Background Profiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), due to the aggressive clinical course of this cancer, which makes obtaining tumor biopsies exceedingly challenging. Methods In this study, we analyzed a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC. We conducted cfDNA low-pass whole genome sequencing (0.1X coverage), comparing it with time-point matched tumor characterized using whole-exome (130X) and transcriptome sequencing. Results A direct comparison of cfDNA and tumor biopsy revealed that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not detected in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Sequencing coverage of plasma DNA fragments around transcription start sites showed distinct treatment-related changes and captured the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors. This allowed for the prediction of SCLC neuroendocrine phenotypes and treatment responses. Conclusions cfDNA captures a comprehensive view of tumor heterogeneity and evolution. These findings have significant implications for the non-invasive stratification of SCLC, a disease currently treated as a single entity.

PURPOSE:There is a need to better understand the molecular features that characterize grade 3 astrocytomas and their significance in predicting clinical outcomes. The aim of this study was to determine the significance of the 2021 World Health Organization (WHO)-defined molecular subgroups, along with MGMT promoter methylation, and other alterations in NRG Oncology/RTOG 9813. METHODS AND MATERIALS:Mutation status was determined by immunohistochemistry and/or next-generation sequencing. Copy number alterations and MGMT methylation were determined by Affymetrix Oncoscan and/or Illumina 450K arrays. Progression-free survival and overall survival were estimated using the Kaplan-Meier method and tested using the log-rank test. Multivariable analyses used Cox proportional hazards models. RESULTS:Application of the 2021 WHO-defined criteria resulted in the reclassification of 26/79 (33%) patients to grade 4 astrocytoma, IDH-mutant or glioblastoma. When looking at newly assigned molecular grade, grade 3 patients experienced longer survival outcomes compared to grade 4 patients. As individual biomarkers, IDH1/2 mutations, MGMT promoter methylation, and ATRX mutations were each associated with longer survival, whereas TERT promoter mutations, EGFR amplification, and gain of chromosome 7/loss of 10 (Chr+7/-10) were associated with shorter survival. Similar survival outcomes were observed for MGMT methylated patients treated with radiation therapy (RT) and temozolomide (TMZ) or RT and BCNU/CCNU, and MGMT unmethylated patients treated with RT and TMZ. Additionally, IDH-mutant patients seemed to respond well to the addition of TMZ. CONCLUSIONS:This study demonstrated the importance of classifying patients according to the 2021 WHO-defined criteria. The majority of IDH-wildtype anaplastic astrocytomas (grade 3) were reclassified as glioblastoma (grade 4). These analyses also shed light on the efficacy of TMZ in certain molecular subgroups, where the addition of TMZ to RT appeared to benefit patients regardless of MGMT methylation status.

PURPOSE:NFE2L2/KEAP1/CUL3 mutations have been validated for radioresistance in cell-based assays and animal models. However, clinical validation of these biomarkers has been challenging because of multimodality treatment regimens. This study aims to investigate the association between NFE2L2/KEAP1/CUL3 mutations and patient outcomes, including local failure, locoregional failure, disease-free survival (DFS), and overall survival, using samples from a phase III trial in which patients were treated with radiation monotherapy at two controlled doses. PATIENTS AND METHODS:We investigated NFE2L2/KEAP1/CUL3 mutations in 250 randomized patients with T2N0 glottic squamous cell carcinoma receiving definitive radiotherapy in the NRG/RTOG 9512 trial. A total of 119 patients had available biospecimens that were subjected to amplicon-based next-generation sequencing to assess for the presence of NFE2L2/KEAP1/CUL3 mutations without regard to outcomes. Mutations in NFE2L2/KEAP1/CUL3 were assessed blinded to clinical outcomes. Cox models (two-sided α = 0.05) were used to evaluate the association with clinical outcomes, performed by an independent statistical team. RESULTS:Nineteen of 119 patients (16.0%) had NFE2L2/KEAP1/CUL3 mutations. Patient, treatment, and tumor characteristics were similar between those with and without mutations. Patients with mutation compared with those without had significantly more local failure [HR = 3.50; 95% confidence interval (CI), 1.56-7.89; P = 0.0025] and locoregional failure (HR = 3.80; 95% CI, 1.80-8.03; P = 0.0005). DFS was significantly worse for the mutated compared with the nonmutated group in the first 2 years (HR = 2.88; 95% CI, 1.46-5.66; P = 0.0022). The median DFS was shorter in the mutation group (10.3 months) versus those with intact NFE2L2/KEAP1/CUL3 (4.2 years). CONCLUSIONS:NFE2L2/KEAP1/CUL3 mutations may predict radiation treatment failure in T2N0 glottic cancer. See related commentary by Rao, p. 1563.

