A proposal for a new PhD level curriculum on quantitative methods for drug development.

This paper provides an overview of "Improving Design, Evaluation and Analysis of early drug development Studies" (IDEAS), a European Commission-funded network bringing together leading academic institutions and small- to large-sized pharmaceutical companies to train a cohort of graduate-level medical statisticians. The network is composed of a diverse mix of public and private sector partners spread across Europe, which will host 14 early-stage researchers for 36 months. IDEAS training activities are composed of a well-rounded mixture of specialist methodological components and generic transferable skills. Particular attention is paid to fostering collaborations between researchers and supervisors, which span academia and the private sector. Within this paper, we review existing medical statistics programmes (MSc and PhD) and highlight the training they provide on skills relevant to drug development. Motivated by this review and our experiences with the IDEAS project, we propose a concept for a joint, harmonised European PhD programme to train statisticians in quantitative methods for drug development.

Impact of Stress Urinary Incontinence After Radical Prostatectomy on Time to Intervention, Quality of Life and Work Status.

OBJECTIVE: To characterize the incidence of stress urinary incontinence (SUI) after radical prostatectomy (RP), its treatment, and impact on quality of life (QoL) and work status 1year after RP. MATERIALS AND METHODS: Prostate cancer patients treated by RP (1998-2016) were selected from CaPSURE. SUI was defined as any pads per day (ppd) 1 year after RP. SUI procedures were tracked by CPT codes (sling and artificial sphincter). Patients reported work status (full-time, part-time, unpaid), UCLA PCa Index urinary function (UF) and bother (UB) and SF36 Index physical function (PF). Associations of incontinence with UF, UB, and PF and work status changes were assessed (ANOVA). Lifetable estimates and Cox proportional hazards regression evaluated risk of undergoing SUI procedures. RESULTS: 664/2989 (22%) men treated with RP reported SUI at 1 year. More men with SUI had ≥GG2, intermediate to high-risk disease and non-nerve-sparing surgery (all P < .01). Cumulative incidence of SUI procedures was 1.4% at 10years after RP. Age (HR 2.68 per 10years, 95% CI 1.41-5.08) and number of ppd at 1 year (HR 3.20, 95% CI 2.27-4.50) were associated with undergoing SUI procedures. UF declined at 1year after RP, while UB and PF remained stable. UF, UB, and PF were inversely associated with number of ppd (all P < .01). Change in work status was not associated with incontinence or QoL scores. CONCLUSION: Incontinence affected QoL without impacting work status, suggesting that men with SUI after RP may continue working and go under-treated despite impact on QoL.

Association Between Common Urologic Medications and Onset of Alzheimer's Disease and Related Dementias in Men With Prostate Cancer Managed by Different Primary Treatment Modalities.

OBJECTIVE: To understand the relationship between common urologic medications phosphodiesterase-5 inhibitors (PDE5i) and anticholinergics (AC) and risk of dementia onset in men who underwent different primary treatments for prostate cancer. MATERIALS AND METHODS: Patients (>50years) with prostate cancer (1998-2022) without Alzheimer's disease or related dementias were selected from Cancer of the Prostatic Strategic Urologic Research Endeavor Registry. Minimum medication use was 3months. Fine-Gray regression was performed to determine the association between medication exposure and dementia onset ≥12months after primary treatment in men matched on age, race, comorbid conditions, smoking, and type of clinical site, with competing risk of death. RESULTS: Among 5937 men (53% PDE5i; 14% AC), PDE5i users were younger (63 vs 70, P < .01) with less CAD, CVA, DM (all P < .01); AC users were older (68 vs 66, P < .01) with higher incidence of comorbidities (P < .01). Median months of use was 24.3 (IQR 12.1, 48.7) for PDE5i and 12.2 (IQR 6.1, 24.3) for AC users. Cumulative incidence of Alzheimer's disease or related dementias was 6.5% at 15years. PDE5i (P = .07) and AC (P = .06) were not associated with dementia regardless of primary treatment modality. CONCLUSION: In this retrospective cohort study, PDE5i and AC use do not appear independently associated with risk of dementia. Notably, our cohort was generally healthy and younger which may limit our ability to detect significance. We recommend prospective investigation into association between PDE5i and dementia and advise continued judicious stewardship of AC in older patient populations.

The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42.

BACKGROUND: Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF. METHODS: Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation. RESULTS: Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16. CONCLUSION: These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.

Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies.

BACKGROUND: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells. METHODS: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action. RESULTS: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells. CONCLUSIONS: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

Long-term follow-up of urethral reconstruction for blunt urethral injury at a young age: urinary and sexual quality of life outcomes.

INTRODUCTION: Little is known about long-term patient-reported outcomes following surgical repair for pediatric blunt urethral trauma. OBJECTIVE: The purpose was to evaluate long-term urinary outcomes, sexual function, and quality of life (QOL) of patients who undergo urethroplasty for blunt urethral trauma in childhood. STUDY DESIGN: After IRB approval, we retrospectively reviewed the records of patients who sustained blunt urethral injury at ≤18 years and underwent urethroplasty at our institution between 1978 and 2013. We then used a web-based survey to assess urinary/sexual/ejaculatory function and overall QOL using validated questionnaires. RESULTS: Of 68 eligible patients, 15 were able to be contacted (table). Median age of injury, age at urethroplasty, and age at follow-up were 17 (4-18), 17 (5-20), and 19 (13.5-21.5) years, respectively. The stricture was membranoprostatic in eight and bulbar in seven patients, with median length of 2 (1.6-2.6) cm. Excision/primary anastomosis was performed in all but three patients who required a buccal graft. Overall, 80% were 'very satisfied' and 20% were 'satisfied' with surgery. One patient reported a subsequent urethral intervention. On urethral stricture surgery patient-reported outcome measurement, the median bother (0 least, 24 worst) was 10 (8-12.5). The force of urine stream (1 strongest, 4 weakest) was 2 (1.5-2), with no report of urinary incontinence. The median Sexual Health Inventory for Men score (0 worst, 25 perfect) was 24 (22.5-24). The median ejaculatory function score (0 worst, 15 normal) was 14 (13-14.75). Six patients had fathered children and none reported infertility. Three patients reported <30° penile curvature not interfering with sex. Median QOL (0 worse, 10 best) was 8 (7.5-8). CONCLUSIONS: Urethroplasty after blunt urethral injury in young adult population is associated with a high long-term success rate with a low rate of long-term urinary and sexual consequences in adulthood.

Defining consensus opinion to develop randomised controlled trials in rare diseases using Bayesian design: An example of a proposed trial of adalimumab versus pamidronate for children with CNO/CRMO.

INTRODUCTION: Chronic nonbacterial osteomyelitis (CNO) is a rare autoinflammatory bone disorder primarily affecting children and adolescents. It can lead to chronic pain, bony deformities and fractures. The pathophysiology of CNO is incompletely understood. Scientific evidence suggests dysregulated expression of pro- and anti-inflammatory cytokines to be centrally involved. Currently, treatment is largely based on retrospective observational studies and expert opinion. Treatment usually includes nonsteroidal anti-inflammatory drugs and/or glucocorticoids, followed by a range of drugs in unresponsive cases. While randomised clinical trials are lacking, retrospective and prospective non-controlled studies suggest effectiveness of TNF inhibitors and bisphosphonates. The objective of the Bayesian consensus meeting was to quantify prior expert opinion. METHODS: Twelve international CNO experts were randomly chosen to be invited to a Bayesian prior elicitation meeting. RESULTS: Results showed that a typical new patient treated with pamidronate would have an 84% chance of improvement in their pain score relative to baseline at 26 weeks and an 83% chance on adalimumab. Experts thought there was a 50% chance that a new typical patient would record a pain score of 28mm (pamidronate) to 30mm (adalimumab) or better at 26 weeks. There was a modest trend in prior opinion to indicate an advantage of pamidronate vs adalimumab, with a 68% prior chance that pamidronate is superior to adalimumab by some margin. However, it is clear that there is considerable uncertainty about the precise relative merits of the two treatments. CONCLUSIONS: The rarity of CNO leads to challenges in conducting randomised controlled trials with sufficient power to provide a definitive outcome. We address this using a Bayesian design, and here describe the process and outcome of the elicitation exercise to establish expert prior opinion. This opinion will be tested in the planned prospective CNO study. The process for establishing expert consensus opinion in CNO will be helpful for developing studies in other rare paediatric diseases.

"Threshold-crossing": A Useful Way to Establish the Counterfactual in Clinical Trials?

A central question in the assessment of benefit/harm of new treatments is: how does the average outcome on the new treatment (the factual) compare to the average outcome had patients received no treatment or a different treatment known to be effective (the counterfactual)? Randomized controlled trials (RCTs) are the standard for comparing the factual with the counterfactual. Recent developments necessitate and enable a new way of determining the counterfactual for some new medicines. For select situations, we propose a new framework for evidence generation, which we call "threshold-crossing." This framework leverages the wealth of information that is becoming available from completed RCTs and from real world data sources. Relying on formalized procedures, information gleaned from these data is used to estimate the counterfactual, enabling efficacy assessment of new drugs. We propose future (research) activities to enable "threshold-crossing" for carefully selected products and indications in which RCTs are not feasible.

A minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.

INTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.

Rational management of malignant colon polyps based on long-term follow-up.

