Novel Machine Learning Analysis Algorithm ofDNA Methylation Patterns Identifies CerebralPalsy with Concurrent Epilepsy.

Background Spastic cerebral palsy, the most common pediatric-onset disabling condition with an estimated prevalence of 0.2% in children, is a complex condition characterized by stiff movement, muscle contractures, and abnormal gait that can diminish quality of life. Spastic CP accounts for approximately 83% of all CP cases and frequently co-occurs with other complex conditions, like epilepsy. An estimated 42% of spastic CP cases have co-occurring epilepsy. Unfortunately, CP is often difficult to diagnose. Although most children with CP are born with it or acquire it immediately after birth, many are not identified until after 19 months of age with CP diagnosis often not confirmed until 5 years of age. New bioinformatic approaches to identify CP earlier are needed. Recent studies indicate that altered DNA methylation patterns associated with CP may have diagnostic value. The potential confounding effects of co-occurrent epilepsy on these patterns are not known. We evaluated machine learning classification of CP patients with or without co-occurring epilepsy. Results Whole blood samples were collected from 30 study participants diagnosed with epilepsy (n=4), spastic CP (n=10), both (n=8), or neither (n=8). A novel Support-Vector-Machine learning algorithm was developed to identify methylation loci that have ability to classify CP from controls in the presence or absence of epilepsy. This algorithm was also employed to measure classification ability of identified methylation loci. After preprocessing of data, isolation of important methylation loci was performed in a binary comparison between CP and controls, as well as in a 4-way scheme, encapsulating epilepsy diagnoses. The classification ability was similarly assessed. CP Classification performance wasevaluated with and without inclusion of epilepsy as a feature. Median F1 scoreswere 0.67 in 4-class comparison, and 1.0 in the binary classification, outperforming Linear-Discriminant-Analysis (0.57 and 0.86, respectively). Conclusion This novel algorithm was able to classify study participants with spastic CPand/or epilepsy from controls with significant performance. The algorithm shows promise for rapid identification in methylation data of diagnostic methylation loci. In this model, Support Vector Machines outperformed Linear Discriminant Analysis in classification. In the evaluation of epigenetics-based diagnostics for CP, epilepsy may not be a significant confounding factor.

DNA Methylation Analysis Reveals Distinct Patterns in Satellite Cell-Derived Myogenic Progenitor Cells of Subjects with Spastic Cerebral Palsy.

Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient-specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC-derived myoblasts (MB) and 1774 CpG sites in differentiating SC-derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo- and hyper-methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.

An engineered dual function peptide to repair fractured bones.

Targeted drug delivery, often referred to as "smart" drug delivery, is a process whereby a therapeutic drug is delivered to specific parts of the body in a manner that increases its concentration at the desired sites relative to others. This approach is poised to revolutionize medicine as exemplified by the recent FDA approval of Cytalux (FDA approves pioneering drug for ovarian cancer surgery - Purdue University News) which is a folate-receptor targeted intraoperative near infrared (NIR) imaging agent that was developed in our laboratories. Fracture-associated morbidities and mortality affect a significant portion of world population. United states, Canada and Europe alone spent $48 billion in treating osteoporosis related fractures although this number doesn't count the economic burden due to loss in productivity. It is estimated that by 2050 ca 21 million hip fractures would occur globally which will be leading cause of premature death and disability. Despite the need for improvement in the treatment for fracture repair, methods for treating fractures have changed little in recent decades. Systemic delivery of fracture-homing bone anabolics holds great promise as a therapeutic strategy in this regard. Here we report the design of a fracture-targeted peptide comprised of a payload that binds and activates the parathyroid hormone receptor (PTHR1) and is linked to a targeting ligand comprised of 20 D-glutamic acids (D-Glu20) that directs accumulation of the payload specifically at fracture sites. This targeted delivery results in reduction of fracture healing times to <1/2 while creating repaired bones that are >2-fold stronger than saline-treated controls in mice. Moreover, this hydroxyapatite-targeted peptide can be administered without detectable toxicity to healthy tissues or modification of healthy bones in dogs. Additionally, since similar results are obtained upon treatment of osteoporotic and diabetic fractures in mice, and pain resolution is simultaneously accelerated by this approach, we conclude that this fracture-targeted anabolic peptide displays significant potential to revolutionize the treatment of bone fractures.

Effect of Celecoxib vs Placebo as Adjuvant Therapy on Disease-Free Survival Among Patients With Breast Cancer: The REACT Randomized Clinical Trial.

