Investigation of Gastrointestinal Toxicities Associated with Concurrent Abdominal Radiation Therapy and the Tyrosine Kinase Inhibitor Sunitinib in a Mouse Model.

Tyrosine kinase inhibitors (TKIs) may be combined with radiation therapy (RT) to enhance tumor control; however, increased incidences of gastrointestinal (GI) toxicity have been reported with this combination. We hypothesize that toxicity is due to compromised intestinal healing caused by inhibition of vascular repair and proliferation pathways. This study explores underlying tissue toxicity associated with abdominal RT and concurrent sunitinib in a mouse model. Four groups of CD-1 mice were treated with 12 Gy abdominal RT, oral sunitinib, abdominal RT + sunitinib, or sham treatment. Mice received oral sunitinib or the vehicle via gavage for 14 days. On day 7, mice were irradiated with 12 Gy abdominal RT or sham treated. Mice were euthanized on day 14 and intestinal tract was harvested for semiquantitative histopathologic evaluation and immunohistochemical quantification of proliferation (Ki67) and vascular density (CD31). Non-irradiated groups had stable weights while abdominal irradiation resulted in weight loss, with mice receiving RT + SUN having greater weight loss than mice receiving RT alone. Semiquantitative analysis showed significant increases in inflammation in irradiated groups. The difference in the density of CD31+ cells was significantly increased in RT alone compared to SUN alone. Ki67+ density was not significant. In summary, we identify a lack of angiogenic response in irradiated GI tissues when abdominal RT is combined with a TKI, which may correlate with clinical toxicities seen in canine and human patients receiving combined treatment.

Outcomes of 35 dogs with craniomaxillofacial osteosarcoma treated with stereotactic body radiation therapy.

Canine craniomaxillofacial osteosarcoma (OSA) is most commonly treated surgically; however, in cases where surgery is not feasible or non-invasive treatment is desired, stereotactic body radiation therapy (SBRT) may be elected for local tumour control. In this study, we evaluated 35 dogs treated with SBRT. Nine dogs (26%) had calvarial, seven (20%) had mandibular and 19 (54%) had maxillary OSA. Median time to first event (TFE) was 171 days, and overall median survival time (MST) was 232 days. Site-specific MSTs were 144 days for mandible, 236 days for calvarium and 232 days for maxilla (p = .49). Pulmonary metastatic disease was observed in 12/35 (34%) patients and was detected pre-SBRT in six dogs (17%) and post-SBRT in the remaining six dogs (17%). Eighteen adverse events post-SBRT were documented. Per veterinary radiation therapy oncology group criteria, five were acute (14%) and three were late (9%) grade 3 events. Neurological signs in two dogs were suspected to be early-delayed effects. Cause of death was local progression for 22/35 (63%) patients, metastasis for 9/35 (26%) patients and unknown for four. On univariate analysis, administration of chemotherapy was associated with a longer TFE (p = .0163), whereas volume of gross tumour volume was associated with a shorter TFE (p = .023). Administration of chemotherapy and five fractions versus single fraction of SBRT was associated with increased survival time (p = .0021 and .049). Based on these findings, a treatment protocol incorporating chemotherapy and five fractions of SBRT could be considered for dogs with craniomaxillofacial OSA electing SBRT with careful consideration of normal tissues in the field.

Retrospective study evaluating the efficacy of stereotactic body radiation therapy for the treatment of confirmed or suspected primary pulmonary carcinomas in dogs.

