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Purpose/Objective(s) The role of PORT for patients with resected stage IIIA (N2) NSCLC is controversial. Recently, the Phase III LungART trial reported a significant improvement in mediastinal control with PORT but failed to show an overall survival (OS) benefit, due in part to cardiopulmonary (CP) toxicity (7% early and 20% late grade (G) 3-5). However, LungART employed 3D conformal radiotherapy (3D-CRT) in 90% of patients as well as elective treatment of mediastinal nodal stations. We evaluated the risk of CP toxicity associated with use of postoperative IMRT for this population. Materials/Methods We conducted a retrospective study of consecutive patients with NSCLC treated with postoperative IMRT between 2008 and 2019. Patients were eligible if they were found to have incidental pN2 or an R1/2 resection. Targets of PORT for pN2 were bronchial stump, ipsilateral hilum, and involved mediastinal station(s). The primary objective was incidence of CTCAE (v4) G3+ CP toxicities. Secondary objectives were major adverse cardiac events (MACE), defined as cardiac death, myocardial infarction, coronary revascularization or hospitalization due to heart failure; disease-free survival (DFS); and OS. Cumulative incidence and Kaplan-Meier method were used as appropriate. Results 73 patients received PORT with IMRT. Median follow-up was 54 months. Median age at diagnosis was 65 years (range 36 - 81); 33 patients (45%) had pre-existing cardiovascular disease and 48 (65.8%) were current/former smokers. 50 patients had pN2 disease and 23 patients had R1/R2 resection. Among patients with pN2, the median number of involved mediastinal nodes was 1 (range 1 – 12). All patients with pN2 and 70% of patients with R1/R2 resection received chemotherapy, the majority of which was sequential (n = 52, 71.2%). Median RT dose was 54 Gy (range 45 – 66 Gy) in 27 fractions for patients with pN2 and 59.4 Gy (range 45 – 66 Gy) in 30 fractions for patients with R1/R2 resection. 3-year cumulative incidence of G3+ CP toxicity was 2.7%, with an overall incidence of 6.8% (5 events in 73 patients). The rate of G3+ CP toxicity in patients with pN2 was 8%. Three patients developed ≥1 MACE: one died of a cardiac arrest and two underwent coronary revascularization. Two of these patients had a history of coronary artery disease. Two episodes of G3+ toxicity involved valve replacements in patients with pre-existing valvular disease. 3-year DFS was 51.4% (95% CL 46.9 – 55.1%). Two patients with pN2 developed mediastinal failures. 3-year OS was 62.3% (95% CL 50.2 – 74.4%). Conclusion PORT for pN2 NSCLC using IMRT and no elective nodal irradiation is associated with a low rate of G3+ CP toxicity in patients with pN2, comparable to the chemotherapy only arm of the LungART trial. Our data suggest that highly conformal radiation techniques should be prospectively assessed for their potential to translate the known local control benefit of PORT into a survival advantage.

