PROTACs Targeting BRM (SMARCA2) Afford Selective In Vivo Degradation over BRG1 (SMARCA4) and Are Active in BRG1 Mutant Xenograft Tumor Models.

The identification of VHL-binding proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) in SW1573 cell-based experiments is described. These molecules exhibit between 10- and 100-fold degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). They also selectively impair the proliferation of the H1944 "BRG1-mutant" NSCLC cell line, which lacks functional BRG1 protein and is thus highly dependent on BRM for growth, relative to the wild-type Calu6 line. In vivo experiments performed with a subset of compounds identified PROTACs that potently and selectively degraded BRM in the Calu6 and/or the HCC2302 BRG1 mutant NSCLC xenograft models and also afforded antitumor efficacy in the latter system. Subsequent PK/PD analysis established a need to achieve strong BRM degradation (>95%) in order to trigger meaningful antitumor activity in vivo. Intratumor quantitation of mRNA associated with two genes whose transcription was controlled by BRM (PLAU and KRT80) also supported this conclusion.

Identification of GDC-1971 (RLY-1971), a SHP2 Inhibitor Designed for the Treatment of Solid Tumors.

Protein tyrosine phosphatase SHP2 mediates RAS-driven MAPK signaling and has emerged in recent years as a target of interest in oncology, both for treating with a single agent and in combination with a KRAS inhibitor. We were drawn to the pharmacological potential of SHP2 inhibition, especially following the initial observation that drug-like compounds could bind an allosteric site and enforce a closed, inactive state of the enzyme. Here, we describe the identification and characterization of GDC-1971 (formerly RLY-1971), a SHP2 inhibitor currently in clinical trials in combination with KRAS G12C inhibitor divarasib (GDC-6036) for the treatment of solid tumors driven by a KRAS G12C mutation.

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC-MS/MS Approach.

KRAS is one of the most frequently mutated oncogenes, with KRAS G12C recently becoming an actionable target for small molecule intervention. GDC-6036 is an investigational KRAS G12C inhibitor that acts by irreversibly binding to the switch II pocket of KRAS G12C when in the inactive GDP-bound state, thereby blocking GTP binding and activation. Assessing target engagement is an essential component of clinical drug development, helping to demonstrate mechanistic activity, guide dose selection, understand pharmacodynamics as it relates to clinical response, and explore resistance. Here, we report the development of an ultra-sensitive approach for assessing KRAS G12C engagement. Immunoaffinity enrichment with a commercially available anti-RAS antibody was combined with a targeted 2D-LC-MS/MS technique to quantify both free and GDC-6036-bound KRAS G12C proteins. A KRAS G12C-positive non-small cell lung cancer xenograft model was dosed with GDC-6036 to assess the feasibility of this assay for analyzing small core needle biopsies. As predicted, dose-dependent KRAS G12C engagement was observed. To date, a sensitivity of 0.08 fmol/µg of total protein has been achieved for both free and GDC-6036-bound KRAS G12C with as little as 4 µg of total protein extracted from human tumor samples. This sub-fmol/µg level of sensitivity provides a powerful potential approach to assess covalent inhibitor target engagement at the site of action using core needle tumor biopsies from clinical studies.

No decrease in incidence of arthroscopic meniscectomy in a Canadian province.

PURPOSE: Arthroscopic meniscectomy (APM) is the most common procedure in orthopedic surgery, despite increasing evidence questioning its benefit over conservative management for treatment of degenerative meniscal tears. The purpose of this study is to determine the epidemiology and trends of APM in Saskatchewan, a Canadian province, over a 20 year period. METHODS: Physician billing codes were used to identify patients who underwent APM in Saskatchewan between January 1, 1998 and December 31, 2017. Records were obtained from eHealth Saskatchewan, a provincial health database. Data was analyzed for overall incidence and age-specific trends of APM. RESULTS: A total of 35,099 APMs were performed during the study period. The population of Saskatchewan ranged from 992,314 to 1,150,782 (median 1,017,368) during this time interval, with 81 orthopedic surgeons performing APM. Overall incidence rate of APM did not change significantly over time. No decrease was observed in patients presumed to have degenerative tears (≥ 50 years). The number of meniscectomies in patients ≥ 50 years was significantly greater during the second decade of study compared to the first (OR 1.48, p < 0.01). Conversely, the increase in incidence rate among older patients was not statistically significant (R2 = 0.125, n.s.). CONCLUSION: Overall incidence rate of APM in Saskatchewan has not decreased during the last 20 years. Furthermore, APM frequency increased over time for individuals ≥ 50 years. Several regional factors may have contributed to these findings, including the large proportion of Saskatchewan residents engaged in physically demanding work and barriers to accessing physiotherapy services. Given recent evidence disputing the benefit of APM over conservative measures, this study highlights the need for improved dissemination of evidence, as well as the importance of an individualized treatment plan to address patient-specific factors. LEVEL OF EVIDENCE: Level IV.