Canine primary pulmonary carcinomas (PCCs) are commonly treated with surgery with overall median survival times (MST) around a year; however, due to extent of disease, prognosis, or client preference, alternative treatments have been considered. Stereotactic body radiation therapy (SBRT) has been utilized in human cancer patients for local control of lung tumours as a surgical alternative. Twenty-one PCCs in 19 dogs that received SBRT for local control were retrospectively evaluated. Dogs were staged according to the canine lung carcinoma stage classification (CLCSC) system with three as Stage 1, five as Stage 2, three as Stage 3, and eight as Stage 4. Overall MST was 343 days with 38% of patients alive at 1 year. Stage did not significantly impact survival time (p = .72). Five (26%) dogs had lymphadenopathy and MST was not significantly different from dogs without lymphadenopathy (343 vs. 353 days; p = .54). Five out of 18 evaluable dogs (28%) experienced acute lung VRTOG effects and 2 of 12 dogs (17%) experienced late lung VRTOG effects. Median lung dose, V5, V20, and D30 to the lung did not correlate significantly with the development of adverse radiation events. Twelve dogs had follow-up imaging and the best response included a complete response (17%), partial response (42%), and stable disease (42%). Progressive disease was noted in seven dogs a median of 229 days after SBRT. SBRT was documented to be a safe and effective alternative to surgery and may have survival advantages for Stage 3 or 4 dogs according to the CLCSC.

Efficacy of stereotactic radiation therapy for the treatment of confirmed or presumed canine glioma.

Intracranial gliomas are the second most common brain tumour in dogs. Radiation therapy provides a minimally invasive treatment option for this tumour type. Earlier publications reporting on the use of non-modulated radiation therapy suggested a poor prognosis for dogs with glioma, with median survival times ranging between 4 and 6 months; more recent literature utilizing stereotactic radiation therapy (SRT) demonstrates that the prognosis for canine gliomas may be more promising, with survival times closer to 12 months. A single institution retrospective study was performed between 2010 and 2020 investigating the outcomes of dogs with biopsy-confirmed glioma or a presumptive diagnosis of intra-cranial glioma based on MRI characteristics that were treated with SRT. Twenty-three client-owned dogs were included. Brachycephalic breeds were overrepresented, totalling 13 dogs (57%). SRT protocols included 16 Gy single fraction (n = 1, 4%), 18 Gy single fraction (n = 1, 4%), 24 Gy in 3 daily fractions (n = 20, 91%), or 27 Gy in four daily fractions (n = 1, 4%). Twenty-one dogs (91%) had improvement of their presenting clinical signs following SRT. Median overall survival time (MST) was 349 days (95% CI, 162-584). Median disease specific survival time was 413 days (95% CI, 217-717). When SRT is incorporated into the management plan for dogs with confirmed or presumed intracranial glioma, a median survival time of approximately 12 months may be achievable.

Canine salivary gland carcinoma treated with stereotactic body radiation therapy: a retrospective case series.

Objective: The aim of this study was to describe the therapeutic outcomes of dogs with locally advanced salivary gland carcinomas (SGC) following stereotactic body radiation therapy (SBRT). Methods: A single institution retrospective study was conducted of client-owned dogs with macroscopic SGC treated with SBRT. Patient signalment, clinical characteristics, and treatment parameters were recorded. Clinical benefit was determined based on follow-up physical examination and medical history. Progression-free interval (PFI), median survival time (MST), and disease-specific survival (DSS) were calculated using Kaplan-Meier analysis. Acute and late toxicity were recorded according to Veterinary Radiation Therapy Oncology Group (VRTOG) criteria. Results: Six patients were included in the study. Tumor origins were mandibular (n = 3), parotid (n = 2), and zygomatic (n = 1) salivary glands. The SBRT prescription was 10 Gy × 3 daily or every other day. All patients (100%) experienced clinical benefit from treatment at a median time of 34 days (range 28-214). No local or regional nodal failure was reported following SBRT. Progressive pulmonary metastatic disease was documented in three dogs (50%). The median PFI was 260 days (range 43-1,014) and the MST was 397 days (range 185-1,014). Median DSS was 636 days (range 185-1,014). Four dogs (66.6%) died of confirmed or suspected metastatic SGC. The reported acute side effects included grade 2 mucositis (n = 1) and vision loss (n = 1). No late side effects were recorded. Conclusion: This study suggests that SBRT may provide durable local control for invasive SGC in dogs. Further investigation in a larger cohort of patients is warranted. The incidence of reported acute and late toxicity was low.

Elective nodal irradiation mitigates local and systemic immunity generated by combination radiation and immunotherapy in head and neck tumors.

