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Background: Our study aims to assess the feasibility and the reproducibility of fluorescent confocal microscopy (FCM) real-time assessment of urethral and ureteral margins during open radical cystectomy (ORC) for bladder cancer (BCa). Methods: From May 2020 to January 2022, 46 patients underwent ORC with intraoperative FCM evaluation. Each specimen was intraoperatively stained for histopathological analysis using FCM, analyzed as a frozen section (FSA), and sent for traditional H&E examination. Sensitivity, specificity, positive predictive value (PPV), and the negative predictive value (NPV) of FCM and FSA were assessed and compared with H&E for urethral and ureteral margins separately. Results: The agreement was evaluated through Cohen’s κ statistic. Urethral diagnostic agreement between FCM and FSA showed a κ = 0.776 (p < 0.001), while between FCM and H&E, the agreement was κ = 0.691 (p < 0.001). With regard to ureteral margins, an overall agreement of κ = 0.712 (p < 0.001) between FCM and FSA and of κ = 0.481 (p < 0.001) between FCM and H&E was found. Conclusions: FCM proved to be a safe, feasible, and reproducible method for the intraoperative assessment of urethral and ureteral margins during ORC. Compared to standard FSA, FCM showed adequate diagnostic performance in detecting urethral and ureteral malignant involvement.

Supplementary Table S1. AML patients. Supplementary Table S2. RT-qPCR primers. Supplementary Table S3. IC50 concentrations (nM) of FLT3 inhibitors, PP2A activators and GSK-3beta inhibitors. Supplementary Figure S1. Concurrent PP2A-activating drug treatment increases cytotoxicity of FLT3 inhibitors in Ba/F3-ITD cells with FLT3-ITD and in an MV4-11 orthotopic mouse model. Supplementary Figure S2. Chou-Talalay analysis of FLT3-WT cell lines. Supplementary Figure S3. Images of all mice in the in vivo experiment. Supplementary Figure S4. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor does not induce apoptosis in wild-type FLT3 AML, AML complete remission (CR) or AML complete remission with incomplete platelet recovery (CRp) marrows. Supplementary Figure S5. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor downregulates c-Myc and Pim-1 protein expression in cells with FLT3-ITD. Supplementary Figure S6. p-c-Myc (T58) and p-c-Myc (S62) expression in Ba/F3-ITD and MV4-11 cells treated with gilteritinib and/or FTY720. Supplementary Figure S7. Concurrent treatment with PP2A-activating drug and FLT3 inhibitor produces variable changes in c-Myc and Pim-1 mRNA expression in cells with FLT3-ITD. Supplementary Figure S8. c-Myc and Pim-1 expression in FLT3-WT AML blasts treated with gilteritinib and/or FTY720. Supplementary Figure S9. Proteasome inhibition inhibits downregulation of c-Myc and Pim-1 expression by concurrent PP2A-activating drug and FLT3 inhibitor treatment. Supplementary Figure S10. Concurrent PP2A-activating drug and FLT3 inhibitor treatment increases c-Myc and Pim-1 protein turnover in cells with FLT3-ITD. Supplementary Figure S11. Concurrent PP2A activator and FLT3 inhibitor treatment downregulates p-ERK later than p-AKT in cells with FLT3-ITD. Supplementary Figure S12. The GSK-3beta inhibitor TWS119 prevents c-Myc and Pim-1 downregulation and apoptosis induction by PP2A-activating drug and FLT3 inhibitor combination in Ba/F3-ITD cells.

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD–expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD–expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo. PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD–expressing cells through a novel mechanism involving AKT inhibition–dependent GSK-3β–mediated increased c-Myc and Pim-1 proteasomal degradation.

AZD1208 and quizartinib co-treatment was well tolerated in the orthotopic model.

The USP9X inhibitor WP1130 enhances induction of apoptosis of Ba/F3-ITD and MV4-11 cells by quizartinib in a concentration-dependent manner.

Pim kinase and FLT3 inhibition does not enhance induction of apoptosis of cells with FLT3-WT.

Quizartinib, sorafenib, crenolanib and gilteritinib cytotoxicity in cell lines with FLT3-ITD or FLT3-WT

Post-transcriptional regulation of Mcl-1 expression.

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition.Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo.Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML.Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Supplementary figure legends

AZD1208 and quizartinib co-treatment does not increase celluar ROS generation, but increases mitochondrial ROS generation.

Enhanced apoptosis of Ba/F3-ITD and 32D/ITD cells treated with FLT3 inhibitors at serial concentrations in the presence of AZD1208.

Combined AZD1208 and quizartinib treatment does not abrogate growth of KG-1a cells, expressing FLT3-WT, in a xenograft model.

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed.

