PURPOSE:Tumor motion during respiration presents a challenge in delivering high-precision stereotactic ablative radiation therapy (SABR) for lung cancer. While inspiratory breath-hold techniques can minimize motion, they are often limited by the patients' ability to sustain prolonged breath-holds and the variability that comes with repeat breath-holds. We present the first-in-human demonstration of the feasibility and safety of percussive ventilation breath-hold (PVB) to eliminate the uncertainty of breath-hold reproducibility between confirmatory volumetric image guidance and delivery of lung tumor SABR by completing both within the same assisted breath-hold. Here, we report that 4 patients were successfully treated as initial observations from a prospective clinical trial of PVB-SABR. METHODS AND MATERIALS:Four patients with lung tumors were treated in a prospective, institutional review board-approved study in which they underwent an education and evaluation session to assess their tolerance for PVB, followed by treatment with PVB-SABR. RESULTS:All 4 patients successfully completed PVB-SABR. The mean PVB duration per fraction from the beginning of the final cone beam computed tomography image verification to the end of SABR delivery was 6 minutes 56 seconds in a single PVB. Within that time, the mean delivery time per fraction was 2 minutes 22 seconds and the mean duty cycle was 93.5%. CONCLUSIONS:These initial observations in a first prospective trial of PVB-SABR demonstrate the feasibility of PVB to enable completion of SABR delivery within the same effective breath-hold as confirmatory volumetric imaging, eliminating the uncertainty of interbreath-hold reproducibility.

Abstract Cancer therapies are limited by normal tissue toxicity. Radiation pneumonitis (RP) is a dose-limiting toxicity that commonly occurs in cancer patients who receive thoracic radiation therapy. RP is caused by inflammation and increased vascular permeability in the lungs and can lead to severe pulmonary symptoms and sometimes death. Currently, the strongest predictors of RP are radiation dose metrics, such as the volume of normal lung receiving ≥20 Gy (V20) or mean lung dose (Dmean); however, these have only modest predictive accuracy. Therefore, it is currently not possible to accurately identify patients who might benefit from RP-directed therapy prior to developing symptoms and there remains an unmet need for sensitive, non-invasive biomarkers to identify patients at risk for RP before symptoms develop. Plasma cell-free RNA (cfRNA) is a promising analyte that enables non-invasive profiling of gene expression in diverse tissues. Our group recently developed an ultrasensitive method called RARE-Seq to detect low abundance transcriptional signatures in cfRNA (Nesselbush et al. Nature 2025). In this study, we applied RARE-Seq to analyze cfRNA from 160 plasma samples from 56 lung cancer patients, 34 of whom developed RP, collected before, during, and after radiation therapy. We observed enrichment of lung- and airway-specific gene transcripts in cfRNA from patients with symptomatic RP compared to those who received radiation but did not develop RP. Additionally, pre-symptomatic samples from patients who later developed RP were enriched for cfRNA signatures of lung pneumocytes, suggesting early molecular changes preceding clinical symptoms. Lastly, we identified cfRNA signatures that distinguished symptomatic RP from non-RP samples with an AUC of 0.85 and pre-symptomatic RP from non-RP samples with an AUC of 0.77, significantly outperforming the classic dosimetric predictors lung V20 and Dmean (V20 P = 4.9e-5, Dmean P = 7.4e-5, paired DeLong test). Collectively, these findings demonstrate proof of concept that cfRNA profiling can predict radiation-induced toxicities before onset of symptoms, establishing it as a potentially transformative biomarker for improving monitoring and personalized management of patients treated with radiation therapy. Citation Format: Isabel Jabara, Noah Kastelowitz, Monica Nesselbush, Nick Phillips, Michael S. Binkley, Rene F. Bonilla, Kevin Liu, Alice Jiang, Nataliya Kovalchuk, Ash A. Alizadeh, Maximilian Diehn. Cell-free RNA changes precede symptomatic radiation pneumonitis in cancer patients receiving thoracic radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5270.