In the setting of conventional radiation therapy, even when combined with immunotherapy, head and neck cancer often recurs locally and regionally. Elective nodal irradiation (ENI) is commonly employed to decrease regional recurrence. Given our developing understanding that immune cells are radio-sensitive, and that T cell priming occurs in the draining lymph nodes (DLNs), we hypothesize that radiation therapy directed at the primary tumor only will increase the effectiveness of immunotherapies. We find that ENI increases local, distant, and metastatic tumor growth. Multi-compartmental analysis of the primary/distant tumor, the DLNs, and the blood shows that ENI decreases the immune response systemically. Additionally, we find that ENI decreases antigen-specific T cells and epitope spreading. Treating the primary tumor with radiation and immunotherapy, however, fails to reduce regional recurrence, but this is reversed by either concurrent sentinel lymph node resection or irradiation. Our data support using lymphatic sparing radiation therapy for head and neck cancer.

Outcome of stereotactic body radiation for treatment of nasal and nasopharyngeal lymphoma in 32 cats.

BACKGROUND: The safety and efficacy of stereotactic body radiation therapy (SBRT) in the treatment of localized nasal lymphoma in cats has not been described. HYPOTHESIS: Stereotactic body radiation therapy with or without adjuvant chemotherapy is an effective and well-tolerated treatment for localized nasal lymphoma in cats. ANIMALS: Thirty-two client owned cats referred to Colorado State University for the treatment of nasal lymphoma. METHODS: Retrospective study of cats treated with SBRT between 2010 and 2020 at Colorado State University. Diagnosis of nasal lymphoma was obtained via cytology or histopathology. Signalment, radiation protocol, concurrent treatments, adverse effects, and survival were recorded. RESULTS: Progression free survival was 225 days (95% CI 98-514) and median survival time (MST) was 365 days (95% CI 123-531). No significant difference in survival was identified between cats that received 1 versus greater than 1 fraction (MST 427 vs. 123 days, P = 0.88). Negative prognostic factors included cribriform lysis (MST 121 vs. 876 days, P = 0.0009) and intracalvarial involvement (MST 100 vs. 438 days, P = 0.0007). Disease progression was noted in 38% (12/32), locally in 22% (7/32), and systemically in 16% (5/32). No cats developed acute adverse effects. Ten cats developed late adverse effects: keratitis/keratitis sicca (n = 2), alopecia (n = 4), and leukotrichia (n = 4). Twenty-four cats (75%) had signs consistent with chronic rhinitis. CONCLUSIONS: SBRT is effective and well tolerated for treating localized nasal lymphoma in cats. Outcomes for cats with lower stage disease (canine modified Adam's stage 3 and lower) are comparable to historic data of cats treated with fractionated radiation therapy.

Immunologic Effects of Stereotactic Body Radiotherapy in Dogs with Spontaneous Tumors and the Impact of Intratumoral OX40/TLR Agonist Immunotherapy.

Stereotactic body radiotherapy (SBRT) is known to induce important immunologic changes within the tumor microenvironment (TME). However, little is known regarding the early immune responses within the TME in the first few weeks following SBRT. Therefore, we used the canine spontaneous tumor model to investigate TME responses to SBRT, and how local injection of immune modulatory antibodies to OX40 and TLR 3/9 agonists might modify those responses. Pet dogs with spontaneous cancers (melanoma, carcinoma, sarcoma, n = 6 per group) were randomized to treatment with either SBRT or SBRT combined with local immunotherapy. Serial tumor biopsies and serum samples were analyzed for immunologic responses. SBRT alone resulted at two weeks after treatment in increased tumor densities of CD3+ T cells, FoxP3+ Tregs, and CD204+ macrophages, and increased expression of genes associated with immunosuppression. The addition of OX40/TLR3/9 immunotherapy to SBRT resulted in local depletion of Tregs and tumor macrophages and reduced Treg-associated gene expression (FoxP3), suppressed macrophage-associated gene expression (IL-8), and suppressed exhausted T cell-associated gene expression (CTLA4). Increased concentrations of IL-7, IL-15, and IL-18 were observed in serum of animals treated with SBRT and immunotherapy, compared to animals treated with SBRT. A paradoxical decrease in the density of effector CD3+ T cells was observed in tumor tissues that received combined SBRT and immunotherapy as compared to animals treated with SBRT only. In summary, these results obtained in a spontaneous large animal cancer model indicate that addition of OX40/TLR immunotherapy to SBRT modifies important immunological effects both locally and systemically.