Prostate cancer (PCa) surgery has a strong impact on men’s social and sexual lives. For this reason, many patients ask for robotic surgery. To assess the rate of lost patients due to the lack of a robotic platform (RPl) at our center, we retrospectively selected 577 patients who underwent prostate biopsy between 2020 and 2021 who were eligible for radical prostatectomy (RP) (ISUP ≥ 2; age ≤ 70 yr). Patients eligible for surgery who decided to be operated received a phone call interview asking the reason for their choice. Overall, 230 patients (31.7%) underwent laparoscopic-assisted radical prostatectomy (LaRP) at our center, while 494 patients (68.3%) were not treated in our hospital. Finally, 347 patients were included: 87 patients (25.1%) underwent radiotherapy; 59 patients (17%) were already under another urologist’s care; 113 patients (32.5%) underwent robotic surgery elsewhere; and 88 patients (25.4%) followed the suggestion of friends or relatives based on their surgical experience. Despite no surgical technique for RP having shown superiority in terms of oncological or functional outcomes, patients eligible for PCa treatment decided to be operated on elsewhere because of the lack of an RPl. Our results show how the presence of an RPl may increase the case volume of RP by 49% at our center.

Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD–expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD–expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo. PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD–expressing cells through a novel mechanism involving AKT inhibition–dependent GSK-3β–mediated increased c-Myc and Pim-1 proteasomal degradation.

Introduction: Prostate cancer (PCa) at its early stages is treated with surgery or androgen-deprivation therapy, but PCa can turn castration resistant possibly due to pre-existing stem cell-like cells that reinitiate tumor growth and lead to metastasis. Given that human tumors express high levels of the oncofetal Cripto, our hypothesis is that Cripto might play a role in PCa initiation and progression, so we conditionally knock out Cripto in GEMMs models representative of early and late metastatic PCa to study its oncogenic potential. Methods: Conditional Cripto knock out (CRIPTOflox/flox) was performed on N (Nkx3.1CreERT2, R26 LSL-YFP/LSL-YFP), NP (Nkx3.1CreERT2; Ptenflox/flox, R26 LSL-YFP/LSL-YFP) and NPK (Nkx3.1CreERT2; Ptenflox/flox; KrasLSL-G12D/+, R26 LSL-YFP/LSL-YFP) to generate respectively NC, NPC and NPKC. Normal epithelium is mirrored by N animals, whereas first stage of the disease, high-grade prostatic intraepithelial neoplasia (PIN)/carcinoma lesions with local invasive epithelium, are seen in NP. The advanced stage of PCa with metastasis formation is developed in NPK. In vivo experiments presented the following workflow: 8-weeks old mice were castrated and induced one month later with 5 daily injections of tamoxifen, after 2.5 weeks animals were weekly treated with testosterone (10 weeks). Single cells were isolated from prostate tissue and the YFP+/− population recovered by FACS sorting and cultured as organoids. Results: Published PCa models N, NP and NPK were confirmed. The histopathological evaluation of newly generated NPC and NPKC shows presence of mPIN (100%, nNPC= 6, nNPKC=5), with a dominant cribriform morphology. NPKC presents invasive PCa with an extent dominant pattern of 100%. Invasive portions in NPKC present dense stroma forming whorl-like structures and occasional sheet-like accumulations of polygonal in stroma of regions with mPIN. Compared to the published models, NPC and NPKC feature a more reactive stroma with a mild/moderate inflammation. OrganoidsYFP+ recapitulate molecular prostate tissue features, expressing luminal and basal markers. Organoids’ morphology varies consistently: N and NC organoids present a more cystic morphology, with lower densities which is consistent with low-grade PIN phenotypes, whereas NP and NPK organoids are smaller and denser which mimics an oncogenic transformation. In general, NC, NPC and NPKC organoids present a higher percentage of hollow organoids compared to N, NP and NPK respectively. Conclusions: Histopathological evaluation of NPC and NPKC phenotype specifically, showed important stromal alterations suggesting that Cripto might play a role not only on the epithelial compartment as well as in the stroma. For this reason, studies on secreted Cripto inhibition with ALK4-Fc are on-going. Our finding support that organoids are an efficient in vitro model replicating different phenotypes seen in vivo. Citation Format: Elisa Rodrigues Sousa, Eugenio Zoni, Mario Scarpa, Marta De Menna, Allen Abey Alexander, Simone De Brot, George N. Thalmann, Marianna Kruithof-de Julio. A new transgenic mouse model to explore the role of Cripto signaling in lethal prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 22.

Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase is present in AML cells in 25% of patients, causing constitutive activation and aberrant signaling. FLT3-ITD+ AML patients have a high relapse rate following chemotherapy and short disease-free and overall survival. Incorporation of FLT3 inhibitors (FLT3is) into therapy for FLT3-ITD+ AML has improved outcomes, but FLT3i efficacy is limited by onset of resistance, which occurs by diverse mechanisms. The tumor suppressor protein phosphatase 2A (PP2A) is inactivated in FLT3-ITD+ AML, and we previously showed that PP2A-activating drugs (PADs), including DT-061 and FTY720, enhance FLT3i efficacy through enhanced proteasomal degradation of c-Myc and Pim-1, mediated by activation of the serine/threonine kinase GSK-3β (Mol Cancer Ther 20:676, 2021). Here we sought to determine whether concurrent treatment with PADs overcomes resistance to FLT3is. We studied MOLM-14 human AML cells, with heterozygous FLT3-ITD, and FLT3i-resistant MOLM-14 cells with D835Y tyrosine kinase domain and F691L gatekeeper FLT3 mutations and M14(R)701 MOLM-14 cells (from Dr. Donald Small, Johns Hopkins) with a G12D NRAS activating mutation. IC50s, determined by WST-1 cytotoxicity assays, of Type 1 FLT3i gilteritinib in MOLM-14, D835Y, F691L and M14(R)701 cells were 9.2, 18.6, 56 and 57.5 nM, and the Type II FLT3i quizartinib, 2.8, 211, 659 and >4,000 nM. Both DT-061 and FTY720 sensitized MOLM-14 D835Y cells to quizartinib (IC50s Citation Format: Moaath Mustafa Ali, Aditi Chatterjee, Jonelle K. Lee, Mario Scarpa, Maria R. Baer. Effects of PP2A-activating drugs on FLT3 inhibitor resistance mediated by diverse mechanisms in acute myeloid leukemia with FLT3-ITD. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5013.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of

INTRODUCTION AND OBJECTIVE:Prostate cancer (PCa) is the most frequent malignancy detected in men and the second leading cause of cancer-related male death in Europe. Current markers frequently fail...

INTRODUCTION AND OBJECTIVE:Multiparametric MRI (mpMRI) represents a useful step for the diagnosis, staging and preoperative planning of prostate cancer (PCa). However, the interpretation of mpMRI i...

Aim: To investigate the prognostic role of neutrophil percentage-to-albumin ratio (NPAR) in muscle-invasive bladder cancer (MIBC) patients treated with neoadjuvant chemotherapy (NAC) and radical cystectomy (RC). Patients & methods: 213 patients were included. Inclusion criteria: Nonmetastatic, MIBC (cT2-T4aN0M0), at least three cycles of NAC, undergone RC and with blood count within 30 days before NAC. Results: Five-years overall survival (OS) with NPAR >18 was 34.06% (95% CI: 18.3–50.5) and 65.37% (95% CI: 52.4–75.6) with NPAR 18 was 42.9% (95% CI: 23.9–60.7) and 74.5% (95% CI: 62.6–83.1) with NPAR, Lay abstract This research demonstrated that presence of a high neutrophil percentage-to-albumin ratio in patients with muscle-invasive bladder cancer prior neoadjuvant chemotherapy and radical cystectomy is associated with worse outcomes and reduced overall and cancer-specific survival. Thus, neutrophil percentage-to-albumin ratio could be a useful biological marker in clinical practice to help predict outcomes for patients with muscle-invasive bladder cancer.