Experimental validation of copy-number of the MYC locus in NCI-H889, DMS-114, NCI-H446 and RA022 patient derived cell lines and number of ecDNA and HSR positive cells in the RA022 patient derived cell lines

PurposeChanges in quantitative magnetic resonance imaging (qMRI) are frequently observed during chemotherapy or radiation therapy (RT). It is hypothesized that qMRI features are reflective of underlying tissue responses. It's unknown what underlying genomic characteristics underly qMRI changes. We hypothesized that qMRI changes may correlate with DNA damage response (DDR) capacity within human tumors. Therefore, we designed the current study to correlate qMRI changes from daily RT treatment with underlying tumor transcriptomic profiles.Methods and MaterialsStudy participants were prospectively enrolled (National Clinical Trial 03500081). RNA expression levels for 757 genes from pretreatment biopsies were obtained using a custom panel that included signatures of radiation sensitivity and DDR. Daily qMRI data were obtained from a 1.5 Tesla MR linear accelerator. Using these images, d-slow, d-star, perfusion, and apparent diffusion coefficient-mean values in tumors were plotted per-fraction, over time, and associated with genomic pathways.ResultsA total of 1022 qMRIs were obtained from 39 patients and both genomic data and qMRI data from 27 total patients. For 20 of those patients, we also generated normal tissue transcriptomic data. Radio sensitivity index values most closely associated with tissue of origin. Multiple genomic pathways including DNA repair, peroxisome, late estrogen receptor responses, KRAS signaling, and UV response were significantly associated with qMRI feature changes (P < .001).ConclusionsGenomic pathway associations across metabolic, RT sensitivity, and DDR pathways indicate common tumor biology that may correlate with qMRI changes during a course of treatment. Such data provide hypothesis-generating novel mechanistic insight into the biologic meaning of qMRI changes during treatment and enable optimal selection of imaging biomarkers for biologically MR-guided RT.

TPS8130 Background: Small-cell lung cancer (SCLC) is the most fatal type of lung cancer characterized by exquisite chemo-sensitivity at diagnosis and chemoresistance at relapse. Despite a highly mutated genome, patients with SCLC derive little benefit from immunotherapy. EZH2 (enhancer of zeste homolog 2) is a master epigenetic regulator of SCLC neuroendocrine cell fate and plasticity. EZH2 inhibition 1) promotes upregulation of Schlafen 11 (SLFN11) which irreversibly blocks replication in response to DNA damaging agents and 2) enhances intrinsic immune signaling, leading to constitutive MHC I recovery, sensitizing resistant SCLC models to DNA damaging chemotherapy and immunotherapy. Tazemetostat is a selective oral EZH2 inhibitor. Methods: This is an investigator-initiated, NCI Cancer Therapy Evaluation Program (CTEP) sponsored, phase I dose escalation and dose expansion study which will evaluate safety and tolerability of combination of tazemetostat with topotecan, a selective TOP1 inhibitor, and programmed cell death protein 1 (PD-1) inhibitor antibody pembrolizumab. Adult patients with relapsed/recurrent SCLC after at least platinum doublet (limited stage-SCLC) or chemo-immunotherapy (extensive stage-SCLC) and ECOG performance 0-1 are eligible. The regimen design involves a 7-day “run-in” of oral tazemetostat BID followed by 21-day cycles of tazemetostat (1-21 days), intravenous (IV) topotecan (day 1-5) and IV pembrolizumab (Day 1). The dose escalation cohort aims to determine safety and optimal doses of tazemetostat and topotecan (with standard dose of pembrolizumab) using a 3+3 design by assessing for dose limiting toxicities. The dose expansion cohort aims to assess safety, tolerability and preliminary efficacy of the combination in 15 additional patients with relapsed SCLC. The study involves collection of mandatory biopsies at pre-treatment and post-treatment (cycle 1) to gain insights into mechanism of action and resistance of the combination using single cell and spatial transcriptomic approaches. For more questions regarding enrollment and eligibility please contact Rasa.vilimas@nih.gov or anish.thomas@nih.gov . Clinical trial information: NCT05353439 .