PURPOSE:Thoracic stereotactic ablative radiation therapy (SABR) is an effective treatment for lung tumors. We evaluated the association between tumor control and (a) tumor respiratory motion and motion management approach and (b) single-fraction dose metrics in patients treated on a prospective clinical trial. METHODS AND MATERIALS:We evaluated 235 patients with 277 thoracic tumors treated on the iSABR trial. Motion management approaches included motion inclusive (MI, 41%), MI with extreme breaths excluded (13%), expiratory gating (Exp Gating, 24%), and inspiratory breath hold (IBH, 22%). The association between tumor motion, motion management technique, and local recurrence (LR) was evaluated using Fine-Gray Analysis. Among the cohort of patients treated in a single fraction (150 tumors), we performed a tumor control probability analysis for dose to the gross tumor (GTV) and planning treatment volumes. RESULTS:There was no significant difference in LR by tumor motion when dichotomized to <1 cm or ≥1 cm (3-year LR of 5.8% vs 6.3%, P = .98). Similarly, there was no difference in LR between patients treated with MI, MI with extreme breaths excluded, Exp Gating, and IBH with 24-month estimates of 5.5%, 5.9%, 4.7%, and 3.7%, respectively (P = .75). For tumors treated with single-fraction SABR, GTV D68.3% and planning treatment volume D65.3% had the strongest correlation with local control (LC). Tumor control probability analysis demonstrated a statistically significant association with GTV D99.7%, D95%, and D68.3%. Rates of LC at 3 years were greater than 90% with a GTV D68.3% > 29.4 Gy and a GTV D95% > 28.1 Gy. CONCLUSIONS:These findings suggest that motion management techniques including Exp Gating and IBH can adequately control respiratory motion. Furthermore, single-fraction SABR with 25 Gy resulted in high rates of LC for small tumors when using heterogeneous dosimetry, including 29.4 Gy to 68.3% of the GTV and 28.1 Gy to 95% of the GTV.

Abstract Introduction: Mutations in KEAP1 and NFE2L2 (NFR2) occur in approximately 20% of non-small cell lung cancer (NSCLC) patients and are associated with resistance to radiotherapy and systemic treatments. In previous research, we found that not all KEAP1 mutations found in NSCLC patients are pathogenic. These benign mutations exhibit a wild-type phenotype that does not confer therapy resistance. With only roughly 100 of 13,000 (<1%) potential single amino acid substitution mutations in KEAP1 characterized thus far, here we comprehensively evaluated all possible point mutations in KEAP1 using deep mutational scanning (DMS). Methods: The High-throughput Mutagenesis by Integrated TilEs (MITE) method was used to generate the KEAP1 DMS library. In short, the KEAP1 sequence was segmented into 21 variable regions (tiles), each comprising a 90-base pair variable region flanked by a 30-base pair constant region. Each codon position included the 20 possible natural amino acids plus a stop codon, for a total of 13,076 KEAP1 variants. The tiles were then assembled to ensure that each full-length KEAP1 construct contained only a single amino acid substitution. The KEAP1 DMS lentivirus library was transduced into H1299-KEAP1NULLcells, followed by selective pressure with H2O2, radiation, or vehicle control. The KEAP1 ORF was then amplified from surviving cell genomic DNA, and mutations were quantified using next-generation sequencing. Functional annotations were derived through mutation enrichment analysis. Results: Both H2O2 and radiation selective pressure of the KEAP1 DMS library induced a bimodal distribution of KEAP1 mutations, with cells harboring KEAP1 nonsense mutations being enriched and cells with silent mutations being depleted. By setting a threshold of 95% specificity for silent mutations to differentiate between benign and pathogenic mutations, we identified 94% of nonsense mutations and 88% of previously characterized pathogenic mutations as pathogenic. While most KEAP1 mutations were categorized as benign (64%), analysis of mutations in TCGA NSCLC patients showed pathogenic mutations were enriched (72%). Mapping mutation phenotypes onto a KEAP1 structural model revealed an enrichment of pathogenic mutations at critical functional sites, including the NRF2 binding pocket of the Kelch domain, the BTB homodimerization interface, and the hydrophobic core of the oxidative stress-sensing IVR domain. Conclusions: Through deep mutational scanning experiments, we generated a comprehensive list of pathogenic KEAP1 single amino acid substitutions. These results align with previously known biological functions and prior single amino acid substitutions characterized in the literature. These findings will facilitate the identification of benign and pathogenic KEAP1 mutations in clinical settings, enabling personalized radiotherapeutic and systemic therapy strategies for patients harboring KEAP1 mutations. Citation Format: Noah Kastelowitz, Shashank Shrishrimal, Soyeong Jun, Anni MY Zhang, Rui Wang, Ilayda Ilerten, Maximilian Diehn. Deep mutational scanning of KEAP1 to identify mutations resistant to radiation therapy and oxidative stress. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr BO23.

