Androgen receptor enhancer amplification in matched patient-derived xenografts of primary and castrate-resistant prostate cancer.

Amplifications of the androgen receptor (AR) occur in up to 80% of men with castration-resistant prostate cancer (CRPC). Recent studies highlighted that these amplifications not only span the AR gene but usually encompass a distal enhancer. This represents a newly recognised, non-coding mechanism of resistance to AR-directed therapies, including enzalutamide. To study disease progression before and after AR amplification, we used tumour samples from a castrate-sensitive primary tumour and castrate-resistant metastasis of the same patient. For subsequent functional and genomic studies, we established serially transplantable patient-derived xenografts (PDXs). Whole genome sequencing showed that alterations associated with poor prognosis, such as TP53 and PTEN loss, existed before androgen deprivation therapy, followed by co-amplification of the AR gene and enhancer after the development of metastatic CRPC. The PDX of the primary tumour, without the AR amplification, was sensitive to AR-directed treatments, including castration, enzalutamide, and apalutamide. The PDX of the metastasis, with the AR amplification, had higher AR and AR-V7 expression in castrate conditions, and was resistant to castration, apalutamide, and enzalutamide in vivo. Treatment with a BET inhibitor outperformed the AR-directed therapies for the metastasis, resulting in tumour regression for some, but not all, grafts. Therefore, this study provides novel matched PDXs to test potential treatments that target the overabundance of AR in tumours with AR enhancer amplifications. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Establishing a cryopreservation protocol for patient-derived xenografts of prostate cancer.

BACKGROUND: Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. METHODS: One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. RESULTS: Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. CONCLUSION: This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the practicality of maintaining PDX models. Cryopreservation may also increase the accessibility of these important resources and provide new opportunities for preclinical studies on a broader spectrum of prostate tumors.

Establishment of primary patient-derived xenografts of palliative TURP specimens to study castrate-resistant prostate cancer.

BACKGROUND: Fresh patient specimens of castrate-resistant prostate cancer (CRPC) are invaluable for studying tumor heterogeneity and responses to current treatments. They can be used for primary patient-derived xenografts (PDXs) or serially transplantable PDXs, but only a small proportion of samples grow successfully. To improve the efficiency and quality of PDXs, we investigated the factors that determine the initial engraftment of patient tissues derived from TURP specimens. METHODS: Fresh tissue was collected from castrate patients who required a TURP for urinary symptoms. Tissue was grafted under the renal capsule of immune-compromised mice for up to 14 weeks. The abundance of cancer in ungrafted and grafted specimens was compared using histopathology. Mice were castrated or implanted with testosterone pellets to determine the androgen-responsiveness of CRPC PDXs from TURP tissue. RESULTS: Primary PDXs were successfully established from 7 of 10 patients that underwent grafting. Of the 112 grafts generated from these 10 patients, 21% contained cancer at harvest. Grafts were most successful when the original patient specimens contained high amounts of viable cancer, defined as samples with (i) at least 50% cancer cells, (ii) no physical damage, and (iii) detectable Ki67 expression. PDX grafts survived in castrated hosts and proliferated in response to testosterone, confirming that they were castrate resistant but androgen-responsive. CONCLUSIONS: Primary PDXs of CRPC can be established from TURP specimens with modest success. The take rate can be increased if the original tissues contain sufficient numbers of actively proliferating cancer cells. Selecting specimens with abundant viable cancer will maximize the rate of engraftment and increase the efficiency of establishing PDXs that can be serially transplanted.

68 Ga-prostate-specific membrane antigen (PSMA) PET/CT as a clinical decision-making tool in biochemically recurrent prostate cancer.

OBJECTIVE: PSMA PET/CT has demonstrated superior sensitivity over conventional imaging in the detection of local and distant recurrence in biochemically relapsed (BCR) prostate cancer. We prospectively investigated the management impact of 68 Ga-PSMA PET/CT imaging in men with BCR, with the aim of identifying baseline clinicopathological predictors for management change. PATIENTS AND METHODS: Men with BCR who met eligibility criteria underwent 68 Ga-PSMA-11 PET/CT at Monash Health (Melbourne, Australia). Intended management plans were prospectively documented before and after 68 Ga-PSMA PET/CT imaging. Binary logistic regression analysis was performed to identify potential clinicopathological predictors of management change. Descriptive statistics were used to characterize the nature of these changes. RESULTS: Seventy men underwent 68 Ga-PSMA-11 PET/CT imaging. Median age was 67 years (IQR 63-72) and median PSA was 0.48 ng/ml (IQR 0.21-1.9). PSMA-avid disease was observed in 56% (39/70) of patients. Pre-scan management plan was altered following scanning in 43% (30/70) of patients. Management changes were significantly more common in patients with higher baseline PSA levels (PSA≥2 ng/ml, p = 0.01). 18/36 (50%) of the patients initially planned for watchful waiting had their management changed, including the use of salvage pelvic radiotherapy (n = 7) and stereotactic ablative body radiotherapy to oligometastatic disease (n = 6). CONCLUSION: Management change after 68 Ga-PSMA PET/CT for BCR is common and typically resulted in treatment intensification strategies in those planned for a watchful waiting approach. This study adds to the growing pool of evidence supporting the clinical utility of PSMA PET/CT imaging in the care of patients with BCR after definitive therapy.

