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INTRATUMORAL ACETIC ACID INJECTION ERADICATES HUMAN PROSTATE CANCER TUMORS IN A MURINE MODEL Jasneet Singh Bhullar MD1, Gokulakkrishna Subhas MD1, Erina Kansakar MD1, Jasper Gill BS2 , Boris Silberberg MD2, Jacqueline Tilak BS2, Lee Andrus LVT BIS2, Milessa Decker LVT2, Patrick Hurley MD1, Jeffrey O’Connor MD1 and Vijay K. Mittal MD FACS1 Departments of 1Surgery and 2Patient Care Research. Providence Hospital and Medical Centers, Southfield, MI, USA Address for correspondence Jasneet Singh Bhullar MD Department of Surgery Providence Hospital & Medical Centers 16001 West Nine Mile Road, Southfield, MI-48075, USA Phone -248-436-8001 Fax – 248-849-5380 Email- [email protected] Key words: Acetic acid, intratumoral injection, human prostate cancer cell lines, prostate cancer, xenograft, murine model. ABSTRACT Purpose: Prostate cancer treatment is associated with substantial morbidity. Ideal treatment of localized prostate cancer would be an effective local therapy with minimal morbidity. Direct injections have been used to treat benign prostatic hyperplasia without major complications, but in limited cases. We evaluated the local oncotoxic effects of acetic acid in a prostate cancer xenograft murine model. Methods: PC3 and LNCaP human prostate cancer cell lines were used to grow subcutaneous tumors in SCID mice. For each cell line, 14 mice underwent intratumor injection with 25% acetic acid (0.05 ml/100 cm3 of tumor) after the tumor was >300 mm3. Post treatment one mouse/group was euthanized after 2 hrs, 24 hrs, 1 week and 2 weeks; remaining mice (n=10) were sacrificed at 120 days. Control mice (8/group) were euthanized after they met the humane criteria for tumor burden and overall health. Results: Tumor necrosis was noted immediately post injection; by 24 hrs, ulceration and crusting of overlying skin were noted, which healed into scars by 23±5 days. Histological examination showed tumor degeneration and necrosis with blood vessel obstruction. Ten treated mice in both groups survived for 120 days, which was much longer than the mean survival of PC3 (40±9 days) and LNCaP (56±10) control mice. Conclusions: Direct injection of acetic acid successfully eradicated both tumors. This treatment option could potentially be used in humans for treatment of early localized prostate cancer and non-operative management of locally advanced cases. This is the first report of successful local chemical therapy for prostate cancer. Introduction: In the United States alone, 200,000 men are diagnosed with prostate cancer each year and one out of six men will be diagnosed in their lifetime (1). With increasing awareness and regular cancer screening, the majority of newly diagnosed prostatic cancers are localized and found at an early stage. In spite of an earlier diagnosis, treatment of these cancers is associated with substantial morbidity. More commonly, radical prostatectomy, brachytherapy, or external beam radiotherapy are used to treat the early localized prostate cancer, but each modality has its morbidity. During early phases of the disease, the malignant prostatic lesions tend to remain focal and restricted to the prostate gland itself. This, combined with the anatomic accessibility of the prostate gland, makes direct intratumoral injection of carcinotoxic and carcinostatic agents a real possibility for effective and relatively noninvasive treatment (2). Direct transurethral injection of dehydrated ethanol into the prostate under endoscopic guidance and transperineal injection under transrectal ultrasound guidance have been used to treat benign prostatic hyperplasia and prostatic adenocarcinoma (3) in limited cases, resulting in improved micturition without major complications. Historically many chemicals - ethanol, carbolic acid, acetic acid and glycerin have been used alone or in combination to successfully chemo-ablate benign prostatic hyperplasia by direct prostatic injections (4, 5, 6, 7). Ethanol injection therapy for locally invasive prostatic cancer has been reported in a few cases (8) as well as in a murine xenograft model, which showed regressed prostatic cancer tumors (9). Currently, acetic acid is used to ablate hepatic carcinomas and other malignancies (10, 11). This led