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Background: HR+ early stage breast cancer (ESBC) is associated with suboptimal pathologic complete response rate (pCR, ~10%) following neoadjuvant cytotoxic chemotherapy. Neoadjuvant endocrine therapy with aromatase inhibitors (AI) may serve as an effective alternative as gauged using the surrogate Ki67 cell proliferation histologic marker. Patients with poor Ki67 response (defined as Ki67>10%) following neoadjuvant AI exhibit poor prognosis and therapeutic resistance to both endocrine therapy and chemotherapy. In a genomic analysis of Ki67-high HR+ tumors, we identified 8-fold upregulation of BIRC5 (survivin), a gene that regulates apoptosis and the cell cycle and is associated with poor clinical outcome. Maveropepimut-S (MVP-S) leverages the non-aqueous, lipid-based DPX delivery platform to educate a specific and persistent T cell-based immune response to 5 HLA-restricted peptides from Survivin. Treatment with MVP-S and intermittent, low-dose cyclophosphamide (CPA) has shown to increase tumor infiltration of survivin-specific T cells. Previous clinical trials have shown that MVP-S is well-tolerated, immunogenic, and could lead to clinical response in several cancer indications. Further exploration of the regimen in breast cancer could extend the application of this immunotherapy for the unmet medical need of improving clinical response in high ki67 HR+ ESBC prior to surgery. Trial Design: NCT04895761 is phase I trial evaluating the safety and immunologic effects of neoadjuvant MVP-S plus letrozole (arm A, n=6), with/without tumor-directed MR-guided radiotherapy (arm B, n=6), or intermittent low-dose cyclophosphamide (CPA, arm C, n=6). Postmenopausal patients with T1c+ HR+HER2- breast cancer with Ki67>10% will receive two doses of MVP-S and 7 weeks of neoadjuvant letrozole prior to surgery (all arms), arm B will be treated additionally with concurrent 10Gy x 2 tumor boost radiation to facilitate immunogenic cell death, and arm C (n=6) will be treated additionally with intermittent low-dose CPA (50mg BID) to facilitate regulatory T cell depletion. Specific Aims: The primary objective is safety. Biomarker objectives are to evaluate for each treatment arm: 1) systemic type I survivin-specific immune response, as measured by IFN-γ ELISPOT; 2) changes in immune environment by GeoMx digital spatial genomic profiling; 3) and changes in tumor infiltrating lymphocytes (TILs) and Ki67. These data will be used to identify the most immunogenic MVP-S combination therapy for study in phase II trial powered to assess clinical outcome (pCR). Accrual: 3 of 6 patients in the MVP-S+ letrozole arm have been enrolled. Arm B and C will enroll after completion of arm A. Citation Format: Sasha E. Stanton, Lisa D. MacDonald, Stephan Fiset, Staci Mellinger, Nicole Moxon, Heather Hirsch, Tracy L. Kelly, Kristina H. Young, David B. Page. Neoadjuvant survivin immunotherapy maveropepimut-S (MVP-S) to increase Th1 immune response in Ki67-high hormone receptor positive (HR+) early-stage breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-20-01.

SM16 efficacy and radiosensitivity of CT26 and Panc02.

Supplemental Figure legends from TGFβ Inhibition Prior to Hypofractionated Radiation Enhances Efficacy in Preclinical Models

Quantified effect of tumor size and epithelial-to-mesenchymal transition.

The immune infiltrate in colorectal cancer has been correlated with outcome, such that individuals with higher infiltrations of T cells have increased survival independent of the disease stage. For patients with lower immune infiltrates, overall survival is limited. Because the patients with colorectal cancer studied have received conventional cancer therapies, these data may indicate that the pretreatment tumor environment increases the efficacy of treatments such as chemotherapy, surgery, and radiotherapy. This study was designed to test the hypothesis that an improved immune environment in the tumor at the time of treatment will increase the efficacy of radiotherapy. We demonstrate that inhibition of TGFβ using the orally available small-molecule inhibitor SM16 improved the immune environment of tumors in mice and significantly improved the efficacy of subsequent radiotherapy. This effect was not due to changes in radiosensitivity, epithelial–mesenchymal transition, or changes in vascular function in the tumor; rather, this effect was dependent on adaptive immunity and resulted in long-term protective immunity in cured mice. These data demonstrate that immunotherapy is an option to improve the immune status of patients with poor tumor infiltrates and that pretreatment improves the efficacy of radiotherapy. Cancer Immunol Res; 2(10); 1011–22. ©2014 AACR.

Radiation has been utilized as a direct cytotoxic tumorcidal modality, however, the effect of radiation on tumor vasculature influences response to anticancer therapies. Although numerous reports have demonstrated vascular changes in irradiated tumors, the findings and implications are extensive and at times contradictory depending on the radiation dose, timing, and models used. In this review, we focus on the radiation-mediated effects on tumor vasculature with respect to doses used, timing postradiation, vasculogenesis, adhesion molecule expression, permeability, and pericyte coverage, including the latest findings.