Purpose/Objective(s) The COVID-19 pandemic posed challenges in resource allocation and breast cancer (BC) treatment decisions. Our study aims to understand changes in practice patterns of United States radiation oncologists (RO) treating BC during the COVID-19 pandemic. Materials/Methods An IRB-approved 58-question survey with 6 clinical scenarios was distributed between July 17 and November 8, 2020 to ASTRO directory members. The cases included 1) Low-grade ductal carcinoma in situ (DCIS), 2) Low-risk BC treated with lumpectomy, 3) Low-risk BC treated with mastectomy with reconstruction 4) BC treated with neoadjuvant chemotherapy and mastectomy with reconstruction 5) BC treated with mastectomy and adjuvant chemotherapy but without reconstruction 6) Metastatic BC with enlarging breast mass. RO were surveyed about treatment recommendations if cases were seen pre-pandemic (PP) and hypothetically during the peak of pandemic (DTPP). Chi-square and McNemar-Bowker tests were used to examine the significance of changes. Results A total of 285 respondents from 48 states completed the survey and reported treating at least one patient with BC in the past 12 months. 45% primarily practice in university affiliated hospitals and 43% in private practice. 22% reported treating ≥ 1 COVID-positive BC patients. Moderate hypofractionation (2.31 - 3 Gy per fraction) in the PMRT and immediate reconstruction setting was recommended by 0.7% PP compared to 10.5% DTPP. In the low-risk PMRT setting, recommendation of no further treatment increased from 13% PP to 20% DTPP. Further, 56% changed their DCIS recommendations if the patient was seen DTPP. For low-risk BC, whole breast RT was preferred by 83.5% PP compared to 46.7% DTPP, and 35.1% recommended delay of RT DTPP compared to 0.4% PP (P < 0.05). Increase in ultra-hypofractionation (> 5 Gy per fraction) was significant for low-risk BC after lumpectomy as 0.4% reported its use PP compared to 3.8% DTPP. In addition, utilization of brachytherapy as PBI modality decreased from 23.9% to 17% among respondents PP and DTPP respectively. The Florence fractionation schedule for PBI was recommended by 46.2% for early-stage BC and by 51.7% for DCIS DTPP compared to 20% and 34.4% PP. Finally, 68.1% reported the use of 10-25 fractions PP for the palliative scenario. However, of those who would change their recommendation (48.8%), 62.8% reported recommendation of ≤ 5 fractions DTPP. Additional subset analysis by geographic region and practice type were notable for variable changes in treatment recommendations, and will be presented. Conclusion This large survey of Breast RO clinical decision making demonstrates significant differences in recommendations and rapid adoption of unique fractionation. While likely reflective of intent to optimize resource allocations during the pandemic, maintenance of new practice patterns remains subject to future investigation.

Simple Summary Malignant pleural mesothelioma (MPM) is characterized by the lack of effective long-term treatments and highly prevalent drug resistance. The paucity of potential therapeutic targets has led to dismal prognosis. We have examined the functional role of the signal transducer and activator of transcription 3 (STAT3) transcription factor in MPM. Even though highly specific STAT3 inhibitors have not yet come to fruition, we performed experiments targeting STAT3 expression and subsequently supported these experiments with small molecule drugs that were previously validated to target STAT3-dependent activation mechanisms. These drugs are United States Food and Drug Administration (FDA)-approved and showed efficacy in preclinical models of MPM at concentrations that can safely be achieved in humans. We also identified genes that strongly support the essential role of STAT3 in cell growth and are consistent with a role of STAT3 in immune suppression. Overall, the results establish a central role for STAT3 in tumor growth and encourage further expedient development of STAT3 pathway inhibitors for clinical use. Abstract Malignant pleural mesothelioma (MPM) is an aggressive cancer defined by loss-of-function mutations with few therapeutic options. We examined the contribution of the transcription factor Signal transducer and activator of transcription 3 (STAT3) to cell growth and gene expression in preclinical models of MPM. STAT3 is activated in a variety of tumors and is thought to be required for the maintenance of cancer stem cells. Targeting STAT3 using specific small hairpin RNAs (shRNAs) or with the pharmacologic inhibitors atovaquone or pyrimethamine efficiently reduced cell growth in established cell lines and primary-derived lines while showing minimal effects in nontransformed LP9 mesothelial cells. Moreover, atovaquone significantly reduced viability and tumor growth in microfluidic cultures of primary MPM as well as in an in vivo xenotransplant model. Biological changes were linked to modulation of gene expression associated with STAT3 signaling, including cell cycle progression and altered p53 response. Reflecting the role of STAT3 in inducing localized immune suppression, using both atovaquone and pyrimethamine resulted in the modulation of immunoregulatory genes predicted to enhance an immune response, including upregulation of ICOSLG (Inducible T-Cell Costimulator Ligand or B7H2). Thus, our data strongly support a role for STAT3 inhibitors as anti-MPM therapeutics.

