In Relation to HIFU Treatment on the Growth of Dunning Tumors: Results of a Preliminary Study

Influence of the docetaxel administration period (neoadjuvant or concomitant) in relation to HIFU treatment on the growth of Dunning tumors: results of a preliminary study

P Paparel1,2, JY Chapelon2, A Bissery3, S Chesnais2, L Curiel2 and A Gelet2,4 1Department of Urology, Lyon Sud Hospital, Pierre Be´nite, France; 2INSERM U 556, Lyon, France; 3Department of Biostatistics, Hospices civils de Lyon, Lyon, France and 4Department of Urology, Edouard Herriot Hospital, Lyon, France

The objective of this study was to evaluate mechanisms of the synergy between high intensity- focused ultrasound (HIFU) and docetaxel and to determine the best sequence of chemotherapy administration in relation to HIFU treatment for obtaining optimum control of tumoral growth. A total of 15 days after s.c. implantation of the tumor, 52 Copenhagen rats studied were randomized in 4 groups of 13: controls, docetaxel alone (group 1), HIFU and docetaxel concomitant (group 2) and HIFU and docetaxel administered 24 h before treatment (group 3). The number of HIFU shots was calculated in order to cover 75% of the tumor volume. The effects of docetaxel, HIFU and their interaction on tumor volumes were analyzed using a linear regression. The distributions of the tumor volumes were significantly greater in the control group than in the group 1 (P ¼ 0.002) and than in both groups 2 and 3 (Po0.0001 and P ¼ 0.0001). These volumes were also significantly greater in group 1 than in both groups 2 and 3 and there was no difference between the groups 2 and 3. The tumor doubling times were 7.8 days for the group 1, 43.8 days for the group 2, 16.1 days for the group 3 and 5.9 days for the controls. The mechanism of the synergy between HIFU and docetaxel on the growth of Dunning tumors is apparently multifaceted. The results are encouraging because in the two groups of rats treated with the combination of HIFU and docetaxel, the percentage of complete remission was approximately 30%. Prostate Cancer and Prostatic Diseases (2008) 11, 181–186; doi:10.1038/sj.pcan.4501005; published online 21 August 2007

Keywords: HIFU; docetaxel; Dunning adenocarcinoma

Introduction back to the beginning of the 1990s when studies proved that in vivo, this combination was synergic and superior High intensity-focused ultrasound (HIFU) is used in the to treatment with HIFU alone or chemotherapy alone.6–9 clinical setting to treat localized prostate cancer in This was notably demonstrated with adriamycin on C patients who are either not candidates for surgical 1300 neuroblastoma, doxorubicin and cyclophospha- treatment (because of their age or comorbidity) or they mide on Morris hepatoma or more recently with do not wish to be treated surgically or with radio- mitomycin on urothelial carcinoma of the urinary therapy.1 With an actuarial 5-year disease-free survival bladder.10 In 2005, we demonstrated for the first time rate at 66%, most authors still report 6–17% residual that this HIFU/chemotherapy synergic effect also exists cancerous tissue in the patients treated.2–4 The persist- with the combination of paclitaxel and estramustin ence of tumor cells after treatment is most often phosphate on Dunning tumors implanted subcuta- explained by the presence of a locally advanced under- neously in Copenhagen rats.5 We found that tumoral staged prostate cancer with extracapsular extensions, growth in the HIFU-alone and chemotherapy-alone arm and given that the HIFU treatment is localized in the was superimposable and statistically different from the prostate these extraprostatic cells are therefore not HIFU/chemotherapy arm which seemed to be the destroyed.5 From these diagnostic findings the impo- optimal treatment. However the mechanisms of this rtance was raised of adding chemotherapy to the HIFU synergy are not well understood. Two concepts might treatment with the aim of destroying residual cells. The explain this phenomenon: the first is the belief that HIFU concept of combining HIFU and chemotherapy dates acts on tumor cells that are already weakened by chemotherapy administered 24 h before treatment;5,8 the second belief is that it is the concomitant application Correspondence: Dr P Paparel, Department of Urology, Lyon Sud of HIFU and chemotherapy that allows better intrace- Hospital, 36 route de Givry, 71100 Saint Remy, France. llular diffusion of the chemotherapy and therefore an E-mail: [email protected] 6–8,11 Received 15 February 2007; revised 11 June 2007; accepted 16 July accrued cytotoxicity. To distinguish the role of these 2007; published online 21 August 2007 two mechanisms we developed an experimental protocol Synergy between HIFU and docetaxel P Paparel et al

182 that included an arm in which chemotherapy was administered 24 h before treatment by HIFU and an arm in which chemotherapy was administered concomitantly with HIFU treatment. The chemotherapy used in our experiment was docetaxel for which the half-life is slightly longer than 90 min when administered by i.p. route.12 The objective of this study was to demonstrate the predominant mechanism of this synergy and in this way determine the best sequence of chemotherapy administration in relation to HIFU treatment for obtaining optimum control of tumoral growth.

