Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

Durvalumab in combination with chemoradiotherapy for patients with unresectable stage III non-small-cell lung cancer: Results from the phase 1 CLOVER study.

INTRODUCTION: For patients with unresectable, stage III non-small-cell lung cancer (NSCLC), current standard of care is concurrent chemoradiotherapy (cCRT) followed by consolidation durvalumab. However, earlier initiation of durvalumab simultaneously with cCRT may increase antitumor activity relative to initiation after cCRT. The phase 1 CLOVER study (NCT03509012) evaluated durvalumab combined with cCRT in patients with advanced solid tumors; we report findings from the NSCLC cohort. METHODS: CLOVER comprised a dose-limiting toxicity (DLT) assessment part, followed by an expansion part. In the NSCLC cohort, patients with previously untreated, unresectable, stage III NSCLC were enrolled in three treatment arms: durvalumab every 4 weeks (Q4W) + cisplatin + etoposide + radiotherapy (Arm 1); durvalumab Q4W + carboplatin + paclitaxel + radiotherapy (Arm 2); or durvalumab Q4W + carboplatin or cisplatin + pemetrexed + radiotherapy (non-squamous histology only; Arm 3). Patients received durvalumab until disease progression or unacceptable toxicity. The primary endpoint was safety and tolerability. RESULTS: Sixty-four patients were enrolled: 21, 22, and 21 in Arms 1, 2, and 3, respectively. One patient in Arm 1 had DLT (grade 3 aspartate aminotransferase increase and grade 4 alanine aminotransferase increase); no DLTs were observed in Arms 2 or 3. Grade 3/4 adverse events occurred in 76.6 % of patients overall; the most common were neutropenia (51.6 %), leukopenia (20.3 %), and anemia (17.2 %). In a post-hoc analysis, 7.8 % of patients had grade 3 pneumonitis/radiation pneumonitis (grouped term) events. Overall, the objective response rate was 60.9 % (95 % confidence interval [CI], 47.9-72.9); median duration of response was 15.8 months (95 % CI, 9.0-not estimable [NE]). Median progression-free survival was 13.4 months (95 % CI, 8.8-20.1) and median overall survival was not reached (95 % CI, 21.9-NE). CONCLUSION: Durvalumab in combination with cCRT was well tolerated, with a manageable safety profile and showed encouraging antitumor activity in patients with unresectable, stage III NSCLC.

Durvalumab in combination with chemoradiotherapy in patients with head and neck squamous cell carcinoma: Results from the Phase 1 CLOVER study.

BACKGROUND: The Phase 1 CLOVER study (NCT03509012) assessed durvalumab in combination with concurrent chemoradiotherapy (cCRT) in patients with advanced solid tumors; we report results from the head and neck squamous cell carcinoma (HNSCC) cohort. METHODS: Patients with histologically/cytologically confirmed locally advanced HNSCC, eligible for definitive cCRT and not considered for primary surgery, received durvalumab plus cisplatin and concurrent external beam radiation. Objectives were to assess safety/tolerability and preliminary efficacy. RESULTS: Eight patients were enrolled. The most frequent any-cause adverse events (AEs) were nausea and radiation skin injury (each n = 5); most frequent grade 3/4 AEs were lymphopenia and stomatitis (each n = 3). No patients had dose-limiting toxicities. Objective response rate was 71.4% (5/7 patients; four complete responses, one partial response); disease control rate was 85.7% at 18 weeks and 83.3% at 48 weeks. CONCLUSIONS: Durvalumab plus cCRT was tolerable and active in patients with unresected, locally advanced HNSCC.

IL7 in combination with radiotherapy stimulates a memory T-cell response to improve outcomes in HNSCC models.

Clinically approved head and neck squamous cell carcinoma (HNSCC) immunotherapies manipulate the immune checkpoint blockade (ICB) axis but have had limited success outside of recurrent/metastatic disease. Interleukin-7 (IL7) has been shown to be essential for effector T-cell survival, activation, and proliferation. Here, we show that IL7 in combination with radiotherapy (RT) is effective in activating CD8 + T-cells for reducing tumor growth. Our studies were conducted using both human papillomavirus related and unrelated orthotopic HNSCC murine models. Immune populations from the tumor, draining lymph nodes, and blood were compared between treatment groups and controls using flow cytometry, proteomics, immunofluorescence staining, and RNA sequencing. Treatment with RT and IL7 (RT + IL7) resulted in significant tumor growth reduction, high CD8 T-cell tumor infiltration, and increased proliferation of T-cell progenitors in the bone marrow. IL7 also expanded a memory-like subpopulation of CD8 T-cells. These results indicate that IL7 in combination with RT can serve as an effective immunotherapy strategy outside of the conventional ICB axis to drive the antitumor activity of CD8 T-cells.

PPARα Agonism Enhances Immune Response to Radiotherapy While Dietary Oleic Acid Results in Counteraction.

PURPOSE: Head and neck cancer (HNC) improvements are stagnant, even with advances in immunotherapy. Our previous clinical trial data show that altered fatty acid (FA) metabolism correlates with outcome. We hypothesized that pharmacologic and dietary modulation of FA catabolism will affect therapeutic efficacy. EXPERIMENTAL DESIGN: We performed in vivo and in vitro experiments using PPARα agonism with fenofibrate (FF) or high oleic acid diets (OAD) with radiotherapy, generating metabolomic, proteomic, stable isotope tracing, extracellular flux analysis, and flow-cytometric data to investigate these alterations. RESULTS: FF improved antitumor efficacy of high dose per fraction radiotherapy in HNC murine models, whereas the OAD reversed this effect. FF-treated mice on the control diet had evidence of increased FA catabolism. Stable isotope tracing showed less glycolytic utilization by ex vivo CD8+ T cells. Improved efficacy correlated with intratumoral alterations in eicosanoid metabolism and downregulated mTOR and CD36. CONCLUSIONS: Metabolic intervention with increased FA catabolism improves the efficacy of HNC therapy and enhances antitumoral immune response.

Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs.

BACKGROUND: Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. METHODS: We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. FINDINGS: Calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 T cell activity in vivo. CONCLUSIONS: Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 T cells and activated CD8 T cells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. FUNDING: 1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).

Cancer immunotherapy responses persist after lymph node resection.

Surgical removal of lymph nodes (LNs) to prevent metastatic recurrence, including sentinel lymph node biopsy (SLNB) and completion lymph node dissection (CLND), are performed in routine practice. However, it remains controversial whether removing LNs which are critical for adaptive immune responses impairs immune checkpoint blockade (ICB) efficacy. Here, our retrospective analysis demonstrated that stage III melanoma patients retain robust response to anti-PD1 inhibition after CLND. Using orthotopic murine mammary carcinoma and melanoma models, we show that responses to ICB persist in mice after TDLN resection. Mechanistically, after TDLN resection, antigen can be re-directed to distant LNs, which extends the responsiveness to ICB. Strikingly, by evaluating head and neck cancer patients treated by neoadjuvant durvalumab and irradiation, we show that distant LNs (metastases-free) remain reactive in ICB responders after tumor and disease-related LN resection, hence, persistent anti-cancer immune reactions in distant LNs. Additionally, after TDLN dissection in murine models, ICB delivered to distant LNs generated greater survival benefit, compared to systemic administration. In complete responders, anti-tumor immune memory induced by ICB was systemic rather than confined within lymphoid organs. Based on these findings, we constructed a computational model to predict free antigen trafficking in patients that will undergo LN dissection.

The dichotomy of regulatory B cells in cancer versus allergic disease.