Canine comparative oncology for translational radiation research.

PURPOSE: Laboratory and clinical research are essential for advancing radiation research; however, there is a growing awareness that conventional laboratory animal models and early-phase clinical studies in patients have not improved the low success rates and late-stage failures in new cancer therapy efforts. There are considerable costs and inefficiencies in moving preclinical research into effective cancer therapies for patients. Canine translational models of radiation research can fill an important niche between rodent and human studies, ultimately providing valuable, predictive, translational biological and clinical results for human cancer patients. Companion dogs naturally and spontaneously develop cancers over the course of their lifetime. Many canine tumor types share important similarities to human disease, molecularly and biologically, with a comparable clinical course. Dogs receive state-of-the-art medical care, which can include radiotherapy, experimental therapeutics, and novel technologies, offering an important opportunity for radiobiology and radiation oncology research. Notably, the National Cancer Institute has developed the Comparative Oncology Program to promote this area of increased research interest. CONCLUSION: In this review, the benefits and limitations of performing translational radiation research in companion dogs will be presented, and current research utilizing the canine model will be highlighted, including studies across research areas focusing on common canine tumor types treated with radiotherapy, comparative normal tissue effects, radiation and immunology research, and alternative radiation therapy approaches involving canine cancer patients.

Therapeutic Intervention Using a Smad7-Based Tat Protein to Treat Radiation-Induced Oral Mucositis.

PURPOSE: Recent studies reported therapeutic effects of Smad7 on oral mucositis in mice without compromising radiation therapy-induced cancer cell killing in neighboring oral cancer. This study aims to assess whether a Smad7-based biologic can treat oral mucositis in a clinically relevant setting by establishing an oral mucositis model in dogs and analyzing molecular targets. METHODS AND MATERIALS: We created a truncated human Smad7 protein fused with the cell-penetrating Tat tag (Tat-PYC-Smad7). We used intensity modulated radiation therapy to induce oral mucositis in dogs and applied Tat-PYC-Smad7 to the oral mucosa in dose-finding studies after intensity modulated radiation therapy. Clinical outcomes were evaluated. Molecular targets were analyzed in biopsies and serum samples. RESULTS: Tat-PYC-Smad7 treatment significantly shortened the duration of grade 3 oral mucositis based on double-blinded Veterinary Radiation Therapy Oncology Group scores and histopathology evaluations. Topically applied Tat-PYC-Smad7 primarily penetrated epithelial cells and was undetectable in serum. NanoString nCounter Canine IO Panel identified that, compared to the vehicle samples, top molecular changes in Tat-PYC-Smad7 treated samples include reductions in inflammation and cell death and increases in cell growth and DNA repair. Consistently, immunostaining shows that Tat-PYC-Smad7 reduced DNA damage and neutrophil infiltration with attenuated TGF-ß and NFκB signaling. Furthermore, IL-1ß and TNF-α were lower in Tat-PYC-Smad7 treated mucosa and serum samples compared to those in vehicle controls. CONCLUSIONS: Topical Tat-PYC-Smad7 application demonstrated therapeutic effects on oral mucositis induced by intensity modulated radiation therapy in dogs. The local effects of Tat-PYC-Smad7 targeted molecules involved in oral mucositis pathogenesis as well as reduced systemic inflammatory cytokines.

Increased incidence of gastrointestinal toxicity in canine cancer patients treated with concurrent abdominal radiation therapy and toceranib phosphate.