Materials and methods

Animals and tumor model Fischer Copenhagen rats, obtained from Harlan (India- napolis, IN, USA), were housed in our laboratory.5 Only male rats, 10–12 weeks old, were used in all experiments. R3327 Dunning prostatic adenocarcinoma subline AT2 was used. Cells were kept in liquid nitrogen. For our experiments, cellular samples of the same pool were warmed and cultured under standard conditions. A rat was given an abdominal s.c. injection of 2 Â 106 cells and after 15 days, a solid tumor was obtained, excised and minced; 20-mg pieces were then implanted in the experimental animals and 15 days later, the tumor was ready to be treated (Figure 1a and b). For the AT2 subline, 7–8 weeks are necessary to induce death.

HIFU treatment Material and treatment parameters. The prototype device used for treatment consisted of three integrated components. Figure 1 Every rat was given an s.c. implantation of a 20 mg tumor (a) and after 15 days, a solid tumor was obtained and ready to be (1) An ultrasound treatment system located in a tank treated (b). filled with degassed and deionized water consisting of a spherical transducer with a geometrical focus at 45 mm, an active diameter aperture of 50 mm and a ultrasound shots, the animal received an i.p. fentanyl central frequency of 3 MHz. injection (0.01 mg/kg). (2) An imaging system consisting of an ultrasound Under this anesthesia, the animal was placed on a scanner (Combison 311, Kretz, Austria) coupled with specially designed Plexiglas gantry with an opening a stereotaxic localizing arm. below the abdominal area for the penetration of (3) A computer which controlled the firing sequence and ultrasound (Figure 3). The tumor was immobilized with the movements of the firing head through a three- a silicone ring fastened to the gantry. This gantry was ˆ dimensional positioning system (Microcontrole, then mounted on the x-y-z positioning system in order to France). grossly position the animal over the generator at the In order to achieve a treatment, multiple shots were approximate focal distance. After the tank was filled made following a spiral model. Each shot was made at with warm (351C) degassed and deionized water, an an acoustical power of 13 W for 5 s with a 5 s waiting time initial localization of the target area with a 5-MHz between shots and were separated by a step of 1.6 mm ultrasound probe was performed. Using the locating arm (Figure 2). of the device, the coordinates of the target volume were sent to the instrument computer which controlled the movements of the therapeutic ultrasound transducer. Treatment session HIFU-lesion modeling was used to predict the lesion Before treatment the rats were anesthetized with isoflur- volume under the actual conditions and the number of an with the use of a vaporizer (Minerve, Esternay, shots was calculated to cover 75% of the tumor volume. France). Isofluran is a halogenic volatile anesthetic. We determined that treating 75% of the tumor volume Anesthesia induction was done in an induction cage would allow us to measure the effects of chemotherapy which prepares the animal for the anesthesia (% of on residual tumor cells outside of the HIFU treatment isofluran: 3–5% for 2–5 min). Anesthesia maintenance zone.13,14 Programming the computer software produces was ensured by the administration of 1.5–2.5 percent of the appropriate number of shots according to the target isofluran depending on the animal. Just before the volume which was set to 75% of the tumor volume.

Prostate Cancer and Prostatic Diseases Synergy between HIFU and docetaxel P Paparel et al 183

Figure 2 HIFU effects on the tumor tissue and modification of the local heating by treatment.

