ANTICANCER RESEARCH 29: 4051-4056 (2009)

In Vivo Model of Pseudomyxoma Peritonei for Novel Candidate Drug Discovery

TERENCE C. CHUA, JAVED AKTHER, PENG YAO and DAVID L. MORRIS

Department of Surgery, University of New South Wales, St. George Hospital, Kogarah, Sydney, NSW 2217, Australia

Abstract. Pseudomyxoma peritonei (PMP) is a clinical mucinous implants accumulating in the pelvis and condition characterized by diffuse intraabdominal spread of subphrenic regions are often daunting to general surgeons mucinous tumor implants that form on peritoneal surfaces. To (2). Traditional treatments include repeated abdominal complement the efficacy of cytoreductive surgery, an effective lavage of mucin, limited debulking of tumor and systemic cytotoxic drug is required as an intraperitoneal agent for chemotherapy (3). However, these treatments are unable to microscopic cytoreduction locoregionally. To develop a model adequately combat the reaccumulation of mucin that for drug discovery, human pseudomyxoma peritonei tumor characterizes the failure of these treatments. implants consisting of a mixture of mucin and epithelial cells Over the last two decades, cytoreductive surgery and were injected into the peritoneal cavity of CBH/rnu/rnu rats. perioperative intraperitoneal chemotherapy comprising of A fixed quantity of tumor (2 ml) was implanted in each of the hyperthermic intraperitoneal chemotherapy (HIPEC) using 9 male rats. Over 90 days, all rats developed PMP indicating mitomycin C with or without postoperative intraperitoneal that this in vivo animal model may become useful platform in chemotherapy (EPIC) using 5-fluorouracil has emerged to a drug discovery program to allow drug efficacy testing become the standard of care in specialized centers (4, 5). A against human PMP. systematic review reporting the results of this treatment in 10 specialized institutions of 863 patients showed a 5-year Pseudomyxoma peritonei (PMP) is a clinical syndrome that survival ranging between 52 to 96% from the time of is characterised by diffuse spread of intra-abdominal tumor cytoreductive surgery (6). Ten-year overall survival rates implants that follow from the perforation of a mucinous have also been reported to be about 85% (7, 8). The survival appendiceal or ovarian neoplasm (1). These mucinous outcomes of this combined modality treatment is based implants form on the parietal and serosal peritoneum and largely on the concept of locoregional therapy where ranges in its viscosity from purely liquid to a semi-solid like extensive surgery to remove the tumor implants together with texture. The epithelial cells within the mucin proliferate and the administration of heated chemotherapy serves to target results in continuous production of mucin. Despite the microscopic residual disease. extensive intraperitoneal spread of this tumor, it rarely Despite the success of this treatment on prolonging invades into organs and almost never metastasizes through survival, treatment failure is common and is often associated the blood stream or lymphatics. This mucinous material with the grade of the tumor (9). To improve the recurrence- remains intra-abdominally and accumulates to eventually free survival, it is of utmost importance that effective cause severe abdominal distension and anorexia from cytotoxic therapy be developed to complement the surgical compression of the small bowel, which are the main cause procedure. This will reduce the need for repeated treatment of morbidity and mortality in untreated patients. The and the risk of morbidity from subsequent cytoreductive intraoperative findings of extensive carcinomatosis with surgeries. Here, we report the outcomes of our efforts at developing an in vivo PMP model from our laboratory’s drug discovery program.

Correspondence to: Professor David L. Morris, Department of Materials and Methods Surgery, University of New South Wales, St. George Hospital, Kogarah, Sydney, NSW 2217, Australia. Tel: +61 02 91132070, Animals. Male CBH/rnu/rnu rats (n=9) weighing between 200-250 Fax: +61 02 91133997, e-mail: [email protected] g were purchased from the Biological Resources Centre of the University of New South Wales and were housed in the PC2 facility Key Words: Pseudomyxoma peritonei, experimental model, at the St. George Hospital. The purchase of the rats and the peritoneal carcinomatosis, intraperitoneal chemotherapy. experimental conduct of this study was approved by the University

