Search

Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFβ blockade during radiotherapy in metastatic breast cancer patients.Experimental Design: Prospective randomized trial comparing two doses of TGFβ blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum.Results: Twenty-three patients were randomized, median age 57 (range 35–77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02–7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool.Conclusions: TGFβ blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493–504. ©2018 AACR.

Supplementary Table S1. Antibodies and dextramers used for flow cytometric analysis Supplementary Table S2. Patients with one or more treatment positively-related (maximum grade possible, probable, definite) AEs by treatment arm Supplementary Table S3. Levels of circulating tumor-specific CD8+ T cells with reactivity for JARID1B, Muc1 or Her2 in breast cancer patients with HLA-A*0201 status Supplementary Table S4. Humoral immune responses were not significantly affected following TGFβ blockade and radiation Supplementary Figure S1: Study schema Supplementary Figure S2. Gating strategy in panel 1. Supplementary Figure S3. Gating strategy in panel 2. Supplementary Figure S4. Changes in PBMC, survivin- and JARID1B-reactive CD8 T cells in response to TGFβ blockade and radiation. Supplementary Figure S5. Diametrically opposing dynamics in the myeloid suppressor compartment versus the suppressor T cell pool in response to TGFβ blockade and radiation. Supplementary Figure S6. Changes in plasma levels of tryptophan and kynurenine in response to TGFβ blockade and radiation.

Supplementary Figure S1 from T-Cell Responses to Survivin in Cancer Patients Undergoing Radiation Therapy

Purpose: The goal of this study was to determine if radiation therapy (RT) of human cancer enhances or diminishes tumor-specific T-cell reactivity. This is important if immunotherapy is to be harnessed to improve the outcome of cancer radiotherapy.Experimental Design: Lymphocytes were isolated from colorectal cancer (CRC) patients before, during, and after presurgical chemoradiotherapy. Similar samples were taken from prostate cancer patients receiving standard RT. The level of CD8+ T cells capable of binding tetramers for the tumor-associated antigen survivin, which is overexpressed in both cancer types, was enumerated in HLA-A*0201 patient samples. CD4+, CD25high, Foxp3+ cells were also enumerated to evaluate therapy-induced changes in Tregulatory cells. For CRC patients, most of whom were enrolled in a clinical trial, pathologic response data were available, as well as biopsy and resection specimens, which were stained for cytoplasmic and intranuclear survivin.Results: Survivin-specific CD8+ T lymphocytes were detected in the peripheral blood of CRC and prostate cancer patients and increased after therapy in some, but not all, patients. Increases were more common in CRC patients whose tumor was downstaged after chemoradiotherapy. Biopsy specimens from this cohort generally had higher nuclear to cytoplasmic survivin expression. Tregulatory cells generally increased in the circulation following therapy but only in CRC patients.Conclusion: This study indicates that RT may increase the likelihood of some cancer patients responding to immunotherapy and lays a basis for future investigations aimed at combining radiation and immunotherapy.

BackgroundHundreds of ongoing clinical trials combine radiation therapy, mostly delivered as stereotactic body radiotherapy (SBRT), with immune checkpoint blockade. However, our understanding of the effect of radiotherapy on the intratumoral immune balance is inadequate, hindering the optimal design of trials that combine radiation therapy with immunotherapy. Our objective was to characterize the intratumoral immune balance of the malignant prostate after SBRT in patients.MethodsSixteen patients with high-risk, non-metastatic prostate cancer at comparable Gleason Grade disease underwent radical prostatectomy with (n = 9) or without (n = 7) neoadjuvant SBRT delivered in three fractions of 8 Gy over 5 days completed 2 weeks before surgery. Freshly resected prostate specimens were processed to obtain single-cell suspensions, and immune-phenotyped for major lymphoid and myeloid cell subsets by staining with two separate 14-antibody panels and multicolor flow cytometry analysis.ResultsMalignant prostates 2 weeks after SBRT had an immune infiltrate dominated by myeloid cells, whereas malignant prostates without preoperative treatment were more lymphoid-biased (myeloid CD45+ cells 48.4 ± 19.7% vs. 25.4 ± 7.0%; adjusted p-value = 0.11; and CD45+ lymphocytes 51.6 ± 19.7% vs. 74.5 ± 7.0%; p = 0.11; CD3+ T cells 35.2 ± 23.8% vs. 60.9 ± 9.7%; p = 0.12; mean ± SD).ConclusionSBRT drives a significant lymphoid to myeloid shift in the prostate-tumor immune infiltrate. This may be of interest when combining SBRT with immunotherapies, particularly in prostate cancer.

Normal tissue responses to ionizing radiation have been a major subject for study since the discovery of X‐rays at the end of the 19th century. Shortly thereafter, time–dose relationships were established for some normal tissue endpoints that led to investigations into how the size of dose per fraction and the quality of radiation affected outcome. The assessment of the radiosensitivity of bone marrow stem cells using colony‐forming assays by Till and McCulloch prompted the establishment of in situ clonogenic assays for other tissues that added to the radiobiology toolbox. These clonogenic and functional endpoints enabled mathematical modeling to be performed that elucidated how tissue structure, and in particular turnover time, impacted clinically relevant fractionated radiation schedules. More recently, lineage tracing technology, advanced imaging and single cell sequencing have shed further light on the behavior of cells within stem, and other, cellular compartments, both in homeostasis and after radiation damage. The discovery of heterogeneity within the stem cell compartment and plasticity in response to injury have added new dimensions to the consideration of radiation‐induced tissue damage. Clinically, radiobiology of the 20th century garnered wisdom relevant to photon treatments delivered to a fairly wide field at around 2 Gy per fraction, 5 days per week, for 5–7 weeks. Recently, the scope of radiobiology has been extended by advances in technology, imaging and computing, as well as by the use of charged particles. These allow radiation to be delivered more precisely to tumors while minimizing the amount of normal tissue receiving high doses. One result has been an increase in the use of schedules with higher doses per fraction given in a shorter time frame (hypofractionation). We are unable to cover these new technologies in detail in this review, just as we must omit low‐dose stochastic effects, and many aspects of dose, dose rate and radiation quality. We argue that structural diversity and plasticity within tissue compartments provides a general context for discussion of most radiation responses, while acknowledging many omissions. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

PURPOSE: This review is focused on radium and radionuclides in its decay chain in honor of Marie Curie, who discovered this element. MATERIALS AND METHODS: We conglomerated current knowledge regarding radium and its history predating our present understanding of this radionuclide. RESULTS: An overview of the properties of radium and its dose assessment is sown followed by discussions about both the negative detrimental and positive therapeutic applications of radium with this history and its evolution reflecting current innovations in medical science. CONCLUSIONS: We hope to remind all those who are interested in the progress of science about the vagaries of the process of scientific discovery. In addition, we raise the interesting question of whether Marie Curie’s initial success was in part possible due to her tight alignment with her husband Pierre Curie who pushed the work along.

Background We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP). Methods The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression. Results At baseline, a significantly higher level of expression (p

Purpose Glioblastoma (GBM) remains an incurable disease despite extensive treatment with surgical resection, irradiation, and temozolomide. In line with many other forms of aggressive cancers, GBM is currently under consideration as a target for immunotherapy. However, GBM tends to be nonimmunogenic and exhibits a microenvironment with few or no effector T cells, a relatively low nonsynonymous somatic mutational load, and a low predicted neoantigen burden. GBM also exploits a multitude of immunosuppressive strategies. Methods and Materials A number of immunotherapeutic approaches have been tested with disappointing results. A rationale exists to combine immunotherapy and radiation therapy, which can induce an immunogenic form of cell death with T-cell activation and tumor infiltration. Results Various immunotherapy agents, including immune checkpoint modulators, transforming growth factor beta receptor inhibitors, and indoleamine-2,3-dioxygenase inhibitors, have been evaluated with irradiation in preclinical GBM models, with promising results, and are being further tested in clinical trials. Conclusions This review aims to present the basic rationale behind this emerging complementary therapeutic approach in GBM, appraise the current preclinical and clinical data, and discuss the future challenges in improving the antitumor immune response.

PURPOSE: As part of the special issue on ‘Women in Science’, this review offers a perspective on past and ongoing work in the field of normal (non-cancer) tissue radiation biology, highlighting the work of many of the leading contributors to this field of research. We discuss some of the hypotheses that have guided investigations, with a focus on some of the critical organs considered dose-limiting with respect to radiation therapy, and speculate on where the field needs to go in the future. CONCLUSIONS: The scope of work that makes up normal tissue radiation biology has and continues to play a pivotal role in the radiation sciences, ensuring the most effective application of radiation in imaging and therapy, as well as contributing to radiation protection efforts. However, despite the proven historical value of preclinical findings, recent decades have seen clinical practice move ahead with altered fractionation scheduling based on empirical observations, with little to no (or even negative) supporting scientific data. Given our current appreciation of the complexity of normal tissue radiation responses and their temporal variability, with tissue- and/or organ-specific mechanisms that include intra-, inter- and extracellular messaging, as well as contributions from systemic compartments, such as the immune system, the need to maintain a positive therapeutic ratio has never been more urgent. Importantly, mitigation and treatment strategies, whether for the clinic, emergency use following accidental or deliberate releases, or reducing occupational risk, will likely require multi-targeted approaches that involve both local and systemic intervention. From our personal perspective as five ‘Women in Science’, we would like to acknowledge and applaud the role that many female scientists have played in this field. We stand on the shoulders of those who have gone before, some of whom are fellow contributors to this special issue.

