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Short course radiation-based total neoadjuvant therapy can improve disease-free survival for patients with high-risk locally advanced rectal cancer. Tumors that involve or threaten the circumferential resection margin have a particularly high risk of local recurrence. Intraoperative radiation therapy enables treatment escalation at the threatened or involved margin at the time of surgery.Patients with rectal adenocarcinoma treated with preoperative short course radiotherapy-based total neoadjuvant therapy and intraoperative radiation at the time of surgery were identified. All patients had a threatened or involved circumferential resection margin on magnetic resonance imaging at the time of diagnosis. Treatment details, radiation toxicities, postoperative complications and oncologic outcomes were recorded.Ten patients received intraoperative radiation after short course radiation-based total neoadjuvant therapy. All patients had an involved or threatened circumferential resection margin, 60% had extramural venous invasion, and 60% had positive lateral pelvic lymph nodes. Seven patients had negative surgical margins (≥ 2 mm), and 3 patients had an R1 resection with radial margins2 mm. The median [IQR] length of hospitalization after surgery was 11 [7-14] days. Three patients required readmission and 2 patients required reoperation due to complications including anastamotic leak and abscess. With a median follow up of 19.5 months postoperatively, no patient developed a pelvic recurrence, and 6 patients developed distant recurrences.The use of intraoperative radiation after a short course radiotherapy-based neoadjuvant therapy is safe and feasible. Further data are needed to determine whether the addition of intraoperative radiation improves local recurrence rates over preoperative radiation alone.

Background Although intensity-modulated radiation therapy (IMRT) is considered the standard of care for the treatment of squamous cell carcinoma of the anus (SCCA), few large series have reported oncologic outcomes and toxicities. In this retrospective report, we aim to describe outcomes and toxicities after IMRT-based chemoradiation (CRT) for the treatment of SCCA, evaluate the impact of dose escalation (>54 Gy), and compare concurrent fluoropyrimidine in combination with either mitomycin or with cisplatin as chemosensitizers. Methods Patients treated at The University of Texas MD Anderson Cancer Center between January 1, 2003 and December 31, 2018 with IMRT-based CRT were included. Median time to locoregional recurrence, time to colostomy, and overall survival were estimated using the Kaplan–Meier method. Results A total of 428 patients were included; median follow-up was 4.4 years. Three hundred and thirty-four patients (78.0%) were treated with concurrent cisplatin and fluoropyrimidine, and 160 (37.4%) with >54 Gy. Two- and 5-year freedom from locoregional failure, freedom from colostomy failure, and overall survival were 86.5% and 81.2%, respectively, 90.0% and 88.3%, respectively, and 93.6% and 85.8%, respectively. Neither dose escalation nor mitomycin-based concurrent chemotherapy resulted in improved outcomes. Mitomycin-based concurrent chemotherapy was associated with in approximately 2.5 times increased grade 3 or greater acute toxicity. Radiation dose >54 Gy was associated with approximately 2.6 times increased Grade 3 or greater chronic toxicity. Conclusions Our results suggest IMRT-based CRT with concurrent fluoropyrimidine and cisplatin is a safe and feasible option for patient with SCCA and may cause less acute toxicity. The role for radiation dose escalation is unclear and requires further study.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)–expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti–PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti–PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti–PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

SUMMARYThis study identifies a novel pathotype of cervical cancer-associated Lactobacillus iners (L. iners) that results in chemoradiation resistance in vitro and is associated with poor patient survival. Cervical cancer affects over half a million women a year around the world. Treatment for women with locally advanced cancer is delivered with definitive chemoradiation (CRT) but is curative for only 60% of patients. There are few validated molecular markers to identify patients who will respond poorly to treatment. Tumor microbiome features are associated with treatment resistance in patients with colon and pancreatic cancers, and thus we investigated their role in the response of cervical cancer to therapy. We identified a strong association between poor clinical response to CRT and tumors dominated by L. iners. Cancer-associated L. iners promoted in vitro resistance of cervical cancer cells and modified the local tumor immunologic microenvironment, while non-cancer-associated L. iners did not. Assembly of genomes from cancer-derived L. iners also demonstrated pathogenic, metabolic, and immune functions not found in healthy patients.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic stroma which induces profound intratumoral hypoxia within its tumor microenvironment (TME). PDAC has low levels of effector T cell infiltration and increased numbers of immunosuppressive macrophages and regulatory T cells, which is thought to be a consequence of the hypoxic TME. We recently demonstrated that cancer-associated fibroblasts (CAFs) within spontaneous pancreatic tumors regulate macrophage migration and polarization through a paracrine mechanism dependent upon hypoxia inducible factor-2 (HIF2) expression. Here we use genetic and biochemical techniques to identify the putative secreted factors mediating this HIF2-dependent CAF-macrophage crosstalk. Methods: We used a dual-recombinase mouse model to generate spontaneous pancreatic tumors with a conditional allele of HIF2 expressed in alpha-SMA+ fibroblasts that was abrogated by ex vivo infection with an adenovirus to overexpress Cre (AdCre) or GFP (AdGFP). HIF2 was also inhibited with the small molecule PT2399. Hypoxia experiments were performed in a chamber which maintained 0.5% O2. Results: Following Cre overexpression via adenovirus, HIF2 was confirmed to be deleted in CAFs through PCR, qPCR, and western blot. HIF2-KO (AdCre) CAFs and HIF2-WT (AdGFP) CAFs were then cultured under normoxia (21% O2) or hypoxia (0.5% O2) for 48 hours and conditioned media (CM) was collected. We analyzed cytokines and chemokines within the CM using a cytokine array to identify proteins secreted in a hypoxia and HIF2-dependent fashion. Among several proteins, insulin-like growth factor-binding protein 3 (IGFBP3) was significantly increased in CM from HIF2-WT CAFs cultured in hypoxia compared to HIF2-KO CAFs. We confirmed this HIF2-dependent secretion in patient-derived pancreatic stellate cells (hPSCs) in hypoxia with either vehicle (DMSO) or 2µM PT2399. IGFBP3 was greatly increased in CM from hPSCs treated with vehicle compared to PT2399 treatment (1166 vs 413.7 pg/mL, p=0.0052). qPCR analysis revealed that IGFBP3 was the most hypoxia-responsive and HIF2-dependent member of the IGFBP family (IGFBP1-IGFBP7). Furthermore, macrophages cultured with CM from HIF2-WT CAFs increased Mrc1 gene expression, a marker of M2 macrophage polarization, while CM from HIF2-KO CAFs did not increase Mrc1. Conclusion: Our results suggest that HIF2-dependent secretion of IGFBP3 from CAFs in the hypoxic pancreatic cancer TME contributes to its immunosuppressive nature. Future work will focus on verifying that IGFBP3 mediates HIF2-dependent modulation of macrophages and will explore other paracrine factors which may be involved in CAF-macrophage crosstalk. Citation Format: Matthew T. Cribb, Sahar Fattani, Ayeisha Colón Ortiz, Natividad R. Fuentes, Cullen Taniguchi. Cancer-associated fibroblasts modulate macrophage phenotype in PDAC through hypoxia inducible factor-2(HIF2)-dependent IGFBP3 secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2351.

Pancreatic cancer is the third leading cause of cancer-related deaths in the U.S. and is most frequently diagnosed between the ages of 65-74 years. Only 11.5% of patients are expected to survive 5 years after diagnosis. There is extensive evidence that tumor microenvironment (TME) plays a critical role in cancer progression and metastasis. Despite the fact that most patients are diagnosed over the age of 60, most preclinical murine tumor models utilize young, healthy, adolescent mice, failing to account for structural and/or functional changes in stroma and immune cells that occur in tissues over time and contribute to tumor progression. Marked differences in lifespan between mice and humans preclude natural development of many age-related physiologic changes in mice that occur in humans over the course of decades. We performed a pilot study to test our hypothesis that KPC pancreatic tumors grown orthotopically in the pancreas of genetically engineered, C57BL/6-Tg (LMNA*G608G) HClns/J mice, a model of Hutchinson-Gilford progeria syndrome with features of accelerated aging, would have altered tumor growth in comparison to tumors in age-matched wild type controls. We found that tumor growth in LMNA mice outpaced that of WT mice. Quantitative digital image analysis showed significantly reduced intratumoral infiltration of CD45+ leukocytes in LMNA mice in comparison to WT (p=0.0098). There was a significant reduction in tumor-infiltrating CD3+ T cells (p=0.038) and F4/80+ macrophages (p=0.0102). Our data supports the hypothesis that functional, age-related, immune and/or stromal changes in the TME impact pancreatic tumor growth and progression. Innovative strategies that better model age-related changes in the TME may better recapitulate the aggressive course of pancreatic cancer in aged patients. Citation Format: Morgan M. Green, Tara Fujimoto, Cullen Taniguchi, Natalie Fowlkes. Modeling age-related changes in the tumor microenvironment in an orthotopic immunocompetent murine pancreatic cancer model - a novel approach. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5191.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dependence on highly active mitochondria to grow and metastasize. We previously found that mitochondrial fusion suppressed PDAC growth and metastasis, leading to improved survival in a preclinical model. The tumor suppressive mechanisms of mitochondrial fusion remain unclear, and thus we explored the role of redox homeostasis through uncoupling protein 2 (UCP2). Methods/Results: Murine pancreatic cancer cells (KPC) with enhanced mitochondrial fusion (sgDrp1) exhibited higher levels of mitochondrial ROS compared to controls targeting GFP (sgGFP), suggesting altered redox homeostasis. We performed RNAseq on these two cell lines and found that sgDRP1 cells exhibited reduced levels of UCP2. The UCP2 protein is known to have indirect antioxidant effects by reducing mitochondrial reactive oxygen species via uncoupling of ATP production from the electron transport chain. We found that abrogating UCP2 in KPC cells reduced its capacity for OXPHOS. Moreover, rescue of UCP2 in sgDrp1 cells restored their mitochondrial activity. Conclusions: Mitochondrial fusion may suppress tumor growth and induce mitophagy by altering redox homeostasis. UCP2 may be a mediator of the tumor suppressive phenotype induced by mitochondrial fusion, thus linking mitochondrial dynamics and PDAC antioxidant strategies. Future Work: These results will be explored for their translation in vivo and the effects of pharmacological inhibition of UCP2, both in vitro and in vivo, via the specific inhibitor Genipin, will be explored. Citation Format: Ariana Carolina Acevedo-Diaz, Emily G. Caggiano, Meifang Yu, Cullen Taniguchi. Uncoupling protein 2 (UCP2) loss of function mediates PDAC tumor suppression by mitochondrial fusion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4830.

Patients with locally advanced pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options with their disease being defined as neither metastatic nor amenable to surgical resection. Although stereotactic body radiation therapy (SBRT) is a viable treatment option in this context, disease progression remains far too common in PDAC. Understanding the interaction between radiation, cancer cells, and the tumor microenvironment (TME) may lead to an understanding of novel resistance pathways that can be exploited. In order to identify these potential nodes of therapeutic actionability, we leveraged a phase I/II dose escalation clinical trial of SBRT with a radiomodulating agent, GC4419, undertaken in patients with locally advanced PDAC. In this trial, core biopsies were obtained following neoadjuvant chemotherapy, but before and after SBRT with 50-60Gy in 5 fractions. A total of 16 biopsies underwent single cell RNA sequencing with 7 patients with matched samples before and after radiation. Of the 7 matched patients, a total of 3 experienced early local progression following SBRT, and were deemed to be nonresponders. We observed a significant increase in NK-like cells and a decrease in B-cells and Mast cells following SBRT in nonresponders. On the other hand, we also observed a significant enrichment in pathways related to downregulation of innate immunity within macrophage populations following SBRT suggesting that an immunosuppressive microenvironment may prevail in these patients. Subsequently, we observed functionally distinct cancer-associated fibroblasts (CAFs) subtypes, including myofibroblastic, inflammatory, and antigen presenting CAFs (apCAF). ApCAFs are believed to be involved in antigen presentation to CD4 T cells, but lack the costimulatory molecules to activate immune response, and are thus presumed to contribute to an immunosuppressive microenvironment through anergy. Interestingly, the population of apCAFs decreased significantly following SBRT in non-responders, and increased in those patients deemed to be responders. Thus whether apCAFs have an additional inflammatory role in the setting of patients receiving radiation remains to be further understood. We have shown how single cell RNA sequencing can be used to profile the kinetics of cell populations following SBRT. This has revealed an interplay between members of the tumor microenvironment whereby immunosuppressive pathways appear to prevail over immunostimulatory signals in nonresponders. Understanding which pathways are enriched in those patients at highest risk for local recurrence may provide insight into novel therapeutic strategies that can be implemented concurrently with radiation therapy. Citation Format: Vincent Bernard, Adnan Elhammali, Daniel Lin, Ching-Wei Tzeng, Manoop S. Bhutani, Cullen Taniguchi. Single cell transcriptomic sequencing of pancreatic ductal adenocarcinoma reveals an enrichment of immunosuppressive pathways in patients at high risk for early local progression following stereotactic body radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1096.

Precision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. However, the long-term fidelity of recently deployed ex vivo preclinical platforms, such as patient-derived organoids (PDOs), remains unknown. Through single-cell RNA sequencing (scRNA-seq), we identify substantial transcriptomic evolution of PDOs propagated from the parental tumor, which may alter predicted drug sensitivity. In contrast, scRNA-seq is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes. Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast (CAF) subclasses, and predict a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches. While PDOs continue to enable prospective therapeutic prediction, our analysis also demonstrates the complementarity of using orthogonal de novo biopsies from PDAC patients paired with scRNA-seq to inform clinical decision-making.

Purpose: To assess the feasibility of skin electronic brachytherapy (EBT) and present the dosimetric and clinical characteristics of our initial experience. Materials and Methods: From May 2013 to November 2013, a total of 211 lesions in 191 patients were treated with primary EBT for skin cancer on a prospective registry. All patients received radiation therapy using the XOFT Axxent HDR EBT system to a dose of 40Gy in 8 fractions of 5Gy each given twice weekly with a minimum interval of 48 hours. Patient selection criteria included Age over 60 years, lesion size less than 4 cm diameter, less than 5mm depth, and basal or squamous cell carcinoma histopathology. Results: All patients successfully completed their planned treatment course. The clinical parameters of Age, lesion location, lesion surface diameter, lesion depth, histopathology and comorbidities as well as the dosimetric parameters of applicator cone size, prescribed depth dose, surface dose, dwell time, use of cutouts and correlating cutout factors will be presented. Conclusion: In our experience, the use of EBT for select skin cancers is highly feasible. A prospective registry trial is ongoing that will allow careful clinical and dosimetric correlation with toxicities and outcomes.