0250-7005/2009 $2.00+.40 4051 ANTICANCER RESEARCH 29: 4051-4056 (2009) of New South Wales Animal Ethics Committee (approval no. 06- Histopathological findings. Figure 2 is a representative 114B). The animals were kept under specific pathogen-free histological display of the mucinous tumor obtained from the conditions with free access to autoclaved food and water. rat that is characterized by pools of mucin with strips of cellular tissue of columnar mucinous epithelium with goblet Tumor collection. Three patients with histologically proven PMP consented to retrieval of tumor specimen at the time of operation. cells on the surface of the bowel mucosa. Accompanying Mucinous tumors were collected after a laparotomy but before serosal fibrinous material and minimal acute inflammatory cytoreductive surgery in sterile containers and immediately response to the pool of mucin is also evident in the brought to the animal facility. The tumor was processed under hematoxylin and eosin staining at ×10 magnifications. sterile conditions to remove cellular debris such that the remaining tumor extract contained pure mucin with the Discussion accompanying epithelial cells.

Tumor implantation. Nude rats were given buprenorphine (0.01 We demonstrate the feasibility of establishing an in vivo mg/kg) 30 min prior to surgery for pain management. model of pseudomyxoma peritonei in immune-compromised Anaesthesia for surgery was performed using isoflurane. Mini- nude rats which may serve as a platform for drug discovery laparotomy was performed using a lower middle incision of 3 whereby intraperitoneal efficacy of various compounds may cm. Intraperitoneal tumor inoculation was performed by injecting be subjected to in vivo experimenting after proving its effects the patient’s tumor specimen under the right lower quadrant of in vitro. To our knowledge, this model represents the first of the abdomen (1 mL) and the left lower quadrant of the abdomen an animal model for DPAM type PMP, which from our (1 mL). Each patient’s tumor was implanted in three rats. After the procedure the wound was closed by sutures. Animals were institution’s experience accounts for almost 70% of all PMP monitored daily for a maximum of 90 days for the development histological subtypes (10). of intraperitoneal tumor. A previous PMP model was reported by Flatmark et al. (11) using a similar method as reported in this study where Post-mortem. At the end of 90 days, all 9 rats underwent a human tumor tissue were implanted in the peritoneal cavity laparotomy with an extended incision from the xiphisternum to the of nude mice to establish the orthotopic models. In their pelvis. Intraabdominal inspection was performed. Tumors in rats study however, peritoneal mucinous carcinomatosis were obtained and sent for routine histology analysis. The specimens were fixed in 10% buffered formalin, embedded in intermediate (PMCA-I) subtype tumor were used. This paraffin, cut with a microtome to 4 μm and stained with histological subtype is one of three as described by Ronnett hematoxylin and eosin. et al., comprising DPAM, PMCA-I and PMCA. DPAM arises from a low-grade appendiceal mucinous tumor and Results is characterized by widespread peritoneal deposits of abundant pools of extracellular mucin with strips of Patients. Tumor specimens were taken from three patients: proliferative mucinous columnar epithelium with little a 66-year-old female, 72-year-old male, and a 56-year-old cytologic atypia or mitotic activity. This subtype has been male with biopsy-proven pseudomyxoma peritonei arising shown to be the most indolent type of PMP and following from an appendiceal mucinous neoplasm during cytoreductive surgery and perioperative intraperitoneal cytoreductive surgery combined with perioperative chemotherapy, long-term survival with 5-year survival rates intraperitoneal chemotherapy. All patients had extensive between 75% to 90% may be reached (10, 7, 8). PMCA intraperitoneal mucinous tumor with a score of 22, 24 and consists of abundant mucinous epithelium, forming glands 38 on the peritoneal cancer index respectively. All patients and/or signet ring cells and commonly have severe underwent a complete cytoreduction (CC0/1). Final cytological atypia and are poorly differentiated histopathological examination of the tumor from all three architecturally. Histological variants with intermediate patients revealed disseminated peritoneal adeno-mucinosis features of both DPAM and PMCA are termed PMCA- (DPAM). intermediate, which are essentially DPAM but have additional cellular foci containing well differentiated Gross pathological findings. All 9 rats developed visible mucinous carcinoma. Clinical outcome studies following evidence of peritoneal mucinous implants. Tumor implants treatment of this less common group have been shown to were widespread and the distribution in the peritoneal cavity be poorer as compared to patients with DPAM (8, 13-16). included the parietal and serosal peritoneum, small bowel Hence, the use of a DPAM model for biological and mesentry, omentum, paracolic gutters, liver, kidney and spleen. therapeutic studies in PMP would appear to be more useful A representative image is shown in Figure 1. Compression of as it has been shown to be more common. Targets for the small bowel by the mucinous tumor was identified in 6 of therapeutic studies may be achieved through identifying 9 rats and appeared to be causing a partial bowel obstruction. cytotoxic drugs that may target the atypical cells and also This had caused diarrhea and weight loss in these rats. dissolve the mucinous component within the tumor.

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Figure 1. Representative depiction of the development of pseudomyxoma peritonei in the immune-compromised rats.

Figure 2. Section of mucinous PMP located on the bowel. ×10 H&E stain.

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Importantly, there is a need to recognise that conventional features, site of origin, prognosis, and relationship to DNA-damaging cytotoxics, though effective at targeting rapidly “pseudomyxoma peritonei”. Am J Surg Pathol 19(12): 1390- dividing cancer cells, have limited efficacy in PMP because the 1408, 1995. majority of PMP are DPAM containing largely mucin pools and 2 Sugarbaker PH, Ronnett BM, Archer A, Averbach AM, Bland R, Chang D et al: Pseudomyxoma peritonei syndrome. Adv are acellular. Hence, basic research have focused on the mucin Surg 30: 233-280, 1996. component of the tumor, with an emphasis on determining the 3 Mann WJ, Wagner J, Chumas J and Chalas E: The etiology of mucin gene expression (17) and where enteric management of pseudomyxoma peritonei. Cancer 66(7): 1636- bacteria were detected in all PMP tumor specimens, with 1640, 1990. correlation between the bacterial density and MUC2 expression, 4 Moran B, Baratti D, Yan TD, Kusamura S and Deraco M: hence suggesting the bacteria present in PMP as contributing to Consensus statement on the locoregional treatment of the role of mucinous ascites and perhaps promoting appendiceal mucinous neoplasms with peritoneal dissemination (pseudomyxoma peritonei). J Surg Oncol 98(4): carcinogenesis. A study by Bibi et al. (18) of factors affecting 277-282, 2008. tumor invasion and metastasis, in particular cell adhesion- 5 Sugarbaker PH: New standard of care for appendiceal related molecules (N- and E-cadherin, vimentin), showed that epithelial neoplasms and pseudomyxoma peritonei syndrome? PMP demonstrated a higher and specific pattern of adhesion- Lancet Oncol 7(1): 69-76, 2006. related protein expression of increased N- cadherin, reduced E- 6 Yan TD, Black D, Savady R and Sugarbaker PH: A systematic cadherin, and increased vimentin in PMP tumor compared to review on the efficacy of cytoreductive surgery and normal colonic mucosa. This suggests a possible epithelial- perioperative intraperitoneal chemotherapy for pseudomyxoma peritonei. Ann Surg Oncol 14(2): 484-492, 2007. mesenchymal transition state that may characterize the distinct 7 Baratti D, Kusamura S, Nonaka D, Cabras AD, Laterza B and non-metastasizing behaviour of PMP. In translation research, Deraco M: Pseudomyxoma peritonei – biological features are expression of mucin gene markers MUC2 and MUC5AC, which the dominant prognostic determinants after complete were analyzed by in situ hybridization, immunocytochemistry, cytoreduction and hyperthermic intraperitoneal chemotherapy. and digital image analysis showed a distinct overexpression of Ann Surg 249: 243-249, 2009. both MUC2 and MUC5AC in all cases of PMP which may 8 Yan TD, Bijelic L and Sugarbaker PH: Critical analysis of potentially serve as a reliable molecular marker for treatment failure after complete cytoreductive surgery and pseudomyxoma peritonei (19). Analysis of such molecular perioperative intraperitoneal chemotherapy for peritoneal dissemination from appendiceal mucinous neoplasms. Ann markers including CK20, CDX-2, and MUC-2 have been Surg Oncol 14(8): 2289-2299, 2007. shown to correlate with prognosis in prospective series of 102 9 Smeenk RM, Verwaal VJ, Antonini N and Zoetmulder FA: patients who underwent complete cytoreduction and HIPEC Progression of pseudomyxoma peritonei after combined from the National Cancer Institute, Milan, Italy (12). modality treatment: management and outcome. Ann Surg Progress has been made in basic and translational research Oncol 14(2): 493-499, 2007. for PMP. To advance this field, drug discovery with a focus on 10 Chua TC, Yan TD, Smigielski ME, Zhu KJ, Ng KM, Zhao J targeting the mucinous component of the tumor through et al: Long-term survival in patients with pseudomyxoma peritonei treated with cytoreductive surgery and perioperative dissolution of mucin may improve resectability and reduce the intraperitoneal chemotherapy: 10 years of experience from a morbidity from treatment of patients with PMP. This may also single institution. Ann Surg Oncol 16(7): 1903-1911, 2009. alleviate the symptoms related to the tumor burden of peritoneal 11 Flatmark K, Reed W, Halvorsen T, Sorensen O, Wiig JN, carcinomatosis imposed by specific solid components in mucin. Larsen SG et al: Pseudomyxoma peritonei – two novel The results of our study appear encouraging and may allow the orthotopic mouse models portray the PMCA-I histopathologic replication of the model development in larger animals. The subtype. BMC Cancer 7: 116, 2007. tumors resulting from intraperitoneal implantation of PMP 12 Baratti D, Kusamura S, Nonaka D, Cabras AD, Laterza B and Deraco M: Pseudomyxoma peritonei: biological features are behaved similarly to those observed clinically in humans and the dominant prognostic determinants after complete have a similar histological appearance. The intraabdominal cytoreduction and hyperthermic intraperitoneal chemotherapy. distribution of the tumor paralleled the pattern of spread that has Ann Surg 249(2): 243-249, 2009. been characteristically described in the clinical syndrome of 13 Baratti D, Kusamura S, Nonaka D, Langer M, Andreola S, PMP. This in vivo model will facilitate drug discovery for cure Favaro M et al: Pseudomyxoma peritonei: clinical pathological of the mucinous tumor of PMP. and biological prognostic factors in patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC). Ann Surg Oncol 15(2): 526-534, References 2008. 14 Elias D, Honore C, Ciuchendea R, Billard V, Raynard B, Dico 1 Ronnett BM, Zahn CM, Kurman RJ, Kass ME, Sugarbaker PH RL et al: Peritoneal pseudomyxoma: results of a systematic and Shmookler BM: Disseminated peritoneal adenomucinosis policy of complete cytoreductive surgery and hyperthermic and peritonealmucinous carcinomatosis. A clinicopathologic intraperitoneal chemotherapy. Br J Surg 95(9): 1164-1171, analysis of 109 cases with emphasis on distinguishing pathologic 2008.