Urolithiasis--medical therapies.

UNLABELLED: This review paper provides a summary of medical therapies available for urolithiasis. The summary includes general medical advice, prophylactic medications, dissolution therapy and medical expulsion therapy. The paper is designed to provide a management strategy for all physicians who treat urolithiasis, from general practitioners, to emergency physicians, to urologists. OBJECTIVE: • To provide an up to date review of the literature in relation to the medical management of stone disease. This will encompass prophylaxis, dissolution therapy and medical expulsion therapy. PATIENTS AND METHODS: • First-time stone formers do not regularly have a full urine and electrolyte evaluation due to the low incidence of a reversible metabolic cause. • However, stone disease is common and over a lifetime urolithiasis can affect up to 10-15% of the population. RESULTS: • Medical management of stone disease encompasses preventative measures, medical dissolution and medical expulsion therapy. CONCLUSIONS: • Recurrent stone formers should have dietary optimization to decrease the risk of further stones. • Furthermore, the correct use of prophylactic and therapeutic medications can decrease the morbidity associated with ureteric calculi.

The MURAL collection of prostate cancer patient-derived xenografts enables discovery through preclinical models of uro-oncology.

Preclinical testing is a crucial step in evaluating cancer therapeutics. We aimed to establish a significant resource of patient-derived xenografts (PDXs) of prostate cancer for rapid and systematic evaluation of candidate therapies. The PDX collection comprises 59 tumors collected from 30 patients between 2012-2020, coinciding with availability of abiraterone and enzalutamide. The PDXs represent the clinico-pathological and genomic spectrum of prostate cancer, from treatment-naïve primary tumors to castration-resistant metastases. Inter- and intra-tumor heterogeneity in adenocarcinoma and neuroendocrine phenotypes is evident from bulk and single-cell RNA sequencing data. Organoids can be cultured from PDXs, providing further capabilities for preclinical studies. Using a 1 x 1 x 1 design, we rapidly identify tumors with exceptional responses to combination treatments. To govern the distribution of PDXs, we formed the Melbourne Urological Research Alliance (MURAL). This PDX collection is a substantial resource, expanding the capacity to test and prioritize effective treatments for prospective clinical trials in prostate cancer.

Patient-derived Models of Abiraterone- and Enzalutamide-resistant Prostate Cancer Reveal Sensitivity to Ribosome-directed Therapy.

BACKGROUND: The intractability of castration-resistant prostate cancer (CRPC) is exacerbated by tumour heterogeneity, including diverse alterations to the androgen receptor (AR) axis and AR-independent phenotypes. The availability of additional models encompassing this heterogeneity would facilitate the identification of more effective therapies for CRPC. OBJECTIVE: To discover therapeutic strategies by exploiting patient-derived models that exemplify the heterogeneity of CRPC. DESIGN, SETTING, AND PARTICIPANTS: Four new patient-derived xenografts (PDXs) were established from independent metastases of two patients and characterised using integrative genomics. A panel of rationally selected drugs was tested using an innovative ex vivo PDX culture system. INTERVENTION: The following drugs were evaluated: AR signalling inhibitors (enzalutamide and galeterone), a PARP inhibitor (talazoparib), a chemotherapeutic (cisplatin), a CDK4/6 inhibitor (ribociclib), bromodomain and extraterminal (BET) protein inhibitors (iBET151 and JQ1), and inhibitors of ribosome biogenesis/function (RNA polymerase I inhibitor CX-5461 and pan-PIM kinase inhibitor CX-6258). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Drug efficacy in ex vivo cultures of PDX tissues was evaluated using immunohistochemistry for Ki67 and cleaved caspase-3 levels. Candidate drugs were also tested for antitumour efficacy in vivo, with tumour volume being the primary endpoint. Two-tailed t tests were used to compare drug and control treatments. RESULTS AND LIMITATIONS: Integrative genomics revealed that the new PDXs exhibited heterogeneous mechanisms of resistance, including known and novel AR mutations, genomic structural rearrangements of the AR gene, and a neuroendocrine-like AR-null phenotype. Despite their heterogeneity, all models were sensitive to the combination of ribosome-targeting agents CX-5461 and CX-6258. CONCLUSIONS: This study demonstrates that ribosome-targeting drugs may be effective against diverse CRPC subtypes including AR-null disease, and highlights the potential of contemporary patient-derived models to prioritise treatment strategies for clinical translation. PATIENT SUMMARY: Diverse types of therapy-resistant prostate cancers are sensitive to a new combination of drugs that inhibit protein synthesis pathways in cancer cells.

