Durability of Response to Primary Chemoablation of Low-Grade Upper Tract Urothelial Carcinoma Using UGN-101, a Mitomycin-Containing Reverse Thermal Gel: OLYMPUS Trial Final Report.

PURPOSE: Our goal was to evaluate long-term safety and durability of response to UGN-101, a mitomycin-containing reverse thermal gel, as primary chemoablative treatment for low-grade upper tract urothelial carcinoma. MATERIALS AND METHODS: In this open-label, single-arm, multicenter, phase 3 trial (NCT02793128), patients ≥18 years of age with primary or recurrent biopsy-proven low-grade upper tract urothelial carcinoma received 6 once-weekly instillations of UGN-101 via retrograde catheter to the renal pelvis and calyces. Those with complete response (defined as negative ureteroscopic evaluation, negative cytology and negative for-cause biopsy) 4-6 weeks after the last instillation were eligible for up to 11 monthly maintenance instillations and were followed for ≥12 months with quarterly evaluation of response durability. Durability of complete response was determined by ureteroscopic evaluation; duration of response was estimated by the Kaplan-Meier method. Treatment-emergent adverse events (TEAEs) were monitored. RESULTS: Of 71 patients who initiated treatment, 41 (58%) had complete response to induction therapy and consented to long-term followup; 23/41 patients (56%) remained in complete response after 12 months (95% CI 40, 72), comprising 6/12 (50%) who did not receive any maintenance instillations and 17/29 (59%) who received ≥1 maintenance instillation. Kaplan-Meier analysis of durability was estimated as 82% (95% CI 66, 91) at 12 months. Ureteric stenosis was the most frequently reported TEAE (31/71, 44%); an increasing number of instillations appeared to be associated with increased incidence of urinary TEAEs. CONCLUSIONS: Durability of response to UGN-101 with or without maintenance treatment is clinically meaningful, offering a kidney-sparing therapeutic alternative for patients with low-grade disease.

Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

Primary chemoablation of low-grade upper tract urothelial carcinoma using UGN-101, a mitomycin-containing reverse thermal gel (OLYMPUS): an open-label, single-arm, phase 3 trial.

BACKGROUND: Most patients with low-grade upper tract urothelial cancer are treated by radical nephroureterectomy. We aimed to assess the safety and activity of a non-surgical treatment using instillation of UGN-101, a mitomycin-containing reverse thermal gel. METHODS: In this open-label, single-arm, phase 3 trial, participants were recruited from 24 academic sites in the USA and Israel. Patients (aged ≥18 years) with primary or recurrent biopsy-proven, low-grade upper tract urothelial cancer (measuring 5-15 mm in maximum diameter) and an Eastern Cooperative Oncology Group performance status score of less than 3 (Karnofsky Performance Status score >40) were registered to receive six instillations of once-weekly UGN-101 (mitomycin 4 mg per mL; dosed according to volume of patient's renal pelvis and calyces, maximum 60 mg per instillation) via retrograde catheter to the renal pelvis and calyces. All patients had a planned primary disease evaluation 4-6 weeks after the completion of initial therapy, in which the primary outcome of complete response was assessed, defined as negative 3-month ureteroscopic evaluation, negative cytology, and negative for-cause biopsy. Activity (complete response, expected to occur in >15% of patients) and safety were assessed by the investigator in all patients who received at least one dose of UGN-101. Data presented are from the data cutoff on May 22, 2019. This study is registered with ClinicalTrials.gov, NCT02793128. FINDINGS: Between April 6, 2017, and Nov 26, 2018, 71 (96%) of 74 enrolled patients received at least one dose of UGN-101. 42 (59%, 95% CI 47-71; p<0·0001) patients had a complete response at the primary disease evaluation visit. The median follow-up for patients with a complete response was 11·0 months (IQR 5·1-12·4). The most frequently reported all-cause adverse events were ureteric stenosis in 31 (44%) of 71 patients, urinary tract infection in 23 (32%), haematuria in 22 (31%), flank pain in 21 (30%), and nausea in 17 (24%). 19 (27%) of 71 patients had study drug-related or procedure-related serious adverse events. No deaths were regarded as related to treatment. INTERPRETATION: Primary chemoablation of low-grade upper tract urothelial cancer with intracavitary UGN-101 results in clinically significant disease eradication and might offer a kidney-sparing treatment alternative for these patients. FUNDING: UroGen Pharma.

Monocyte subpopulations and their differentiation patterns during infection.