BACKGROUND Internal tandem duplication in the fms-like tyrosine kinase 3 receptor tyrosine kinase (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients, associated with poor treatment outcomes due to rapid relapse. FLT3 inhibitors are used in the clinic, but with incomplete efficacy and development of resistance. Further treatment options are needed. The serine/threonine kinase proviral integration site for Moloney murine leukemia virus (Pim-1) is upregulated downstream of FLT3-ITD; it directly stimulates cell growth and inhibits apoptosis, and also phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual targeting of Pim-1 and FLT3 is a promising treatment strategy. The c-Myc transcription factor contributes to dysregulation of cell growth and apoptosis in cancers, including AML. In addition to transcriptional regulation, c-Myc is regulated post-translationally by T58 phosphorylation by the serine/threonine kinase glycogen synthase kinase-3- β (GSK-3β). Here we show that concurrent treatment of cells with FLT3-ITD with Pim and FLT3 inhibitors activates GSK-3β, which phosphorylates and post-translationally downregulates c-Myc. METHODS Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and FLT3-ITD AML patient blasts were cultured with the pan-Pim inhibitor AZD1208 (1 μM) and/or the FLT3 inhibitors gilteritinib or quizartinib (15 nM, 1 nM), with and without the GSK-3β inhibitor TCG-24 (20 μM). c-Myc, p-GSK3-α/β (S21/9) and GSK3-α/β protein expression was measured by immunoblotting. c-Myc mRNA was measured by qPCR. Cells were also cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM) to measure protein half-life and proteasomal degradation. To study the role of c-Myc overexpression and activation, Ba/F3-ITD cells were infected with retroviral estrogen receptor (ER)-Myc plasmid, causing c-Myc nuclear translocation when activated by 4-hydroxytamoxifen (4-OHT; 300 nM). To study the role of c-Myc phosphorylation at T58, Ba/F3-ITD cells were infected with MycT58A plasmid, preventing c-Myc phosphorylation at T58. Apoptosis was detected by Annexin V and propidium iodide staining, measured by flow cytometry. RESULTS Treatment with Pim inhibitor AZD1208 and FLT3 inhibitor gilteritinib or quizartinib combination rapidly downregulated c-Myc protein expression in Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and in primary FLT3-ITD AML patient blasts, compared to quizartinib or gilteritinib alone. Pim inhibitor and FLT3 inhibitor combination treatment did not decrease c-Myc mRNA levels, but markedly decreased c-Myc protein half-life, from 36 mins without drugs and 24 mins with gilteritinib to 18 mins with combination. Half-life did not decrease when cells were pre-treated with the proteasome inhibitor MG-132, consistent with post-translational downregulation through proteasomal degradation. Apoptosis induction by Pim inhibitor and FLT3 inhibitor combination decreased by more than 50% in Ba/F3-ITD cells infected with ER-Myc plasmid and treated with 4-OHT, demonstrating the major role of c-Myc downregulation in apoptosis induction by combination treatment. GSK-3b is inactivated by phosphorylation, and combination treatment rapidly decreased p-GSK-3b levels, while total GSK-3b levels were unchanged, indicating activation of GSK-3b. Treatment of cells with FLT3-ITD with the GSK-3b inhibitor TCG-24 in addition to Pim and FLT3 inhibitors abrogated c-Myc protein downregulation, demonstrating that Pim and FLT3 inhibitor combination downregulates c-Myc through activation of GSK-3b. Finally, Pim and FLT3 inhibitor combination treatment did not downregulate c-Myc in Ba/F3-ITD cells transfected with c-Myc T58A, preventing c-Myc phosphorylation at T58, showing that c-Myc phosphorylation at T58 is necessary for its downregulation by combination treatment. CONCLUSIONS Concurrent treatment of cells with FLT3-ITD with Pim kinase inhibitor enhances the efficacy of FLT3 inhibitors through activation of GSK-3β and GSK-3β-mediated phosphorylation of c-Myc at T58, with resulting c-Myc downregulation through increased proteasomal degradation. This work and previous work in our laboratory on PP2A activating drugs and FLT3 inhibitor combination (Mol Cancer Ther 20:676, 2021) support GSK-3β activation as a mechanism for enhancing efficacy of FLT3 inhibitors in AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

We compare bipolar vs monopolar transurethral prostate resection safety/secondary outcomes including efficacy in patients with large prostate volume or severe lower urinary tract symptoms. From July 2006 to June 2009 candidates for transurethral prostate resection were recruited at 4 centers, randomized 1:1 into monopolar/bipolar transurethral prostate resection arms and followed up to 36 months. Post hoc data analysis from patients with large prostate volume or severe lower urinary tract symptoms is presented. Patients with large prostate volume or severe lower urinary tract symptoms were defined as those with transrectal ultrasound based prostate volume greater than 80 ml or International Prostate Symptom Score greater than 19. Safety was estimated using sodium/hemoglobin changes immediately after surgery, complications during the early postoperative period (up to 6 weeks), and short-term (up to 12 months) and midterm (up to 36 months) followup. Secondary outcomes included, among others, efficacy quantified by changes in maximum urine flow rate, post-void residual urine volume and International Prostate Symptom Score compared with baseline. A total of 279 patients were randomized. Post hoc analysis of data from patients with a large prostate volume or severe lower urinary tract symptoms was based on analysis A-in 62 of 279 participants (22.3%) (monopolar transurethral prostate resection 32, bipolar transurethral prostate resection 30) or analysis B-in 126 of 279 participants (45.2%) (monopolar transurethral prostate resection 57, bipolar transurethral prostate resection 69). Mean (SD) prostate volume was 108.0 (25.9) ml for monopolar transurethral prostate resection and 108.9 (23.4) ml for bipolar transurethral prostate resection (p=0.756). Mean International Prostate Symptom Score was 25.0 (4.2) for monopolar transurethral prostate resection and 25.3 (3.7) for bipolar transurethral prostate resection (p=0.402). Neither safety nor any secondary outcome differed significantly between the arms throughout followup. The only exception was the decrease in sodium (analysis A), which was significantly greater after monopolar transurethral prostate resection (-4.2 vs -0.7 mmol/l, p=0.023) and did not translate into a significant difference in transurethral resection syndrome rates (monopolar transurethral prostate resection 1 of 32 vs bipolar transurethral prostate resection 0 of 30, p=1.000). Bipolar and monopolar transurethral prostate resection show similar safety/efficacy in these patient subpopulations

BACKGROUND New approaches have been developed to further reduce the invasiveness of laparoscopic pyeloplasty (P) as treatment for uretero-pelvic junction obstruction (UPJO). Aim of the study was to compare perioperative, functional and cosmetic results of mini-laparoscopic (mL-P) versus robot-assisted laparoendoscopic single-site P (rLESS-P). METHODS Since April 2009 to June 2010, 12 adult patients with primary UPJO, BMI

Introduction fms-like tyrosine like kinase 3 internal tandem duplication (FLT3-ITD), present in acute myeloid leukemia (AML) cells of 30% of patients, results in constitutive and aberrant FLT3 signaling and, clinically, short disease-free survival. Efficacy of FLT3 inhibitors is limited and transient, but may be enhanced by dual targeting of FLT3-ITD signaling pathways. The tumor suppressor protein phosphatase 2A (PP2A) is inhibited in cells with FLT3-ITD. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated and also stabilized by PP2A inhibition in cells with FLT3-ITD. Pim-1 contributes directly to FLT3-ITD proliferative and anti-apoptotic effects, and also phosphorylates and stabilizes FLT3-ITD in a positive feedback loop. Moreover FLT3-ITD, PP2A and Pim-1 all regulate the transcription factor c-Myc. PP2A-activating drugs enhance efficacy of FLT3 inhibitors. We sought to identify mechanisms underlying the efficacy of this combination. Methods Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, and blasts from patients with AML with FLT3-ITD were cultured with the FLT3 inhibitors gilteritinib (15 nM) or quizartinib (1 nM) and/or the PP2A-activating drugs FTY720 (2-4 µM) or DT-061 (10 µM), or with DMSO control. Pim-1, c-Myc, p-AKT (S473 and T308) and AKT protein expression was measured by immunoblotting, along with p-STAT5 (Y694), STAT5, p-PP2A (Y307) and PP2A expression. To study post-translational regulation, cells were cultured with cycloheximide (100 µg/mL) with and without the proteasome inhibitor MG-132 (20 µM). Ubiquitinated c-Myc was measured by co-immunoprecipitation and immunoblotting with c-Myc and ubiquitin antibodies. Ba/F3-ITD cells were stably transfected with estrogen receptor (ER)-c-Myc, kinase-dead Pim-1 or myristoylated AKT plasmids or corresponding empty vectors. Apoptosis was detected by Annexin V and propidium iodine staining, measured by flow cytometry. Cells were also cultured with the pan-Pim kinase inhibitor AZD1208 (1 µM), the Myc inhibitor 10058-F4 (100 µM) or the pan-AKT inhibitor MK-2206 (5 µM). Results Concurrent treatment of Ba/F3-ITD and MV4-11 cells and primary AML cells with FLT3-ITD with a FLT3 inhibitor (gilteritinib or quizartinib) and a PP2A-activating drug (FTY720 or DT-061) decreased growth and increased apoptosis induction, relative to treatment with single drugs. Concurrent FLT3 inhibitor and PP2A-activating drug treatment decreased expression of both Pim-1 and c-Myc protein. Concurrent treatment decreased Pim-1 half-life from 15 to 5 minutes, and c-Myc half-life from 30 to 5 minutes, while half-lives were restored by concurrent treatment with the proteasome inhibitor MG-132. Concurrent treatment was also shown to increase c-Myc ubiquitination. Effects of concurrent treatment on Pim-1 and c-Myc were independent, as transfection with kinase-dead Pim-1 or treatment with Pim inhibitor AZD1208 did not alter c-Myc downregulation, and c-Myc overexpression or treatment with Myc inhibitor 10058-F4 did not alter Pim-1 downregulation. Concurrent treatment with FLT3 inhibitor and PP2A-activating drug did not alter expression of c-Myc deubiquitinases, but rapidly decreased AKT S473 and T308 phosphorylation. FLT3 inhibitor and PP2A activator co-treatment did not induce downregulation or increased turnover of Pim-1 and c-Myc protein or apoptosis in cells with constitutive AKT activation caused by transfection of myristoylated AKT. Moreover, AKT inhibition downregulated Pim-1 and c-Myc protein expression, decreased Pim-1 and c-Myc protein half-lives from 15 to 5 minutes and 30 to 10 minutes, respectively, and induced apoptosis of cells with FLT3-ITD, replicating the effects of FLT3 inhibitor and PP2A activator co-treatment. Conclusion PP2A activators enhance the efficacy of FLT3 inhibitors in AML cells with FLT3-ITD through AKT inactivation-dependent increased Pim-1 and c-Myc proteasomal degradation, which is a novel mechanism. The data support further preclinical and clinical testing of this dual targeting approach to treatment of AML with FLT3-ITD. Disclosures Baer: Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

Since ancient times, sutures have been a fundamental part of the surgeon's practice. Beside the vegetable and animal origin materials, in the last decades different refined synthetic materials have been introduced in the clinical practice. More recently, different devices, such as staplers and tissue sealers have been developed. Thus, more than ever, urologists are faced with a multitude of suture materials, and research of safe, effective materials and devices seems to be continuously growing. The introduction and widespread diffusion of laparoscopic and robotic surgery has further boosted this research. Given there is no single material that is ideal for all situations, the surgeon must choose the best material for each particular case. The aim of this non-systematic review is to summarize the more innovative suture materials and devices and to describe the different surgical methods to utilize them both in general and in urologic surgery.

Objectives To compare the occurrence of depression, anxiety, self body image perception, sleep disturbances, and diminished quality of life in prostate cancer patients undergoing adjuvant androgen-deprivation therapy (ADT) as opposed to patients in follow-up alone. Methods and materials Hospital Anxiety and Depression Scale, Pittsburgh Sleep Quality Index, Restless Legs Syndrome Study Group essential diagnostic criteria, Body Image Scale and Functional Assessment of Cancer Therapy Prostate were administered to consecutive prostate cancer patients who underwent radical prostatectomy or radiation therapy and are presently either under adjuvant ADT or included in a follow-up program. Results Of the 103 patients enrolled, 49 (47.6%) were receiving adjuvant ADT and 54 (52.4%) were not. Compared with the controls, the patients undergoing ADT showed higher levels of depression ( P = 0.002), worse self body image perception ( P = 0.001), worse quality of life ( P = 0.0001) and worse sleep quality ( P = 0.04). ADT was significantly associated with depression at multivariate analysis after adjustment for age, stage, Gleason score, as well as demographic and social variables ( P = 0.001). Depression scores showed a strong inverse correlation with quality of life scores ( P Conclusions Adjuvant ADT is associated with depression, worse quality of life, and altered self body image in prostate cancer patients.

Background Pooled data from randomised controlled trials (RCTs) with short-term follow-up have shown a safety advantage for bipolar transurethral resection of the prostate (B-TURP) compared with monopolar TURP (M-TURP). However, RCTs with follow-up >12 mo are scarce. Objective To compare the midterm safety/efficacy of B-TURP versus M-TURP. Design, setting, and participants From July 2006 to June 2009, TURP candidates with benign prostatic obstruction were consecutively recruited in four centres, randomised 1:1 into the M-TURP or the B-TURP arm and regularly followed up to 36 mo postoperatively. A total of 295 patients were enrolled. Intervention M-TURP or B-TURP using the AUTOCON II 400 electrosurgical unit. Outcome measurements and statistical analysis Safety was estimated by complication rates with a special emphasis on urethral strictures (US) and bladder neck contractures (BNCs) recorded during the short-term (up to 12 mo) and midterm (up to 36 mo) follow-up. Efficacy quantified by changes in maximum urine flow rate, postvoid residual urine volume, and International Prostate Symptom Score was compared with baseline, and reintervention rates in each arm were also evaluated. Results and limitations A total of 279 patients received treatment after allocation. Mean follow-up was 28.8 mo. A total of 186 of 279 patients (66.7%) completed the 36-mo follow-up. Posttreatment withdrawal rates did not differ significantly between arms. Safety was assessed in 230 patients (82.4%) at a mean follow-up of 33.4 mo. Ten US cases were seen in each arm (M-TURP vs B-TURP: 9.3% vs 8.2%; p =0.959); two versus eight BNC cases (M-TURP vs B-TURP: 1.9% vs 6.6%; p =0.108) were collectively detected at the midterm follow-up. Resection type was not a significant predictor of the risk of US/BNC formation. Efficacy was similar between arms and durable. A total of 10 of 230 patients (4.3%) experienced failure to cure and needed reintervention without significant differences between arms. High overall reintervention rates, withdrawal rates, and sample size determination not based on US/BNC rates represent potential limitations. Conclusions The midterm safety and efficacy of B-TURP and M-TURP are comparable. Trial registration Netherlands Trial Register, NTR703 (http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=703).

What's known on the subject? and What does the study add? The effect of TURP on overall sexual function and particularly erectile function (EF) is controversial with conflicting results based on a low level of evidence. The effects of monopolar and bipolar TURP (M-TURP and B-TURP, respectively) on EF are similar, as has been shown in a few non-focused randomized control trials (RCTs). For the first time, the present study offers focused results of a comparative evaluation of the effects of B-TURP and M-TURP on overall sexual function, as quantified with the International Index of Erectile Function Questionnaire (IIEF-15) in an international, multicentre, double-blind RCT setting. Objective To compare monopolar and bipolar transurethral resection of the prostate (M-TURP and B-TURP, respectively) using a true bipolar system, for the first time in an international multicentre double-blind randomized controlled trial focusing on the overall sexual function quantified with the International Index of Erectile Function Questionnaire (IIEF-15). Other baseline/perioperative parameters potentially influencing erectile function (EF) after TURP were secondarily investigated. Materials and Methods From July 2006 to June 2009, consecutive TURP candidates with benign prostatic obstruction were prospectively recruited in four academic urological centres, randomized 1:1 into M-TURP/B-TURP arms and followed up at 6 weeks, 6 and 12 months after surgery. In all, 295 eligible patients were enrolled. Overall sexual function was quantified using self-administered IIEF-15 at baseline and at each subsequent visit. Total IIEF/domain scores were calculated and EF score classified erectile dysfunction severity. Differences in erectile dysfunction severity at each visit compared with baseline (EF evolution), classified patients into ‘improved’, ‘stable’ or ‘deteriorated’. Pre-postoperative IIEF/domain scores and differences in the distribution of EF evolution were compared between arms throughout follow-up. Results In all, 279 patients received the allocated intervention; 218/279 patients (78.1%) provided complete IIEF-15 data at baseline and were considered in sexual function analysis. Complete IIEF-15 data were available from 193/218 (88.5%), 186/218 (85.3%) and 179/218 (82.1%) patients at 6 weeks, 6 months and 12 months, respectively. Sexual function did not differ significantly between arms during follow-up (scores: IIEF, P = 0.750; EF, P = 0.636; orgasmic function, P = 0.868; sexual desire, P = 0.735; intercourse satisfaction, P = 0.917; overall satisfaction, P = 0.927). Resection type was not a predictor of any sexual function changes observed. Distribution of EF evolution did not differ between arms at any time (M-TURP vs B-TURP at 12 months: improved, 23/87 [26.4%] vs 18/92 [19.6%]; stable, 53/87 [60.9%] vs 56/92 [60.8%]; deteriorated, 11/87 [12.7%] vs 18/92 [19.6%]; P = 0.323). Conclusion There were no differences between M-TURP/B-TURP in any aspect of sexual function.

INTRODUCTION: Percutaneous nephrolithotomy (PNL) is usually performed worldwide with a rigid-only antegrade approach. Daily practice suggests that adding flexible nephroscopy and/or ureteroscopy to conventional rigid PNL might improve its efficacy and safety, but available evidence is weak. Appraisal of reliable outcomes of such PNL techniques would better guide intraoperative choices and optimize surgical results. Therefore our objective was to systematically review relevant literature comparing the outcomes of rigid-only PNL and combined flexible PNLs (adding flexible nephroscopy and/or flexible ureteroscopy) for the treatment of large and/or complex upper urinary tract calculi, with regard to efficacy and safety.EVIDENCE ACQUISITION: Ovid MedLine (R), PubMed, Scopus and Web of Science databases were searched in August 2016 to identify relevant studies. Article selection was performed according to the Preferred Reporting Items for Systematic Review and Meta-analysis criteria.EVIDENCE SYNTHESIS: Six articles reporting on 666 patients were included: two randomized controlled trials, two retrospective comparative studies and two case series > 50 patients (one prospective and one retrospective). A narrative synthesis of minor evidences was also prepared. The adjunct of flexible nephroscopy and/or ureteroscopy provided better stone-free rates (range 86.7-96.97%), through a single percutaneous access most of the times and in any position, reducing the need for second-look procedures. Safety of the combined flexible procedures was improved to a variable degree, with a consensual reduction of the mean hospital stay (range 5.1-7 days).CONCLUSIONS: The current evidence suggests that patients with large and/or complex urolithiasis might benefit from the adjunct of flexible nephroscopy and/or ureteroscopy to rigid PNL.

Osteoporosis is a complication of androgen deprivation therapy (ADT) in men with prostate carcinoma. This is a multicenter, randomized, double-blind prospective study on use of denosumab versus alendronate in the therapy of secondary osteoporosis related to ADT.A total of 234 patients with diagnosis of osteoporosis underwent ADT for prostate cancer were enrolled. Patients were randomly assigned 1:1 to receive denosumab 60 mg subcutaneously every 6 months or alendronate (70 mg/week) for 2 years. All patient received supplemental vitamin D (600 IU/day) and supplemental calcium to maintain a calcium intake of 1200 mg per day. Effectiveness of therapy in both groups (denosumab group and alendronate group) was assessed by changes in bone turnover markers (BTMs), bone mineral density, fracture incidence, Visual Analogue Scale score for back pain, and Short Form-8 health survey score for health-related quality of life.In the denosumab study group, level of BTMs for bone formation were significantly increased from baseline at all time points during the study (P0.001); in the alendronate study group level of BTMs for bone formation were increased too (P0.05). Mean changes in BMD at final follow-up differed significantly between two groups. BMD changes at the lumbar spine at 24 months were 5.6% with denosumab vs. -1.1% with alendronate (P0.001).Denosumab and alendronate showed similar clinical efficacy in the therapy of ADT-related osteoporosis in men with prostate carcinoma; both drugs provided significant improvements in back pain and general health conditions. Denosumab showed significant increase of BTMs and BMD than alendronate with lower rate of new vertebral fractures.

Background Renal cell carcinoma (RCC) is among the ten most common cancers in both men and women. It comprises 2-3% of all malignancies. The most common site for metastasis from RCC is the lung (50% of patients), followed by the skeleton (20% to 50% of patients). Methods We retrospectively reviewed our consecutive series of 63 patients surgically treated for spinal metastases from RCC. The surgical treatment for spinal metastases varied in each patient. Factors influencing the choice of surgery included age, disease status, symptoms, morbidity of the patient, the location, the number of repetitive lesions and the existence of extra-osseous metastases. For 42 patients (66.66%) the treatment included radiation two weeks after the surgical procedure. Results A total of 68 surgical procedures were performed in 63 patients. For all patients we collected data about type of RCC, locations of metastasis, type of spine metastasis, type of surgery and complications. In this way we had an exahustive vision of the natural history of this kind of patients. Conclusions Spine metastatic RCC has a poor prognosis. The favorable prognostic factor is solitary spinal metastasis without involvement of visceral organs where the gold standard treatment is corpectomy en bloc. The negative prognostic factors are the presence of one or more pathological fractures and neurological deficits. Bisphosphonate as zoledronic acid reduce significantly skeletal morbidity and significantly prolong time to bone lesion progression in patients with spine metastases from renal cell carcinoma.

The feasibility of a transvaginal hybrid natural orifice transluminal endoscopic surgery (NOTES) nephrectomy has already been demonstrated using standard laparoscopic ports through the abdominal wall. We evaluated the feasibility of a transvaginal NOTES-assisted minilaparoscopic nephrectomy (mLN). The patient is positioned in a semilumbotomy position with legs separated to allow for vaginal access. A 3.5-mm port is placed at the umbilicus for a 30° laparoscope; two 3.5-mm ports are placed in the flank in the same location as for a standard transperitoneal nephrectomy; and a 12-mm port is placed through the vagina, perforating the vaginal wall. Kidney dissection is performed following the steps of a traditional nephrectomy. The renal pedicle is dissected and secured with Hem-o-Lok clips through the vaginal access port. The specimen is then extracted through an extended incision in the posterior wall of the vagina. We treated five patients. The average operative time was 120 min, blood loss was 160 ml, and no complications were recorded. Our initial experience suggests that transvaginal NOTES-assisted mLN is feasible and appears to be safe. It is simpler than a pure NOTES procedure and ensures excellent cosmetic results.

Study Type – Therapy (RCT) Level of Evidence 1b What’s known on the subject? and What does the study add? Short-term efficacy is similar but B-TURP is preferable due to a more favourable safety. a) first multicentre RCT, b) adequate quality, c) experience with a new bipolar device, d) morbidity standardize using the modified Clavien classification system. OBJECTIVE • To compare the perioperative efficacy and safety of bipolar (B-) and monopolar transurethral resection of the prostate (M-TURP) in an international multicentre double-blind randomized controlled trial using the bipolar system AUTOCON® II 400 ESU for the first time. PATIENTS AND METHODS • From July 2006 to June 2009, consecutive transurethral resection of the prostate (TURP) candidates with benign prostatic obstruction were prospectively recruited in four academic urological centres, randomized 1:1 into an M-TURP or B-TURP arm and followed up for 6 weeks after surgery. • A total of 295 eligible patients were enrolled. • Of these, 279 patients received treatment (M-TURP, n= 138; B-TURP, n= 141) and were analysed for immediate postoperative outcomes and perioperative safety. In all, 268 patients (M-TURP, n= 129; B-TURP, n= 139) were analysed for efficacy, which was quantified using changes in maximum urinary flow rate, postvoid residual urine volume and International Prostate Symptom Score at 6 weeks compared with baseline. Safety was estimated using sodium and haemoglobin level changes immediately after surgery and perioperative complication occurrence graded according to the modified Clavien classification system. • Secondary outcomes included operation-resection time, resection rate, capsular perforation and catheterization time. RESULTS • No significant differences were detected between each study arm except that postoperative decreases in sodium levels favoured B-TURP (–0.8 vs –2.5 mmol/L, for B-TURP and M-TURP, respectively; P= 0.003). The lowest values were 131 mmol/L (B-TURP) and 106 mmol/L (M-TURP). Nine patients ranged between 125 and 130 mmol/L and the values for three patients were

Percutaneous nephrolithotomy (PNL), although practiced for almost 30 years, is still the most appropriate treatment modality for several forms of renal stones. We analysed a number of very recent advances in PNL technique, contributing to the continuous improvement of its efficacy and safety.A thorough review of the recent literature identifies five major progressing areas, inspiring fruitful innovations in PNL technique: imaging (computed tomography being the standard tool) before, during and after the procedure; patient positioning (the traditional prone position being now challenged with the emerging supine positions); endoscopic combined intrarenal surgery (ECIRS), a versatile antero-retrograde approach to the upper urinary tract and a new comprehensive attitude of the urologist toward the various PNL steps; intracorporeal lithotripsy (improved by new devices combining ultrasonic and pneumatic lithotripsy, or ultrasonic and high-power holmium: YAG laser lithotripsy); and post-PNL tube management (namely nephrostomy-free and totally tubeless procedures).Urologists dedicate significant efforts to the improvement of the PNL procedure, with the aim of further increasing its stone-free outcomes and reducing patient morbidity. Large-scale multicentre prospective trials are needed to define benefits and identify possible drawbacks of the described innovations in percutaneous stone management.