Regulatory B cells (Bregs) are an immunosuppressive cell phenotype that affects the immune system by limiting the inflammatory cascade. Dysregulation of Bregs can interestingly play a dichotomous role in the pathophysiology of many diseases and is especially highlighted when examining cancer pathology compared to allergic disease. This study reviews the existing literature on Bregs and compares their role in allergic disease in contrast to cancer development. Upregulation of Bregs in cancer states has been associated with poor prognostic outcomes across various cancer types, and Breg proliferation was associated with chronic interferon signaling, activation of the BCR-BTK (B cell receptor-Bruton's tyrosine kinase) pathway, and release of C-X-C motif ligand 13. In contrast, Breg dysfunction has been identified as a key mechanism in many allergic diseases, such as allergic asthma, allergic rhinitis, atopic dermatitis, and contact dermatitis. Development of Breg-targeted immunotherapies is currently at the preclinical level, but strategies differentially focus on Breg depletion in cancer versus Breg stimulation in allergy. Our review highlights the divergent functions that Bregs play in cancer compared to allergy. We conclude that natural homeostasis hinges on a fine balance between the dichotomous role of Bregs-over or underactivation can result in a pathological state.

Pancreatic Ductal Adenocarcinoma Cells Regulate NLRP3 Activation to Generate a Tolerogenic Microenvironment.

Defining feature of pancreatic ductal adenocarcinoma (PDAC) that participates in the high mortality rate and drug resistance is the immune-tolerant microenvironment which enables tumors to progress unabated by adaptive immunity. In this study, we report that PDAC cells release CSF-1 to induce nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) activation in myeloid cells. Increased NLRP3 expression was found in the pancreas of patients with PDAC when compared with normal pancreas which correlated with the formation of the NLRP3 inflammasome. Using human primary cells and an orthotopic PDAC mouse model, we show that NLRP3 activation is responsible for the maturation and release of the inflammatory cytokine IL1ß which selectively drives Th2-type inflammation via COX2/PGE2 induction. As a result of this inflammation, primary tumors were characterized by reduced cytotoxic CD8+ T-cell activation and increased tumor expansion. Genetic deletion and pharmacologic inhibition of NLRP3 enabled the development of Th1 immunity, increased intratumoral levels of IL2, CD8+ T cell­mediated tumor suppression, and ultimately limited tumor growth. In addition, we observed that NLRP3 inhibition in combination with gemcitabine significantly increased the efficacy of the chemotherapy. In conclusion, this study provides a mechanism by which tumor-mediated NLRP3 activation exploits a distinct adaptive immunity response that facilitates tumor escape and progression. Considering the ability to block NLRP3 activity with safe and small orally active molecules, this protein represents a new promising target to improve the limited therapeutic options in PDAC. SIGNIFICANT: This study provides novel molecular insights on how PDAC cells exploit NLRP3 activation to suppress CD8 T-cell activation. From a translational perspective, we demonstrate that the combination of gemcitabine with the orally active NLRP3 inhibitor OLT1177 increases the efficacy of monotherapy.

Selective targeting of IL2Rßγ combined with radiotherapy triggers CD8- and NK-mediated immunity, abrogating metastasis in HNSCC.

The implementation of cancer immunotherapies has seen limited clinical success in head and neck squamous cell carcinoma (HNSCC). Interleukin-2 (IL-2), which modulates the survival and functionality of lymphocytes, is an attractive target for new immunotherapies but one that is limited by presence of regulatory T cells (Tregs) expressing the high-affinity IL-2Rα. The bispecific immunocytokine PD1-IL2v preferentially delivers IL-2 signaling through IL-2Rßγ on PD-1-expressing cells. Selectively targeting the intermediate-affinity IL-2Rßγ can be leveraged to induce anti-tumor immune responses in effector T cells and natural killer (NK) cells while limiting the negative regulation of IL-2Rα activation on Tregs. Using radiation therapy (RT) in combination with PD1-IL2v improves local tumor control and survival, and controls metastatic spread in orthotopic HNSCC tumor models. PD1-IL2v drives systemic activation and expansion of circulating and tumor-infiltrating cytotoxic T cells and NK cells while limiting Treg-mediated immunosuppression. These data show that PD1-L2v induces durable systemic tumor control in HNSCC.