TGF-β is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-β blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-β type I receptor kinase inhibitor galunisertib to neoadjuvant chemoradiotherapy would improve pathological complete response rates in patients with locally advanced rectal cancer.This was an investigator-initiated, single-arm, phase 2 study done in two medical centres in Portland (OR, USA). Eligible patients had previously untreated, locally advanced, rectal adenocarcinoma, stage IIA-IIIC or IV as per the American Joint Committee on Cancer; Eastern Cooperative Oncology Group status 0-2; and were aged 18 years or older. Participants completed two 14-day courses of oral galunisertib 150 mg twice daily, before and during fluorouracil-based chemoradiotherapy (intravenous fluorouracil 225 mg/mBetween Oct 19, 2016, and Aug 31, 2020, 38 participants were enrolled. 25 (71%) of the 35 patients who completed chemoradiotherapy proceeded to total mesorectal excision surgery, five (20%) of whom had pathological complete responses. Ten (29%) patients had non-operative management, three (30%) of whom ultimately chose to have total mesorectal excision. Two (67%) of those three patients had pathological complete responses. Of the remaining seven patients in the non-operative management group, five (71%) had clinical complete responses at 1 year after their last modified FOLFOX6 infusion. In total, 12 (32% [one-sided 95% CI ≥19%]) of 38 patients had a complete response. Common grade 3 adverse events during treatment included diarrhoea in six (16%) of 38 patients, and haematological toxicity in seven (18%) patients. Two (5%) patients had grade 4 adverse events, one related to chemoradiotherapy-induced diarrhoea and dehydration, and the other an intraoperative ischaemic event. No treatment-related deaths occurred.The addition of galunisertib to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer improved the complete response rate to 32%, was well tolerated, and warrants further assessment in randomised trials.Eli Lilly via ExIST program, The Providence Foundation.

A malignant tumor consists of malignant cells as well as a wide array of normal host tissues including stroma, vasculature, and immune infiltrate. The interaction between cancer and these host tissues is critical as these host tissues play a variety of roles in supporting or resisting disease progression. Radiotherapy (RT) has direct effects on malignant cells, but, also, critically important effects on these other components of the tumor microenvironment (TME). Given the growing role of immune checkpoint inhibitors and other immunotherapy strategies, understanding how RT affects the TME, particularly the immune compartment, is essential to advance RT in this new era of cancer therapy. The interactions between RT and the TME are complex, affecting the innate and adaptive arms of the immune system. RT can induce both proinflammatory effects and immune suppressive effects that can either promote or impede antitumor immunity. It is likely that the initial proinflammatory effects of RT eventually lead to rebound immune-suppression as chronic inflammation sets in. The exact kinetics and nature of how RT changes the TME likely depends on timing, dose, fractionation, site irradiated, and tumor type. With increased understanding of the effects of RT on the TME, in the future it is likely that we will be able to personalize RT by varying the dose, site, and timing of intervention to generate the desired response to partner with immunotherapy strategies.

BackgroundCheckpoint inhibitors targeting programmed death receptor-1 (PD-1) have been tested in the neoadjuvant setting for the treatment of locoregionally advanced head and neck squamous cell carcinoma (HNSCC); however, response rates are modest. We hypothesized that adding stereotactic body radiation therapy (SBRT) to anti-PD-1 would be safe prior to definitive surgical resection and would enhance pathological response compared with historical cohorts of patients with locoregionally advanced HNSCC treated with checkpoint inhibitor alone.MethodsThe Neoadjuvant Immuno-Radiotherapy Trial was an investigator-initiated single institution phase Ib clinical trial that enrolled patients with previously untreated locally advanced HPV-positive and HPV-negative HNSCC between 2018 and 2019. Eligible patients were treated with neoadjuvant SBRT at a total dose of either 40 Gy in 5 fractions or 24 Gy in 3 fractions, delivered in a 1-week timespan, with or without nivolumab, prior to definitive surgical resection. Patients were then planned for treatment with adjuvant nivolumab for 3 months. The primary safety endpoint was unplanned delay in surgery considered to be at least possibly related to neoadjuvant treatment. The primary efficacy endpoints included pathological complete response (pCR), major pathological response (mPR), and the rate of clinical to pathological downstaging after neoadjuvant treatment.ResultsTwenty-one patients underwent neoadjuvant treatment, which was well tolerated and did not delay surgery, thus meeting the primary endpoint. Tissue responses were characterized by robust inflammatory infiltrates in the regression bed, plasma cells and cholesterol clefts. Among the entire study group, the mPR and pCR rate was 86% and 67%, respectively. Clinical to pathological downstaging occurred in 90% of the patients treated.ConclusionThese data demonstrate that radiation delivered only to the gross tumor volume combined with immunotherapy was safe, resulted in a high rate of mPR and should be further evaluated as a locally focused neoadjuvant therapy for patients with head and neck cancer.Trial registration numberThis study is registered with clinicaltrials.gov (NCT03247712) and is active, but closed to patient accrual.

Pancreatic ductal adenocarcinoma (PDAC) has traditionally been thought of as an immunologically quiescent tumor type presumably because of a relatively low tumor mutational burden (TMB) and poor responses to checkpoint blockade therapy. However, many PDAC tumors exhibit T cell inflamed phenotypes. The presence of tertiary lymphoid structures (TLS) has recently been shown to be predictive of checkpoint blockade response in melanomas and sarcomas, and are prognostic for survival in PDAC. In order to more comprehensively understand tumor immunity in PDAC patients with TLS, we performed RNA-seq, single and multiplex IHC, flow cytometry and predictive genomic analysis on treatment naïve, PDAC surgical specimens. Forty-six percent of tumors contained distinct T and B cell aggregates reflective of “early-stage TLS” (ES-TLS), which correlated with longer overall and progression-free survival. These tumors had greater CD8+ T cell infiltration but were not defined by previously published TLS gene-expression signatures. ES-TLS+ tumors were enriched for IgG1 class-switched memory B cells and memory CD4+ T cells, suggesting durable immunological memory persisted in these patients. We also observed the presence of active germinal centers (mature-TLS) in 31% of tumors with lymphocyte clusters, whose patients had long-term survival (median 56 months). M-TLS-positive tumors had equivalent overall T cell infiltration to ES-TLS, but were enriched for activated CD4+ memory cells, naive B cells and NK cells. Finally, using a TCGA-PDAC dataset, ES-TLS+ tumors harbored a decreased TMB, but M-TLS with germinal centers expressed significantly more MHCI-restricted neoantigens as determined by an in silico neoantigen prediction method. Interestingly, M-TLS+ tumors also had evidence of increased rates of B cell somatic hypermutation, suggesting that germinal centers form in the presence of high-quality tumor neoantigens leading to increased humoral immunity that confers improved survival for PDAC patients. AbbreviationsTLS: tertiary lymphoid structures; GC: germinal center(s); PDAC: pancreatic ductal adenocarcinoma; RNA-seq: RNA sequencing; BCRseq: B cell receptor sequencing; HEV: high endothelial venule; PNAd: peripheral node addressin; TMB: tumor mutational burden; TCGA: the cancer genome atlas; PAAD: pancreatic adenocarcinoma; FFPE: formalin fixed paraffin embedded; TIME: tumor immune microenvironment.

In many applications based on kinetic evaluation analysis and model fitting, quantitative mapping retrieved on data series from modalites such as MRI is completed on a voxel-by-voxel basis, where motion and low signal to noise ratio (SNR) would considerably degenerate the reliability of estimations. The coherence of image series in space and time can be used as prior knowledge to mitigate this occurrence. In this study, spatial and temporal higher-order total variations (HOTVs) are applied on a data series of MRI signal (e.g. dynamic contrast-enhanced (DCE) MRI and intravoxel incoherent motion (IVIM) MRI) to exploit the coherence of signal in space and time to minimize the variabilities caused by motion as well as improving quality of images with low SNR while retaining the physical details of original data properly. Simultaneously applying spatial and temporal HOTVs on images is non-trivial in implementation since it is a non-smooth optimization problem with multiple regularizers. Therefore, we use the proximal gradient method as well as a primal-dual split proximal mechanism to address the problem properly. In addition to increase the reliability of quantitative parametric map estimations, this preprocessing procedure can be included into many existing map estimation algorithms and pipelines effortlessly. We demonstrate our method on the parametric maps estimation for DCE MRI and IVIM MRI.

Immunotherapies are rapidly entering the clinic as approved treatments for diverse cancer pathologies. Radiation therapy is an integral partner in cancer therapy, commonly as part of complicated multimodality approaches that optimize patient outcomes. Preclinical studies have demonstrated that the success of radiation therapy in tumor control is due in part to immune mechanisms, and that outcomes following radiation therapy can be improved through combination with a range of immunotherapies. However, preclinical models of cancer are very different from patient tumors, and the way these preclinical tumors are treated is often very different from standard of care treatment of patients. This review examines the preclinical and clinical data for the role of the immune system in radiation therapy outcomes, and how to integrate preclinical findings into clinical trials, using ongoing studies as examples.

Transforming growth factor beta (TGFβ) is a multipotent immunosuppressive cytokine. TGFβ excludes immune cells from tumors, and TGFβ inhibition improves the efficacy of cytotoxic and immune therapies. Using preclinical colorectal cancer models in cell type-conditional TGFβ receptor I (ALK5) knockout mice, we interrogate this mechanism. Tumor growth delay and radiation response are unchanged in animals with Treg or macrophage-specific ALK5 deletion. However, CD8αCre-ALK5flox/flox (ALK5ΔCD8) mice reject tumors in high proportions, dependent on CD8+ T cells. ALK5ΔCD8 mice have more tumor-infiltrating effector CD8+ T cells, with more cytotoxic capacity. ALK5-deficient CD8+ T cells exhibit increased CXCR3 expression and enhanced migration towards CXCL10. TGFβ reduces CXCR3 expression, and increases binding of Smad2 to the CXCR3 promoter. In vivo CXCR3 blockade partially abrogates the survival advantage of an ALK5ΔCD8 host. These data demonstrate a mechanism of TGFβ immunosuppression through inhibition of CXCR3 in CD8+ T cells, thereby limiting their trafficking into tumors., TGFβ has a role in cancer immunosuppression but the exact mechanisms haven’t been fully elucidated. Here, using mouse models deficient in TGFβ-signaling, the authors show that loss of ALK5 in CD8 + T cells enhances their tumour trafficking and cytotoxicity suggesting that ALK5 inhibitors may have clinical utility.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes.

Purpose The purpose of this article is to assemble, review, and provide a synopsis of the historical and current literature regarding optimal sequencing of radiation (RT) and immunotherapy combination treatments. Materials and methods A review of the literature was performed using PubMed with the query “radiation” and “Immunotherapy”, “PD1”, “PDL1”, “CTLA4”, “OX40”, “checkpoint”, “vaccine”, “macrophage”, “STING”, and “TGFbeta”. Studies that included sequencing of therapy were evaluated and the studies were included at the authors discretion. Results A paucity of primary literature exists examining the best order of radiation and immunotherapy, most of which was performed in the pre-clinical setting. The observations are that optimal sequencing of various radiation plus immune therapy combinations is dependent on the mechanism(s) of activation by the combination treatment. Immunosuppressive molecules tend to be better inhibited prior to RT while engagement of costimulatory genes is better activated concomitantly with RT. Conclusions These data should compel more basic research into both the direct investigation of sequencing efficacy and studies on the mechanisms of immune mediated cell death potentiated by radio-therapy.

In this study we determine the survival in patients with HPV-positive oropharyngeal carcinoma treated with transoral robotic surgery (TORS), neck dissection and risk-adapted adjuvant therapy.We retrospectively identified 122 patients with HPV-positive oropharyngeal carcinoma treated with TORS and neck dissection between 2011 and 2018. Survival probability was calculated. We determined the effect of the type of neck dissection performed (modified radical neck dissection-MRND vs. selective neck dissection - SND), extranodal extension (ENE), margin status, and presence of ≥ 5 metastatic nodes on survival.Our patient population had a five-year overall survival of 91.0% (95% C.I. 85-97%). The five-year probability of recurrence or cancer-associated death was 0.0977 (95% C.I. 0.0927-0.1027). The five-year probability of cancer-associated death was 0.0528 (95% C.I. 0.048-0.0570). All patients who died of their disease had distant metastasis. Our PEG dependence rate was 0%. Patients with ENE and positive margins who underwent adjuvant chemoradiation did not have worse survival. Presence of ≥ 5 metastatic nodes portended worse survival after controlling for age, positive ENE and margins. Low yield (18 nodes) on neck dissection worsened DFS on multivariable analysis. Furthermore, patients who underwent SND did not have worse OS than those who underwent MRND.Our study demonstrates that surgery could be simplified by performing TORS with SND rather than MRND. The one true poor prognostic factor in HPV-positive oropharyngeal carcinoma patients who undergo surgery is high nodal burden. Patients with high nodal burden are much more likely to die from their disease.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic stroma with a poor lymphocyte infiltrate, in part driven by cancer-associated fibroblasts (CAFs). CAFs, which express fibroblast activation protein (FAP), contribute to immune escape via exclusion of anti-tumor CD8(+) T cells from cancer cells, upregulation of immune checkpoint ligand expression, immunosuppressive cytokine production, and polarization of tumor infiltrating inflammatory cells. FAP is a post-proline peptidase selectively expressed during tissue remodeling and repair, such as with wound healing, and in the tumor microenvironment by cancer-associated fibroblasts. We targeted FAP function using a novel small molecule inhibitor, UAMC-1110, and mice with germline knockout of FAP and concomitant knock-in of E. coli beta-galactosidase. We depleted CAFs by adoptive transfer of anti-beta gal T cells into the FAP knockout animals. Established syngeneic pancreatic tumors in immune competent mice were targeted with these 3 strategies, followed by focal radiotherapy to the tumor. FAP loss was associated with improved antigen-specific tumor T cell infiltrate and enhanced collagen deposition. However, FAP targeting alone or with tumor-directed radiation did not improve survival even when combined with anti-PD1 therapy. Targeting of CAFs alone or in combination with radiation did not improve survival. We conclude that targeting FAP and CAFs in combination with radiation is capable of enhancing anti-tumor T cell infiltrate and function, but does not result in sufficient tumor clearance to extend survival.

Aim Small, solitary hepatocellular carcinoma is curable with stereotactic radiation or other methods of tumor ablation, however, regional and systemic tumor recurrence occurs in over 70% of patients. Here we describe the ability of immunoradiotherapy to induce an antitumor immune response and delay the growth of tumors in immunocompetent mice. Methods A syngeneic hepatocellular carcinoma cell line (Hep-55.1c) was injected directly into the livers of C57BL/6 mice using ultrasound guidance, then tumors were treated with stereotactic radiation using a Small Animal Radiation Research Platform with computed tomography guidance. Results Delivery of three doses of 250 μg anti-programmed cell death protein-1 (αPD-1) antibody concurrently with 30 Gy stereotactic body radiation therapy in three fractions reduced the growth rate of tumors and improved survival (P

// Marka R. Crittenden 1, 2 , Jason Baird 1 , David Friedman 1 , Talicia Savage 1 , Lauren Uhde 1 , Alejandro Alice 1 , Benjamin Cottam 1 , Kristina Young 1, 2 , Pippa Newell 1, 3 , Cynthia Nguyen 1 , Shelly Bambina 1 , Gwen Kramer 1 , Emmanuel Akporiaye 1 , Anna Malecka 4 , Andrew Jackson 4 , Michael J. Gough 1 1 Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center, Portland OR, USA 2 The Oregon Clinic, Portland OR, USA 3 Providence Hepatobiliary and Pancreatic Cancer Program, Providence Portland Medical Center, Portland OR, USA 4 Host-Tumour Interactions Group, Division of Cancer and Stem Cells, University of Nottingham, UK Correspondence to: Michael J. Gough, email: michael.gough@providence.org Keywords: radiation, macrophage, tumor, phagocytosis, apoptosis Received: July 16, 2016 Accepted: August 25, 2016 Published: September 2, 2016 ABSTRACT Radiation therapy provides a means to kill large numbers of cancer cells in a controlled location resulting in the release of tumor-specific antigens and endogenous adjuvants. However, by activating pathways involved in apoptotic cell recognition and phagocytosis, irradiated cancer cells engender suppressive phenotypes in macrophages. We demonstrate that the macrophage-specific phagocytic receptor, Mertk is upregulated in macrophages in the tumor following radiation therapy. Ligation of Mertk on macrophages results in anti-inflammatory cytokine responses via NF-kB p50 upregulation, which in turn limits tumor control following radiation therapy. We demonstrate that in immunogenic tumors, loss of Mertk is sufficient to permit tumor cure following radiation therapy. However, in poorly immunogenic tumors, TGFβ inhibition is also required to result in tumor cure following radiation therapy. These data demonstrate that Mertk is a highly specific target whose absence permits tumor control in combination with radiation therapy.

Hypothesis: Hypofractionated radiation therapy (HFRT) in combination with checkpoint blockade may modulate the tumor microenvironment to induce favorable antitumor immune responses and enhance surgical resection. Purpose: The purpose of this first-in-human phase Ib clinical trial was to test the safety and immunologic effects of neoadjuvant immunoradiotherapy prior to surgery in patients with head and neck squamous cell carcinoma (HNSCC) (NCT03247712). Methods: Patients with previously untreated stage I-III (AJCC 8th Ed) p16 positive squamous cell carcinoma of the oropharynx (OPSCC) or cervical lymph nodes from an unknown primary (SCCUP) were eligible to enroll. Neoadjuvant treatment consisted of sandwich nivolumab in combination with HFRT to gross tumor volume in one of two dose-finding cohorts of 8Gy x5 (Monday-Friday) or 8Gy x3 (Monday-Wednesday-Friday), followed five weeks later by definitive surgical resection. The primary endpoint was < 33% unplanned surgical delay; secondary endpoint was pathologic response. Surgical complications were assessed using the Clavien-Dindo scale, a validated scoring tool for surgical complications, applied for 90 days postoperatively. Serial blood and tissue specimens were obtained longitudinally for immunogenomic changes over time. Results: 10 patients (stage: T0N1M0, N=3; T1N1M0, N=1; T2N1M0, N=6) enrolled and underwent neoadjuvant immunoradiotherapy prior to surgery with curative intent, which consisted of selective neck dissection (level II-IV) and transoral robotic assisted oropharyngectomy for patients with mucosal disease. Surgery was safely performed in all patients without delay, thus meeting the primary endpoint. 7 patients demonstrated a partial radiologic response (PR) by RECIST criteria prior to surgery and 3 patients had stable disease (SD). The pathologic complete response rate (pCR) was 90% (8GyX5 cohort=5/5; 8GyX3 cohort=4/5) with the remaining patient achieving a major pathologic response (MPR; Conclusions: Neoadjuvant HFRT in combination with nivolumab is safe, does not delay definitive surgical resection, and results in significant downstaging with major pathologic response. A lower toxicity profile favors a radiation dose of 8Gy x3 for future development in both human papilloma virus (HPV)-related and HPV-unrelated HNSCC. Citation Format: R. Bryan Bell, Rom Leidner, Marcus Couey, Ashish Patel, Amber Watters, Hong Ziao, Carlo Bifulco, Brian Piening, George Morris, Lessli Rushforth, Dawn Brucker, Shoren Nemeth, Kristina Young, Michael Gough, Marka Crittenden. Surgical outcomes following neoadjuvant hypofractionated radiation in combination with nivolumab: High rate of pathologic complete response in patients with p16+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B01.