Sensitive methods for detection of cell-free RNA (cfRNA) could facilitate non-invasive gene expression profiling and monitoring of diseases1-6. Here we describe RARE-seq (random priming and affinity capture of cfRNA fragments for enrichment analysis by sequencing), a method optimized for cfRNA analysis. We demonstrate that platelet contamination can substantially confound cfRNA analyses and develop an approach to overcome it. In analytical validations, we find RARE-seq to be approximately 50-fold more sensitive for detecting tumour-derived cfRNA than whole-transcriptome RNA sequencing (RNA-seq), with a limit of detection of 0.05%. To explore clinical utility, we profiled 437 plasma samples from 369 individuals with cancer or non-malignant conditions and controls. Detection of non-small-cell lung cancer expression signatures in cfRNA increased with stage (6 out of 20 (30%) in stage I; 5 out of 8 (63%) in stage II; 10 out of 15 (67%) in stage III; 80 out of 96 (83% sensitivity) in stage IV at 95% specificity) and RARE-seq was more sensitive than tumour-naive circulating tumour DNA (ctDNA) analysis. In patients with EGFR-mutant non-small-cell lung cancer who developed resistance to tyrosine kinase inhibitors, we detected both histological transformation and mutation-based resistance mechanisms. Finally, we demonstrate the potential utility of RARE-seq for determination of tissue of origin, assessing benign pulmonary conditions and tracking response to mRNA vaccines. These results highlight the potential value of ultrasensitive cfRNA analysis and provide proof of concept for diverse clinical applications.

INTRODUCTION:The aim of this study was to investigate the clinical significance of inter-fraction volume changes during stereotactic ablative body radiotherapy (SABR) for early-stage NSCLC. The prevalence and impact on disease control remain poorly studied. This is the largest study examining the association between inter-fraction volume changes during lung SABR and clinical outcomes. PATIENTS AND METHODS:121 NSCLC patients treated between April 2009 and July 2019 were included. Gross tumor volume (GTV) was calculated from planning-CT, and inter-fraction volumes from cone-beam CT images taken before treatment. Univariable and multivariable linear regression analyzed relationships between volumetric changes and baseline characteristics. Fine-Gray models assessed volume change associations with local, regional, and distant recurrence, considering death as a competing risk. RESULTS:61.9% and 11.1% of tumors demonstrated a > 10% increase and decrease, respectively, in volume from the start to end of treatment. There was a negative correlation between initial tumor volume and inter-fraction volume increase on univariable analysis. Fine-Gray models showed no significant correlation between volume increase and local recurrence but found a negative association between volume increase and regional as well as distant recurrence. CONCLUSIONS:Notable radiographic volume changes occurred in over half of lung SABR patients. This phenomenon did not appear to adversely affect local control. Increased radiographic volume correlated with reduced regional and distant recurrence, possibly representing an immune effect. Our findings suggest volume changes are likely a consequence of acute inflammatory reactions rather than tumor growth, and modest PTV expansions may be sufficient for tumor coverage.

BACKGROUND:Hyperfractionated reirradiation (re-RT) has been shown to mitigate late toxicity for some cancers, but data on safety and efficacy for thoracic tumors are limited. METHODS:We evaluated a cohort of 28 consecutive cases among 25 patients treated with re-RT to either primary or metastatic thoracic tumors, using 60 Gy in 50 fractions delivered twice daily. Incidence rates of toxicity, local recurrence (LR), and any disease progression were evaluated using competing risk analysis. Cumulative dose to organs at risk (OARs) was calculated (EQD2 [alpha/beta = 3]), correlated with toxicity outcomes, and compared to published constraints for re-RT. RESULTS:There was direct overlap of 100 % isodose lines between treatment courses in 90.5 % of cases. Most patients had tumors abutting the proximal bronchial tree (PBT, 89 %). The median re-RT PTV volume was 67 cm3. The overall rate of ≥G2 toxicity was 46 % and the rate of ≥ G3 toxicity was 15 %. The rate of local recurrence and overall survival at 12 months was 33 % and 80 %, respectively. The lung volume receiving a cumulative dose of 20 Gy was kept under 40 % (V20 Gy < 40 %) for nearly all patients (91 %). Lung V5Gy ≥ 60 % was associated with higher rates of ≥ G2 pulmonary toxicity (69 vs. 18 %, p < 0.01). Cumulative Dmax exceeded previously published constraints in most cases for the PBT (110 Gy, 57 % of cases) and great vessels (120 Gy, 52 % of cases) without observed cases of ≥G2 pulmonary hemorrhage, stenosis, fistula, or great vessel toxicity. CONCLUSIONS:Hyperfractionated re-RT for thoracic tumors resulted in favorable rates of local control and toxicity in a high-risk cohort and is worthy of prospective evaluation. Putative dose constraints were not able to be met for many cases of definitive re-RT, particularly for the great vessels and PBT, without excessive observed toxicity.