The term "monocyte" implies a single, homogenous population of cells with uniform physiology. Recent evidence from a number of laboratories indicates that it is likely that blood monocytes may consist of several subpopulations of cells, which differ in size, nuclear morphology, granularity, and functionality. The aim of this review is to give a summary of the new findings in the emerging field of monocyte heterogeneity. We provide a short description of the differentiation patterns of blood monocyte subpopulations, with an emphasis on how these subpopulations can be influenced by infection. We provide a comparison among the main monocyte subpopulations in humans, mice, and rats and illustrate some of the common features of these cells and some of the important interspecies distinctions. We will also discuss the bone marrow precursors of these cells and the differentiation patterns of these subsets in different tissues in response to infection. Most of the data about monocyte trafficking during infection are necessarily derived from murine models, and comparisons between mouse and man must be made with caution. However, these models may provide interesting springboards to permit us to speculate about the topic of monocyte heterogeneity in humans.

Assessment of the efficacy of repeated instillations of mitomycin C mixed with a thermosensitive hydrogel in an orthotopic rat bladder cancer model.

BACKGROUND: We investigated a thermoreversible hydrogel that is highly viscous at body temperature, while fluid-like at a low temperature, thus aiming for a slow and prolonged intravesical drug release. Our study purposed to assess antitumor efficacy of mitomycin C (MMC) mixed with hydrogel in an orthotopic rat bladder cancer model. METHODS: Bladders of female Fischer F344 rats were grafted with 1.5 × 106 AY-27 urothelial carcinoma cells. On day 5, tumor presence was assessed by cystoscopy and rats were divided into six groups (five treatment, one control, n = 10/group). Intravesical treatments (0.5 mg or 1 mg MMC-H2O or MMC-hydrogel, or 2 mg MMC-hydrogel) were administered on days 5, 8 and 11. Rats were sacrificed at day 14 and bladders were evaluated. RESULTS: Rats with tumor at cystoscopy (47/60) were evaluated for efficacy. At necropsy, all control animals (8/8) had tumors. No microscopic tumors were present in the 0.5 mg and 1 mg MMC-hydrogel groups compared with 2/8 and 1/8 rats in the 0.5 mg and 1 mg MMC-H2O groups (p = 0.47 and p = 1.00, respectively).Greater toxicity was seen in animals treated with MMC-hydrogel compared with MMC-H2O, as demonstrated by lower body weights at necropsy (p = 0.000) and a tendency for more severe clinical signs in the 1 and 2 mg MMC-hydrogel groups. Rats that died prematurely received 1 mg (4/10) or 2 mg (9/10) of MMC-hydrogel. CONCLUSIONS: Under the current model conditions it is unclear whether instillation of MMC-hydrogel is more effective than MMC-H2O. Nonetheless, the observed difference in toxicity, acting as a surrogate marker for systemic MMC exposure in the MMC-hydrogel-treated rats, supports the prolonged drug release mechanism of the hydrogel.

Serial retrograde instillations of sustained release formulation of mitomycin C to the upper urinary tract of the Yorkshire swine using a thermosensitive polymer: Safety and feasibility.

BACKGROUND: MitoGel is a novel drug formulation intended for the treatment of upper tract urothelial cancer with proven feasibility and safety in an animal model. OBJECTIVE: To evaluate the feasibility, safety, toxicokinetics, and histologic changes associated with serial retrograde MitoGel instillations to the upper urinary tract in a swine model. DESIGN, SETTING, AND PARTICIPANTS: Overall, 27 Yorkshire swine underwent 6 once-weekly unilateral retrograde instillations of MitoGel. Doses of 14, 28, or 56-mg mitomycin C (respective concentrations of 2, 4, and 8mg/ml with 9 animals per group) were evaluated. Additionally, 6 animals received sterile water as a procedure control, and 9 received gel alone (without mitomycin C), as a vehicle control. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Blood and urine samples were collected for determination of MMC toxicokinetics and for hematology, biochemistry, coagulation, and urinalysis throughout the study. Two-thirds of the cohort were euthanized 24 hours after final instillation, and one-third was euthanized 1 month after final instillation. Necropsy was performed to evaluate the histologic effects of treatments. RESULTS AND LIMITATIONS: All animals received all 6 doses of agents per protocol. No mortality, clinical adverse events, or meaningful changes in hematology, chemistry, coagulation, or urinalysis were attributable to MitoGel, RTGel alone, or water instillations. Peak plasma levels of MMC were 2 orders of magnitude less than known toxicity thresholds. MitoGel-related dose-dependent microscopic findings were seen in the treated kidneys and ureters, but were of limited severity, lacked associated clinical adverse findings, and decreased over time. CONCLUSIONS: Serial retrograde instillations of MitoGel to the pyelocaliceal system were technically feasible, and produced no observable adverse clinical, laboratory, or histologic effects.