Receptor tyrosine kinase inhibitors (TKIs) are used to treat human and canine cancers and may be combined with radiation therapy (RT) to enhance tumor control due their anticancer and antiangiogenic effects; however, recent case reports have emerged describing incidences of gastrointestinal toxicity when antiangiogenic therapies are combined with hypofractionated radiotherapy in human cancer patients. We evaluated the incidence of gastrointestinal (GI) toxicity in dogs receiving concurrent hypofractionated abdominal RT and the TKI toceranib (TOC) compared to those receiving abdominal RT alone, TOC alone, or concurrent non-abdominal RT and TOC. Medical records of canine cancer patients were retrospectively reviewed and identified dogs were included in the following treatment categories: dogs which received RT to a portion of the abdomen and concurrent TOC (n = 19), abdominal RT alone (n-29), TOC alone (n = 20), or non-abdominal RT plus TOC (n = 9). Toxicities were graded using the Veterinary Cooperative Oncology Group - Common Terminology Criteria for Adverse Events criteria and compared to published data on TOC-associated GI toxicity. Patients receiving TOC while undergoing abdominal RT had significantly increased rates of any grade of diarrhea (p = 0.002), hyporexia (p = 0.0045), and vomiting (p = 0.003), as well as severe hyporexia (p = 0.003) when compared across the treatment groups. This retrospective study reveals significantly increased incidences of GI toxicity when abdominal RT is combined with TOC in canine patients. These findings are in-line with the clinical concerns reported for increased normal tissue toxicity in human patients when antiangiogenics are combined with RT.

Comparative study of the collapsed cone convolution and Monte Carlo algorithms for radiation therapy planning of canine sinonasal tumors reveals significant dosimetric differences.

Computer-based radiation therapy requires high targeting and dosimetric precision. Analytical dosimetric algorithms typically are fast and clinically viable but can have increasing errors near air-bone interfaces. These are commonly found within dogs undergoing radiation planning for sinonasal cancer. This retrospective methods comparison study is designed to compare the dosimetry of both tumor volumes and organs at risk and quantify the differences between collapsed cone convolution (CCC) and Monte Carlo (MC) algorithms. Canine sinonasal tumor plans were optimized with CCC and then recalculated by MC with identical control points and monitor units. Planning target volume (PTV)air , PTVsoft tissue , and PTVbone were created to analyze the dose discrepancy within the PTV. Thirty imaging sets of dogs were included. Monte Carlo served as the gold standard calculation for the dosimetric comparison. Collapsed cone convolution overestimated the mean dose (Dmean ) to PTV and PTVsoft tissue by 0.9% and 0.5%, respectively (both P < 0.001). Collapsed cone convolution overestimated Dmean to PTVbone by 3% (P < 0.001). Collapsed cone convolution underestimated the near-maximum dose (D2 ) to PTVair by 1.1% (P < 0.001), and underestimated conformity index and homogeneity index in PTV (both P < 0.001). Mean doses of contralateral and ipsilateral eyes were overestimated by CCC by 1.6% and 1.7%, respectively (both P < 0.001). Near-maximum doses of skin and brain were overestimated by CCC by 2.2% and 0.7%, respectively (both P < 0.001). As clinical accessibility of Monte Carlo becomes more widespread, dose constraints may need to be re-evaluated with appropriate plan evaluation and follow-up.

Outcomes of dogs with thymoma treated with intensity modulated stereotactic body radiation therapy or non-modulated hypofractionated radiation therapy.

Canine thymomas are routinely treated with radiotherapy (RT). In this study, we investigate the response and toxicity of canine thymoma treated with intensity-modulated stereotactic body radiation therapy (SBRT) relative to dogs treated with hypofractionated non-modulated radiation therapy (NMRT). A retrospective study was performed of dogs with thymoma treated with RT (total: n = 15; SBRT: n = 8, NMRT: n = 7). Tumour response was evaluated in six dogs (40%); following SBRT, three dogs (100%) experienced stable disease (SD); following NMRT, one dog (33%) had a PR, and two dogs (67%) had SD. Median PFS was 116 days (range 66-727 days) for the SBRT group and 134 days (range 10-405 days) for the NMRT group. The MST for the SBRT group was 250 days (range 1-727 days) and 155 days (range 10-405 days) for NMRT. Median disease-specific survival was 250 days (range 1-727 days) for the SBRT group and 169 days (range 20-405 days) for the NMRT group. No significant differences in survival data were found between the treatment groups, however the results from the small number of dogs analysed are likely underpowered for statistical comparisons. Reported acute and late side effects were limited to the lungs and heart and were statistically significantly more common in the NMRT (71%) compared to the SBRT group (25%) (p = .04). We suggest similar treatment efficacy may be provided for canine thymoma treated with either approach, but SBRT could provide the clinical benefit of reduced incidence of radiation-induced toxicity and completion of RT in a shorter time frame.

