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Neoantigens generated by somatic nonsynonymous mutations are key targets of tumor-specific T cells, but only a small number of mutations predicted to be immunogenic are presented by MHC molecules on cancer cells. Vaccination studies in mice and patients have shown that the majority of neoepitopes that elicit T cell responses fail to induce significant antitumor activity, for incompletely understood reasons. We report that radiotherapy upregulates the expression of genes containing immunogenic mutations in a poorly immunogenic mouse model of triple-negative breast cancer. Vaccination with neoepitopes encoded by these genes elicited CD8+ and CD4+ T cells that, whereas ineffective in preventing tumor growth, improved the therapeutic efficacy of radiotherapy. Mechanistically, neoantigen-specific CD8+ T cells preferentially killed irradiated tumor cells. Neoantigen-specific CD4+ T cells were required for the therapeutic efficacy of vaccination and acted by producing Th1 cytokines, killing irradiated tumor cells, and promoting epitope spread. Such a cytotoxic activity relied on the ability of radiation to upregulate class II MHC molecules as well as the death receptors FAS/CD95 and DR5 on the surface of tumor cells. These results provide proof-of-principle evidence that radiotherapy works in concert with neoantigen vaccination to improve tumor control.

Radiation therapy (RT) increases tumor response to CTLA-4 inhibition (CTLA4i) in mice and in some patients, yet deep responses are rare. To identify rational combinations of immunotherapy to improve responses we use models of triple negative breast cancer highly resistant to immunotherapy in female mice. We find that CTLA4i promotes the expansion of CD4 + T helper cells, whereas RT enhances T cell clonality and enriches for CD8 + T cells with an exhausted phenotype. Combination therapy decreases regulatory CD4 + T cells and increases effector memory, early activation and precursor exhausted CD8 + T cells. A combined gene signature comprising these three CD8 + T cell clusters is associated with survival in patients. Here we show that targeting additional immune checkpoints expressed by intratumoral T cells, including PD1, is not effective, whereas CD40 agonist therapy recruits resistant tumors into responding to the combination of RT and CTLA4i, indicating the need to target different immune compartments. Radiation therapy (RT) has been shown to improve responses to immunotherapy in preclinical cancer models, but deep responses in patients are still rare. Here the authors provide immunological insights into the response to RT and CTLA4 inhibition in tumor bearing mice and show that agonistic CD40 therapy improves response to the combination of RT and immune checkpoint inhibition.

Glioblastoma (GBM), the most common adult primary brain tumor, has an average survival of only 15–18 months. Recently, the combination of immune checkpoint blockers paired with radiotherapy has shown promise in preclinical murine GBM models. Human clinical trials have largely failed. One reason for this may be the discrepancy between radiation protocols utilized in preclinical models versus clinical practice. For translational relevance, defining correct and comparable radiation dosages and schedules to achieve optimal synergy with immunotherapeutic drugs, is essential. We used the GL261-based syngeneic mouse GBM model to compare the effects of two radiation regimens on tumor cell growth and survival. We assessed the in vivo effects of a single dose of 10 Gy (10Gyx1) or five consecutive doses of 2 Gy (2Gyx5) on the tumor immune microenvironment over time and compared their efficacy when combined with anti-PD-1 in vivo . Our data show that the 10Gyx1 regimen is more effective than 2Gyx5 at inhibiting tumor cell proliferation and growth in vitro and in vivo . Both regimens preserved the antigen-presenting ability of both dendritic cells and local microglia, but 10Gyx1 led to the highest lymphocyte infiltration. The combination of radiation with the checkpoint blocker anti-PD-1 was advantageous for both radiation regimens with animals treated with the 10Gyx1 regimen surviving the longest. Our study highlights how radiation regimen choices may impact the translation of preclinical findings, and in particular, the effects of radiation and immunotherapy in GBM. This work and literature data on the effects of positive hypofractionation in human GBM patients suggest that applying fewer, higher-dose radiation fractions may benefit GBM patients and lead to tumoricidal effects without sacrificing favorable anti-tumor immune responders.