Sustained-release Formulation of Mitomycin C to the Upper Urinary Tract Using a Thermosensitive Polymer: A Preclinical Study.

OBJECTIVE: To evaluate the safety and feasibility of single and serial instillations of MitoGel into the upper urinary tract using a preclinical swine animal model. MitoGel is a novel sustained release formulation of mitomycin C (MMC) based on RTGel, a proprietary thermosensitive hydrogel technology. MitoGel is liquid at cold temperatures and solidifies to gel state at body temperature. It is intended as a treatment for upper tract urothelial carcinoma, given its ability to provide sustained release of MMC in the upper urinary tract. MATERIALS AND METHODS: We utilized 23 pigs in a 3-phase design. All animals underwent bilateral nephrostomy tube placement. During phase 1, 3 animals underwent antegrade RTGel instillation, imaging, and euthanasia within 12 hours. In phase 2, 10 pigs underwent single antegrade instillation, unilateral nephrectomy 3 days following instillation, and contralateral nephrectomy and euthanasia 30 days following instillation. During phase 3, 10 animals underwent 6 instillations over 3 weeks, followed by bilateral nephrectomy and necropsy 30 days postinstillation. MitoGel (2 mg/mL and 4 mg/mL), aqueous MMC (2 mg/mL and 4 mg/mL), and RTGel alone were evaluated. RESULTS: MitoGel remained visible within the pelvicalyceal system on fluoroscopic and computed tomography imaging for 4-6 hours. MMC plasma levels were well within acceptable safety thresholds. There was no evidence of urinary obstruction, acute kidney injury, sepsis, or myelosuppression. Histologic changes in the urinary system were mild and transient. CONCLUSION: Antegrade MitoGel delivery to the pelvicalyceal system of Yorkshire swine is feasible and safe. Further evaluation of MitoGel in human clinical trials is warranted.

Interleukin-12 augments a Th1-type immune response manifested as lymphocyte proliferation and interferon gamma production in Leishmania infantum-infected dogs.

The dog is the major reservoir for human visceral leishmaniasis caused by Leishmania infantum. Interleukin-12 is considered to have an essential role in the development of both innate and adaptive immunity to Leishmania spp. and other intracellular pathogens. This study focused on the influence of IL-12 in experimental and natural canine visceral leishmaniasis. Responses of peripheral blood mononuclear cells to IL-12, interleukin-10 and Leishmania soluble antigen were evaluated in L. infantum experimentally infected oligosymptomatic beagles, uninfected beagles, naturally infected polysymptomatic dogs, and their matched uninfected controls. Leishmania soluble antigen induced strong peripheral blood mononuclear cells proliferation both in experimentally infected dogs (median stimulation index [SI]=15.01), and in naturally infected dogs (SI=8.86), but not by cells from the control groups. IL-12 addition further enhanced cell proliferation in naturally (SI=14.95), but not in experimentally infected animals. Peripheral blood mononuclear cells from experimentally infected dogs were able to produce significant amounts of IFN-gamma (3.39 ng/ml) upon LSA stimulation, but no such production was detected in cells from naturally infected or control animals. Interestingly, addition of IL-12 reversed the inhibitory effect of LSA on IFN-gamma production by cells from polysymptomatic naturally infected dogs and the uninfected beagles (4.84 and 7.45 ng/ml, respectively), and further increased IFN-gamma production by peripheral blood mononuclear cells from experimentally infected oligosymptomatic dogs (29.28 ng/ml). IFN-gamma mRNA expression correlated well with IFN-gamma production. Addition of IL-10 to Leishmania soluble antigen stimulated peripheral blood mononuclear cells inhibited proliferation and IFN-gamma production in experimentally infected dogs. Thus, the ability of IL-12 to augment IFN-gamma production by peripheral blood mononuclear cells from dogs with experimental or natural symptomatic canine visceral leishmaniasis makes it a good candidate for cytokine therapy in dogs that are refractory to current therapy.