Timing of syrinx reduction and stabilization after posterior fossa decompression for pediatric Chiari malformation type I.

OBJECTIVE: The aim of this study was to determine the timeline of syrinx regression and to identify factors mitigating syrinx resolution in pediatric patients with Chiari malformation type I (CM-I) undergoing posterior fossa decompression (PFD). METHODS: The authors conducted a retrospective review of records from pediatric patients (< 18 years old) undergoing PFD for the treatment of CM-I/syringomyelia (SM) between 1998 and 2015. Patient demographic, clinical, radiological, and surgical variables were collected and analyzed. Radiological information was reviewed at 4 time points: 1) pre-PFD, 2) within 6 months post-PFD, 3) within 12 months post-PFD, and 4) at maximum available follow-up. Syrinx regression was defined as ≥ 50% decrease in the maximal anteroposterior syrinx diameter (MSD). The time to syrinx regression was determined using Kaplan-Meier analysis. Multivariate analysis was conducted using a Cox proportional hazards model to determine the association between preoperative, clinical, and surgery-related factors and syrinx regression. RESULTS: The authors identified 85 patients with CM-I/SM who underwent PFD. Within 3 months post-PFD, the mean MSD regressed from 8.1 ± 3.4 mm (preoperatively) to 5.6 ± 2.9 mm within 3 months post-PFD. Seventy patients (82.4%) achieved ≥ 50% regression in MSD. The median time to ≥ 50% regression in MSD was 8 months (95% CI 4.2-11.8 months). Using a risk-adjusted multivariable Cox proportional hazards model, the patients who underwent tonsil coagulation (n = 20) had a higher likelihood of achieving ≥ 50% syrinx regression in a shorter time (HR 2.86, 95% CI 1.2-6.9; p = 0.02). Thirty-six (75%) of 45 patients had improvement in headache at 2.9 months (IQR 1.5-4.4 months). CONCLUSIONS: The maximum reduction in syrinx size can be expected within 3 months after PFD for patients with CM-I and a syrinx; however, the syringes continue to regress over time. Tonsil coagulation was associated with early syrinx regression in this cohort. However, the role of surgical maneuvers such as tonsil coagulation and arachnoid veil identification and sectioning in the overall role of CM-I surgery remains unclear.

Risk of Periprosthetic Joint Infection in Patients With Multiple Arthroplasties.

BACKGROUND: Risk of subsequent periprosthetic joint infection (PJI) in a second prosthetic joint following initial PJI has been shown to be 19%-20%. We sought to identify (1) the risk of developing a second PJI for our patients with multiple prosthetic joints and (2) the effect of bacteremia on development of a subsequent PJI. METHODS: We retrospectively reviewed all patients treated surgically for PJI by a single surgeon from 2003 to 2014. Time between initial and subsequent infection, bacteremia, and risk factors for PJI were identified. RESULTS: Of 167 patients treated for PJI, 76 had multiple prosthetic joints. Thirteen percent (10/76) developed a PJI in a second location. Excluding simultaneous infections, the rate was 8.3% (6/72), despite having a 57% incidence of immunosuppression, diabetes, renal failure, smoking, or steroid use. Average follow-up for patients with 1 PJI was 4.6 years (range 0.03-13.6). Seventy percent (7/10) of patients with multiple infections were bacteremic at the time of initial infection compared to 18.1% (12/66) of patients with a single infection (P = .0004). Excluding the 4 simultaneous infections (all bacteremic), the risk of developing an infection in a second joint was 20% if bacteremic and 5.2% if not bacteremic. CONCLUSION: Our study identified the risk of developing a subsequent PJI to be one half of previous studies. Bacteremia at the time of PJI is an important factor for developing subsequent PJI. Multiple prosthetic joints may be less hazardous than previously thought for patients with PJI suggesting that suppressive antibiotics may only be necessary in cases with bacteremia.