An accumulating body of evidence demonstrates that radiation therapy can generate adaptive immune responses that contribute to tumor control. However, in the absence of additional immune therapy, the adaptive immune response is insufficient to prevent tumor recurrence or affect distant disease. It has been shown in multiple models that tumor-infiltrating myeloid cells exhibit alternative activation phenotypes and are able to suppress adaptive immune responses, and recent data suggests that the myeloid response in tumors treated with cytotoxic therapy limits tumor control. We hypothesized that tumor myeloid cells inhibit the adaptive immune response after radiation therapy through expression of the enzyme arginase I. Using a myeloid cell-specific deletion of arginase I in mice, we demonstrate an improved tumor control after radiation therapy. However, tumors still recurred despite the conditional knockdown of arginase I. Since multiple alternative factors may combine to inhibit adaptive immunity, we propose that targeting macrophage differentiation may be a more effective strategy than targeting individual suppressive pathways.

Background: Standard treatment of locally advanced HPV-associated oropharyngeal HNSCC includes definitive chemoRT or surgery followed by risk-adapted adjuvant RT +/- chemo. These approaches provide high rates of long term survival, but are associated with significant subacute as well as long term morbidity. Therefore, increasing efforts have been directed at exploring alternative approaches. Combined SBRT and PD-1 blockade may have particularly favorable properties for anti-tumor immune response in HNSCC (Gough 2009). We present phase I safety & efficacy data from the NIRT trial of neoadjuvant nivolumab/SBRT in oropharyngeal p16+ HNSCC (NCT03247712). Methods: Patients with p16+ oropharyngeal or unknown primary HNSCC, with clinical indications for adjuvant RT, or upfront TORS ineligible due to tumor size, could enroll. Patients received Nivo 240mg IV q2wks x3 prior to surgery, with SBRT to GTV+3mm delivered between Nivo doses 1 & 2, in two dose finding cohorts (n=5 each): 8Gy x5 daily (M-F) and de-escalated 8Gy x3 (M,W,F). Surgery was performed 5 weeks post-SBRT followed by adjuvant Nivo 480mg IV q4wks x3, starting 4 weeks post-op. The primary endpoint was < 33% unplanned surgical delay; the secondary endpoint was pathologic response by irPRC (Cottrell 2018). Results: There were no unplanned surgical delays. All patients had radiologic evidence of decreased tumor size prior to surgery, but none showed a CR by RECIST. Remarkably, the pathologic CR rate was 100% in the 8Gy x 5 cohort (5/5) and 80% In the 8 Gy x 3 cohort (4/5), with the remaining patient achieving MPR (major pathologic response; Conclusions: Neoadjuvant combined PD-1/SBRT to GTV+3mm dosed at either 8Gy x5 (M-F) or 8Gy x3 (M,W,F) did not delay HNSCC surgery in this Phase I trial (n=10; five per dose). Potent anti-tumor response was observed in all cases (CR=9 + MPR=1). A high response rate and lower toxicity profile favors the 8Gy x3 cohort for development. This study represents a major paradigm shift in the approach to treatment of locally advanced p16+ oropharyngeal cancers. Citation Format: Rom Leidner, R. Bryan Bell, Kristina Young, Brendan Curti, Marcus Couey, Ashish Patel, Amber Watters, Hong Xiao, Carlo Bifulco, Brian Piening, George Morris, Lessli Rushforth, Dawn Brucker, Shorin Nemeth, Michael Gough, Marka Crittenden. Neoadjuvant immuno-radiotherapy (NIRT) in head and neck cancer: Phase I/Ib study of combined PD-1/SBRT prior to surgical resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT182.

Objective Radiation for brain metastases is typically delivered by whole brain radiation (WBRT), stereotactic radiosurgery (SRS), or a combination thereof. There are patients with lesions that are not candidates for SRS owing to a maximum target diameter >3 cm, for which avoiding WBRT may provide a quality of life benefit. Here, we report our early experience with stereotactic hypofractionated radiation therapy for brain metastases or resection cavities (SBRT brain) as an alternative to WBRT.

// Gwendolyn J. McGinnis 1, 2, 3 , David Friedman 4 , Kristina H. Young 4 , Eileen Ruth S. Torres 2 , Charles R. Thomas Jr 3 , Michael J. Gough 3, 4 , Jacob Raber 2, 3, 5, 6 1 Howard Hughes Medical Institute, Oregon Health and Science University, Portland, OR 2 Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, OR 3 Department of Radiation Medicine, Oregon Health and Science University, Portland, OR 4 Earle A. Chiles Research Institute, Providence Cancer Center, Portland, OR 5 Department of Neurology, Oregon Health and Science University, Portland, OR 6 Division of Neuroscience, Oregon National Primate Research Center, Portland, OR Correspondence to: Jacob Raber, email: raberj@ohsu.edu Keywords: neuroinflammation, cancer-related neurological dysfunction, microglia, immunotherapy, radiotherapy Received: June 21, 2016 Accepted: November 14, 2016 Published: November 24, 2016 ABSTRACT Background: Cancer patients often report behavioral and cognitive changes following cancer treatment. These effects can be seen in patients who have not yet received treatment or have received only peripheral (non-brain) irradiation. Novel treatments combining radiotherapy (RT) and immunotherapy (IT) demonstrate remarkable efficacy with respect to tumor outcomes by enhancing the proinflammatory environment in the tumor. However, a proinflammatory environment in the brain mediates cognitive impairments in other neurological disorders and may affect brain function in cancer patients receiving these novel treatments. Currently, gaps exist as to whether these treatments impact the brain in individuals with or without tumors and with regard to the underlying mechanisms. Results: Combined treatment with precision RT and checkpoint inhibitor IT achieved control of tumor growth. However, BALB/c mice receiving combined treatment demonstrated changes in measures of anxiety levels, regardless of tumor status. C57BL/6J mice with tumors demonstrated increased anxiety, except following combined treatment. Object recognition memory was impaired in C57BL/6J mice without tumors following combined treatment. All mice with tumors showed impaired object recognition, except those treated with RT alone. Mice with tumors demonstrated impaired amygdala-dependent cued fear memory, while maintaining hippocampus-dependent context fear memory. These behavioral alterations and cognitive impairments were accompanied by increased microglial activation in mice receiving immunotherapy alone or combined with RT. Finally, based on tumor status, there were significant changes in proinflammatory cytokines (IFN-γ, IL-6, IL-5, IL-2, IL-10) and a growth factor (FGF-basic). Materials and Methods: Here we test the hypothesis that IT combined with peripheral RT have detrimental behavioral and cognitive effects as a result of an enhanced proinflammatory environment in the brain. BALB/c mice with or without injected hind flank CT26 colorectal carcinoma or C57BL/6J mice with or without Lewis Lung carcinoma were used for all experiments. Checkpoint inhibitor IT, using an anti-CTLA-4 antibody, and precision CT-guided peripheral RT alone and combined were used to closely model clinical treatment. We assessed behavioral and cognitive performance and investigated the immune environment using immunohistochemistry and multiplex assays to analyze proinflammatory mediators. Conclusions: Although combined treatment achieved tumor growth control, it affected the brain and induced changes in measures of anxiety, cognitive impairments, and neuroinflammation.

Small, solitary hepatocellular carcinoma is curable with stereotactic radiation or other methods of tumor ablation, however, regional and systemic tumor recurrence occurs in over 70% of patients. Here we describe the ability of immunoradiotherapy to induce an antitumor immune response and delay the growth of tumors in immunocompetent mice.A syngeneic hepatocellular carcinoma cell line (Hep-55.1c) was injected directly into the livers of C57BL/6 mice using ultrasound guidance, then tumors were treated with stereotactic radiation using a Small Animal Radiation Research Platform with computed tomography guidance.Delivery of three doses of 250 μg anti-programmed cell death protein-1 (αPD-1) antibody concurrently with 30 Gy stereotactic body radiation therapy in three fractions reduced the growth rate of tumors and improved survival (P 0.05). Combined treatment was associated with increased CD8Tumor response to stereotactic radiation can be augmented by concurrent treatment with αPD-1. The efficacy of this combination therapy was transient, however, and treatment induced markers of adaptive immune resistance. These data are promising, but also indicate that mechanisms of immune resistance will need to be durably overcome for this combination to generate lasting immunity to protect against tumor recurrence.

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

Background Preclinical studies have shown synergy between radiation therapy and immunotherapy. However, in almost all preclinical models, radiation is delivered in single doses or short courses of high doses (hypofractionated radiation). By contrast in most clinical settings, radiation is delivered as standard small daily fractions of 1.8-2 Gy to achieve total doses of 50–54 Gy (fractionated radiation). We do not yet know the optimal dose and scheduling of radiation for combination with chemotherapy and immunotherapy. Methods To address this, we analyzed the effect of neoadjuvant standard fractionated and hypofractionated chemoradiation on immune cells in patients with locally advanced and borderline resectable pancreatic adenocarcinoma. Results We found that standard fractionated chemoradiation resulted in a significant and extended loss of lymphocytes that was not explained by a lack of homeostatic cytokines or response to cytokines. By contrast, treatment with hypofractionated radiation therapy avoided the loss of lymphocytes associated with conventional fractionation. Conclusion Hypofractionated neoadjuvant chemoradiation is associated with reduced systemic loss of T cells. Trial registration ClinicalTrials.gov NCT01342224, April 21, 2011; NCT01903083, July 2, 2013. Electronic supplementary material The online version of this article (doi:10.1186/s40425-016-0149-6) contains supplementary material, which is available to authorized users.

Human papilloma virus positive (HPV+) tumors represent a large proportion of anal, vulvar, vaginal, cervical and head and neck squamous carcinomas (HNSCC) and late stage invasive disease is thought to originate from a premalignant state. Cyclic dinucleotides that activate STimulator of INterferon Genes (STING) have been shown to cause rapid regression of a range of advanced tumors. We aimed to investigate STING ligands as a novel treatment for papilloma. We tested therapies in a spontaneous mouse model of papilloma of the face and anogenital region that histologically resembles human HPV-associated papilloma. We demonstrate that STING ligands cause rapid regression of papilloma, associated with T cell infiltration, and are significantly more effective than Imiquimod, a current immunotherapy for papilloma. In humans, we show that STING is expressed in the basal layer of normal skin and lost during keratinocyte differentiation. We found STING was expressed in all HPV-associated cervical and anal dysplasia and was strongly expressed in the cancer cells of HPV+ HNSCC but not in HPV-unrelated HNSCC. We found no strong association between STING expression and progressive disease in non-HPV oral dysplasia and oral pre-malignancies that are not HPV-related. These data demonstrate that STING is expressed in basal cells of the skin and is retained in HPV+ pre-malignancies and advanced cancers, but not in HPV-unrelated HNSCC. However, using a murine HNSCC model that does not express STING, we demonstrate that STING ligands are an effective therapy regardless of expression of STING by the cancer cells.

The tumor immune environment has been linked to prognosis in patients with a range of malignancies. Recently, we demonstrated in pre-clinical models that modifying the tumor immune environment using a small-molecule inhibitor of TGFb significantly improved outcome to subsequent radiation therapy. These data suggest that this and other immunotherapies may be used to remodel the tumor before conventional cancer therapies to improve outcomes.

The immune infiltrate in colorectal cancer has been correlated with outcome, such that individuals with higher infiltrations of T cells have increased survival independent of the disease stage. For patients with lower immune infiltrates, overall survival is limited. Because the patients with colorectal cancer studied have received conventional cancer therapies, these data may indicate that the pretreatment tumor environment increases the efficacy of treatments such as chemotherapy, surgery, and radiotherapy. This study was designed to test the hypothesis that an improved immune environment in the tumor at the time of treatment will increase the efficacy of radiotherapy. We demonstrate that inhibition of TGFβ using the orally available small-molecule inhibitor SM16 improved the immune environment of tumors in mice and significantly improved the efficacy of subsequent radiotherapy. This effect was not due to changes in radiosensitivity, epithelial–mesenchymal transition, or changes in vascular function in the tumor; rather, this effect was dependent on adaptive immunity and resulted in long-term protective immunity in cured mice. These data demonstrate that immunotherapy is an option to improve the immune status of patients with poor tumor infiltrates and that pretreatment improves the efficacy of radiotherapy. Cancer Immunol Res; 2(10); 1011–22. ©2014 AACR.

Increasingly, positron emission tomography (PET) scans are being utilized in radiation medicine departments for staging, treatment planning, and follow-up. Positron emission tomography scans use signals emitted from the radioactive tracer [F]-fluoro-2-deoxy-Dglucose (FDG-glucose) that is preferentially taken up by cancer cells. After image processing including fusion with simultaneously obtained computed tomography (CT) scans, the oncologist is able to identify tumors as they “light up.” Positron emission tomography scans are becoming a part of standard oncologic evaluation for many cancers for which their sensitivity and specificity for cancer detection is superior to CT scans. Positron emission tomography is used frequently at the time of simulation because treatment planning can be tailored by targeting areas of metabolically active tumor. Given the sensitivity of PET in cancer detection, it is being used increasingly as part of standard followup to evaluate response to treatment, to detect early recurrent or persistent disease, and to help differentiate radiation necrosis from tumor. With all these useful applications in our cancer patients, the number of PET scans ordered has risen significantly in the last few years. For example, taking just the US Veterans Administration patients treated in our department, the number of PET scans ordered increased from 100 scans in 2008 to 811 in 2012. This presents a new and unique problem in radiation protection. The FDG-glucose is administered intravenously at a typical dose of 5-10 mCi directly before scanning, and it remains in a patient’s circulation for several hours after administration, with a physical half-life of 110 minutes. The biologic half-life is dependent on urinary excretion. Given patient anxiety in cancer detection and the fact that PET scanners are often located in close proximity to the ordering radiation oncologist, physician visits are

Radiation therapy is showing potential as a partner for immunotherapies in preclinical cancer models and early clinical studies. As has been discussed elsewhere, radiation provides debulking, antigen and adjuvant release, and inflammatory targeting of effector cells to the treatment site, thereby assisting multiple critical checkpoints in antitumor adaptive immunity. Adaptive immunity is terminated by inflammatory resolution, an active process which ensures that inflammatory damage is repaired and tissue function is restored. We discuss how radiation therapy similarly triggers inflammation followed by repair, the consequences to adaptive immune responses in the treatment site, and how the myeloid response to radiation may impact immunotherapies designed to improve control of residual cancer cells.

e23117Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic stroma and poor immune infiltrate, in part driven by cancer-associated fibroblasts (CAFs). CAFs contribute t...