Modified supine versus prone percutaneous nephrolithotomy: Surgical outcomes from a tertiary teaching hospital.

PURPOSE: The traditional prone positioning of percutaneous nephrolithotomy (PCNL) is associated with various anesthetic and logistic difficulties. We aimed to compare the surgical outcomes of PCNLs performed using our modified supine position with those performed in the standard prone position. MATERIALS AND METHODS: A prospective group of 236 renal units (224 patients) undergoing PCNL were included in this 2 site study: 160 were performed in the modified supine position were compared with 76 undergoing PCNL in the prone position. The outcomes of radiation dose, radiation time, stone free rate, body mass index (BMI), stone size, operative time, length of stay (LOS), in hospital and complications were compared. Chi-square and t-tests were used. RESULTS: There were no significant differences in mean radiation time, radiation dose or stone size between the modified supine and prone groups. The supine group had a higher mean BMI (31 kg/m(2) vs. 28 kg/m(2), p=0.03), shorter mean surgical time (93 minutes vs. 123 minutes, p<0.001), shorter mean LOS (2 days vs. 3 days, p=0.005) and higher stone free rate (70% vs. 50%, p=0.005). There were no differences in septic or bleeding complications but the prone group had a higher rate of overall complications. CONCLUSIONS: Modified supine PCNL has significantly lower operative time, shorter LOS and higher stone-free rate compared with prone in our series, while remaining a safe procedure.

Endoscopic management of symptomatic caliceal diverticula: a retrospective comparison of percutaneous nephrolithotripsy and ureteroscopy.

BACKGROUND AND PURPOSE: A myriad of minimally invasive options exist for managing symptomatic caliceal diverticula, including shockwave lithotripsy, percutaneous surgery, retrograde ureteroscopy, and laparoscopy. Yet no direct comparisons have been made in the literature of the relative treatment efficacy of ureteroscopy (URS) and percutaneous nephrolithotripsy (PNL). A retrospective review of our patients was performed to determine the most appropriate endoscopic management option for patients with symptomatic caliceal diverticula. PATIENTS AND METHODS: Between November of 1994 and April 2001, 39 patients presented with symptomatic caliceal diverticula, 37 of which contained calculi. Twenty-two patients (56%) underwent PNL, and 17 patients (44%) were managed by URS. Of the PNL group, 82% required the creation of a neoinfundibulotomy. The stone burden in the PNL group averaged 11.4 x 12.0 mm and that in the URS group 12.7 x 13.0 mm (p > 0.05). Pain, recurrent urinary tract infections, and nausea and vomiting were the presenting complaints in both subgroups of patients, with pain being by far the most common symptom. The average hospital stay was 2.8 days for the PNL group. All the URS procedures were performed on a same-day-surgery basis. Results, including stone-free, symptom-free, and complication rates, were compared for the two groups. RESULTS: Thirty-five percent of the URS group were symptom free at 6 weeks' follow-up, with an additional 29% reporting an improvement in pain, whereas 86% of the PNL group was completely symptom free at 6 weeks' follow-up. Only 19% of the URS group were stone free on follow-up intravenous urography v 78% of those undergoing PNL (three patients failed to return for follow-up imaging). It was not possible to identify the ostium of the stenotic infundibulum in 4 patients (24%) undergoing URS, and 7 patients (41%) eventually went on to PNL with ultimate success. The PNL was statistically better than URS in producing stone-free results for diverticula located in the upper pole and for stones <11 mm (p < 0.05). No complications occurred in the URS group; however, complications were identified in four patients after PNL. One patient developed clot urinary retention necessitating Foley catheterization and manual bladder irrigation; one patient experienced significant bleeding necessitating early cessation of the procedure. Two patients sustained intrathoracic complications, one a pneumothorax and the other a pneumohemothorax after supra-11(th) rib access. Both were managed successfully with tube thoracostomy. CONCLUSIONS: Our review clearly suggests an advantage of percutaneous management over ureteroscopy for complex posterior symptomatic caliceal diverticula, although with a slightly increased risk of complications. Therefore, PNL should be considered the primary modality for managing these difficult processes. In cases where the stenotic infundibulum cannot be traversed with a guidewire, creation of a neoinfundibulotomy permitted secure access to the collecting system while providing effective results.