(iv) Group 3: HIFU and docetaxel administrated 24 h before treatment (docetaxel was administrated 24 h before HIFU treatment). The tumor volume was measured weekly. Over the course of the observation of tumor growth in the weeks following treatment, the rats were killed if the tumor exceeded 60 mm in diameter or if the tumor volume was such that the rats were no longer able to move.5 The rats were monitored and fed daily. They were placed in cages in groups of three and all of the cages were located in an air-conditioned area of the animal house approved by the faculty of medicine. Each rat was inspected daily to ensure first of all that it was alive and that good mobility was maintained in the cage despite the tumor. The Figure 3 Photography of the rat Cop, just before HIFU treatment, abdominal wall was also inspected and in case of an placed on a specially designed Plexiglas gantry with an opening abdominal perforation or a tumor with a diameter that below the abdominal area for the penetration of ultrasound. surpassed 60 mm, the rat was immediately taken from the cage and euthanized. The experiments were con- ducted with approval from our Animal care and use committee. Chemotherapy The tumors were photographed upfront and in profile Docetaxel (Aventis pharmaceuticals, Bridgewarer, NJ, each week following a rule that allowed precise USA) was administrated at a dose of 10 mg/kg as a measurement in mm. The formula utilized to calculate single i.p. injection. The choice of the doses and the tumor volume was length Â width Â height Â 0.5236 protocol of docetaxel administration were made accord- (expressed in cm3).16 ing to an optimal balance of efficacy and tolerance, as previously tested in the litterature.15 Criteria of evaluation A rat was considered to be cured if on day 60, the date of Experimental design the end of the study, there was no trace of tumor on its A total of 15 days after s.c. implantation of the tumor, abdominal wall and no obvious sign of local recurrence. with the tumor measuring approximately 10 cc, the 52 The main judgment criterion for treatment response was rats studied were randomized in 4 groups of 13: the change in tumoral volume and the secondary criterion was the survival of the rats in each group. (i) Controls, (ii) Group 1: docetaxel alone, (iii) Group 2: HIFU and docetaxel concomitant (doce- Statistical methods taxel was administrated 30 min before HIFU The distributions of the tumor volumes at day 29 of treatment) and follow-up were globally compared using a nonpara-

Prostate Cancer and Prostatic Diseases Synergy between HIFU and docetaxel P Paparel et al 184 Table 1 Distribution per group of the tumoral volumes on days 15 and 29 Group Median First quartile Third quartile Minimum Maximum No. of rats

J15 J29 J15 J29 J15 J29 J15 J29 J15 J29 J15 J29

Control 1.15 19.5 1 17.3 1.26 5.7 0.56 7.1 2.66 39.8 13 13 Group 1 1.28 12 1.14 5 2.02 1.9 0.64 3.2 2.9 22.2 13 13 Group 2 1.51 0 1.27 0 1.73 15.3 0.91 0 2.21 7 13 13 Group 3 1.4 0.7 1.15 0 1.44 27.1 0.84 0 2.63 13.4 13 13

At day 15, there was no difference in the tumoral volumes between the groups (P ¼ 0.37, Kruskall–Wallis test). At day 29, these volumes were significantly greater in the control group than in group 1 (P ¼ 0.002) and than in groups 2 and 3 (Po0.0001 and P ¼ 0.0001); these volumes were also significantly greater in the group 1 than in groups 2 and 3 (P ¼ 0.0001 and 0.0064); there was no difference between the groups 2 and 3 (P ¼ 0.21).

metric analysis of variance (Kruskal–Wallis test); then, more specifically, each group was compared to each of the others (Mann–Whitney test) with a Bonferroni correction so as to keep the overall significance level of the comparison tests at Po0.05 (using the correction, one of the six paired test is significant if Po0.0083). The effects of chemotherapy, HIFU and their interaction on tumor volumes was analyzed using a linear regression for repeated measurements after a logarithmic transformation of the volume values (initially expressed in cm3). To deal with the logarithmic transformation of volume equal to 0, we added 0.5 cm3 to all volume. The results of this regression analysis were expressed as percentages of volume augmentation per day and as tumor doubling times in days. All analyses were performed using R 2.0.1 software. Figure 4 Box plots of tumor volume at day 29 according to treatment group.

Results Table 2 Estimated daily percentage of increase in tumoral volume according to the groups The treatment groups Overall, 52 rats were used for this study: 13 underwent Group Estimation (%) 95% CI P-value docetaxel only (group 1), 13 underwent HIFU and docetaxel the same day (group 2), 13 underwent HIFU Control 12.6 (10.6; 14.7) o0.0001 Group 1 9.4 (7.4; 11.4) o0.0001 and a single administration of docetaxel 24 h before Group 2 1.6 (À0.3; 3.5) 0.09 treatment (group 3) and 13 were used as controls. Group 3 4.4 (2.5; 6.3) o0.0001