Validation of algorithms to determine incidence of Hirschsprung disease in Ontario, Canada: a population-based study using health administrative data.

OBJECTIVE: Incidence rates of Hirschsprung disease (HD) vary by geographical region, yet no recent population-based estimate exists for Canada. The objective of our study was to validate and use health administrative data from Ontario, Canada to describe trends in incidence of HD between 1991 and 2013. STUDY DESIGN: To identify children with HD we tested algorithms consisting of a combination of diagnostic, procedural, and intervention codes against the reference standard of abstracted clinical charts from a tertiary pediatric hospital. The algorithm with the highest positive predictive value (PPV) that could maintain high sensitivity was applied to health administrative data from April 31, 1991 to March 31, 2014 (fiscal years 1991-2013) to determine annual incidence. Temporal trends were evaluated using Poisson regression, controlling for sex as a covariate. RESULTS: The selected algorithm was highly sensitive (93.5%) and specific (>99.9%) with excellent predictive abilities (PPV 89.6% and negative predictive value >99.9%). Using the algorithm, a total of 679 patients diagnosed with HD were identified in Ontario between 1991 and 2013. The overall incidence during this time was 2.05 per 10,000 live births (or 1 in 4,868 live births). The incidence did not change significantly over time (odds ratio 0.998, 95% confidence interval 0.983-1.013, p = 0.80). CONCLUSION: Ontario health administrative data can be used to accurately identify cases of HD and describe trends in incidence. There has not been a significant change in HD incidence over time in Ontario between 1991 and 2013.

Unplanned readmission within 90 days after pediatric neurosurgery.

OBJECTIVE Readmission and return to operating room after surgery are increasingly being used as a proxy for quality of care. Nearly 60% of these readmissions are unplanned, which translates into billions of dollars in health care costs. The authors set out to analyze the incidence of readmission at their center, to define causes of unplanned readmission, and to determine the preoperative and surgical variables associated with readmissions following pediatric neurosurgery. METHODS A total of 536 children who underwent operations for neurosurgical diagnoses between 2012 and 2015 and who were later readmitted were included in the final analysis. Unplanned readmissions were defined to have occurred as a result of complications within 90 days after index surgery. Patient records were retrospectively reviewed to determine the primary diagnosis, surgery indication, and cause of readmission and return to operating room. The cost for index hospitalization, readmission episode, and total cost were derived based on the charges obtained from administrative data. Bivariate and multivariable analyses were conducted. RESULTS Of 536 patients readmitted in total, 17.9% (n = 96) were readmitted within 90 days. Of the overall readmissions, 11.9% (n = 64) were readmitted within 30 days, and 5.97% (n = 32) were readmitted between 31 and 90 days. The median duration between discharge and readmission was 20 days (first quartile [Q1]: 9 days, third quartile [Q3]: 36 days). The most common reason for readmission was shunt related (8.2%, n = 44), followed by wound infection (4.7%, n = 25). In the risk-adjusted multivariable logistic regression model for total 90-day readmission, patients with the following characteristics: younger age (p = 0.001, OR 0.886, 95% CI 0.824-0.952); "other" (nonwhite, nonblack) race (p = 0.024, OR 5.49, 95% CI 1.246-24.2); and those born preterm (p = 0.032, OR 2.1, 95% CI 1.1-4.12) had higher odds of being readmitted within 90 days after discharge. The total median cost for patients undergoing surgery in this study cohort was $11,520 (Q1: $7103, Q3: $19,264). For the patients who were readmitted, the median cost for a readmission episode was $8981 (Q1: $5051, Q3: $18,713). CONCLUSIONS Unplanned 90-day readmissions in pediatric neurosurgery are primarily due to CSF-related complications. Patients with the following characteristics: young age at presentation; "other" race; and children born preterm have a higher likelihood of being readmitted within 90 days after surgery. The median cost was > $8000, which suggests that the readmission episode can be as expensive as the index hospitalization. Clearly, readmission reduction has the potential for significant cost savings in pediatric neurosurgery. Future efforts, such as targeted education related to complication signs, should be considered in the attempt to reduce unplanned events. Given the single-center, retrospective study design, the results of this study are primarily applicable to this population and cannot necessarily be generalized to other institutions without further study.