Canine oral squamous cell carcinoma as a spontaneous, translational model for radiation and immunology research.

Introduction: Improving outcomes for oral squamous cell carcinoma (OSCC) patients has been hindered by a lack of effective predictive animal models. Spontaneously occurring canine OSCC could help fill this gap. The objective of this study was to characterize the immune landscape of canine OSCC to advance understanding of how dogs could serve as a surrogate for human OSCC. Methods/Results: Canine OSCC contains a heterogenous tumor immune microenvironment. CD3+ T cells were the predominant tumor infiltrating immune cell population; however, there was a wide range CD3+ T cell density across samples. The most common CD3+ T cell micro-anatomical distribution was defined as "pre-existing immunity", but the remaining 20% of tumors were characterized as "immunologically ignorant" or "excluded infiltrates" patterns. When compared to normal oral mucosa, the tumor gene expression pattern suggests that canine OSCC microenvironment is highly inflamed and characterized by the presence of an anti-tumor immune response dominated by cytotoxic\effector T cells and NK cells (CD8a, GZMA, OX40, and HLA-A); however, overexpression of genes associated with effector T cell exhaustion and microenvironmental immunosuppression was also identified (PD-1, LAG3, CXCL2). Correlations between CD3+ T cell density and immune gene expression revealed key genes associated with cytotoxic anti-tumor T cell responses (GZMA, GZMB, PRF1), co-stimulation of T cells (CD27, CD28, ICOS), and other immune processes, including Type I IFN response (TNF, TNFSF10), and T cell exhaustion (CTLA4, PD-1). CD3+ T cell density in canine OSCC was significantly correlated with a cytolytic activity score (mean PRF1 and GZMA expression), suggestive of active effector CD8 T cell function. CD204+ macrophages were the second most abundant tumor infiltrating immune cell, and when comparing to normal oral mucosa, two differently expressed genes linked to tumor associated macrophages and myeloid derived suppressor cells (MDSC) were identified: CXCL2, CD70. Overexpression of CXCL2 was also identified in canine OSCC "T cell-high" tumors compared to "T cell-low" tumors. Discussion: This study identified actionable immunotherapy targets which could inform future comparative oncology trials in canine OSCC: CTLA-4, PD-1, CXCL2. These data provide a good first step towards utilizing spontaneous canine OSCC as a comparative model for human OSCC radiation and immuno-oncology research.

Reprogramming the Canine Glioma Microenvironment with Tumor Vaccination plus Oral Losartan and Propranolol Induces Objective Responses.

Purpose: Malignant gliomas have a highly immune suppressive tumor microenvironment (TME) which renders them largely unresponsive to conventional therapeutics. Therefore, the present study evaluated a therapeutic protocol designed overcome the immune barrier by combining myeloid cell targeted immunotherapy with tumor vaccination. Experimental Design: We utilized a spontaneously occurring canine glioma model to investigate an oral TME modifying immunotherapy in conjunction with cancer stem cell (CSC) vaccination. Dogs were treated daily with losartan (monocyte migration inhibitor) and propranolol (myeloid-derived suppressor cell depleting agent) plus anti-CSC vaccination on a bi-weekly then monthly schedule. Tumor volume was monitored by MRI and correlated with patient immune responses. Results: Ten dogs with histologically confirmed gliomas were enrolled into a prospective, open-label clinical trial to evaluate the immunotherapy protocol. Partial tumor regression was observed in 2 dogs, while 6 dogs experienced stable disease, for an overall clinical benefit rate of 80%. Overall survival times (median = 351 days) and progression-free intervals (median = 163 days) were comparable to prior studies evaluating surgical debulking followed by immunotherapy. Dogs with detectable anti-CSC antibody responses had an increased overall survival time relative to dogs that did not generate antibody responses (vaccine responder MST = 500 days; vaccine non-responder MST = 218 days; p = 0.02). Conclusions: These findings suggest that combining myeloid cell targeted oral immunotherapy with tumor vaccination can generate objective tumor responses, even in the absence of conventional therapy. Overall, this approach has promise as a readily implemented therapeutic strategy for use in brain cancer patients.