to our hypothesis that intratumoral administration of acetic acid could be an effective treatment for prostate cancer, and could be safely used to inject the prostate in humans as it has been used to treat benign hyperplasia. In this study, based in part on promising in vitro results from our laboratory, we explored the effectiveness of direct intratumoral injection of acetic acid in malignant prostatic tumors. To our knowledge, this is the first examination of intratumoral acetic acid as a treatment strategy for prostate cancer, and we feel that these data form powerful preliminary evidence indicating that such a minimally invasive strategy could be efficacious. Material and Methods: Cell lines: PC3 and LNCaP human prostate cancer cells were obtained from ATCC (Rockville, Maryland, USA). Cells were maintained at 37°C, 5% CO2 and 95% humidity in DMEM medium (CellGro,Herndon, Virginia, USA) supplemented with 10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville, Maryland, USA. Mice: Hairless male scid mice at 8 weeks of age were purchased from Charles River Laboratories (Wilmington, Massachusetts, USA). Animals were allowed to acclimate for 2 weeks prior to experimentation. The animals were cared and managed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Xenograft: There were 22 mice in each of PC3 and LNCaP groups. In each cell line group 8 were control mice while 14 underwent treatment with acetic acid. PC3 and LNCaP cells were grown to 70% confluence, harvested and injected in the dorsum of animals subcutaneously. Each inoculum consisted of 100 μL of cell suspension with 107 cells suspended in matrigel. Tumors were measured biweekly. Tumor volume was determined using the following formula: Volume = Length × Width2. Direct intratumoral injection was done in 14 mice for each cell line group. Tumors were injected with 25% acetic acid, 0.05ml/100cm3 of tumor after it attained a size > 300mm3. Post treatment one mouse in each group was euthanized after 2 hours, 24 hours, 1 week and 2 weeks; the remaining mice (n=10) were allowed to survive for 120 days. As a final measure of the potential usefulness of the treatment as a component of prostate cancer management, we assayed the ability of the intratumoral acetic acid injection protocol to extend the life of animals in our prostate cancer xenograft model. Because they are growing subcutaneously rather than orthotopically, xenograft tumors may grow to significant size without causing animal death. For humane reasons, a scoring system was established to assess animal welfare and animals not able to meet any requirement were euthanized. The scoring system consisted of the following: 1. Maintenance of normal weight (Weight loss >15%) 2. Normal ambulation 3. Normal grooming 4. Normal feeding Importantly, the decision to remove an animal from the protocol due to extreme tumor burden was made by an animal care technician unaware of the treatment group of the particular animal at the time of the decision. Thus, humane removal of an animal from the protocol was recorded as a death event, and with these data we evaluated survival. After euthanizing, specimens from all animals were sent for histopathology and the hematoxylin & eosin-stained slides were reviewed by a board certified pathologist. Results: Control mice: There were eight control mice each in the PC3 and LNCaP groups. The control mice were monitored for tumor growth rate and overall health and were euthanized according to the humane criteria described earlier, which was recorded as a death event. The PC3 tumors grew faster and more solid tumors, while the LNCaP tumors showed a slower rate of tumor growth and grew spherical soft tumors (Picture -1). The mean survival was 40±9 days and 56±10 in the PC3 and LNCaP group control mice, respectively. Survival in the PC3 group was reduced because of the faster tumor growth leading to greater tumor volume and its consequent effects on the overall health of the mouse. No changes were noted on the skin over the tumor during the course of observation in any of the mice. Treatment group: There were 14 mice each in the treatment arms of PC3 and LNCaP groups. Post treatment one mouse/group was euthanized after 2 hrs, 24 hrs, 1 week and 2 weeks. The remaining mice (n=10) were sacrificed at 120 days. Post injection, gross changes in tumor of greenish –grey color in the tumor and overlying skin were noted by 2 hours (Picture -3), and became prominent by 6 hours. Ulceration and crusting of overlying skin were seen by 24 hours. The scabs contracted and healed into scars by 23±5 days (Picture 2). Gross tumor eradication was noticed in 17 (n=9 in PC3 and n=8 in LNCaP) (Picture 4) mice at 120 days. Gross tumor recurrence was seen in 1 of PC3 and 2 of LNCaP mice. The recurrences were retreated successfully with a second intratumoral injection. Microscopic tumor recurrence was noted in scar of 1 mouse of PC3 group on histopathology at 120 days. Histopathological examination of the tumors from the mice euthanized after 2 hours post injection showed tumor degeneration and ischemic necrosis with intravascular aggregation of red blood cells inside the tumor (Picture 5 and 6). After 24 hours, areas of necrotic tissue with coagulative necrosis were noticed, and the extent of cellular damage and tissue necrosis became more extensive. After 1 and 2 weeks, severe degeneration with infiltration of fibroblasts and granulation tissue was noted. Histopathology of the scars showed no evidence of tumor cells, except in 1 specimen of PC3 group (Picture 7). Discussion: Prostate cancer represents a unique clinical problem with respect to treatment options. Ninety percent of men will present with localized disease (12). For localized prostate cancer, the current treatment paradigm is prostatectomy or radiotherapy (external beam or bachytherapy). For men with advanced disease, androgen therapy offers the best opportunity for long term survival. Given the increased age at which many men present with prostate cancer and the slow growing nature of this cancer, in many cases, the treatment options may have equivalent morbidity in comparison to the cancer itself. Hence, less invasive methods of treatment with fewer side effects would be very advantageous for men presenting with localized disease. However, chemoablation of the prostate cancer is not yet established (3). There is much evidence in the literature to suggest that intratumoral treatment with acetic acid for hepatocellular carcinoma has good results while being systemically safe (10, 11). Also intra prostatic injection of acetic acid has been used in humans to relieve the urinary obstruction secondary to benign prostatic hyperplasia (4). In this study significant tumor eradication was achieved with 25% acetic acid injection into the PC3 and LNCaP tumors implanted in scid mice. Fifty percent acetic acid has been used in humans safely. An initial pilot study was done to find the acetic acid concentration which would achieve prostate tumor eradication without causing any significant damage to peri tumoral tissues. Different acetic concentrations of 5%, 10%, 25% and 50% were tried in 5 mice each. A direct relation of peri tumoral tissue necrosis was observed with increasing acetic acid concentration, with 5% causing the least and 50% causing maximal destruction of surrounding tissues. A direct relationship of acetic acid concentration on tumor recurrences was also noted, 5% group having more tumor recurrences while no tumor recurrence was see in the group treated with 25% and 50%. Based on the results of the pilot study 25% was selected as it caused tumor eradication with minimal peri tumoral tissue damage. Acetic acid should be correctly and carefully injected into the target site, since it can degenerate and necrotize both the prostatic and adjunctive tissues. In this study, complications such as skin ulceration and crust formation were observed when acetic acid was injected at too large a volume ratio compared to the tumor volume, or when the injection injured many capillaries to the skin since this tumor was located subcutaneously. Pulling out the needle immediately after the end of the injection might also have caused leakage to the surrounding tissue due to backflow. In the clinical practice of local ethanol injection in patients with benign prostate hypertrophy, the needle is removed after leaving it in the injection site for one minute after injection. The reason is that if the needle is removed before the ethanol permeates, there is a risk that the ethanol may