Measurement of mouse head and neck tumors by automated analysis of CBCT images.

Animal experiments are often used to determine effects of drugs and other biological conditions on cancer progression, but poor accuracy and reproducibility of established tumor measurement methods make results unreliable. In orthotopic mouse models of head and neck cancer, tumor volumes approximated from caliper measurements are conventionally used to compare groups, but geometrical challenges make the procedure imprecise. To address this, we developed software to better measure these tumors by automated analysis of cone-beam computed tomography (CBCT) scans. This allows for analyses of tumor shape and growth dynamics that would otherwise be too inaccurate to provide biological insight. Monitoring tumor growth by calipers and imaging in parallel, we find that caliper measurements of small tumors are weakly correlated with actual tumor volume and highly susceptible to experimenter bias. The method presented provides a unique window to sources of error in a foundational aspect of preclinical head and neck cancer research and a valuable tool to mitigate them.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

A novel preclinical model of craniospinal irradiation in pediatric diffuse midline glioma demonstrates decreased metastatic disease.

Background: Diffuse midline glioma (DMG) is an aggressive pediatric central nervous system tumor with strong metastatic potential. As localized treatment of the primary tumor improves, metastatic disease is becoming a more important factor in treatment. We hypothesized that we could model craniospinal irradiation (CSI) through a DMG patient-derived xenograft (PDX) model and that CSI would limit metastatic tumor. Methods: We used a BT245 murine orthotopic DMG PDX model for this work. We developed a protocol and specialized platform to deliver craniospinal irradiation (CSI) (4 Gy x2 days) with a pontine boost (4 Gy x2 days) and compared metastatic disease by pathology, bioluminescence, and MRI to mice treated with focal radiation only (4 Gy x4 days) or no radiation. Results: Mice receiving CSI plus boost showed minimal spinal and brain leptomeningeal metastatic disease by bioluminescence, MRI, and pathology compared to mice receiving radiation to the pons only or no radiation. Conclusion: In a DMG PDX model, CSI+boost minimizes tumor dissemination compared to focal radiation. By expanding effective DMG treatment to the entire neuraxis, CSI has potential as a key component to combination, multimodality treatment for DMG designed to achieve long-term survival once novel therapies definitively demonstrate improved local control.

Simultaneous targeting of PD-1 and IL-2Rßγ with radiation therapy inhibits pancreatic cancer growth and metastasis.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rß and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.

Short-term topiramate treatment prevents radiation-induced cytotoxic edema in preclinical models of breast-cancer brain metastasis.

BACKGROUND: Brain edema is a common complication of brain metastases (BM) and associated treatment. The extent to which cytotoxic edema, the first step in the sequence that leads to ionic edema, vasogenic edema, and brain swelling, contributes to radiation-induced brain edema during BM remains unknown. This study aimed to determine whether radiation-associated treatment of BM induces cytotoxic edema and the consequences of blocking the edema in preclinical models of breast-cancer brain metastases (BCBM). METHODS: Using in vitro and in vivo models, we measured astrocytic swelling, trans-electric resistance (TEER), and aquaporin 4 (AQP4) expression following radiation. Genetic and pharmacological inhibition of AQP4 in astrocytes and cancer cells was used to assess the role of AQP4 in astrocytic swelling and brain water intake. An anti-epileptic drug that blocks AQP4 function (topiramate) was used to prevent cytotoxic edema in models of BM. RESULTS: Radiation-induced astrocytic swelling and transient upregulation of AQP4 occurred within the first 24 hours following radiation. Topiramate decreased radiation-induced astrocytic swelling and loss of TEER in astrocytes in vitro, and acute short-term treatment (but not continuous administration), prevented radiation-induced increase in brain water content without pro-tumorigenic effects in multiple preclinical models of BCBM. AQP4 was expressed in clinical BM and breast-cancer cell lines, but AQP4 targeting had limited direct pro-tumorigenic or radioprotective effects in cancer cells that could impact its clinical translation. CONCLUSIONS: Patients with BM could find additional benefits from acute and temporary preventive treatment of radiation-induced cytotoxic edema using anti-epileptic drugs able to block AQP4 function.