Purpose/Objective(s) Treatment with external beam radiation therapy (RT) using a 1.5 magnetic resonance linear accelerator (MRL) is a novel treatment modality that allows for real time, adaptive RT while taking advantage of improved contrast afforded by MR. Reported outcomes for MR guided RT (MRgRT) are scarce, generally low-field MRgRT (i.e. 0.35 T magnet), and have limited follow up of a few disease sites. We hypothesize good completion rate, toxicity, patient reported quality of life outcomes (PROs) and clinical outcomes for all patients treated at a single institution in the United States on the MRL. Materials/Methods Patients enrolled on the prospective MOMENTUM Study (NCT04075305) from July 2019 – March 2023 and treated at a single institution were included in the analysis. Prospective clinical data, technical data, Common Terminology Criteria for Adverse Events (CTCAEs) and PROs were obtained. Data was collected at baseline, 3, 6, 12, and 24 month follow-up. PROs collected included the EuroQol Five Dimension Five Level (EQ5D5L) and European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaires (QLQ). Results 186 total patients were enrolled, of which 96.5% completed treatment. Follow-up was completed by 91.4%, 78.0%, 62.9%, and 34.4% of patients at 3, 6, 12, and 24 months. The most frequently treated disease sites were pancreas (26%), liver (24%), prostate (15%), and brain (11%). Other treated sites (24%) include breast, lung, esophagus, head/neck, vagina, nodal regions, retroperitoneum, adrenal glands, bones, spinal cord, gallbladder, and pelvis. Adapt to position (ATP) was used in 69%, 29%, 36%, and 100% of liver, pancreas, prostate and brain patients. A total of 18 grade 3+ (G3) toxicities possibly or related to RT were reported. Acute G3 toxicity was seen in two liver, two pancreas, two prostate, and seven H&N treatments, and late G3 toxicity was seen in five pancreas treatments. PROs were completed in 51.4%, 45.8%, 48.2%, 46.5%, and 34.1% at baseline, 3, 6, 12, and 24 months follow-up for liver, pancreas, prostate, and brain patients. Statistically significant differences over time were increased pancreas PR25 pain scores, improved brain QLQC30 global health and physical functioning scores, decreased brain BN20 motor dysfunction and bladder control, and increased brain BN20 pruritus. EQ5D5L visual analog scale scores ranged from 70-78, 76-82, and 74-83 for liver, pancreas, and prostate, and improved from 71 at baseline to 79-88 during follow up for brain treatments. Conclusion We have presented single US institution prospective outcomes utilizing the 1.5 MRL, one of few experiences with high-field MRgRT. These results show a high completion rate with overall acceptable toxicity, clinical outcomes, and PROs in a cohort with a broad variety of malignancies, fractionation schedules, and treatment intentions.

Importance In 2018, the first online adaptive magnetic resonance (MR)-guided radiotherapy (MRgRT) system using a 1.5-T MR-equipped linear accelerator (1.5-T MR-Linac) was clinically introduced. This system enables online adaptive radiotherapy, in which the radiation plan is adapted to size and shape changes of targets at each treatment session based on daily MR-visualized anatomy. Objective To evaluate safety, tolerability, and technical feasibility of treatment with a 1.5-T MR-Linac, specifically focusing on the subset of patients treated with an online adaptive strategy (ie, the adapt-to-shape [ATS] approach). Design, Setting, and Participants This cohort study included adults with solid tumors treated with a 1.5-T MR-Linac enrolled in Multi Outcome Evaluation for Radiation Therapy Using the MR-Linac (MOMENTUM), a large prospective international study of MRgRT between February 2019 and October 2021. Included were adults with solid tumors treated with a 1.5-T MR-Linac. Data were collected in Canada, Denmark, The Netherlands, United Kingdom, and the US. Data were analyzed in August 2023. Exposure All patients underwent MRgRT using a 1.5-T MR-Linac. Radiation prescriptions were consistent with institutional standards of care. Main Outcomes and Measures Patterns of care, tolerability, and technical feasibility (ie, treatment completed as planned). Acute high-grade radiotherapy-related toxic effects (ie, grade 3 or higher toxic effects according to Common Terminology Criteria for Adverse Events version 5.0) occurring within the first 3 months after treatment delivery. Results In total, 1793 treatment courses (1772 patients) were included (median patient age, 69 years [range, 22-91 years]; 1384 male [77.2%]). Among 41 different treatment sites, common sites were prostate (745 [41.6%]), metastatic lymph nodes (233 [13.0%]), and brain (189 [10.5%]). ATS was used in 1050 courses (58.6%). MRgRT was completed as planned in 1720 treatment courses (95.9%). Patient withdrawal caused 5 patients (0.3%) to discontinue treatment. The incidence of radiotherapy-related grade 3 toxic effects was 1.4% (95% CI, 0.9%-2.0%) in the entire cohort and 0.4% (95% CI, 0.1%-1.0%) in the subset of patients treated with ATS. There were no radiotherapy-related grade 4 or 5 toxic effects. Conclusions and Relevance In this cohort study of patients treated on a 1.5-T MR-Linac, radiotherapy was safe and well tolerated. Online adaptation of the radiation plan at each treatment session to account for anatomic variations was associated with a low risk of acute grade 3 toxic effects.

Purpose/Objective(s) To determine the maximum-tolerated dose of Dose-Escalated Hypofractionated Adaptive Radiotherapy (DEHART) with atezolizumab in patients with head and neck squamous cell carcinomas (HNSCCs) in a Phase I trial. Materials/Methods Eligible patients were 18 years or older with de novo metastatic HNSCC or patients with localized AJCC 8th edition T3-T4 N0-N3, T0-T4 N1-N3 HNSCC meeting one of the following criteria: 1) not a candidate for concurrent bolus cisplatin, 2) refusal of concurrent cisplatin-based chemoradiation, 3) had unresected oral cavity cancer or 4) had recurrent disease after definitive surgery. DEHART is a novel 15-fraction radiation regimen in which dose is volume adapted weekly to MR-documented regression of gross tumor. Pre-specified dose levels to gross tumor were 50 Gy, 55 and 60 Gy (Dose Levels 1, 2, and 3, respectively) with elective nodal radiation to 40 Gy in 15 fractions. A Time-to-Event Continual Reassessment Methodology (TiTE-CRM) was used for to select the dose level for each newly enrolled patient. All patients were treated on a 1.5T MR Linear Accelerator. For the first five patients, atezolizumab (1680 mg) was delivered on the first day of radiation and q4w for up to 1 year after DEHART. The protocol was subsequently amended to remove concurrent atezolizumab due to observed toxicity, and remaining patients only received adjuvant atezolizumab only. Results Enrollment to this phase I study is complete. 18 patients have enrolled with details in Table 1. Five patients enrolled to Dose Level 1 prior to the amendment. HSV-1 reactivation was noted in three of these patients two weeks after initiating treatment, two of whom experienced dose-limiting toxicity (ICU admission and discontinuation of radiation). The study was amended to remove concurrent atezolizumab and the TITE-CRM was restarted. Subsequently, three additional patients were enrolled to Dose Level 1, three patients were enrolled to Dose Level 2 and seven patients were enrolled to Dose Level 3. No further HSV-1 reactivation or dose-limiting toxicities have occurred. Four of the 18 patients have died, two of disease. Three patients required salvage surgery and are alive without recurrence. The one-year follow-up periods for Dose Levels 1 and 2 are complete. Of seven patients treated at 60 Gy, no locoregional recurrences and no Grade 4+ toxicities have been observed at a median follow-up of 5.8 months (range: 0.2-9.8 months), though two have died (one of distant disease). Conclusion 60 Gy in 15 fractions with elective nodal treatment using weekly MR-adaptation is safe with an early signal of efficacy. In our population, administration of concurrent atezolizumab with hypofractionated radiation resulted in HSV-1 re-activation and excess toxicity.

Introduction PCNSL is an aggressive and rare subtype of extranodal non-Hodgkin Lymphoma affecting the brain, spinal cord, cerebrospinal fluid (CSF), leptomeninges and/or vitreoretinal components. While there have been trials comparing outcomes of consolidation treatment with whole brain radiation therapy (WBRT) versus (vs) autologous hematopoietic cell transplantation (auto-HCT) following different induction regimens in PCNSL, the optimal induction regimen to pair with these forms of consolidation remains controversial. Our group adopted a modified version of the RTOG 0227 induction as our standard PCNSL induction in 2017. This regimen includes 5 cycles of Methotrexate (MTX) at 3.5 g/m 2 and Rituximab (R) at 375-500 mg/m 2, and Temozolomide (TMZ) on cycles 2 and 4. Based on the published phase 2 trial of 53 patients (pts), the regimen is significantly less toxic than more aggressive induction regimens such as MATRiX, especially in older pts or those with comorbidities. We evaluated our real-world outcomes in PCNSL pts treated with RTOG 0227 induction followed by consolidation with either WBRT/TMZ or auto-HCT, at a single academic university hospital. Methods We identified newly diagnosed PCNSL pts treated at a single academic university hospital between 2017 and 2023. Electronic medical records were retrospectively reviewed to collect data including demographics, clinicopathological features, treatment details, response rates, long-term follow up, disease recurrence, and survival. The IELSG prognostic index tool was used for risk assessment. All pts received induction regimen consisting of R (dose between 375- 500 mg/m 2) and 3.5 g/m 2 of MTX with leucovorin on weeks 1, 3, 5, 7 and 9 along with TMZ daily for 5 days on weeks 4 and 8, followed by consolidation with either WBRT/TMZ or auto-HCT. In WBRT cohort, maintenance TMZ was given at 200mg/m 2 daily for 5 days every 28 days, for total of 10 cycles, and dose was reduced if side effects developed. Objective response rate (ORR) and complete response rate (CRR) after induction chemotherapy were calculated. Overall survival (OS) and progression free survival (PFS) at 2 years were estimated with the Kaplan-Meir method (using GraphPad Prism 9.3.1) and compared between the WBRT and auto-HCT cohorts, using the log-rank test. Results A total of 29 PCNSL pts were included in the study. Patient and disease characteristics for the entire cohort, as well as pts treated with WBRT vs auto-HCT consolidation, are shown on the Table. On average (avg), pts received 4 cycles of MTX and 8 cycles of TMZ. Nine (31%) pts developed acute kidney injury and 1 (3.4%) developed transaminitis from MTX. The avg total cumulative TMZ dose was 5913 mg/m 2 and 8 (27.5%) pts required TMZ dose reduction. Treatment related mortality (TRM) following induction was 0%. Eight (27.5%) pts had complete response, 19 (65.5%) had partial response, 1 (3.5%) had stable disease and 1 (3.5%) had disease progression. ORR was 93.1% and CRR was 27.5%. Eighteen (62%) pts received WBRT/TMZ consolidation, while 11 (38%) received auto-HCT consolidation. Median dose of WBRT administered was 36 Gy (23.4 to 36 Gy). Thirteen (72.2%) pts received 36 Gy in 30 fractions (fx) of 1.2 Gy BID, 4 (22.2%) pts received 23.4 Gy in 13 fx of 1.8 Gy OD, and 1 (5.5%) pt received 30.6 Gy in 17 fx of 1.8 Gy OD. Most common conditioning regimen prior to auto-HCT was carmustine/thiotepa (BCNU/TT). With a median follow up of 36 months, 4 (22.2%) pts in WBRT cohort and 1 (9.1%) in auto-HCT cohort had disease recurrence. Two-year PFS was 83% overall, 81% for the WBRT pts and 87% for the auto-HCT pts, with a p value of 0.65 for log-rank comparison of PFS for the WBRT vs auto-HCT pts. Two-year OS was 96% for the entire cohort, 94% for the WBRT pts and 100% for the auto-HCT pts, with a p value of 0.27 for log-rank comparison of OS for the WBRT vs auto-HCT pts. Conclusion In this real-world cohort of PCNSL pts not treated as part of a clinical trial, we observed excellent outcomes using the modified RTOG 0227 induction regimen. PFS and OS at 2 yrs in our cohort exceeds those reported with any prospective clinical trial to date, and with zero TRM to date. This data indicates that the modified RTOG 0227 induction with either WBRT or auto-HCT consolidation represents a very tolerable and highly effective option for first line therapy of PCNSL, for both younger and older pts.

Abstract BACKGROUND The response of cystic brain metastases (BMets) to radiotherapy is poorly understood, with conflicting results regarding local control (LC), overall survival (OS), and treatment-related toxicity. This study aims to examine the role of Gamma Knife (GK) in managing cystic BMets. METHODS Volumetric analysis was conducted to measure tumor and edema volume at the time of GK and follow-up MRI studies. We evaluated the association of 4 variables with survival using Cox regression analysis and used the Kaplan-Meier method to estimate median survival times (MST). RESULTS Between 2016 and 2021, 54 patients with 83 cystic BMets were treated with GK at our institution. Lung cancer was the most common pathology (51.9%), followed by breast (13.0%). The mean target volume was 2.7 cc (range, 0.1-39.0 cc) and the mean edema volume was 13.9 cc (range, 0-165.5 cc). The median prescription dose of single fraction and fractionated GK was 20 Gy (range, 14-27.5 Gy). With a median follow-up of 8.9 months, MST was 11.1 months, OS was 33.3%, and the one-year LC rate was 75.9%. GK was associated with decreased tumor and edema volumes over time, although 68.5% of patients required steroids post-GK. Patients whose tumors grew beyond baseline following GK received significantly more pre-GK whole-brain radiation therapy (WBRT) than those whose tumors declined following GK. Higher age at diagnosis of BMets and pre-GK systemic therapy were associated with worse survival, with an MST of 7.8 months in patients who received it compared to 23.3 months in those who did not. CONCLUSIONS Pre-GK WBRT may select for BMets with increased radioresistance. This study highlights the ability of GK to control cystic BMets with the cost of high post-treatment steroid use.

Supplemental figure 7: (A) GR50 comparison of Mia PaCa2 (top) or Panc-1 (bottom) parental cells, and their respective "chronic" abemaciclib therapy cells once treated with abemaciclib. These "chronic" abemaciclib therapy cells were then liberated from abemaciclib exposure and cultured for 14 and 21 days and GR50 was re-tested after abemaciclib exposure. Mia PaCa 2 parental, Mia PaCa2 "chronic" abemaciclib therapy (denoted as MP CA, without abemaciclib 14 days or 21 days), Panc-1 parental and Panc-1 "chronic" abemaciclib therapy (denoted as P CA, without abemaciclib 14 days or 21 days) cells GR curves are plotted together on the same graph and shown in Supplemental figure 7A. MP CA= Mia PaCa2 "chronic" abemaciclib therapy cells, P CA= Panc-1 "chronic" abemaciclib therapy cells, w/o= without. (B) β-galactosidase staining quantification of Mia PaCa2 parental/ Mia PaCa2 "chronic" abemaciclib therapy cells (top), or Panc-1 parental/ Panc-1 "chronic" abemaciclib therapy cells (bottom), as collected 3, 5, or 7 days after plating with no additional drug treatment. (C) qPCR results of Mia PaCa2 (top) or Panc-1 (bottom) parental or "chronic" abemaciclib therapy cells evaluating for SASP markers. Samples were collected 3, 5, or 7 days after plating and normalized to the same day no treatment. MP CA= Mia PaCa2 "chronic" abemaciclib therapy cells, P CA= Panc-1 "chronic" abemaciclib therapy cells.