Background Ultra-hypofractionated regimens for definitive prostate cancer (PCa) radiotherapy are increasingly utilized due in part to promising safety and efficacy data complemented by greater patient convenience from a treatment course requiring fewer sessions. As such, stereotactic body radiation therapy (SBRT) is rapidly emerging as a standard definitive treatment option for patients with localized PCa. The commercially available magnetic resonance linear accelerator (MR-LINAC) integrates MR imaging with radiation delivery, providing several theoretical advantages compared to computed tomography (CT)-guided radiotherapy. MR-LINAC technology facilitates improved visualization of the prostate, real-time intrafraction tracking of prostate and organs-at-risk (OAR), and online adaptive planning to account for target movement and anatomical changes. These features enable reduced treatment volume margins and improved sparing of surrounding OAR. The theoretical advantages of MR-guided radiotherapy (MRgRT) have recently been shown to significantly reduce rates of acute grade ≥ 2 GU toxicities as reported in the prospective randomized phase III MIRAGE trial, which compared MR-LINAC vs CT-based 5 fraction SBRT in patients with localized PCa (Kishan et al. JAMA Oncol 9:365-373, 2023). Thus, MR-LINAC SBRT–utilizing potentially fewer treatments–is warranted and clinically relevant for men with low or intermediate risk PCa electing for radiotherapy as definitive treatment. Methods/Design A total of 136 men with treatment naïve low or intermediate risk PCa will be randomized in a 1:1 ratio to 5 or 2 fractions of MR-guided SBRT using permuted block randomization. Randomization is stratified by baseline Expanded PCa Index Composite (EPIC) bowel and urinary domain scores. Patients undergoing 5 fractions will receive 37.5 Gy to the prostate over 10–14 days and patients undergoing 2 fractions will receive 25 Gy to the prostate over 7–10 days. The co-primary endpoints are GI and GU toxicities as measured by change scores in the bowel and urinary EPIC domains, respectively. The change scores will be calculated as pre-treatment (baseline) score subtracted from the 2-year score. Discussion FORT is an international, multi-institutional prospective randomized phase II trial evaluating whether MR-guided SBRT delivered in 2 fractions versus 5 fractions is non-inferior from a gastrointestinal (GI) and genitourinary (GU) toxicity standpoint at 2 years post-treatment in men with low or intermediate risk PCa. Trial registration Clinicaltrials.gov identifier: NCT04984343 . Date of registration: July 30, 2021. Protocol version: 4.0, Nov 8, 2022.

Background Preclinical evidence from us and others demonstrates that the anticancer effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors can be enhanced with focal radiation therapy (RT), but only when RT is delivered prior to (rather than after) CDK4/6 inhibition. Depending on tumor model, cellular senescence (an irreversible proliferative arrest that is associated with the secretion of numerous bioactive factors) has been attributed beneficial or detrimental effects on response to treatment. As both RT and CDK4/6 inhibitors elicit cellular senescence, we hypothesized that a differential accumulation of senescent cells in the tumor microenvironment could explain such an observation, i.e., the inferiority of CDK4/6 inhibition with palbociclib (P) followed by RT (P→RT) as compared to RT followed by palbociclib (RT→P). Methods The impact of cellular senescence on the interaction between RT and P was assessed by harnessing female INK-ATTAC mice, which express a dimerizable form of caspase 8 (CASP8) under the promoter of cyclin dependent kinase inhibitor 2A ( Cdkn2a , coding for p16 Ink4 ), as host for endogenous mammary tumors induced by the subcutaneous implantation of medroxyprogesterone acetate (MPA, M) pellets combined with the subsequent oral administration of 7,12-dimethylbenz[a]anthracene (DMBA, D). This endogenous mouse model of HR + mammary carcinogenesis recapitulates key immunobiological aspects of human HR + breast cancer. Mice bearing M/D-driven tumors were allocated to RT, P or their combination in the optional presence of the CASP8 dimerizer AP20187, and monitored for tumor growth, progression-free survival and overall survival. In parallel, induction of senescence in vitro, in cultured human mammary hormone receptor (HR) + adenocarcinoma MCF7 cells, triple negative breast carcinoma MDA-MB-231 cells and mouse HR + mammary carcinoma TS/A cells treated with RT, P or their combination, was determined by colorimetric assessment of senescence-associated β-galactosidase activity after 3 or 7 days of treatment. Results In vivo depletion of p16 Ink4 -expressing (senescent) cells ameliorated the efficacy of P→RT (but not that of RT→P) in the M/D-driven model of HR + mammary carcinogenesis. Accordingly, P→RT induced higher levels of cellular senescence than R→TP in cultured human and mouse breast cancer cell lines. Conclusions Pending validation in other experimental systems, these findings suggest that a program of cellular senescence in malignant cells may explain (at least partially) the inferiority of P→RT versus RT→P in preclinical models of HR + breast cancer.