Response of Canine Soft Tissue Sarcoma to Stereotactic Body Radiotherapy.

Canine soft tissue sarcoma (STS) has served as a preclinical model for radiation, hyperthermia, experimental therapeutics, and tumor microenvironmental research for decades. Stereotactic body radiotherapy (SBRT) demonstrates promising results for the control of various tumors in human and veterinary medicine; however, there is limited clinical data for the management of STS with SBRT. In this retrospective study, we aimed to define overall efficacy and toxicity of SBRT for the treatment of macroscopic canine STS to establish this preclinical model for comparative oncology research. Fifty-two canine patients met inclusion criteria. Total radiation dose prescribed ranged from 20-50 Gy delivered in 1-5 fractions. Median progression-free survival time (PFST) was 173 days and overall survival time (OST) 228 days. Best overall response was evaluable in 46 patients, with 30.4% responding to treatment (complete response n = 3; partial response n = 11). For responders, OST significantly increased to 475 days vs. 201 days (P = 0.009). Prognostic factors identified by multivariable Cox regressions included size of tumor and metastasis at presentation. Dogs were 3× more likely to progress (P = 0.009) or 3.5× more likely to experience death (P = 0.003) at all times of follow up if they presented with metastatic disease. Similarly, every 100-cc increase in tumor volume resulted in a 5% increase in the risk of progression (P = 0.002) and death (P = 0.001) at all times of follow up. Overall, 30.8% of patients developed acute toxicities, 7.7% grade 3; 28.8% of patients developed late toxicities, 11.5% grade 3. Increased dose administered to the skin significantly affected toxicity development. SBRT serves as a viable treatment option to provide local tumor control for canine macroscopic STS, particularly those with early-stage disease and smaller tumors. The results of this study will help to define patient inclusion criteria and to set dose limits for preclinical canine STS trials involving SBRT.

Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis.

BACKGROUND: Numerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT. METHODS: We subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3-/-, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments. RESULTS: In MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence. CONCLUSIONS: Regulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.

Outcome and prognosis for canine appendicular osteosarcoma treated with stereotactic body radiation therapy in 123 dogs.

Canine appendicular osteosarcoma is commonly treated with limb amputation; however, limb-sparing options are frequently desired or necessary for a subset of patients. We evaluated 123 patients and 130 sites treated with stereotactic body radiation therapy (SBRT). Eighty-two out of 98 dogs (84%) had maximum lameness improvement at a median of 3 weeks for a median of 6 months duration. Histopathologic evaluation of available samples from amputation or necropsy revealed >80% tumor necrosis in 50% of limbs consistent with local disease control. Of evaluable patients, 41% fractured and 21% pursued an amputation after treatment. Fine needle aspirate (n = 52) and needle core biopsy (n = 28) did not result in increased fracture risk compared to those without tumor sampling (n = 50). Median survival time (MST) was 233 days and time to first event was 143 days. Gross tumor volume and planned target volume were significantly inversely associated with survival and tumor location was significantly associated with survival. Dogs with salvage amputation had a significantly longer MST compared to those without (346 vs 202 days; P = .04). The presence of metastatic disease at the time of treatment in 15 dogs did not significantly impact survival time (200 vs 237 days without metastasis; P = .58). Skin side effects correlated significantly with dose with 33% of patients with acute grade 3 effects developing consequential late grade 3 effects. While SBRT improves lameness in most patients, further investigation is needed to identify candidates with minimal early fracture risk prior to initiating therapy.

Manganese Porphyrin and Radiotherapy Improves Local Tumor Response and Overall Survival in Orthotopic Murine Mammary Carcinoma Models.