Importance:Patients with relapsed small cell lung cancer (SCLC), a high replication stress tumor, have poor prognoses and few therapeutic options. A phase 2 study showed antitumor activity with the addition of the ataxia telangiectasia and Rad3-related kinase inhibitor berzosertib to topotecan. Objective:To investigate whether the addition of berzosertib to topotecan improves clinical outcomes for patients with relapsed SCLC. Design, Setting, and Participants:Between December 1, 2019, and December 31, 2022, this open-label phase 2 randomized clinical trial recruited 60 patients with SCLC and relapse after 1 or more prior therapies from 16 US cancer centers. Patients previously treated with topotecan were not eligible. Interventions:Eligible patients were randomly assigned to receive topotecan alone (group 1), 1.25 mg/m2 intravenously on days 1 through 5, or with berzosertib (group 2), 210 mg/m2 intravenously on days 2 and 5, in 21-day cycles. Randomization was stratified by tumor sensitivity to first-line platinum-based chemotherapy. Main Outcomes and Measures:The primary end point was progression-free survival (PFS) in the intention-to-treat population. Secondary end points included overall survival (OS) in the overall population and among patients with platinum-sensitive or platinum-resistant tumors. The PFS and OS for each treatment group were estimated using the Kaplan-Meier method. The log-rank test was used to compare PFS and OS between the 2 groups, and Cox proportional hazards models were used to estimate the treatment hazard ratios (HRs) and the corresponding 2-sided 95% CI. Results:Of 60 patients (median [range] age, 59 [34-79] years; 33 [55%] male) included in this study, 20 were randomly assigned to receive topotecan alone and 40 to receive a combination of topotecan with berzosertib. After a median (IQR) follow-up of 21.3 (18.1-28.3) months, there was no difference in PFS between the 2 groups (median, 3.0 [95% CI, 1.2-5.1] months for group 1 vs 3.9 [95% CI, 2.8-4.6] months for group 2; HR, 0.80 [95% CI, 0.46-1.41]; P = .44). Overall survival was significantly longer with the combination therapy (5.4 [95% CI, 3.2-6.8] months vs 8.9 [95% CI, 4.8-11.4] months; HR, 0.53 [95% CI, 0.29-0.96], P = .03). Adverse event profiles were similar between the 2 groups (eg, grade 3 or 4 thrombocytopenia, 11 of 20 [55%] vs 20 of 40 [50%], and any grade nausea, 9 of 20 [45%] vs 14 of 40 [35%]). Conclusions and Relevance:In this randomized clinical trial, treatment with berzosertib plus topotecan did not improve PFS compared with topotecan therapy alone among patients with relapsed SCLC. However, the combination treatment significantly improved OS. Trial Registration:ClinicalTrials.gov Identifier: NCT03896503.

Supplemental figure 5: (A) Loewe synergy plots for Mia PaCa2 and Panc-1 cells treated with abemaciclib with 5-florouracil (5-FU) or irinotecan. (B) Percent viability for various combination therapies, in Mia PaCa2 and Panc-1 cells, after treatment with abemaciclib and HuR or YAP1 inhibitors as shown. This is in correspondence to the Loewe synergism graphs shown in Figure 6A-B.

Supplemental figure 6: (A) Pico Green assay assessing cell viability in Mia PaCa2 HuR CRISPR knock out cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (B) Western blot of Mia PaCa2 HuR CRISPR knock out cells treated with abemaciclib for 3 days and probed for pRb and HuR. Samples were quantified and normalized to untreated parental cell type. (C) Mia PaCa2 HuR CRISPR KO clone validation, both by western demonstrating no HuR product (top) and by Sanger sequencing. Clone 8 has a 476 base pair insertion, and clone 10 has a single base pair deletion in one allele and a two base pair deletion in the other allele, both leading to a frameshift mutation. (D) Pico Green assays in Mia PaCa2 cells assessing cell viability in si negative control, siHuR or siYAP1 transfected cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (E) Western blot in Mia PaCa2 cells of si negative control, siHuR or siYAP1 transfected cells treated with abemaciclib for 3 days. Samples were probed for pRb, cyclin D1, HuR, YAP1 and α-tubulin. Samples were quantified and normalized to untreated si negative control. (F) qPCR results from RNA immunoprecipitation (RIP) and probed for HuR targets cyclin D1, p27 and PIM1 (positive control). Cytoplasmic extraction samples normalized to IgG. Significance is denoted as ***p<0.0001. (G) Pico Green assays in Mia PaCa2 cells assessing growth rate inhibition in si negative control or si cyclin D1 transfected cells treated with abemaciclib. Assay was performed for 5 days prior to collection. (H) Western blot in Mia PaCa2 cells of si negative control or si cyclin D1 transfected cells treated with abemaciclib for 3 days. Samples were probed for pRb, cyclin D1 and α-tubulin. Samples were quantified and normalized to untreated si negative control.

Abstract INTRODUCTION Gamma knife (GK) is a highly precise form of stereotactic radiosurgery (SRS) that delivers high doses of radiation to small, well-defined targets in a variety of tumor and vascular abnormalities. In some frame-based cases, a small translation can occur between the planning MRI and the cone-beam CT acquired at the time of treatment. Measuring the effect of patient translation on the dose distribution may be useful for informing clinical decisions. Materials/ METHODS This retrospective study of 113 planning target volumes (PTV) from 33 patients investigates the effect of translational variation on dose metrics recalculated based on cone-beam CT guided for frame based GK radiosurgery. The approved dose adjustments after the original treatment plan in terms of change in mean dose and dose coverage were compared with corresponding translation variation and the volume of PTV. The magnitude of translational variation was compared with the change in dose distribution. RESULTS We observed about 53% of cases have at least 0.1 Gy to max 1.2 Gy change in mean dose approved for the translation variation of patients while about 20% of cases have dose coverage fraction change in the range of 2-10%. Furthermore, the change in mean dose and coverage fraction is not linear with the translation variation, but a significant number of dose changes is observed when the variation magnitude is >0.5 mm. PTV volume varied between 0.04 to 22 cm3 and the dose change has been most often adopted when the volume is <2.5 cm3 and the change increases for the decreasing PTV volume. CONCLUSION This work provides new insights identifying the suitable tolerance level for translational variations of patients and the resulting impact on the prescribed dose for frame-based SRS.