BACKGROUND Management for men aged ≤55 years with low-risk prostate cancer (LRPC) is debated given quality-of-life implications with definitive treatment versus the potential missed opportunity for cure with conservative management. The objective of this study was to define rates of conservative management for LRPC and associated short-term outcomes in young versus older men in the United States. METHODS The nonpublic Surveillance, Epidemiology, and End Results Prostate with Active Surveillance/Watchful Waiting (AS/WW) Database identified 50,302 men who were diagnosed with LRPC from 2010 through 2015. AS/WW rates in the United States were stratified by age (≤55 vs ≥56 years). Prostate cancer-specific mortality and overall mortality were defined by initial management type (AS/WW vs definitive treatment [referent]) and age. RESULTS AS/WW utilization increased from 8.61% (2010) to 34.56% (2015) among men aged ≤55 years (P for trend

Diverse HPV subtypes are responsible for considerable disease burden worldwide, necessitating safe, cheap, and effective vaccines. The HPV minor capsid protein L2 is a promising candidate to create broadly protective HPV vaccines, though it is poorly immunogenic by itself. To create highly immunogenic and safe vaccine candidates targeting L2, we employed a plant-based recombinant protein expression system to produce two different vaccine candidates: L2 displayed on the surface of hepatitis B core (HBc) virus-like particles (VLPs) or L2 genetically fused to an immunoglobulin capable of forming recombinant immune complexes (RIC). Both vaccine candidates were potently immunogenic in mice, but were especially so when delivered together, generating very consistent and high antibody titers directed against HPV L2 (>1,000,000) that correlated with virus neutralization. These data indicate a novel immune response synergy upon co-delivery of VLP and RIC platforms, a strategy that can be adapted generally for many different antigens.

Summary The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.

Background: The complex interaction between tumor cells and the immune system has led to increased efforts focused on development of ex vivo systems that recapitulate the tumor microenvironment and model responses to immune checkpoint blockade. Recently, we demonstrated successful ex vivo growth of organotypic tumor spheroids and profiling of PD-1 blockade in syngeneic models and patient tumors1. Optimization of tissue processing from the operating room to the lab for patient-derived materials can further aid these efforts. Here, we identify pre-analytical variables that may impact tumor characterization in ex vivo systems. Methods: We conducted an evaluation of pre-analytical variables for tumor tissue in a cohort of mesothelioma and non-small cell lung cancer (NSCLC) cases. Pre-analytical variables included neoadjuvant chemotherapy administration, pleurodesis status, type of surgery, presence of fibrosis, and onset of warm and cold ischemia during tissue retrieval and processing. Prior to surgery, medical records were reviewed for pathology, chemotherapy, and pleurodesis status. On surgery day, time of tissue removal or clamping of the pulmonary artery was recorded as onset of warm ischemia. Tissue was transported to pathology and allocations to research were made. Specimens were placed in culture media and ice; placement on ice was recorded as onset of cold ischemia. Arrival of tissue in lab was recorded. Chart and observational data were correlated with tissue analysis at the bench followed by 4-color immunofluorescence to characterize tumor and immune components of spheroids. Results: A cohort of mesothelioma and NSCLC samples were followed from the operating room to the research lab. The average time between onset of warm and cold ischemia was 60 minutes (range 33-100 min). Mean time for removal of tissue in the OR to arrival in lab was 104 minutes (range 87-137 min). Two patients received neoadjuvant chemotherapy and were among 2 of 3 cases where fibrotic samples were collected. Fibrosis was often associated with more extensive surgery via extrapleural pneumonectomy and lower tumor content per microscopy and immunofluorescence. Conclusions: Prolonged ischemic times were associated with poor tissue viability. In two cases, pre-therapeutic status was linked to tissue fibrosis, extensive surgery, low tumor yield, and failed spheroid generation. More data can help elucidate variable significance. These initial findings highlight the unique challenges associated with performing studies on live tumor tissue, and suggest that further streamlining of processes for tissue collection in the OR can optimize success of ex vivo cancer models in guiding functional precision therapies. Citation Format: Dalia Larios, Amir Aref, Elena Ivanova, Brandon Piel, Andrew Portell, David A. Barbie, Raphael Bueno, Cloud Paweletz. From OR to bench: Identification of variables affecting success in generating patient derived organotypic spheroids (pDOTS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5025.

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.