Purpose To develop a deep learning model to generate synthetic CT for MR‐only radiotherapy of prostate cancer patients treated with 0.35 T MRI linear accelerator. Materials and Methods A U‐NET convolutional neural network was developed to translate 0.35 T TRUFI MRI into electron density map using a novel cost function equalizing the contribution of various tissue types including fat, muscle, bone, and background air in training. The impact of training time, dataset size, image standardization, and data augmentation approaches was also quantified. Mean absolute error (MAE) between synthetic and planning CTs was calculated to measure the goodness of the model. Results With 20 patients in training, our U‐NET model has the potential to generate synthetic CT with a MAE of about 29.68 ± 4.41, 16.34 ± 2.67, 23.36 ± 2.85, and 105.90 ± 22.80 HU over the entire body, fat, muscle, and bone tissues, respectively. As expected, we found that the number of patients used for training and MAE are nonlinearly correlated. Data augmentation and our proposed loss function were effective to improve MAE by ~9% and ~18% in bony voxels, respectively. Increasing the training time and image standardization did not improve the accuracy of the model. Conclusion A U‐NET model has been developed and tested numerically to generate synthetic CT from 0.35T TRUFI MRI for MR‐only radiotherapy of prostate cancer patients. Dosimetric evaluation using a large and independent dataset warrants the validity of the proposed model and the actual number of patients needed for the safe usage of the model in routine clinical workflow.

Patients with gluten-sensitive enteropathy usually have increased numbers of duodenal intraepithelial lymphocytes even if the villous architecture is normal. Some authors advocate the use of CD8 and CD3 immunohistochemical stains to improve detection of intraepithelial lymphocytosis, yet the added value of immunohistochemistry when biopsies appear normal remains unproven. The purpose of this study was to evaluate the utility of CD3 and CD8 immunostains in detecting intraepithelial lymphocytosis among duodenal biopsies originally interpreted to be normal based on routine evaluation. We identified 200 duodenal biopsies from 172 patients, all of which were accompanied by a clinical question of gluten-sensitive enteropathy. Five well-oriented villi from each biopsy were assessed. Intraepithelial lymphocytes present in hematoxylin and eosin (H&E)-stained slides were counted and compared with the number of CD3 and CD8 immunopositive cells present in the villous epithelium. Results were expressed as the mean number of intraepithelial lymphocytes or immunopositive cells present per 20 villous tip enterocytes. Review of H&E-stained slides revealed a mean of 2.1±0.1 intraepithelial lymphocytes, compared with 3.2±0.1 CD3-positive and 2.1±0.1 CD8-positive intraepithelial cells ( P =<0.001 and 1, respectively), although none of the cases displayed sufficient numbers of intraepithelial lymphocytes to be considered abnormal (ie, ≥12/20 enterocytes) by any method. The number of intraepithelial lymphocytes detected by H&E evaluation or immunohistochemistry did not correlate with results of serologic studies for markers of gluten sensitivity. We conclude that immunostains for T cell markers do not improve detection of gluten-sensitive enteropathy when H&E-stained sections are normal.