Novel synthetic compounds, known as manganese porphyrins (MnPs), have been designed to shift the redox status of both normal cells and cancer cells. When MnPs are coupled with cancer therapies, such as radiation, they have been shown to sensitize tumor cells to treatment and protect normal tissues from damage through the modulation of the redox status of various tissue types. Until now, our preclinical studies have focused on local effects of MnPs and radiation; however, we recognize that successful outcomes for cancer patients involve control of tumor cells throughout the body. In this study, using murine orthotopic mammary tumor models, we investigated how MnPs and radiation influence the development of distant metastasis. We hypothesized that the combination of MnP (MnP/RT), such as MnTnBuOE-2-PyP5+ and radiation treatment (RT) would increase local tumor control via a shift in the intratumoral redox environment, leading to subsequent downregulation of HIF-1 in the primary tumor. Secondarily, we hypothesized that these primary tumor treatment effects would result in a reduction in pulmonary metastatic burden. Balb/c mice with orthotopic 4T1 mammary carcinomas were treated with saline, MnP, RT or MnP/RT. We found MnP/RT did extend local tumor growth delay and overall survival compared to controls and was associated with increased intratumoral oxidative stress. However, the primary tumor growth delay observed with MnP/RT was not associated with a reduced pulmonary metastatic burden. Future directions to investigate the effects of MnP/RT on the development of distant metastasis may include modifications to the radiation dose, the experimental timeline or using a murine mammary carcinoma cell line with a less aggressive metastatic behavior. Clinical trials are underway to investigate the clinical utility of MnTnBuOE-2-PyP5+ for patients undergoing radiotherapy for various tumor types. The promising preclinical data from this study, as well as others, provides support that MnP/RT has the potential to improve local tumor control for these patients.

3D-printed bolus improves dose distribution for veterinary patients treated with photon beam radiation therapy.

Commercial bolus is frequently used to increase dose at the patient's surface for superficial radiotherapy; however, uneven surfaces can create air gaps and discrepancies between prescribed and delivered dose. The purpose of this study was to determine if a customizable, 3D-printed bolus would improve dosimetry compared with a commercial bolus. For each patient, a planned bolus was generated within planning software, then created with 3D-printing. The treatment plan was recalculated with each bolus in situ. When evaluating tumor volumes at prescription, the 3D-printed bolus was closer to prescription compared to the commercial bolus. There was a significant difference in air gaps in patients receiving radiotherapy to the head (P < 0.001) but the difference was not significant for air gaps in caudal body sites (P = 0.05). Overall, the 3D-printed bolus resulted in reduced air gaps, dosimetry closer to prescription, and should be considered for superficial treatment areas of high irregularity.

Un bolus obtenu par impression 3D améliore la distribution de la dose de patients vétérinaires traités par radiation de faisceau de photons. Un bolus commercial est fréquemment utilisé pour augmenter la dose à la surface d'un patient lors de radiothérapie de surface; toutefois, des surfaces inégales peuvent créer des espaces d'air et ainsi des différences entre la dose prescrite et la dose livrée. Le but de la présente étude était de déterminer si un bolus sur mesure, obtenu par impression 3D, améliorerait la dosimétrie comparativement à un bolus commercial. Pour chaque patient, un bolus planifié fut généré à l'aide d'un logiciel de planification, puis créé avec une imprimante 3D. Le plan de traitement fut recalculé avec chaque bolus in situ. Lors de l'évaluation du volume des tumeurs à la prescription, le bolus obtenu par impression 3D était plus près de la prescription comparativement au bolus commercial. Il y avait une différence significative dans les espaces d'air chez les patients recevant la radiothérapie à la tête (P < 0,001) mais la différence n'était pas significative pour les espaces d'air sur les sites corporels en partie caudale (P = 0,05). De manière globale, le bolus obtenu par impression 3D a résulté en une diminution des espaces d'air, une dosimétrie plus près de la prescription et devrait être considéré lors du traitement de surfaces superficielles hautement irrégulières.(Traduit par Dr Serge Messier).