Abstract Blood-based liquid biopsies enable non-invasive characterization of cancers. Cell-free RNA (cfRNA) analysis could potentially complement circulating tumor DNA (ctDNA) analysis and allow broader molecular characterization of cancers but has not been extensively explored. Here, we describe RARE-Seq, a novel cfRNA sequencing method designed to maximize sensitivity by targeting rare, tissue-specific transcripts, and simultaneously detecting both tumor-derived expression signatures and somatic mutations in circulating tumor RNA (ctRNA). To interrogate samples for specific gene expression signatures, we developed and optimized a framework to measure tumor- and tissue-specific enrichment scores (ES) within cfRNA. We then empirically determined the limit of detection (LOD95) of RARE-Seq using admixtures of tumor cell line RNA and control cfRNA across a wide range of fractions. We found RARE-Seq to have an estimated LOD95 of 0.05% for cancer-derived RNA, which was 52-fold better than whole transcriptome cfRNA profiling. To explore its potential clinical utility, we profiled 192 samples from 170 subjects with various cancers and non-cancer controls using RARE-Seq. Non-small cell lung cancer (NSCLC) expression signatures were significantly detected in 40 out of 51 NSCLC samples (78% sensitivity at 90% specificity). Detection increased with stage, ranging from 40% in stage I to 87% in stage IV. In a subset of stage IV NSCLC patients with matching ctDNA data (n=16), ctRNA levels were significantly correlated with ctDNA allele frequencies (Pearson R=0.84, P=0.005). Intriguingly, 86% of samples with undetectable ctDNA contained detectable ctRNA, suggesting potential complementarity of ctRNA and ctDNA analysis for cancer detection. We also developed and benchmarked a direct ctRNA genotyping approach, allowing noninvasive detection of driver mutations in 42% of patients with advanced NSCLC. When applied to other tumors, RARE-Seq demonstrated 80-100% sensitivity at 90% specificity for detection of pancreatic adenocarcinomas (PAAD), prostate adenocarcinomas (PRAD), and hepatocellular carcinomas (LIHC). Furthermore, when evaluating tissue of origin (TOO) classification performance, RARE-Seq achieved 85% median accuracy for the top predicted tissue and 96% median accuracy for the top two tissues. These results highlight the potential value of ultrasensitive cfRNA analysis, providing proof-of-principle for detection of multiple tumor types using RARE-Seq. Citation Format: Monica C. Nesselbush, Bogdan A. Luca, Young-Jun Jeon, Isabel Jabara, Catherine B. Meador, Michael S. Binkley, Nova Xu, Angela B. Hui, Andrea Garofalo, William Y. Shi, Kevin J. Liu, Takeshi Sugio, Noah Kastelowitz, Emily G. Hamilton, Rene F. Bonilla, Yi Peng Wang, Alice Jiang, Brianna Lau, Jordan Eichholz, Mandeep Banwait, Joseph Schroers-Martin, Jan Boegeholz, Daniel A. King, Mohammad S. Esfahani, Tyler Raclin, Robert Tibshirani, Sandy Srinivas, Viswam S. Nair, James M. Isbell, Bob T. Li, Zofia Piotrowska, Lecia V. Sequist, Aaron N. Hata, Joel W. Neal, Heather A. Wakelee, Andrew J. Gentles, Ash A. Alizadeh, Maximilian Diehn. Cell-free RNA analysis for non-invasive cancer detection and characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6560.