Routine ureteral stenting is not necessary after ureteroscopy and ureteropyeloscopy: a randomized trial.

BACKGROUND AND PURPOSE: Retrospective studies have suggested that routine stenting can be avoided following ureteroscopy. We prospectively analyzed the need for routine ureteral stent placement in patients undergoing ureteroscopic procedures. PATIENTS AND METHODS: Fifty-five consecutive patients (60 renal units) were randomized into either a stent or a no-stent group following ureteroscopy with either a 7.5F semirigid or a 7.5F flexible ureteroscope for treatment of calculi (holmium laser or pneumatic lithotripsy) or transitional-cell carcinoma (holmium laser). Intraoperative variables assessed included total stone burden, the need for ureteral dilation, and overall operative times. All patients were evaluated by questionnaire on postoperative days 0, 1, and 6 with regard to pain, frequency, urgency, dysuria, and hematuria. RESULTS: Of the 60 renal units treated, 38 received ureteral stents (mean 5.2 days), and 22 were treated without a stent. All 10 patients requiring ureteral balloon dilation had stents placed and were removed from the analysis. There was no significant difference between the groups with regard to age, sex, or stone burden. Operative time was decreased in the no-stent group (43 minutes v 55 minutes; P = 0.013). Flank discomfort was significantly less common in the no-stent group on days 0, 1, and 6 (P = 0.004, P = 0.003, P < 0.001, respectively), as was the incidence of suprapubic pain on day 6 (P = 0.002). There was no difference in urinary frequency, urgency, or dysuria between the groups on postoperative day 1, but all these symptoms were significantly reduced in the no-stent group on day 6 (P < 0.001, P < 0.001, P = 0.002, respectively). There was no significant difference in patient-reported postoperative hematuria in either group. One patient in each group developed a urinary tract infection. One patient in the no-stent group developed ureteral obstruction in the postoperative period that necessitated stenting, and one patient in the stent group experienced stent migration necessitating removal. CONCLUSIONS: Routine ureteral stenting does not appear to be warranted in those patients who do not require ureteral dilation during ureteroscopic procedures. Ureteral stent placement following ureteroscopy may be avoided, thereby reducing operative time, surgical costs, and patient morbidity.

Neoinfundibulotomy for the management of symptomatic caliceal diverticula.

PURPOSE: Direct percutaneous access provides effective treatment for complex caliceal diverticula. Yet, access into the diverticulum alone is usually tenuous and passage of a guide wire across a stenotic infundibulum is often impossible. An alternative technique is described which creates a "neoinfundibulum" to assist in the management of symptomatic caliceal diverticula. MATERIALS AND METHODS: During a 6-year period 22 patients with symptomatic caliceal diverticula were treated via a percutaneous approach, of whom 21 had calculi within the diverticula. After accessing the diverticulum directly, it was impossible to pass a guide wire through the stenotic infundibulum in 18 (82%) patients, prompting advancement of the access needle through the diverticular wall into the renal pelvis. Once secure access was established, balloon dilation was performed to 30Fr to create the "neoinfundibulum." Percutaneous ultrasonic lithotripsy was performed in the usual fashion. A 22Fr Councill catheter was placed to keep the infundibular tract open for 5 to 7 days to allow complete epithelialization and drainage. Stone-free, symptom-free and complication rates were assessed. RESULTS: Pain, recurrent urinary tract infections and hematuria were the presenting complaints in the subgroup of patients undergoing "neoinfundibulotomy." Average stone burden was 11.7 x 12 mm. and average hospital stay was 2.8 days. Of the patients 94% were symptom-free at 6-week followup, and 80% were stone-free on followup excretory urography. The remaining patients had residual stone fragments less than 3 mm. in diameter. Complications related to access were identified in 2 patients who sustained a pneumothorax after a supra-11th rib access, which was successfully managed with tube thoracostomy. CONCLUSIONS: Percutaneous management of complex caliceal diverticula provides a safe and effective option for symptomatic patients. When the stenotic infundibulum cannot be traversed with a guide wire, creation of a new infundibulum offers a secure alternative for accessing the collecting system, while providing equally effective results.