IMPORTANCE: Patients with breast cancer remain at risk of relapse after adjuvant therapy. Celecoxib has shown antitumor effects in preclinical models of human breast cancer, but clinical evidence is lacking. OBJECTIVE: To evaluate the role of celecoxib as an addition to conventional therapy for women with ERBB2 (formerly HER2)-negative primary breast cancer. DESIGN, SETTING, AND PARTICIPANTS: The Randomized European Celecoxib Trial (REACT) was a phase 3, randomized, double-blind study conducted in 160 centers across the UK and Germany testing 2 years of adjuvant celecoxib vs placebo among 2639 patients recruited between January 19, 2007, and November 1, 2012, with follow-up 10 years after treatment completion. Eligible patients had completely resected breast cancer with local and systemic therapy according to local practice. Patients with ERBB2-positive or node-negative and T1, grade 1 tumors were not eligible. Randomization was in a 2:1 ratio between celecoxib or placebo. Statistical analysis was performed from May 5, 2019, to March 5, 2020. INTERVENTIONS: Patients received celecoxib, 400 mg, or placebo once daily for 2 years. MAIN OUTCOMES AND MEASURES: The primary end point was disease-free survival (DFS), analyzed in the intention-to-treat population using Cox proportional hazards regression and log-rank analysis. Follow-up is complete. RESULTS: A total of 2639 patients (median age, 55.2 years [range, 26.8-86.0 years]) were recruited; 1763 received celecoxib, and 876 received placebo. Most patients' tumors (1930 [73%]) were estrogen receptor positive or progesterone receptor positive and ERBB2 negative. A total of 1265 patients (48%) had node-positive disease, and 1111 (42%) had grade 3 tumors. At a median follow-up of 74.3 months (interquartile range, 61.4-93.6 years), DFS events had been reported for 487 patients (19%): 18% for those who received celecoxib (n = 323; 5-year DFS rate = 84%) vs 19% for those who received placebo (n = 164; 5-year DFS rate = 83%); the unadjusted hazard ratio was 0.97 (95% CI, 0.80-1.17; log-rank P = .75). Rates of toxic effects were low across both treatment groups, with no evidence of a difference. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, patients showed no evidence of a DFS benefit for 2 years' treatment with celecoxib compared with placebo as adjuvant treatment of ERBB2-negative breast cancer. Longer-term treatment or use of a higher dose of celecoxib may lead to a DFS benefit, but further studies would be required to test this possibility. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02429427 and isrctn.org Identifier: ISRCTN48254013.

Randomized Prospective Biomarker Trial of ERCC1 for Comparing Platinum and Nonplatinum Therapy in Advanced Non-Small-Cell Lung Cancer: ERCC1 Trial (ET).

Purpose Retrospective studies indicate that expression of excision repair cross complementing group 1 (ERCC1) protein is associated with platinum resistance and survival in non-small-cell lung cancer (NSCLC). We conducted the first randomized trial, to our knowledge, to evaluate ERCC1 prospectively and to assess the superiority of nonplatinum therapy over platinum doublet therapy for ERCC1-positive NSCLC as well as noninferiority for ERCC1-negative NSCLC. Patients and Methods This trial had a marker-by-treatment interaction phase III design, with ERCC1 (8F1 antibody) status as a randomization stratification factor. Chemonaïve patients with NSCLC (stage IIIB and IV) were eligible. Patients with squamous histology were randomly assigned to cisplatin and gemcitabine or paclitaxel and gemcitabine; nonsquamous patients received cisplatin and pemetrexed or paclitaxel and pemetrexed. Primary end point was overall survival (OS). We also evaluated an antibody specific for XPF (clone 3F2). The target hazard ratio (HR) for patients with ERCC1-positive NSCLC was ≤ 0.78. Results Of patients, 648 were recruited (177 squamous, 471 nonsquamous). ERCC1-positive rates were 54.5% and 76.7% in nonsquamous and squamous patients, respectively, and the corresponding XPF-positive rates were 70.5% and 68.5%. Accrual stopped early in 2012 for squamous patients because OS for nonplatinum therapy was inferior to platinum therapy (median OS, 7.6 months [paclitaxel and gemcitabine] v 10.7 months [cisplatin and gemcitabine]; HR, 1.46; P = .02). Accrual for nonsquamous patients halted in 2013. Median OS was 8.0 (paclitaxel and pemetrexed) versus 9.6 (cisplatin and pemetrexed) months for ERCC1-positive patients (HR, 1.11; 95% CI, 0.85 to 1.44), and 10.3 (paclitaxel and pemetrexed) versus 11.6 (cisplatin and pemetrexed) months for ERCC1-negative patients (HR, 0.99; 95% CI, 0.73 to 1.33; interaction P = .64). OS HR was 1.09 (95% CI, 0.83 to 1.44) for XPF-positive patients, and 1.39 (95% CI, 0.90 to 2.15) for XPF-negative patients (interaction P = .35). Neither ERCC1 nor XPF were prognostic: among nonsquamous patients, OS HRs for positive versus negative were ERCC1, 1.11 ( P = .32), and XPF, 1.08 ( P = .55). Conclusion Superior outcomes were observed for patients with squamous histology who received platinum therapy compared with nonplatinum chemotherapy; however, selecting chemotherapy by using commercially available ERCC1 or XPF antibodies did not confer any extra survival benefit.