Abstract Introduction: KEAP1 and NFE2L2 (NFR2) mutations occur in roughly 20% of non-small cell lung cancer (NSCLC) patients and are associated with resistance to a broad range of therapies including radiotherapy, chemotherapy, targeted therapies, and immunotherapy. In prior work, we demonstrated that not all KEAP1 mutations observed in NSCLC patients are pathogenic. These benign mutations have a wild-type phenotype that does not impart therapy resistance. However, only roughly 100 of ~13,000 (<1%) possible KEAP1 single amino acid substitution mutations have been characterized. Here, we performed saturation mutagenesis of KEAP1 to comprehensively evaluate all possible point mutation phenotypes in vitro. Methods: Saturation mutagenesis was accomplished using the High-throughput Mutagenesis by Integrated TilEs (MITE) method. In brief, the KEAP1 sequence was divided into 21 tiles each containing a 90 bp variable region flanked by a 30 bp constant region. At each codon position, mutations representing the 20 possible natural amino acids and a stop codon were included for a total of 13,076 KEAP1 variants. The tiles were assembled such that each full length KEAP1 construct contained a single desired mutation. The KEAP1 MITE lentivirus library was transduced into H1299-KEAP1NULL cells that were then treated with vehicle or H2O2 for 24 days. The KEAP1 ORF was amplified from gDNA and mutations were quantified by next generation sequencing. Mutation enrichment analysis was used to derive functional annotations. Results: Oxidative stress resulted in a bimodal distribution of KEAP1 mutations. Cells with KEAP1 nonsense mutations were enriched while cells with silent mutations were depleted. Using a threshold of 95% specificity for silent mutations to distinguish benign from pathogenic mutations, 94% of nonsense mutations and 88% of previously characterized pathogenic mutations were identified as pathogenic. While the majority of KEAP1 mutations were benign (64%), analysis of mutations found in TCGA NSCLC patients showed an enrichment of pathogenic mutations (72%). Overlaying mutation calls onto the KEAP1 structure showed enrichment of pathogenic mutations in the BTB homodimerization interface, hydrophobic core of the oxidative stress sensing IVR domain, and NRF2 binding pocket of the Kelch domain. Conclusions: Saturation mutagenesis of KEAP1 generated a comprehensive list of pathogenic KEAP1 single amino acid substitutions. Assignments strongly agreed with biologic function and prior literature. The assessment of KEAP1 mutation phenotypes is important as benign mutations occur in a significant subset of NSCLC patients and are not expected to impart therapy resistance. Our results will allow identification of benign and pathogenic KEAP1 mutations in clinical studies, which will enable testing of personalized therapeutic strategies for patients with KEAP1 mutations. Citation Format: Noah Kastelowitz, Shashank Shrishrimal, Soyeong Jun, Anni M. Zhang, Rui Wang, Ilayda Ilerten, Maximilian Diehn. Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 377.

We present a patient with widespread PCGD-TCL of the bilateral arms and legs, who underwent radiotherapy with 34 Gy in 17 fractions using circumferential VMAT and 3-D printed bolus to the four extremities prior to planned stem cell transplant, who was then found to have progression in the liver, lung, and skin, followed by drastic regression of all in and out-of-field lesions on imaging 1.5 months later. The cause of regression may be related to a radiation-induced abscopal effect from the immunomodulatory effects of radiation, or related to immune reactivation in the setting of cessation of systemic immunosuppressive agents.

Background Radiotherapy may cause grade >= 3 cardiac events, necessitating a better understanding of risk factors. The potential predictive role of imaging biomarkers with radiotherapy doses for cardiac event occurrence has not been studied.Objectives The aim of this study was to establish the associations between cardiac substructure dose and coronary artery calcium (CAC) scores and cardiac event occurrence.Methods A retrospective cohort analysis included patients with locally advanced non-small cell lung cancer treated with radiotherapy (2006-2018). Cardiac substructures, including the left anterior descending coronary artery, left main coronary artery, left circumflex coronary artery, right coronary artery, and TotalLeft (left anterior descending, left main, and left circumflex coronary arteries), were contoured. Doses were measured in 2-Gy equivalent units, and visual CAC scoring was compared with automated scoring. Grade >= 3 adverse cardiac events were recorded. Time-dependent receiver-operating characteristic modeling, the log-rank statistic, and competing-risk models were used to measure prediction performance, threshold modeling, and the cumulative incidence of cardiac events, respectively.Results Of the 233 eligible patients, 61.4% were men, with a median age of 68.1 years (range: 34.9-90.7 years). The median follow-up period was 73.7 months (range: 1.6-153.9 months). Following radiotherapy, 22.3% experienced cardiac events, within a median time of 21.5 months (range: 1.7-118.9 months). Visual CAC scoring showed significant correlation with automated scoring (r = 0.72; P < 0.001). In a competing-risk multivariable model, TotalLeft volume receiving 15 Gy (per 1 cc; HR: 1.38; 95% CI: 1.11-1.72; P = 0.004) and CAC score >5 (HR: 2.51; 95% CI: 1.08-5.86; P = 0.033) were independently associated with cardiac events. A model incorporating age, TotalLeft CAC (score >5), and volume receiving 15 Gy demonstrated a higher incidence of cardiac events for a high-risk group (28.9%) compared with a low-risk group (6.9%) (P < 0.001).Conclusions Adverse cardiac events associated with radiation occur in more than 20% of patients undergoing thoracic radiotherapy within a median time of <2 years. The present findings provide further evidence to support significant associations between TotalLeft radiotherapy dose and cardiac events and define CAC as a predictive risk factor.