A serosurvey of Hepatozoon canis and Ehrlichia canis antibodies in wild red foxes (Vulpes vulpes) from Israel.

A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with Ehrlichia canis and Hepatozoon canis antigens in free-ranging red foxes (Vulpes vulpes) in Israel. Of 84 fox sera assayed, 36% were seropositive for E. canis by the indirect fluorescent antibody (IFA) test and 24% were positive for H. canis using an enzyme-linked immunosrbent assay (ELISA). Canine ehrlichiosis and hepatozoonosis appear to be endemic in the wild red fox populations in Israel, and foxes may serve as a reservoir for infection of domestic dogs and other wild canine species.

On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells.

Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGF-based transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6C(hi) monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited "standard" monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.

Acute phase protein response in experimental canine leishmaniasis.

Acute phase proteins (APPs) have been proposed as useful markers for the diagnosis and monitoring of treatment of dogs infected by Leishmania infantum. However, the kinetics and behavior of these proteins in canine leishmaniasis is still unknown. The aim of this study was to monitor the kinetics of APPs in dogs experimentally infected with L. infantum, before, during and after therapy against canine leishmaniasis. Levels of serum haptoglobin, serum amyloid A and C-reactive protein from 6 infected beagles, positive by both PCR and parasite culture, were monitored for 7 months post-infection. The dogs were then treated for 3 months with allopurinol (20 mg mg/kg/day PO), and their response to therapy was followed for 11 additional months. Levels of Immunoglobulins G and M were recorded during these 21 months and compared. Experimental infection with L. infantum amastigotes induced an increase in all APPs studied which was statistically significant 2 months after infection for all proteins. Clinical recovery was accompanied by a significant decrease of all APPs 1 month after the beginning of treatment. However, differences were found between the APPs in both magnitude and duration of serum level elevations. The increase in total IgG and IgM was delayed in comparison to APPs and contrarily to the APPs, these immunoglobulins did not significantly decrease with treatment. In conclusion, the results of this study suggest that APPs could be used as early markers for disease as well as for monitoring the response to treatment in canine leishmaniasis.

Splenic immune responses during canine visceral leishmaniasis.

Dogs are the main reservoir host for zoonotic visceral leishmaniasis caused by Leishmania infantum. In this study we investigated the immune response in spleens of L. infantum-infected dogs by measuring the mRNA expression levels for a wide panel of cytokines, transcription factors and chemokines. mRNA levels and parasite load were followed during 7 months of experimental infection and 14 months post-treatment, and were compared to naturally-infected (NI) dogs. Similarly, serum anti-Leishmania IgG and IgG subclass levels were measured during experimental infection. An increase in IFN-gamma, T-bet, IP-10 and RANTES was found in the experimentally and NI dogs, implicating a substantial type-1 immune response during canine visceral leishmaniasis. IL-4, a type-2 associated cytokine, increased as early as one month after experimental infection, while IL-5 was high at later stages. Interestingly, the expression levels of the Treg-associated cytokines, IL-10 and TGF-beta, did not change during the infection. Total anti-Leishmania IgG and IgG subclasses increased during the experimental infection. However, no association with specific cytokine patterns was observed. Parasite load in the spleens increased as early as one month post-infection and remained high until treatment. The load was higher in the polysymptomatic NI dogs than in the experimentally-infected dogs. This study indicates that both type-1 and type-2 immune responses occur in the spleen during canine L. infantum infection, and suggests that the early elevation of IL-4 might have a role in the persistence of parasites in the presence of high IFN-gamma expression.

Leishmania-derived murine monocyte chemoattractant protein 1 enhances the recruitment of a restrictive population of CC chemokine receptor 2-positive macrophages.

Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites.

Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantum DNA in dogs.

A polymerase chain reaction (PCR) procedure using noninvasively obtained samples, for the identification of Leishmania infantum in canine tissues, was evaluated and compared with serologic testing and culture. A total of 92% of naturally infected, symptomatic, seropositive dogs were found to be positive by use of DNA from conjunctival swabs. Spleen or lymph node aspirates were found to be positive by PCR in 86% and by culture in 74% of these dogs. The sensitivity and specificity of conjunctival PCR were 92% and 100%, respectively. Experimentally infected dogs were found to be positive by conjunctival PCR already at 45 days of infection (83%) and before seroconversion. PCR using noninvasively obtained conjunctival samples will be useful for epidemiological studies and for direct diagnosis of canine visceral leishmaniasis.