500.70.572977480.827023500.750.6299750.870025500.80.689125660.910874550.60.470526660.729473550.650.523943450.776057550.70.578888780.821111550.750.635560660.864439550.80.694285460.905715600.60.476038720.723961600.650.529310040.77069600.70.584044840.815955600.750.640432670.859567600.80.698786040.9012141000.60.503980.696021000.650.556513960.7434861000.70.610181520.7898181000.750.665129510.834871000.80.72160.8784 ................
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