Introduction: The impact of radiation prescription dose on postoperative complications during standard of care trimodality therapy for operable stage II-III esophageal and gastroesophageal junction cancers has not been established.Methods: We retrospectively reviewed 82 patients with esophageal or gastroesophageal junction cancers treated between 2004 and 2016 with neoadjuvant chemoradiation followed by resection at a single institution. Post-operative complications within 30 days were reviewed and scored using the Comprehensive Complication Index (CCI). Results were compared between patients treated with <50 Gy and ≥ 50 Gy, as well as to published CROSS study neoadjuvant chemoradiation group data (41.4 Gy).Results: Twenty-nine patients were treated with <50 Gy (range 39.6–46.8 Gy) and 53 patients were treated with ≥ 50 Gy (range 50.0–52.5 Gy) delivered using IMRT/VMAT (41%), 3D-CRT (46%), or tomotherapy IMRT (12%). Complication rates and CCI scores between our <50 Gy and ≥ 50 Gy groups were not significantly different. Assuming a normal distribution of the CROSS data, there was no significant difference in CCI scores between the CROSS study neoadjuvant chemoradiation, <50 Gy, or ≥ 50 Gy groups. Rates of pulmonary complications were greater in the CROSS group (50%) than our <50 Gy (38%) or ≥ 50 Gy (30%) groups.Conclusions: In selected esophageal and gastroesophageal junction cancer patients, radiation doses ≥ 50 Gy do not appear to increase 30 day post-operative complication rates. These findings suggest that the use of definitive doses of radiotherapy (50–50.4 Gy) in the neoadjuvant setting may not increase post-operative complications.

Purpose: Our institution cancelled all in-person clerkships owing to the coronavirus disease 2019 pandemic. In response, we designed a virtual radiation oncology medical student clerkship. Methods and Materials: We convened an advisory panel to design a virtual clerkship curriculum. We implemented clerkship activities using a cloud-based learning management system, video web conferencing systems, and a telemedicine portal. Students completed assessments pre- and postclerkship to provide data to improve future versions of the clerkship. Results: The virtual clerkship spans 2 weeks and is graded pass or fail. Students attend interactive didactic sessions during the first week and participate in virtual clinic and give talks to the department during the second week. Didactic sessions include lectures, case-based discussions, treatment planning seminars, and material adapted from the Radiation Oncology Education Collaborative Study Group curriculum. Students also attend virtual departmental quality assurance rounds, cancer center seminars, and multidisciplinary tumor boards. The enrollment cap was met during the first virtual clerkship period (April 27 through May 8, 2020), with a total of 12 students enrolling. Conclusions: Our virtual clerkship can increase student exposure and engagement in radiation oncology. Data on clerkship outcomes are forthcoming. (C) 2020 The Author(s). Published by Elsevier Inc. on behalf of American Society for Radiation Oncology.