Background Ultra-hypofractionated image-guided stereotactic body radiotherapy (SBRT) is increasingly used for definitive treatment of localized prostate cancer. Magnetic resonance imaging-guided radiotherapy (MRgRT) facilitates improved visualization, real-time tracking of targets and/or organs-at-risk (OAR), and capacity for adaptive planning which may translate to improved targeting and reduced toxicity to surrounding tissues. Given promising results from NRG-GU003 comparing conventional and moderate hypofractionation in the post-operative setting, there is growing interest in exploring ultra-hypofractionated post-operative regimens. It remains unclear whether this can be done safely and whether MRgRT may help mitigate potential toxicity. SHORTER (NCT04422132) is a phase II randomized trial prospectively evaluating whether salvage MRgRT delivered in 5 fractions versus 20 fractions is non-inferior with respect to gastrointestinal (GI) and genitourinary (GU) toxicities at 2-years post-treatment. Methods A total of 136 patients will be randomized in a 1:1 ratio to salvage MRgRT in 5 fractions or 20 fractions using permuted block randomization. Patients will be stratified according to baseline Expanded Prostate Cancer Index Composite (EPIC) bowel and urinary domain scores as well as nodal treatment and androgen deprivation therapy (ADT). Patients undergoing 5 fractions will receive a total of 32.5 Gy over 2 weeks and patients undergoing 20 fractions will receive a total of 55 Gy over 4 weeks, with or without nodal coverage (25.5 Gy over 2 weeks and 42 Gy over 4 weeks) and ADT as per the investigator’s discretion. The co-primary endpoints are change scores in the bowel and the urinary domains of the EPIC. The change scores will reflect the 2-year score minus the pre-treatment (baseline) score. The secondary endpoints include safety endpoints, including change in GI and GU symptoms at 3, 6, 12 and 60 months from completion of treatment, and efficacy endpoints, including time to progression, prostate cancer specific survival and overall survival. Discussion The SHORTER trial is the first randomized phase II trial comparing toxicity of ultra-hypofractionated and hypofractionated MRgRT in the salvage setting. The primary hypothesis is that salvage MRgRT delivered in 5 fractions will not significantly increase GI and GU toxicities when compared to salvage MRgRT delivered in 20 fractions. Trial registration ClinicalTrials.gov Identifier: NCT04422132. Date of registration: June 9, 2020.

The first changes associated with smoking are in the small airway epithelium (SAE). Given that smoking alters SAE gene expression, but only a fraction of smokers develop chronic obstructive pulmonary disease (COPD), we hypothesized that assessment of SAE genome-wide gene expression would permit biologic phenotyping of the smoking response, and that a subset of healthy smokers would have a \"COPD-like\" SAE transcriptome. SAE (10th-12th generation) was obtained via bronchoscopy of healthy nonsmokers, healthy smokers and COPD smokers and microarray analysis was used to identify differentially expressed genes. Individual responsiveness to smoking was quantified with an index representing the % of smoking-responsive genes abnormally expressed (I(SAE)), with healthy smokers grouped into \"high\" and \"low\" responders based on the proportion of smoking-responsive genes up- or down-regulated in each smoker. Smokers demonstrated significant variability in SAE transcriptome with I(SAE) ranging from 2.9 to 51.5%. While the SAE transcriptome of \"low\" responder healthy smokers differed from both \"high\" responders and smokers with COPD, the transcriptome of the \"high\" responder healthy smokers was indistinguishable from COPD smokers. The SAE transcriptome can be used to classify clinically healthy smokers into subgroups with lesser and greater responses to cigarette smoking, even though these subgroups are indistinguishable by clinical criteria. This identifies a group of smokers with a \"COPD-like\" SAE transcriptome.

Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, P<0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P = 0.04) and estrogen receptor-negative status (P = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (P = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The Axin2(NLSlacZ) mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, Axin2(NLSlacZ) mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia.