Establishment and characterization of NCC-PMP2-C1: a novel patient-derived cell line of pseudomyxoma peritonei with signet ring cells.

Pseudomyxoma peritonei (PMP) is a rare phenomenon, characterized by accumulation of mucus in the abdominal cavity due to a mucinous neoplasm. Histologically, PMP is divided into three prognostic classes, namely low-grade mucinous carcinoma peritonei (LGMCP), high-grade mucinous carcinoma peritonei (HGMCP), and high-grade mucinous carcinoma peritonei with signet ring cells (HGMCP-S); HGMCP-S exhibits the worst prognosis. Complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy have been established as the standard therapy for PMP. However, 50% of patients with PMP experience a recurrence, and 30-40% are unable to receive the standard treatment due to invasive diseases. Therefore, novel therapies are required for their treatment. Although patient-derived cell lines are important tools for basic and pre-clinical research, PMP cell lines derived from patients with HGMCP-S have never been reported. Thus, we established a novel PMP cell line NCC-PMP2-C1, using surgically resected tumor tissue from a patient with HGMCP-S. NCC-PMP2-C1 cells were maintained for more than five months and passaged 30 times under culture conditions. NCC-PMP2-C1 cells exhibited multiple deletions and somatic mutations, slow growth, histological features, and dissemination of tumor cells in nude mice. Screening for the anti-proliferative effects of anti-cancer drugs on cells revealed that bortezomib, mubritinib, and romidepsin had a significant response against NCC-PMP2-C1 cells. Thus, the NCC-PMP2-C1 cell line is the first PMP cell line harboring signet ring cells and will be a valuable resource for basic and preclinical studies of HGMCP-S.

Targeted Next-Generation Sequencing Improves the Prognostication of Patients with Disseminated Appendiceal Mucinous Neoplasms (Pseudomyxoma Peritonei).

BACKGROUND: Appendiceal mucinous neoplasms (AMNs) with disseminated disease (pseudomyxoma peritonei) are heterogeneous tumors with variable clinicopathologic behavior. Despite the development of prognostic systems, objective biomarkers are needed to stratify patients. With the advent of next-generation sequencing (NGS), it remains unclear if molecular testing can improve the evaluation of disseminated AMN patients. METHODS: Targeted NGS was performed for 183 patients and correlated with clinicopathologic features to include American Joint Committee on Cancer/World Health Organization (AJCC/WHO) histologic grade, peritoneal cancer index (PCI), completeness of cytoreduction (CC) score, and overall survival (OS). RESULTS: Genomic alterations were identified for 179 (98%) disseminated AMNs. Excluding mitogen-activated protein kinase genes and GNAS due to their ubiquitous nature, collective genomic alterations in TP53, SMAD4, CDKN2A, and the mTOR genes were associated with older mean age, higher AJCC/WHO histologic grade, lymphovascular invasion, perineural invasion, regional lymph node metastasis, and lower mean PCI (p < 0.040). Patients harboring TP53, SMAD4, ATM, CDKN2A, and/or mTOR gene alterations were found to have lower OS rates of 55% at 5 years and 14% at 10 years, compared with 88% at 5 years and 88% at 10 years for patients without the aforementioned alterations (p < 0.001). Based on univariate and multivariate analyses, genomic alterations in TP53, SMAD4, ATM, CDKN2A, and/or the mTOR genes in disseminated AMNs were a negative prognostic factor for OS and independent of AJCC/WHO histologic grade, PCI, CC score, and hyperthermic intraperitoneal chemotherapy treatment (p = 0.006). CONCLUSIONS: Targeted NGS improves the prognostic assessment of patients with disseminated AMNs and identifies patients who may require increased surveillance and/or aggressive management.

Tumor-stroma ratio as a new prognosticator for pseudomyxoma peritonei: a comprehensive clinicopathological and immunohistochemical study.

BACKGROUND: As a rare clinical tumor syndrome with an indolent clinical course and lack of pathognomonic symptoms, pseudomyxoma peritonei (PMP) is usually diagnosed at an advanced stage. In-depth pathological analysis is essential to assess tumor biological behaviors, assist treatment decision, and predict the clinical prognosis of PMP. The tumor-stroma ratio (TSR) is a promising prognostic parameter based on the tumor and stroma. This study explored the relationship between TSR and the pathological characteristics and prognosis of PMP. METHODS: PMP patients with complete data who underwent cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy were enrolled. The TSR of postoperative pathological images was quantitatively analyzed by Image-Pro Plus. Then the relationship between TSR and the clinicopathological characteristics, immunohistochemical characteristics and prognosis of PMP was analyzed. RESULTS: Among the 50 PMP patients included, there were 27 males (54.0%) and 23 females (46.0%), with a median age of 55 (range: 31-76) years. 25 (50.0%) patients were diagnosed with low-grade PMP (LG-PMP), and 25 (50.0%) were diagnosed with high-grade PMP (HG-PMP). There were 4 (8.0%) patients with vascular tumor emboli, 3 (6.0%) patients with nerve invasion, and 5 (10.0%) patients with lymph node metastasis. The immunohistochemical results showed that the Ki67 label index was < 25% in 18 cases (36.0%), 25 - 50% in 18 cases (36.0%) and > 50% in 14 cases (28.0%). The range of TSR was 2 - 24% (median: 8%). The cutoff value of TSR was 10% based on the receiver operating characteristic (ROC) curve and X-Tile analysis. There were 31 (62.0%) cases with TSR < 10% and 19 (38.0%) cases with TSR ≥ 10%. The TSR was closely related to histopathological type (P < 0.001) and Ki67 label index (P < 0.001). Univariate analysis showed that preoperative carcinoembryonic antigen (CEA), preoperative carbohydrate antigen 19-9, pathological type, vascular tumor emboli and TSR influenced the prognosis of PMP patients (P < 0.05). Multivariate analysis showed that preoperative CEA, vascular tumor emboli and the TSR were independent prognostic factors. CONCLUSIONS: The TSR could be a new independent prognosticator for PMP.

Altered linkage pattern of N-glycan sialic acids in pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a highly mucinous adenocarcinoma growing in the peritoneal cavity and most commonly originating from the appendix. Glycans play an important role in carcinogenesis, and glycosylation is altered in malignant diseases, including PMP. We have previously demonstrated that fucosylation of N-glycans is increased in PMP, but we did not observe modulation of overall sialylation. As sialic acids can be attached to the rest of the glycan via α2,3- or α2,6-linkage, we have now analyzed the linkage patterns of sialic acids in tissue specimens of normal appendices, low-grade appendiceal mucinous neoplasms (LAMN), low-grade (LG) PMP and high-grade (HG) PMP. For the linkage analysis, the enzymatically released acidic N-glycans were first treated with ethyl esterification or α2,3-sialidase digestion followed by MALDI-TOF mass spectrometry. Significant increase in the relative abundance of α2,6-sialylated and decrease in α2,3-sialylated N-glycans was observed in PMP tumors as compared to the normal appendices (P < 0.025). More specifically, increased α2,6-sialylation (P < 0.05) and decreased α2,3-sialylation (P < 0.01) were detected in afucosylated and monofucosylated N-glycans of PMPs, whereas the less abundant multifucosylated glycans, containing terminal fucose, demonstrated increased α2,3-sialylation (P < 0.01). Importantly, the increase in α2,6-sialylation was also detected between PMP and the appendiceal precursor lesion LAMN (P < 0.01). The identified glycosylation alterations produce ligands for sialic acid-binding immunoglobulin-like lectins (Siglecs) and sialofucosylated glycans binding selectins, which play a role in the peritoneal dissemination and progression of the disease.

The mutational landscape and prognostic indicators of pseudomyxoma peritonei originating from the ovary.

Pseudomyxoma peritonei (PMP) is a rare disorder with unique pathological and genetic changes. Although several studies have reported the clinical features and mutational changes of PMP that originates from the appendix, few studies on PMP originating from the ovary have been reported due to its extreme rarity. In order to characterize the somatic mutational landscape and to investigate the prognosis predicting factors of ovary-originating PMP, we examined 830 cases of PMP and identified 16 patients with PMP that originated from the ovary. Whole-exome sequencing (WES) was performed on 12 cases using formalin-fixed, paraffin-embedded (FFPE) tissue samples. We found that 25% (3/12) of the patients carried mutations in cancer driver genes, including TP53, ATM and SETD2, and 16.7% (2/12) of the patients carried mutations in cancer driver genes, including ATRX, EP300, FGFR2, KRAS, NOCR1 and RB1. The MUC16 (58.33%), BSN (41.67%), PCNT (41.67%), PPP2R5A (41.67%), PRSS36 (41.67%), PTPRK (41.67%) and SBF1 (41.67%) genes presented the highest mutational frequencies. The PI3K-Akt signaling pathway, human papillomavirus infection pathway, cell skeleton, cell adhesion, and extracellular matrix and membrane proteins were the major pathways or functions that were affected. Patients were followed up to 174 months (median: 48.26 months). The 5-year OS rate for all patients was 71.2% and the median OS was not reached. PTPRK mutations, presurgical CA199 level, completeness of cytoreduction (CCR) and peritoneal cancer index (PCI) were identified as potential predictive factors for patient survival. In conclusion, the mutational landscape for ovary-originating PMP was revealed and exhibited unique features distinct from appendix-originating PMP. PTPRK, CA199, CCR and PCI may predict patient survival.

Pathological prognostic factors of pseudomyxoma peritonei: comprehensive clinicopathological analysis of 155 cases.

BACKGROUND: Pseudomyxoma peritonei (PMP) is an extremely rare malignancy, characterized by extensive peritoneal implantation and colloidal ascites. This study was to explore the pathological prognostic factors of PMP. METHODS: Specimens from 155 PMP patients were analyzed by H&E and immunohistochemistry. Parameters included primary tumor location, histological grade, lymph node metastasis, tumor emboli in the blood and lymph vessels, perineural invasion, Ki67 labeling index, p53, mismatch repair (MMR) gene mutations, MUC1, MUC2, MUC5AC, and MUC6. Clinicopathological and follow-up data were subjected to univariate and multivariate analyses. RESULTS: The patients included 63.2% (n = 98) low-grade mucinous carcinoma peritonei, 31.6% (n = 49) high-grade mucinous carcinoma peritonei and 5.2% (n = 8) high-grade mucinous carcinoma peritonei with signet ring cells. There were 9.7% (n = 15) with lymph node metastasis; 11.6% (n = 18) with angiolymphatic invasion; 6.3% (n = 8) with defective MMR (dMMR); 35.5% (n = 55) with Ki67 labeling index ≥ 50%; 36.1% (n = 56) with p53 mutation. For PMP from appendiceal origin (n = 140), univariate analysis identified 10 potential prognostic factors. But Multivariate analysis identified only histologic grade was the independent prognostic factor for OS. Mortality risk of high-grade peritoneal mucinous carcinoma or high-grade peritoneal mucinous carcinoma with signet ring cells was 7.056 times (P < .0001, 95% CI: 2.701-18.435) or 27.224 times (P < .0001, 95% CI: 6.207-119.408), respectively, higher than low-grade. CONCLUSIONS: For PMP from the appendiceal origin, histological grade could be the only independent prognostic factor.

Slippery Nanoparticles as a Diffusion Platform for Mucin Producing Gastrointestinal Tumors.

BACKGROUND: Treatment failure in pseudomyxoma peritonei (PMP) is partly attributed to the ineffective delivery of therapeutics through dense mucinous tumor barriers. We modified the surface of Poly (lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG-NPs) with a low-density, second PEG layer (PLGA-TPEG-NPs-20) to reduce their binding affinity to proteins and improve diffusion through mucin. METHODS: Nanoprecipitation was used to fabricate PLGA-PEG-NPs. To construct the second PEG layer of PLGA-TPEG-NPs-20, PEG-Thiol was conjugated to PLGA-PEG-NPs composed of 80% methoxy PLGA-PEG and 20% of PLGA-PEG-Maleimide. DiD-labeled nanoparticles (NPs) were added to the inner well of a trans-well system containing cultured LS174T or human PMP tissue. Diffusion of NPs was measured via fluorescence signal in the bottom well. In an ex vivo rat model, small intestine was treated with DiD-labeled NPs. In an in vivo murine LS174T subcutaneous tumor model, Nu/Nu nude mice received supratumoral injections (subcutaneous injection above the tumor) of DiD-labeled NPs. Thirty minutes after injection, mice were sacrificed, and tumors were collected. All tissue was cryosectioned, mounted with DAPI-containing media, and inspected via confocal microscopy. RESULTS: Diffusion profiles of NPs through PMP and cultured LS174T cells were generated. PLGA-TPEG-NPs-20 diffused faster with ~ 100% penetration versus PLGA-PEG-NPs with ~ 40% penetration after 8 h. Increased diffusion of PLGA-TPEG-NPs-20 was further observed in ex vivo rat small intestine as evidenced by elevated luminal NP fluorescence signal on the luminal surface. Subcutaneous LS174T tumors treated with PLGA-TPEG-NPs-20 demonstrated greater diffusion of NPs, showing homogenous fluorescence signal throughout the tumor. CONCLUSIONS: PLGA-TPEG-NPs-20 can be an effective mucin penetrating drug delivery system.

Appendiceal mucinous neoplasm mimics ovarian tumors: Challenges for preoperative and intraoperative diagnosis and clinical implication.

OBJECTIVE: The aim of this study was to investigate the clinicopathological characteristics of appendiceal mucinous neoplasm that had been preoperatively misdiagnosed as a mucinous ovarian tumor and to discuss the clinical impacts of misdiagnosis. METHODS: Seventy-eight patients with a final pathologic diagnosis of appendiceal mucinous neoplasm during a 10-year period were retrospectively reviewed. All patients were diagnosed with ovarian tumor before treatment. A univariate analysis was performed to evaluate predictors of the diagnostic accuracy of a frozen section. RESULTS: The patients' median age was 61 years (range, 21-82 years), and most were diagnosed as low-grade appendiceal mucinous neoplasm (LAMN) (84.62%). The diagnostic concordance between the frozen section and the final pathology was 56.92%. The most consistent diagnosis was LAMN (64.14%). Univariate analysis indicated that maximal diameter of the ovarian tumor (unilateral), laterality of the ovarian tumors (unilateral or bilateral), and frozen section site (appendix or extra-appendix) significantly correlated with the accuracy of frozen section diagnosis (all p < 0.05). Although the diagnostic discordance between the frozen section and the final pathology was 43.08%, only one patient was clinically impacted because of suboptimal surgery. CONCLUSION: Appendiceal mucinous neoplasm should be considered as a differential diagnosis of pelvic mass in women. For patients who do not require fertility-sparing surgery, excision and frozen section of the bilateral ovaries and appendix regardless of the appearance of the appendix might improve the diagnosis. For older patients with peritoneal dissemination, appropriate cytoreductive surgery is recommended to reduce the clinical impact of misdiagnosis.

Glycomic Profiling Highlights Increased Fucosylation in Pseudomyxoma Peritonei.

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma that most often originates from the appendix, and grows in the peritoneal cavity filling it with mucinous ascites. KRAS and GNAS mutations are frequently found in PMP, but other common driver mutations are infrequent. As altered glycosylation can promote carcinogenesis, we compared N-linked glycan profiles of PMP tissues to those of normal appendix. Glycan profiles of eight normal appendix samples and eight low-grade and eight high-grade PMP specimens were analyzed by mass spectrometry. Our results show differences in glycan profiles between PMP and the controls, especially in those of neutral glycans, and the most prominent alteration was increased fucosylation. We further demonstrate up-regulated mRNA expression of four fucosylation-related enzymes, the core fucosylation performing fucosyltransferase 8 and three GDP-fucose biosynthetic enzymes in PMP tissues when compared with the controls. Up-regulated protein expression of the latter three enzymes was further observed in PMP cells by immunohistochemistry. We also demonstrate that restoration of fucosylation either by salvage pathway or by introduction of an expression of intact GDP-mannose 4,6-dehydratase enhance expression of MUC2, which is the predominant mucin molecule secreted by the PMP cells, in an intestinal-derived adenocarcinoma cell line with defective fucosylation because of deletion in the GDP-mannose 4,6-dehydratase gene. Thus, altered glycosylation especially in the form of fucosylation is linked to the characteristic mucin production of PMP. Glycomic data are available via ProteomeXchange with identifier PXD010086.

[Clinicopathologic characteristics of pseudomyxoma peritonei].

Objective: To analyze the relationship between clinicopathologic characteristics of pseudomyxoma peritonei (PMP) and its prognosis. Methods: Fifty-two cases of PMP collected from 2012 to 2017 at Beijing Shijitan Hospital, Capital Medical University were reviewed using the diagnostic criteria of WHO 2010. The histopathologic features, including original location, neural invasion and calcification were observed; and the relationship with prognosis was analyzed. Immunohistochemical staining for CK7, CK20 and CDX2 was performed on all cases. ER, PR, and p16 were additionally performed on those without clear origin. Results: Patients' mean age was 52.0 years, and included 29 males and 23 females. Thirty-two cases were derived from appendix; among them, 23 were low grade and 29 were high grade. Signet ring-cells, neural invasion and calcification were detected in 15, 12 and 9 cases respectively. Neural invasion was associated with adverse prognosis (P=0.025) and signet ring cell morphology (P<0.01). Prognosis was not related to gender, age, original location or grade. Conclusion: Neural invasion is not uncommon in PMP and predicts a bad prognosis.

Appendix-derived Pseudomyxoma Peritonei (PMP): Molecular Profiling Toward Treatment of a Rare Malignancy.

OBJECTIVES: Pseudomyxoma peritonei (PMP) is a rare malignancy originating from the appendix, characterized by disseminated mucinous tumor implants on peritoneal surfaces. We examined the role of multiplatform molecular profiling to study biomarker-guided treatment strategies for this rare malignancy. METHODS: A total of 54 patients with appendix-derived PMP were included in the study. Tests included one or more of the following: gene sequencing (Sanger or next generation sequencing), protein expression (immunohistochemistry), and gene amplification (C/fluorescent in situ hybridization). RESULTS: Targeted sequencing of 47 genes detected variants in KRAS (81%), GNAS (74%), SMAD4 (16%), and ATM (16%). Mutations were found at low frequencies (n=1 to 2) in APC, BRAF, PIK3CA, MLH1, and TP53. GNAS and KRAS co-occurrence was found in 87%. Protein overexpression was found in epidermal growth factor receptor (83%), cyclooxygenase-2 (73%), cMET (63%), cKIT (58%), and platelet-derived growth factor receptor alpha (58%). Immune checkpoint expression was found in 36% (programmed cell death protein 1) and 18% (programmed death-ligand 1). Surrogate markers of cell proliferation were found at low rates (TLE3 23%, TOP2A 22%), consistent with the slow-growing biology of PMP. Phosophatase and tensin homolog was intact (wild type [100%]) and positive (immunohistochemistry [80%]). Patients exhibited stable microsatellite status and mismatch repair proficiency (93%). Importantly, multidrug resistance protein expression was elevated (100% BCRP, 94% MRP1, 88% PGP). Markers for gemcitabine (RRM1), fluorouracil (TS), oxaliplatin (ERCC1), and irinotecan (TOPO1) chemosensitivities were detected at favorable rates: 93%, 87%, 77% and 65%, respectively. CONCLUSIONS: Molecular profiling by multiple platforms identified potential therapies for the nontargetable KRAS-mutated population. The role of cMET-targeted therapeutics and immune checkpoint inhibitors merits further investigation. Biomarker-guided selection of cytotoxic chemotherapies may facilitate efficacy to systemic treatment.

Bilateral blockade of MEK- and PI3K-mediated pathways downstream of mutant KRAS as a treatment approach for peritoneal mucinous malignancies.

Mucinous colorectal adenocarcinomas (MCAs) are clinically and morphologically distinct from nonmucinous colorectal cancers (CRCs), show a distinct spectrum of genetic alterations (higher KRAS mutations, lower p53, high MUC2), exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement) and are associated with poorer response to chemotherapy with limited treatment options. Here, we report the effectiveness of combinatorial targeting of two KRAS-mediated parallel pathways in reducing MUC2 production and mucinous tumor growth in vitro and in vivo. By knockdown of mutant KRAS we show that, mutant KRAS (a) is necessary for MUC2 production in vitro and (b) synergistically engages PI3K/AKT and MEK/ERK pathways to maintain MUC2 expression in MCA cells. These results define a novel and a previously undescribed role for oncogenic KRAS in mucinous cancers. MCA cells were sensitive to MEK inhibition suggesting cellular dependence ('addiction') of KRAS-mutant MCA cells on hyperactivation of the MEK-driven pathway. Interestingly, MCA cells, though initially sensitive, were later resistant to PI3K single agent inhibition. Our studies suggest that this resistance involves dynamic rewiring of signaling circuits mediated through relief of RTK inhibition and MEK-ERK rebound activation. This resistance however, could be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEK- and PI3K-targeted combination therapy for not only KRAS mutant MCA but also for other related mucinous neoplasms that overproduce MUC2 and have a high rate of KRAS mutations such as pseudomyxoma peritonei.

Multiple components of PKA and TGF-ß pathways are mutated in pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma mainly restricted to the peritoneal cavity and most commonly originating from the appendix. The genetic background of PMP is poorly understood and no targeted treatments are currently available for this fatal disease. While RAS signaling pathway is affected in most if not all PMP cases and over half of them also have a mutation in the GNAS gene, other genetic alterations and affected pathways are, to a large degree, poorly known. In this study, we sequenced whole coding genome of nine PMP tumors and paired normal tissues in order to identify additional, commonly mutated genes and signaling pathways affected in PMP. These exome sequencing results were validated with an ultra-deep amplicon sequencing method, leading to 14 validated variants. The validated results contain seven genes that contribute to the protein kinase A (PKA) pathway. PKA pathway, which also contains GNAS, is a major player of overproduction of mucin, which is the characteristic feature of PMP. In addition to PKA pathway, we identified mutations in six genes that belong to the transforming growth factor beta (TGF-ß) pathway, which is a key regulator of cell proliferation. Since either GNAS mutation or an alternative mutation in the PKA pathway was identified in 8/9 patients, inhibition of the PKA pathway might reduce mucin production in most of the PMP patients and potentially suppress disease progression.

Physical and chemical characteristics of mucin secreted by pseudomyxoma peritonei (PMP).

Background: Pseudomyxoma peritonei (PMP) is a rare disease with excess intraperitoneal mucin secretion. Treatment involves laparotomy, cytoreduction and chemotherapy that is very invasive with patients often acquiring numerous compromises. Hence a mucolytic comprising of bromelain and N-acetyl cystein has been developed to solubilise mucin in situ for removal by catherization. Owing to differences in mucin appearance and hardness, dissolution varies. Therefore the current study investigates the inter-mucin physical and chemical characteristics, in order to reformulate an effective mucolytic for all mucin. Method: PMP mucin, from the three categories (soft, semi hard and hard mucin) was solubilised and then various physical characteristics such as turbidity, density, kinematic viscosity were measured. The water content and the density of solid mucin were also determined. This was followed by the determination of sialic acid, glucose, lipid, Thiol (S-S and S-H) content of the samples. Lastly, the distribution of MUC2, MUC5B and MUC5AC was determined using western blot technique. Results: Both turbidity and kinematic viscosity and sialic acid content increased linearly as the hardness of mucin increased. However, density, hydration, protein, glucose, lipid and sulfhydryl and disulphide content decreased linearly as hardness of mucin increased. The distribution ratio of mucins (MUC2:MUC5B:MUC5AC) in soft mucin is 2.25:1.5:1.0, semi hard mucin is 1:1:1 and hard mucin is 3:2:1. Conclusion: The difference in texture and hardness of mucin may be due to cellular content, hydration, glucose, protein, lipids, thiol and MUC distribution. Soft mucin is solely made of glycoprotein whilst the others contained cellular materials.

Synchronous Ovarian and Appendiceal Mucinous Neoplasms in the Absence of Pseudomyxoma Peritonei.

BACKGROUND: Synchronous ovarian/appendiceal mucinous neoplasms sometimes occur in the absence of clinical pseudomyxoma peritonei (PMP), which raises a question about whether the 2 tumors could be independent. METHODS: We identified 11 cases of synchronous ovarian/appendiceal mucinous neoplasms without PMP and subclassified them into groups 1 and 2 based on the presence or absence of microscopic peritoneal/ovarian surface mucin deposits. A 7-marker panel (CK7, CK20, CDX2, PAX8, MUC1, MUC2, and MUC5AC) immunohistochemistry was performed on both tumors. RESULTS: Between the 2 groups, there were no significant differences in age, laterality, size, and histology of ovarian/appendiceal tumors. In group 1, 2 of 4 cases developed PMP later, and both had ovarian surface and contralateral ovarian involvement and appendiceal perforation with microscopic mucin deposits on the peritoneum. No patients in group 2 developed PMP. All group 1 cases showed a high degree of concordance of immunoprofile between the synchronous tumors, with an identical expression of appendiceal pattern in greater than 90% of the markers. In group 2, only 1 of 7 cases showed concordance in all markers. CONCLUSIONS: If peritoneal mucin deposits present, even microscopic and acellular, the synchronous tumors are most likely of a single appendiceal origin. Otherwise, they are more heterogeneous, and some may be truly dual primaries.

Mucinous Balls Tangled With Mesothelial Cells and MUC2-Positive Cancer Cells in the Ascites of Pseudomyxoma Peritonei.

Pseudomyxoma peritonei (PMP) is characterized by extensive mucinous ascites following rupture of mucinous neoplasms of an intra-abdominal origin, and contain secreted gel-forming mucins such as MUC2 and MUC5AC. We encountered a 66-year-old Japanese man complaining of abdominal distension. Ascites at the site was gelatinous upon gross examination, and needle aspirate smears showed histiocytes and many mucinous balls wrapped in spindle cells, which were positive for vimentin, pan-cytokeratin, and podoplanin. The cell block showed several adenocarcinoma clusters, which were positive for MUC2, MUC5AC, CK20, and CDX-2, and negative for CK7. From these findings, a diagnosis of PMP arising from colon cancer was indicated. Cytoreductive surgery was performed, and the cystic diverticulum was found to be infiltrated by tumor cells in the sigmoid colon that caused PMP. Mucinous balls surrounded by mesothelial cells and MUC2-positive adenocarcinoma cells are useful clues in the diagnosis of PMP. Diagn. Cytopathol. 2016;44:628-631. © 2016 Wiley Periodicals, Inc.

Expression of CEA, CA19-9, CA125, and EpCAM in pseudomyxoma peritonei.

Pseudomyxoma peritonei is a fatal clinical syndrome with mucinous tumor cells disseminated into peritoneal cavity and secreting abundant mucinous ascites. The serum tumor markers CEA, CA19-9, and CA125 are used to monitor pseudomyxoma peritonei remission, but their expression at tissue level has not been well characterized. Herein, we analyzed expression of these proteins and the adenocarcinoma marker EpCAM in 92 appendix-derived pseudomyxoma peritonei tumors by immunohistochemistry. All tumors were found to ubiquitously express CEA and EpCAM. In the majority of the tumors (94.6%), CEA showed polarized immunostaining, but in 5 aggressive high-grade tumors containing numerous signet ring cells, a nonpolarized staining was detected. We found preoperative CEA serum values to correlate with peritoneal cancer index. However, the serum values of the advanced cases with nonpolarized staining pattern were normal, and the patients died within 5 years after diagnosis. Thus, serum CEA measurements did not reflect aggressiveness of these tumors. CA19-9 showed strong immunopositivity in most of the tumors (91.3%), and mutated enzyme FUT3 was demonstrated from the cases showing negative or weak staining. CA125 was infrequently expressed by tumor cells (focal staining in 6.5% of the cases), but in most of the cases (79.3%), adjacent nonneoplastic mesothelial cells showed immunopositivity. As a conclusion, CEA and EpCAM are invariably expressed by pseudomyxoma peritonei tumor cells and could be exploited to targeted therapies against this malignancy.

Patient-derived xenograft mouse models of pseudomyxoma peritonei recapitulate the human inflammatory tumor microenvironment.

Pseudomyxoma peritonei (PMP) is a neoplastic syndrome characterized by peritoneal tumor implants with copious mucinous ascites. The standard of care for PMP patients is aggressive cytoreductive surgery performed in conjunction with heated intraperitoneal chemotherapy. Not all patients are candidates for these procedures and a majority of the patients will have recurrent disease. In addition to secreted mucin, inflammation and fibrosis are central to PMP pathogenesis but the molecular processes that regulate tumor-stromal interactions within the peritoneal tumor microenvironment remain largely unknown. This knowledge is critical not only to elucidate PMP pathobiology but also to identify novel targets for therapy. Here, we report the generation of patient-derived xenograft (PDX) mouse models for PMP and assess the ability of these models to replicate the inflammatory peritoneal microenvironment of human PMP patients. PDX mouse models of low- and high-grade PMP were generated and were of a similar histopathology as human PMP. Cytokines previously shown to be elevated in human PMP were also elevated in PDX ascites. Significant differences in IL-6 and IL-8/KC/MIP2 were seen between human and PDX ascites. Interestingly, these cytokines were mostly secreted by mouse-derived, tumor-associated stromal cells rather than by human-derived PMP tumor cells. Our data suggest that the PMP PDX mouse models are especially suited to the study of tumor-stromal interactions that regulate the peritoneal inflammatory environment in PMP as the tumor and stromal cells in these mouse models are of human and murine origins, respectively. These mouse models are therefore, likely to be useful in vivo surrogates for testing and developing novel therapeutic treatment interventions for PMP.

Targeting hypoxia-mediated mucin 2 production as a therapeutic strategy for mucinous tumors.

Excessive accumulation of mucin 2 (MUC2; a gel-forming secreted mucin) protein in the peritoneal cavity is the major cause of morbidity and mortality in pseudomyxoma peritonei (PMP). Hypoxia (hypoxia-inducible factor-1α; HIF-1α) has been shown to regulate the expression of similar mucins (eg, MUC5AC). We hypothesized that hypoxia (HIF-1α) drives MUC2 expression in PMP and is therefore a novel target to reduce mucinous tumor growth. The regulation of MUC2 by 2% hypoxia (HIF-1α) was evaluated in MUC2-secreting LS174T cells. The effect of BAY 87-2243, an inhibitor of HIF-1α, on MUC2 expression and mucinous tumor growth was evaluated in LS174T cells, PMP explant tissue, and in a unique intraperitoneal murine xenograft model of PMP. In vitro exposure of LS174T cells to hypoxia increased MUC2 messenger RNA (mRNA) and protein expression and increased HIF-1α binding to the MUC2 promoter. Hypoxia-mediated MUC2 protein overexpression was downregulated by transfected HIF-1α small interfering RNA (siRNA) compared with scrambled siRNA in LS174T cells. BAY 87-2243 inhibited hypoxia-induced MUC2 mRNA and protein expression in LS174T cells and PMP explant tissue. In a murine xenograft model of PMP, chronic oral therapy with BAY 87-2243 inhibited mucinous tumor growth and MUC2, HIF-1α expression in the tumor tissue. Our data suggest that hypoxia (HIF-1α) induces MUC2 promoter activity to increase MUC2 expression. HIF-1α inhibition decreases MUC2 production and mucinous tumor growth, providing a preclinical rationale for the use of HIF-1α inhibitors to treat patients with PMP.

A Consensus for Classification and Pathologic Reporting of Pseudomyxoma Peritonei and Associated Appendiceal Neoplasia: The Results of the Peritoneal Surface Oncology Group International (PSOGI) Modified Delphi Process.

Pseudomyxoma peritonei (PMP) is a complex disease with unique biological behavior that usually arises from appendiceal mucinous neoplasia. The classification of PMP and its primary appendiceal neoplasia is contentious, and an international modified Delphi consensus process was instigated to address terminology and definitions. A classification of mucinous appendiceal neoplasia was developed, and it was agreed that "mucinous adenocarcinoma" should be reserved for lesions with infiltrative invasion. The term "low-grade appendiceal mucinous neoplasm" was supported and it was agreed that "cystadenoma" should no longer be recommended. A new term of "high-grade appendiceal mucinous neoplasm" was proposed for lesions without infiltrative invasion but with high-grade cytologic atypia. Serrated polyp with or without dysplasia was preferred for tumors with serrated features confined to the mucosa with an intact muscularis mucosae. Consensus was achieved on the pathologic classification of PMP, defined as the intraperitoneal accumulation of mucus due to mucinous neoplasia characterized by the redistribution phenomenon. Three categories of PMP were agreed-low grade, high grade, and high grade with signet ring cells. Acellular mucin should be classified separately. It was agreed that low-grade and high-grade mucinous carcinoma peritonei should be considered synonymous with disseminated peritoneal adenomucinosis and peritoneal mucinous carcinomatosis, respectively. A checklist for the pathologic reporting of PMP and appendiceal mucinous neoplasms was also developed. By adopting the classifications and definitions that were agreed, different centers will be able to use uniform terminology that will allow meaningful comparison of their results.