Phase 1 Dose Escalation Study of SBRT Using 3 Fractions for Locally Advanced Pancreatic Cancer.

PURPOSE: The optimal dose and fractionation of stereotactic body radiation therapy (SBRT) for locally advanced pancreatic cancer (LAPC) have not been defined. Single-fraction SBRT was associated with more gastrointestinal toxicity, so 5-fraction regimens have become more commonly employed. We aimed to determine the safety and maximally tolerated dose of 3-fraction SBRT for LAPC. METHODS AND MATERIALS: Two parallel phase 1 dose escalation trials were conducted from 2016 to 2019 at Memorial Sloan Kettering Cancer Center and University of Colorado. Patients with histologically confirmed LAPC without distant progression after at least 2 months of induction chemotherapy were eligible. Patients received 3-fraction linear accelerator-based SBRT at 3 dose levels, 27, 30, and 33 Gy, following a modified 3+3 design. Dose-limiting toxicity, defined as grade ≥3 gastrointestinal toxicity within 90 days, was scored by National Cancer Institute Common Terminology Criteria for Adverse Events, version 4. The secondary endpoints included cumulative incidence of local failure (LF) and distant metastasis (DM), as well as progression-free and overall survival PFS and OS, respectively, toxicity, and quality of life (QoL) using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (QLQ-C30) and the pancreatic cancer-specific QLQ-PAN26 questionnaire. RESULTS: Twenty-four consecutive patients were enrolled (27 Gy: 9, 30 Gy: 8, 33 Gy: 7). The median (range) age was 67 (52-79) years, and 12 (50%) had a head/uncinate tumor location, with a median tumor size of 3.8 (1.1-11) cm and CA19-9 of 60 (1-4880) U/mL. All received chemotherapy for a median of 4 (1.4-10) months. There were no grade ≥3 toxicities. Two-year rates (95% confidence interval) of LF, DM, PFS, and OS were 31.7% (8.6%-54.8%), 70.2% (49.7%-90.8%), 20.8% (4.6%-37.1%), and 29.2% (11.0%-47.4%), respectively. Three- and 6-month QoL assessment showed no detriment. CONCLUSIONS: For select patients with LAPC, dose escalation to 33 Gy in 3 fractions resulted in no dose-limiting toxicities, no detriments to QoL, and disease outcomes comparable with conventional RT. Further exploration of SBRT schemes to maximize tumor control while enabling efficient integration with systemic therapy is warranted.

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

Short-term Topiramate treatment prevents radiation-induced cytotoxic edema in preclinical models of breast-cancer brain metastasis.