Supplemental figure 2: (A) Annexin V flow cytometry cellular gating for cells treated with abemaciclib and shown as dot/ contour plots. Gating was done using unlabeled and single labeled positive control cells.

Purpose:Advancing equity, diversity, and inclusion in the physician workforce is essential to providing high-quality and culturally responsive patient care and has been shown to improve patient outcomes. To better characterize equity in the field of radiation oncology, we sought to describe the current academic radiation oncology workforce, including any contemporary differences in compensation and rank by gender and race/ethnicity. Methods and Materials:We conducted a retrospective cohort study using data from the Society of Chairs of Academic Radiation Oncology Programs (SCAROP) 2018 Financial Survey. Multivariable logistic regression models were used to identify factors associated with associate or full professor rank. Compensation was compared by gender and race/ethnicity overall and stratified by rank and was further analyzed using multivariable linear regression models. Results:Of the 858 academic radiation oncologists from 63 departments in the United States in the sample, 33.2% were female, 65.2% were White, 27.2% were Asian, and 7.6% were underrepresented in medicine (URiM). There were 44.0% assistant professors, 32.0% associate professors, and 22.8% full professors. Multivariable logistic regression analysis for factors associated with associate or full professor rank did not reveal statistically significant associations between gender or race/ethnicity with academic rank (odds ratio [OR], 0.86; 95% confidence interval [CI], 0.56-1.32; P = .48 for gender; OR, 0.81; 95% CI, 0.5-1.30; P = .37 for Asian vs White; and OR, 0.69; 95% CI, 0.31-1.55; P = .37 for URiM vs White), but CIs were wide due to sample size, and point estimates were <1. Similarly, multivariable linear regression analysis modeling the log relative total compensation did not detect statistically significant differences between radiation oncologists by gender (-1.7%; 95% CI, -6.8% to 3.4%; P = .51 for female vs male) or race/ethnicity (-1.6%; 95% CI, -7.3% to 4.0%; P = .57 for Asian vs White and -3.0%; 95% CI, -12.1% to 6.0%; P = .51 for URiM vs White). Conclusions:The low numbers of women and faculty with URiM race/ethnicity in this radiation oncology faculty sample limits the ability to compare career trajectory and compensation by those characteristics. Given that point estimates were <1, our findings do not contradict larger multispecialty studies that suggest an ongoing need to monitor equity.

Supplemental figure 1: (A) RT-PCR of human genomic DNA probed for CDKN2A in Mia PaCa2, Panc-1, HS 766T, HPNE and PDXs, compared to positive control cells PA-1[54]. Subsequent RT-PCR of mouse genomic DNA probed for CDKN2A in KPC (KRAS mutant, p53 null) and KTC (KRAS mutant, HuR null) mouse organoids to validate CDKN2A status. (B) Pico green cell viability assay on PDX cell lines treated with palbociclib, abemaciclib or gemcitabine for 5 days, with IC50 tabulated below. (C) Cell titer glow cell viability assay to calculate IC50 for abemaciclib in KPC (IC50 4.9µM) and CDKN2A null organoids (IC50 4.0µM). (D) Top: Pico green assays to assess short-term abemaciclib drug response in Mia PaCa2, which has a deletion of CDKN2A gene, compared to HPNE, which has a preserved CDKN2A gene. IC50 for both cell lines are shown in the parenthesis. Bottom: Drug curves for HPNE cells treated with abemaciclib, gemcitabine or oxaliplatin are graphed with IC50 shown in parenthesis. (E) Crystal violet colony formation images for cells treated with palbociclib or abemaciclib at respective concentrations. (F) PCR primer sequences used for RT-PCR reactions.