flow back along the path of the needle insertion (9). Review of histopathology of the implanted tumors post injection showed ischemic necrosis of the tumor cells starting immediately post injection. It translated to considerable survival advantage in both prostate cancer cell line groups. Goya et al. reported tumor regression with dehydrated ethanol injection (9) but multiple intratumoral injections with high-dose ethanol were needed for tumor regression. Repeated intratumoral injections may not be a desirable treatment modality for human prostate cancer patients to achieve only tumor regression. The present study showed that a single injection of acetic acid into tumors of human prostate carcinoma cells in scid mice caused tumor eradication as well as histological tumor cell injury and necrosis, suggesting that acetic acid injection may be effective against human prostate cancer. This treatment option could potentially be used in humans for treatment of early localized prostate cancer and for non-operative management of symptoms in locally advanced cases. This is the first report of a successful local chemo ablation for prostate cancer. References: 1. Shah MR, Kriedt CL, Lents NH, Hoyer MK, Jamaluddin N, Klein C, Baldassare J. Direct intra-tumoral injection of zinc-acetate halts tumor growth in a xenograft model of prostate cancer. J Exp Clin Cancer Res. 2009 Jun 17;28:84. 2. Springate CM, Jackson JK, Gleave ME, Burt HM.Efficacy of an intratumoral controlled release formulation of clusterin antisense oligonucleotide complexed with chitosan containing paclitaxel or docetaxel in prostate cancer xenograft models.Cancer Chemother Pharmacol. 2005 Sep;56(3):239-47. 3. Kim ED. Ethanol injection for the treatment of benign prostatic hyperplasia. Curr Urol Rep. 2002 Aug; 3(4):276-9. 4. Talwar GL, Pande SK. Injection treatment of enlarged prostate. Br J Surg. 1966 May;53(5):421-7. 5. Grise P, Plante M, Palmer J, Martinez-Sagarra J, Hernandez C, Schettini M, Gonzalez-Martin M, Castiñeiras J, Ballanger P, Teillac P, Rolo F, Baena V, Erdmann J, Mirone V. Evaluation of the transurethral ethanol ablation of the prostate (TEAP) for symptomatic benign prostatic hyperplasia (BPH): a European multi-center evaluation. Eur Urol. 2004 Oct;46(4):496-501; discussion 501-2. 6. Plante MK, Marks LS, Anderson R, Amling C, Rukstalis D, Badlani G, Getlin L, Vang E. Phase I/II examination of transurethral ethanol ablation of the prostate for the treatment of symptomatic benign prostatic hyperplasia. J Urol. 2007 Mar; 177(3):1030-5; discussion 1035. 7. Goya N, Ishikawa N, Ito F, Ryoji O, Tokumoto T, Toma H, Yamaguchi Y. Ethanol injection therapy of the prostate for benign prostatic hyperplasia: preliminary report on application of a new technique. J Urol. 1999 Aug; 162(2):383-6. 8. Amano H, Goya N, Ryoji O, Yagisawa T, Nakazawa H, Toma H.Ethanol injection therapy for locally invasive prostatic adenocarcinoma. Urology. 2002 May; 59(5):771-2. 9. Goya N, Koga S, Tomizawa Y, Onitsuka S, Yamaguchi Y, Toma H. Effects of direct injection of dehydrated ethanol on PC3 human prostate cancer cells in nude mice: preliminary study. Int J Urol. 2007 Aug; 4(8):760-3. 10. Khan NA, Baerlocher MO, Owen RJ, Ho S, Kachura JR, Kee ST, Liu DM. Ablative technologies in the management of patients with primary and secondary liver cancer: an overview. Can Assoc Radiol J. 2010 Oct; 61(4):217-22. 11. Padma S, Martinie JB, Iannitti DA. Liver tumor ablation: percutaneous and open approaches. J Surg Oncol. 2009 Dec 15; 100(8):619-34. 12. Dahm P, Yeung LL, Chang SS, Cookson MS.A critical review of clinical practice guidelines for the management of clinically localized prostate cancer. J Urol. 2008 Aug; 180(2):451-9; discussion 460. Picture 1: A - LNCaP control group 8 weeks B - PC3 control group at 5 weeks Picture 2: A & B- PC3 treatment group 6 days post injection C & D - PC3 treatment group 2 weeks post injection. E & F - PC3 treated group 3-4 weeks post injection Picture 3: A- PC3 tumor at 5 weeks. B - Tumor 2hours, post injection of acetic acid. C - Healed scar after 5 weeks. Picture 4: PC3 treatment group at 120 days, with scars. Picture 5 Picture 6 Picture 7 A & B: Post treatment scar – no recurrence C: PC3 post treatment scar (H&E) –tumor recurrence surrounded by fibrosis.