The impact of curve severity on obstetric complications and regional anesthesia utilization in pregnant patients with adolescent idiopathic scoliosis: a preliminary analysis.

OBJECTIVE Adolescent idiopathic scoliosis (AIS), the most common type of scoliosis, often presents immediately prior to a woman's childbearing years; however, research investigating the impact of AIS on women's health, particularly pregnancy delivery outcomes, is sparse, with existing literature reporting mixed findings. Similarly limited are studies examining the change in scoliotic curve during or after pregnancy. Therefore, this study aims to determine 1) the impact of scoliotic curvature on obstetric complications (preterm births, induction of labor, and urgent/emergency caesarean section delivery), 2) regional anesthetic decision making and success during delivery for these patients, and 3) the effect of pregnancy on curve progression. METHODS Records of all pregnant patients diagnosed with AIS at the authors' institution who delivered between January 2002 and September 2016 were retrospectively reviewed. Demographic information, pre- and postpartum radiographic Cobb angles, and clinical data for each pregnancy and delivery were recorded and analyzed. The Wilcoxon rank-sum test and the Wilcoxon signed-rank test were used for statistical analyses. RESULTS Fifty-nine patients (84 deliveries) were included; 14 patients had undergone prior posterior spinal fusion. The median age at AIS diagnosis was 15.2 years, and the median age at delivery was 21.8 years. Overall, the median major Cobb angle prior to the first pregnancy was 25° (IQR 15°-40°). Most births were by spontaneous vaginal delivery (n = 45; 54%); elective caesarean section was performed in 17 deliveries (20%). Obstetric complications included preterm birth (n = 18; 21.4%), induction of labor (n = 20; 23.8%), and urgent/emergency caesarean section (n = 12; 14.0%); none were associated with severity of scoliosis curve or prior spinal fusion. Attempts at spinal anesthesia were successful 99% of the time (70/71 deliveries), even among the patients who had undergone prior spinal fusion (n = 13). There were only 3 instances of provider refusal to administer spinal anesthesia. In the subset of 11 patients who underwent postpartum scoliosis radiography, there was no statistically significant change in curve magnitude either during or immediately after pregnancy. CONCLUSIONS The results of this study suggest that there was no effect of the severity of scoliosis on delivery complications or regional anesthetic decision making in pregnant patients with AIS. Moreover, scoliosis was not observed to progress significantly during or immediately after pregnancy. Larger prospective studies are needed to further investigate these outcomes, the findings of which can guide the prenatal education and counseling of pregnant patients with AIS.

GNE-781, A Highly Advanced Potent and Selective Bromodomain Inhibitor of Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP).

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

Independent Preoperative Predictors of Prolonged Length of Stay after Laparoscopic Appendectomy in Patients Over 30 Years of Age: Experience from a Single Institution.

Prompt discharge after laparoscopic appendectomy (LA) is a marker of quality of care, fiscally desirable and feasible in select patients. Patients over 30 comprise a more heterogeneous cohort known to experience worse outcomes after LA. We aimed to identify easily available preoperative risk factors portending a postoperative length of stay ≥2 days among patients above age 30. In this investigation, 296 included patients from a single institution who underwent LA for acute appendicitis from 2010 to 2014 were retrospectively reviewed for preoperative demographics, laboratory studies, comorbidities, presentation characteristics, radiographic finding, and other rationally selected factors for association with postoperative length of stay ≥2 days. Bivariate and multivariate analysis was conducted to determine independent risk factors, which were subsequently modeled via receiver-operating characteristic curve generation and Kaplan-Meier analysis. "Classic" presentation [odds ratio (OR) = 0.5, P = .02], elevated red cell distribution width (RDW; OR = 1.5/% increase, P = 0.004) as well as evidence of rupture on CT (OR = 6.9, P < 0.001) were independently associated with postoperative length of stay ≥ 2 days. Modeling length of stay using these factors generated an area under the curve of 0.713 ± 0.037. Kaplan-Meier analysis of "classic" presentation, elevated RDW, and evidence of rupture on CT through the fifth postoperative day generated log-rank P values of 0.02, 0.05, and ≤ 0.001, respectively. In summary, lack of "classic" presentation, elevated RDW, and CT evidence of rupture are novel risk factors for prolonged postoperative length of stay in LA patients over 30. These findings may help target patients most appropriate for prompt discharge.

Multiple roles for the receptor tyrosine kinase axl in tumor formation.

A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA-mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.