Purpose: We evaluated the impact of a virtual radiation oncology clerkship. Methods and Materials: We developed a 2-week virtual radiation oncology clerkship that launched on April 27, 2020. Clerkship components included a virtual clinic with radiation oncology faculty and residents, didactic lectures, student talks, and supplemental sessions such as tumor boards and chart rounds. Medical students completed preand post-clerkship selfassessments. Faculty and resident participants also completed surveys on their experience with virtual lectures and clinics. Preand post-clerkship results were compared using a 2-sided paired t test. An analysis of variance model was used to analyze the clerkship components. Results: Twenty-six medical students, including 4 visiting students, enrolled over 2 clerkship periods (4 weeks). All students completed the preand post-clerkship self-assessments and agreed that the clerkship improved their understanding of radiation oncology. Compared with 3 (11.5%) students who agreed that they understood the daily responsibilities of a radiation oncologist before the clerkship, 22 (84.6%) students agreed and 3 (11.5%) strongly agreed that they understood the daily responsibilities of a radiation oncologist after the clerkship (P < .0001). Although 15 students (57.7%) reported an increased interest in radiation oncology because of the clerkship, the mean level of interest in radiation oncology as a career remained the same, with preand post-clerkship scores of 3.0 (+/- 0.9) and 3.0 (+/- 1.1) on a 5-point scale, respectively (P = .7). Students found virtual clinic and didactic lectures to be the most valuable components of the clerkship. Most respondents agreed (30.8%) or strongly agreed (65.4%) to recommend the clerkship to their classmates. Conclusions: Our virtual clerkship was effective in increasing medical student interest in and knowledge about radiation oncology. These data will help optimize a new paradigm of virtual radiation oncology education for medical students during COVID-19 and beyond. (C) 2020 Elsevier Inc. All rights reserved.

119 Background: Neoadjuvant chemoradiation (nCRT) followed by resection is standard of care for operable stage II-III EC and GEJC; however, it can be associated with significant risk of postoperative complications (POC). The CROSS study group reported no increase in POC severity with nCRT using 41.4 Gy compared to surgery alone as defined by the Comprehensive Complication Index (CCI). We applied the CCI metric to evaluate the impact of nCRT radiation dose of < 50 Gy vs. ≥ 50 Gy on POC rates and compared to the CROSS rates. Methods: We retrospectively reviewed 82 pts (2004-2016) with EC or GEJC treated with nCRT followed by resection at our institution. 29 (35%) pts were treated with < 50 Gy (range 39.6-46.8 Gy) and 53 (65%) were treated with ≥ 50 Gy (range 50.0-52.5 Gy) delivered using IMRT/VMAT (41%), 3D-CRT (46%), or tomotherapy IMRT (12%). Concurrent chemotherapy were carboplatin/paclitaxel (59%), cisplatin/5-FU (17%), or other (24%). Resection was performed by Ivor Lewis esophagectomy (67%), esophagogastrectomy (14%), or trans-hiatal approach (11%). POC within 30 days were graded using the Clavien-Dindo scale and CCI scores were computed and compared between the two dose groups and with the CROSS nCRT group. Results: CCI scores and complication rates between our < 50 Gy and ≥ 50 Gy groups were not significantly different. Assuming a normal distribution of the CROSS CCI scores, there was no significant difference in CCI scores between the CROSS study nCRT, < 50 Gy, or ≥ 50 Gy groups. Rates of pulmonary complications were greater in the CROSS study. Conclusions: In highly selected EC and GEJC pts, definitive nCRT radiation doses do not appear to increase POC rates. Thus, 50 Gy can likely be delivered without increasing toxicity while also achieving a definitive dose for pts not able or willing to undergo subsequent surgery. [Table: see text]

Nanoscale confinement has a strong effect on the phase behavior of water. Studies in the last two decades have revealed a wealth of novel crystalline and quasicrystalline structures for water confined in nanoslits. Less is known, however, about the nature of ice-liquid coexistence in extremely nanoconfined systems. Here, we use molecular simulations to investigate the ice-liquid equilibrium for water confined between two nanoscopic disks. We find that the nature of ice-liquid phase coexistence in nanoconfined water is different from coexistence in both bulk water and extended nanoslits. In highly nanoconfined systems, liquid water and ice do not coexist in space because the two-phase states are unstable. The confined ice and liquid phases coexist in time, through oscillations between all-liquid and all-crystalline states. The avoidance of spatial coexistence of ice and liquid originates on the non-negligible cost of the interface between confined ice and liquid in a small system. It is the result of the small number of water molecules between the plates and has no analogue in bulk water.

Strategies for maximizing the microbial production of bio-based chemicals and fuels include eliminating branched points to streamline metabolic pathways. While this is often achieved by removing key enzymes, the introduction of nonnative enzymes can provide metabolic shortcuts, bypassing branched points to decrease the production of undesired side-products. Pyruvate decarboxylase (PDC) can provide such a shortcut in industrially promising thermophilic organisms; yet to date, this enzyme has not been found in any thermophilic organism. Incorporating nonnative enzymes into host organisms can be challenging in cases such as this, where the enzyme has evolved in a very different environment from that of the host.

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo , we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.