Background: Brain edema is a common complication of brain metastases (BM) and associated treatment. The extent to which cytotoxic edema, the first step in the sequence that leads to ionic edema, vasogenic edema and brain swelling, contributes to radiation-induced brain edema during BM remains unknown. This study aimed to determine whether radiation-associated treatment of BM induces cytotoxic edema and the consequences of blocking the edema in pre-clinical models of breast cancer brain metastases (BCBM). Methods: Using in vitro and in vivo models, we measured astrocytic swelling, trans-electric resistance (TEER) and aquaporin 4 (AQP4) expression following radiation. Genetic and pharmacological inhibition of AQP4 in astrocytes and cancer cells was used to assess the role of AQP4 in astrocytic swelling and brain water intake. An anti-epileptic drug that blocks AQP4 function (topiramate) was used to prevent cytotoxic edema in models of BM. Results: Radiation-induced astrocytic swelling and transient upregulation of AQP4 within the first 24 hours following radiation. Topiramate decreased radiation-induced astrocytic swelling, loss of TEER in astrocytes in vitro , and acute short term treatment (but not continuous administration), prevented radiation-induced increase in brain water content without pro-tumorigenic effects in multiple pre-clinical models of BCBM. AQP4 was expressed in clinical BM and breast cancer cell lines, but AQP4 targeting had limited direct pro-tumorigenic or radioprotective effects in cancer cells that could impact its clinical translation. Conclusions: Patients with BM could find additional benefits from acute and temporary preventive treatment of radiation-induced cytotoxic edema using anti-epileptic drugs able to block AQP4 function. Key points: Radiation induces cytotoxic edema via acute dysregulation of AQP4 in astrocytes in preclinical models of BM. Pharmacologic blockage of AQP4 function prevents water intake, astrocytic swelling and restores TEER in vitro. Pre-treatment with single-dose Topiramate prevents brain radiation-induced brain edema without direct tumor effects in pre-clinical models of BCBM. IMPORTANCE OF THE STUDY: In this study we describe a novel role for astrocytic swelling and cytotoxic edema in the progression of radiation-induced brain edema during BM treatment. While radiation-induced edema has been fully attributed to the disruption of the blood-brain barrier (BBB) and ensuing vasogenic effects, our results suggest that cytotoxic edema affecting astrocytes in the acute setting plays an important role in the progression of brain edema during BM standard of care. Current standard of care for brain edema involves pre-treatment with steroids and the use of bevacizumab only after clinically significant edema develops. Both interventions are presumed to target vasogenic edema. This study suggests that patients with BM could find additional benefits from acute and temporary preventive treatment of radiation-induced cytotoxic edema using an already FDA-approved anti-epileptic drug. Such early prevention strategy can be easily clinically implemented with the goal of minimizing treatment-related toxicities.

Vasculitis, CA19-9, and Perineural Invasion Differentially Predict Response and Surgical Outcome in Pancreatic Ductal Adenocarcinoma.

PURPOSE: Curative intent treatment of pancreatic adenocarcinoma (PDAC) relies on surgical resection. Modern treatment protocols focus on optimizing neoadjuvant therapy to increase resectability and improve oncologic outcomes. To elucidate differences in outcomes, we investigated the relationship between neoadjuvant chemotherapy (NAC), either with or without stereotactic body radiation therapy (SBRT), and vascular inflammation, surgical outcomes, and the resultant transcriptomic changes. METHODS AND MATERIALS: Clinical data were collected from patients with borderline resectable PDAC (clinical T3-T4N0-1) who underwent NAC or NAC-SBRT followed by curative intent resection between 2014 and 2019. Vascular structures on surgical specimens were histologically evaluated for vasculitis. RNA sequencing was used to evaluate differential gene expression and to generate enrichment maps. Multivariate analysis was used to analyze the relationship between patient characteristics and oncological outcome. RESULTS: In total, 46 patients met inclusion criteria (n = 12 NAC, n = 34 NAC-SBRT) with a median follow-up of 20.1 months. All patients underwent curative resection, with 91.3% achieving R0. There was no significant difference in patterns of failure, overall survival, or progression-free survival between NAC and NAC-SBRT groups. Patients with vasculitis had a lower median overall survival compared with those without (14.5 vs 28.3 months; hazard ratio, 12.96; 95% confidence interval, 3.55-47.28; P < .001). There was no significant correlation between inflammation and surgical complications or pathologic response. Neoadjuvant therapy did not have a significant effect on development of vasculitis (odds radio, 1.64 for NAC-SBRT; 95% confidence interval, 0.40-8.43; P = .52). Predictors of poor survival included perineural invasion and high baseline carbohydrate antigen 19-9 (CA19-9) (>191 U/mL). Patients with robust CA19-9 (>20% decrease) responses to neoadjuvant therapy had enrichment in immune response, chemotaxis, and cytotoxic T-cell and natural killer-cell proliferation. CONCLUSIONS: Vasculitis predicts for poor survival outcomes in patients with PDAC; NAC-SBRT did not increase the rate of vasculitis compared with NAC. Perineural invasion and CA19-9 remain strong prognosticators. Understanding and optimizing immune interactions remain a crucial hurdle in achieving response in pancreatic cancer.