Stereotactic ablative body radiotherapy for non-small-cell lung cancer: setup reproducibility with novel arms-down immobilization.

OBJECTIVE: A clinical evaluation of the intrafraction and interfraction setup accuracy of a novel thermoplastic mould immobilization device and patient position in early-stage lung cancer being treated with stereotactic radiotherapy at the Beatson West of Scotland Cancer Centre, Glasgow, UK. METHODS: 35 patients were immobilized in a novel, arms-down position, with a four-point Klarity™ (Klarity Medical Products, Ohio, US) clear thermoplastic mould fixed to a SinMed (CIVCO Medical solutions, lowa, US) head and neck board. A knee support was also used for patient comfort and support. Pre- and post-treatment kilovoltage cone beam CT (CBCT) images were fused with the planning CT scan to determine intra- and interfraction motion. A total of 175 CBCT scans were analysed in the longitudinal, vertical and lateral directions. RESULTS: The mean intrafraction errors were 0.05 ± 0.77 mm (lateral), 0.44 ± 1.2 mm (superior-inferior) and -1.44 ± 1.35 mm (anteroposterior), respectively. Mean composite three-dimensional displacement vector was 2.14 ± 1.2 mm. Interfraction errors were -0.66 ± 2.35 mm (lateral), -0.13 ± 3.11 mm (superior-inferior) and 0.00 ± 2.94 mm (anteroposterior), with three-dimensional vector 4.08 ± 2.73 mm. CONCLUSION: Setup accuracy for lung image-guided stereotactic ablative radiotherapy using a unique immobilization device, where patients have arms by their sides, has been shown to be safe and favourably comparable to other published setup data where more complex and cumbersome devices were utilised. There was no arm toxicity reported and low arm doses. Advances in knowledge: We report on the accuracy of a novel patient immobilization device.

IDEAL-CRT: A Phase 1/2 Trial of Isotoxic Dose-Escalated Radiation Therapy and Concurrent Chemotherapy in Patients With Stage II/III Non-Small Cell Lung Cancer.

PURPOSE: To report toxicity and early survival data for IDEAL-CRT, a trial of dose-escalated concurrent chemoradiotherapy (CRT) for non-small cell lung cancer. PATIENTS AND METHODS: Patients received tumor doses of 63 to 73 Gy in 30 once-daily fractions over 6 weeks with 2 concurrent cycles of cisplatin and vinorelbine. They were assigned to 1 of 2 groups according to esophageal dose. In group 1, tumor doses were determined by an experimental constraint on maximum esophageal dose, which was escalated following a 6 + 6 design from 65 Gy through 68 Gy to 71 Gy, allowing an esophageal maximum tolerated dose to be determined from early and late toxicities. Tumor doses for group 2 patients were determined by other tissue constraints, often lung. Overall survival, progression-free survival, tumor response, and toxicity were evaluated for both groups combined. RESULTS: Eight centers recruited 84 patients: 13, 12, and 10, respectively, in the 65-Gy, 68-Gy, and 71-Gy cohorts of group 1; and 49 in group 2. The mean prescribed tumor dose was 67.7 Gy. Five grade 3 esophagitis and 3 grade 3 pneumonitis events were observed across both groups. After 1 fatal esophageal perforation in the 71-Gy cohort, 68 Gy was declared the esophageal maximum tolerated dose. With a median follow-up of 35 months, median overall survival was 36.9 months, and overall survival and progression-free survival were 87.8% and 72.0%, respectively, at 1 year and 68.0% and 48.5% at 2 years. CONCLUSIONS: IDEAL-CRT achieved significant treatment intensification with acceptable toxicity and promising survival. The isotoxic design allowed the esophageal maximum tolerated dose to be identified from relatively few patients.