We reviewed the long-term results of management of 38 patients with carcinoma in colorectal polyps. Of these, 16 patients demonstrated malignant invasion of the lamina propria but not the muscularis mucosa (group I), and 22 patients showed malignant invasion of the muscularis mucosa (group II). Primary therapy for group I patients consisted of polypectomy in 12, local excision in one, and colonic resection in three. One patient had a subsequent abdominal-perineal resection and was found to have no residual disease and no lymph node involvement. Follow-up of the group I patients showed that 11 were alive and well (mean 5.8 years) and five died of unrelated causes (mean 5.2 years). Of group II patients, 12 underwent polypectomy, six local excision, and four colectomy. Of these 22 patients, 11 underwent further operation, including nine major bowel resections and two local re-excisions. None of these 11 patients had either residual tumor or lymph node metastases. One patient died of complications after abdominal-perineal resection. Follow-up showed that 18/22 group II patients were alive and well 5 to 15 years later (mean 7.5 years); four died of unrelated causes (mean 3.2 years). We then reviewed another group of 220 patients who had undergone resection for invasive colon cancer to relate the presence or absence of lymph node metastases to the depth of malignant invasion in the bowel wall. We found that 44% of this entire group had lymph node involvement. Of 36 patients with tumor confined to the bowel wall, nodal metastases occurred in only 22%. Of eight patients with malignancy superficial to the muscularis propria, only one had nodal involvement. We conclude that colon cancer tends to progress in an orderly fashion and the risk of nodal metastases increases with the depth of invasion. Carcinoma in a polyp represents a very early stage of colon cancer. We therefore recommend polypectomy as primary treatment for pedunculated polyps containing carcinoma either superficial to or invading muscularis mucosa. If histologic review demonstrates incomplete excision, lymphatic invasion, or poor differentiation, patients with lesions invading the muscularis mucosa should undergo formal colonic resection.

Gastric carcinoma: intestinal metaplasia and tumor growth patterns as indicators of prognosis.

We have reviewed 200 cases of gastric carcinoma treated between 1970 and 1980 to assess the value of intestinal metaplasia in the stomach and tumor growth patterns in determining prognosis. Intestinal metaplasia was found to be more frequently associated with early gastric tumors, expanding-type tumors, and tumors located in the antrum. The survival rate was 53% with intestinal metaplasia and 34% without. Sixty-three percent of expanding tumors with metaplasia survived. If the lymph nodes were not involved, the survival rate with metaplasia was 81%. We conclude that intestinal metaplasia and growth patterns are valuable in predicting outcome. Preoperative evaluation of gastric tumors should include multiple endoscopic mucosal biopsy specimens. If intestinal metaplasia is present, the improved possibility of survival should influence the surgeon in the choice of operative treatment.

Prognosis in colon cancer: a pathologic reassessment.

One hundred forty-eight cases of colon carcinoma were subjected to further pathologic study. Survival was correlated with stage and grade of the tumor and with the number of involved lymph nodes. In addition, cases were assessed as to the extent of local chronic inflammatory reaction about the lesion and the degree of sinus histiocytosis in draining lymph nodes. A correlation was possible between grading, staging, extent of lymph node involvement, and survival. A substantial difference in five-year survival was shown when local inflammatory reaction was present and when sinus histiocytosis was observed. The presence of both of these factors further improved survival. An adequate evaluation of these factors, both individually and in combination, should improve our ability to assess prognosis in colon cancer.

Mortality and prognosis of obstructing carcinoma of the large bowel.

In a series of 760 patients with adenocarcinoma of the colon and rectum, 103 patients presented with acute obstruction requiring urgent surgical decompression. Obstructed patients were slightly older and had slightly more advanced tumors than the total group. Obstructing lesions of the left colon treated primarily by staged procedures had relatively low mortality and five year survival figures comparable to unobstructed cases. Obstructing lesions of the right colon had a much poorer absolute five year survival rate, mainly because of the high operative mortality associated with primary resection in our institution. Five year survival after curative resection in patients with obstructing tumors of the right colon was considerably less than in patients with nonobstructing tumors. A suggestion is made for consideration of proximal external bowel decompression in association with resection of the right colon.

The role of HPV type in Recurrent Respiratory Papillomatosis.

OBJECTIVE: Human Papillomavirus (HPV) 6 and 11 are the aetiological agents responsible for Recurrent Respiratory Papillomatosis (RRP). There is general consensus that HPV11 results in more aggressive disease compared to HPV6. METHOD: Pubmed was searched using the terms respiratory papillomatosis, HPV 6 and HPV11. Comparisons were made in the outcomes of HPV6 versus HPV11 positive RRP disease. RESULTS: There are numerous sub-types or variants of both HPV6 and HPV11. These sub-types have different activities at least in-vitro. The numbers of different HPV types within RRP tissue may be more extensive than initially appeared. This depends specifically upon the HPV types tested for. CONCLUSION: The clinical differences between HPV6 and HPV11 disease may not be accurately predictable as these viruses exist in numerous sub-types. Also, RRP tissue may contain more than one subtype or even be co-infected with other viruses that may influence outcome. In-vitro studies upon cell lines are a reasonable starting point for evaluation of these differences.