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15 Smeenk RM, Verwaal VJ, Antonini N and Zoetmulder FAN: 18 Bibi R, Pranesh N, Saunders MP, Wilson MS, O’Dwyer ST, Survival analysis of pseudomyxoma peritonei patients treated Stern PL et al: A specific cadherin phenotype may characterise by cytoreductive surgery and hyperthermic intraperitoneal the disseminating yet non-metastatic behaviour of chemotherapy. Ann Surg 245(1): 104-109, 2007. pseudomyxoma peritonei. Br J Cancer 95(9): 1258-1264, 16 Sugarbaker PH and Chang D: Results of treatment of 385 2006. patients with peritoneal surface spread of appendiceal 19 O’Connell JT, Hacker CM and Barsky SH: MUC2 is a malignancy. Ann Surg Oncol 6(8): 727-731, 1999. molecular marker for pseudomyxoma peritonei. Mod Pathol 17 Semino-Mora C, Liu H, McAvoy T, Nieroda C, Studeman K, 15(9): 958-972, 2002. Sardi A et al: Pseudomyxoma peritonei: is disease progression related to microbial agents? A study of bacteria, MUC2 AND MUC5AC expression in disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis. Ann Received June 30, 2009 Surg Oncol 15(5): 1414-1423, 2008. Accepted July 24, 2009

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