Tumor volume at day 15 of follow-up the controls, the group 1 and the group 3. As to the group (day of randomization) of rats treated by concomitant HIFU/chemotherapy There was no difference in the tumoral volumes between (group 2), the average augmentation in tumor volume the groups on day 15 (P ¼ 0.37, Kruskal–Wallis test) was 1.6 percent per day; however, that augmentation was (Table 1). not significantly different from zero (P ¼ 0.09). The estimations of the average tumor volume augmentation in each treatment group are shown in Table 2 and Tumor volumes at day 29 of follow-up Figure 5. The distributions of the tumor volumes according to the The augmentation of the tumor volume may also be treatment group were significantly different (Po0.0001) expressed in terms of tumor doubling time. Thus, the (Table 1 and Figure 4). These volumes were significantly tumor doubling times were 7.8 days for the group 1, 43.8 greater in the control group than in group 1 (P ¼ 0.002) days for the group 2, 16.1 days for the group 3 and 5.9 and than in groups 2 and 3 (Po0.0001 and P ¼ 0.0001). days for the controls. These volumes were also significantly greater in the The effect of HIFU/chemotherapy on the reduction of group 1 than in groups 2 and 3 (P ¼ 0.0001 and the tumor growth was statistically significant (Po0.0001 P ¼ 0.0064). There was no difference between the groups for group 2 and Po0.0001 for group 3 versus controls), 2 and 3 (P ¼ 0.21). but the tumor growth in group 1 was not statistically different from that in the control group, after Bonferroni correction (P ¼ 0.027 versus controls). Modification in tumor size In the whole lot of 52 rats at day 15 of follow-up, the mean tumor volume and the standard deviation were Study of the survival 1.44 and 0.56 cm3, respectively. That volume augmented In the group 2, four rats were cured, one was rapidly in three out of the four treatment groups (Table 2): disapproved on day 44 because of death related to a

Prostate Cancer and Prostatic Diseases Synergy between HIFU and docetaxel P Paparel et al

as one moves further away from the focal area.14 The 185 hyperthermia in the non treated area might thus facilitate the intracellular penetration of docetaxel. Mohamed et al.22 demonstrated an in vivo improvement in docetaxel efficacy at 41.51C. Studies have shown that tumor cells were more sensitive to hyperthermia (from 41.5 to 43.51C) than normal cells.8 Moreover, hyperthermia and docetaxel have a common action since hyperthermia itself favors the disorganization of the microtubular system.20 In the group 3, chemotherapy was administered 24 h before HIFU treatment. After a single dose peritoneal injection of docetaxel the concentration in peritoneal fluid progressively increases and reaches a plateau from the 90th minute.12 The molecule then progressively diffuses into the surrounding tissue (colon, stomach, Figure 5 Estimation of the average tumor volume augmentation in liver, heart) reaching maximum concentration in the each group. anterior abdominal wall from the 90th minute.12 Dun- ning tumors implanted subcutaneously in the anterior abdominal wall are therefore completely exposed to the 12 perforation of the abdominal wall by necrosis and eight treatment. Docetaxel blocks the cell cycle in the G2/M rats were euthanized because tumors exceeded 60 mm in phase.15,23 Lokeshwar et al.16 demonstrated that 80% of diameter. all tumor cells are blocked in the G2/M phase 24 h after In the group 3, four rats were cured, one was the administration of i.p. paclitaxel in Dunning tumors disapproved on day 22 because of death related to implanted in Copenhagen rats. In this study the authors unknown cause and eight rats were euthanized because demonstrated a potentiation of radiotherapy action by tumors exceeded 60 mm in diameter. paclitaxel because the effect of radiotherapy is at a 16 In the control group and group 1, all rats were maximum on cells in the G2/M phase. No data are euthanized because tumors exceeded 60 mm in diameter. currently available to ascertain whether treatment with HIFU is at a maximum on cells blocked in the G2/M phase; however this mechanism might intervene in this group. Treatment with HIFU acts on tumor cells Discussion weakened by chemotherapy.5,23 The hyperthermia that is generated by the HIFU is produced 24 h after the Docetaxel is a chemotherapy that belongs to the taxan administration of docetaxel which has a half-life of a few family of drugs developed from paclitaxel. It is a mitotic hours; it therefore has little influence on the intracellular spindle poison which facilitates in vitro polymerization penetration of the chemotherapy in this group. of tubulin into microtubules and stabilizes microtubules Our results do not show a statistically significant in vivo which results in a blockage of the G2/M phase of difference between the two HIFU/chemotherapy groups the cell cycle.16 An apoptosis inducer, it stimulates the (groups 2 and 3). The synergy mechanism between HIFU phosphorylation of bcl-2 which is a protein with and docetaxel is therefore apparently multifaceted with- antiapoptic properties.12 In addition, docetaxel is used out any absolutely predominant mechanism. The combi- in the clinical setting in patients with hormone refractory nation of HIFU and docetaxel in the two groups was prostate cancer.17,18 more efficient than treatment with chemotherapy alone In the group 2, chemotherapy was administered or in the control group. This synergic combination is concomitantly with HIFU treatment. The mechanism potentially curative since the rate of total response was mentioned to explain the synergy between the two higher than 30%. A total of 6 weeks after the application treatments is based on the increase in intracellular of the different treatments there was no further sign of penetration of the chemotherapy, thanks to the direct tumoral progression in these rats. The tumor was entirely and indirect effects of HIFU: the direct effect concerns the necrosed even though only 75% of the tumoral volume area treated by HIFU (75% of the tumoral volume) where was treated. In the group 1, docetaxel slowed down there is an increase in blood flow and in the permeability tumoral growth without actually stopping it. Yang et al.7 of the tumor cell membrane thus facilitating the supply cured 12 mice out of 15 with the C1300 neuroblastoma and intracellular penetration of docetaxel.8 The cavita- cells, thanks to the combination of HIFU and adriamycin tion generated by HIFU takes place with creating a administered concomitantly. This cure rate that is higher circulating fluid flow (‘microstreaming’) in the interstitial than ours is explained by the fact that the entire tumor tissue exposed and inducing shear forces who can was treated and not only 75% as was the case in our created gaps between endothelial cells.11,19 The indirect study. effect that concerns the nontreated tumoral area (25% of Our study has a number of limitations. It is not certain the tumoral volume) might be related to the regional whether the results observed with the Dunning model hyperthermia caused by the HIFU and the diffusion of implanted subcutaneously can be adapted to prostatic the ultrasound beam.5,10,14,20,21 At the focal area the tumor cells developing in an orthotopic position. We did temperature of the treated tissue can reach 1001C.13 The not administer any corticoids in the rats receiving remaining tumoral ring that is nontreated by HIFU is the docetaxel as is done in the clinical setting in order to focus of a rise in temperature that decreases in intensity evaluate only the effects of docetaxel without potentia-

Prostate Cancer and Prostatic Diseases Synergy between HIFU and docetaxel P Paparel et al

186 17 tion by another molecule. Finally, we did not have an 8 Yang R, Reilly CR, Rescorla FJ, Faught PR, Sanghvi NT, Fry FJ HIFU-alone arm because the objective of this study was et al. High-intensity focused ultrasound in the treatment of above all to compare tumoral growth between the two experimental liver cancer. Arch Surg 1991; 126: 1002–1009. HIFU/docetaxel arms in order to determine the best 9 Fry FJ, Johnson LK. Tumor irradiation with intense ultrasound. method of administration of these two treatments. Ultrasound Med Biol 1978; 4: 337–341. 10 Yu T, Huang X, Hu K, Bai J, Wang Z. Treatment of transplanted adriamycin-resistant ovarian cancers in mice by combination of adriamycin and ultrasound exposure. Ultrason Sonochem 2004; 11: 287–291. Conclusion 11 Frenkel V, Li KC. Potential role of pulsed-high intensity focused ultrasound in gene therapy. Future Oncol 2006; 2: 111–119. The mechanism of the synergy between HIFU and 12 Marchettini P, Stuart OA, Mohamed F, Yoo D, Sugarbaker PH. docetaxel on the growth of Dunning tumors is appar- Docetaxel: pharmacokinetics and tissue levels after intraperito- ently multifaceted. More studies are necessary in order to neal and intravenous administration in a rat model. Cancer understand all the mechanisms which explain the Chemother Pharmacol 2002; 49: 499–503. synergy between these two treatments. The results 13 Chapelon JY, Margonari J, Vernier F, Gorry F, Ecochard R, are encouraging because in the two groups of rats Gelet A. In vivo effects of high-intensity ultrasound on prostatic treated with the combination of HIFU and docetaxel adenocarcinoma Dunning R3327. Cancer Res 1992; 52: 6353–6357. 14 Paparel P, Chapelon JY, Curiel L, Rabilloud M, Chesnais S, Gelet the percentage of complete remission was approxi- A. Potentiation of focal ultrasound treatment of prostate mately 30%. adenocarcinoma by concomitant chemotherapy with estramustine phosphate and paclitaxel. Prog Urol 2004; 14: 40–46. 15 Oudard S, Legrier ME, Boye K, Bras-Goncalves R, De Pinieux G, De Cremoux P et al. Activity of docetaxel with or without References estramustine phosphate versus mitoxantrone in androgen dependent and independent human prostate cancer xenografts. 1 Colombel M, Gelet A. Principles and results of high-intensity JUrol2003; 169: 1729–1734. focused ultrasound for localized prostate cancer. Prostate Cancer 16 Lokeshwar BL, Ferrell SM, Block NL. Enhancement of radiation Prostatic Dis 2004; 7: 289–294. response of prostatic carcinoma by taxol: therapeutic potential 2 Gelet A, Chapelon JY, Bouvier R, Rouviere O, Lasne Y, Lyonnet for late-stage malignancy. Anticancer Res 1995; 15: 93–98. D et al. 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