Background Powerful constitutive and inducible transgenic / bitransgenic / tritransgenic murine models of breast cancer have been used over the past two decades to shed light on the molecular mechanisms by which the given transgenic oncogenes have interacted with other cellular genes and set in motion breast cancer initiation and progression. However, these transgenic models, as in vivo models only, are expensive and restrictive in the opportunities they provide to manipulate the experimental variables that would enable a better understanding of the molecular events related to initial transformation and the target cell being transformed. Methods To overcome some of these limitations, we derived oncogene-containing induced pluripotent stem cell (iPSC) clones from tail vein fibroblasts of these transgenic mice and manipulated them both in vitro and in vivo in non-transgenic background mice. We created the iPSC clones with a relatively low M.O.I, producing retroviral integrations which averaged only 1 to 2 sites per retroviral plasmid construct used. Results Most iPSC clones derived from each group displayed an essentially normal murine karyotype, strong expression of the exogenous reprogrammable genes and significant expression of characteristic endogenous murine surface stem cell markers including SSEA-1 (CD15), PECAM-1 (CD31), Ep-Cam (CD326), and Nectin (CD112), but no expression of their transgene. A majority (75%) of iPSC clones displayed a normal murine karyotype but 25% exhibited a genomically unstable karyotype. However, even these later clones exhibited stable and characteristic iPSC properties. When injected orthotopically, select iPSC clones, either constitutive or inducible, no longer expressed their exogenous pluripotency reprogramming factors but expressed their oncogenic transgene (PyVT or ErbB2) and participated in both breast ontogenesis and subsequent oncogenesis. When injected non-orthotopically or when differentiated in vitro along several different non-mammary lineages of differentiation, the iPSC clones failed to do so. Although many clones developed anticipated teratomas, select iPSC clones under the appropriate constitutive or inducible conditions exhibited both breast ontogenesis and oncogenesis through the same stages as exhibited by their transgenic murine parents and, as such, represent transgenic surrogates. Conclusions The iPSC clones offer a number of advantages over transgenic mice including cost, the ability to manipulate and tag in vitro, and create an in vitro model of breast ontogeny and oncogenesis that can be used to gain additional insights into the differentiated status of the target cell which is susceptible to transformation. In addition, the use of these oncogene-containing iPSC clones can be used in chemopreventive studies of breast cancer.

International audience; Ionizing radiation interacts with the immune system in many ways with a multiplicity that mirrors thecomplexity of the immune system itself: namely the need to maintain a delicate balance between differentcompartments, cells and soluble factors that work collectively to protect, maintain, and restore tissue function inthe face of severe challenges including radiation damage. The cytotoxic effects of high dose radiation are lessrelevant after low dose exposure, where subtle quantitative and functional effects predominate that may gounnoticed until late after exposure or after a second challenge reveals or exacerbates the effects. For example,low doses may permanently alter immune fitness and therefore accelerate immune senescence and pave the wayfor a wide spectrum of possible pathophysiological events, including early-onset of age-related degenerativedisorders and cancer. By contrast, the so called low dose radiation therapy displays beneficial, anti-inflammatoryand pain relieving properties in chronic inflammatory and degenerative diseases. In this review, epidemiological,clinical and experimental data regarding the effects of low-dose radiation on the homeostasis and functionalintegrity of immune cells will be discussed, as will be the role of immune-mediated mechanisms in the systemicmanifestation of localized exposures such as inflammatory reactions. The central conclusion is that ionizingradiation fundamentally and durably reshapes the immune system. Further, the importance of discovery ofimmunological pathways for modifying radiation resilience amongst other research directions in this field isimplied.

Purpose This study aimed to evaluate the feasibility and safety of prostate stereotactic body radiation therapy (SBRT) neoadjuvant to radical prostatectomy (RP) in a phase 1 trial. The primary endpoint was treatment completion rate without severe acute surgical complications. Secondary endpoints included patient-reported quality of life and physician-reported toxicities. Methods and Materials Patients with nonmetastatic high-risk or locally advanced prostate cancer received 24 Gy in 3 fractions to the prostate and seminal vesicles over 5 days, completed 2 weeks before RP. Patients with pN1 disease were treated after multidisciplinary discussion and shared decision making. Patient-reported quality of life (International Prostate Symptom Score and Expanded Prostate Cancer Index Composite 26-item version questionnaires) and physician-reported toxicity (Common Terminology Criteria for Adverse Events, version 4.03) were assessed before SBRT, immediately before surgery, and at 3-month intervals for 1 year. Results Twelve patients were enrolled, and 11 completed treatment (1 patient had advanced disease on prostate-specific membrane antigen positron emission tomography after enrollment but before treatment). There were no significant surgical complications. After RP, 2 patients underwent additional radiation therapy to nodes with androgen suppression for pN1 disease. Median follow-up after completion of treatment was 20.1 months, with 9 of 11 patients having a follow-up period of >12 months. Two patients had biochemical recurrence (prostate-specific antigen ≥0.05) within the first 12 months, with an additional 2 patients found to have biochemical recurrence after the 12-month period. The highest Common Terminology Criteria for Adverse Events genitourinary grades were 0, 1, 2, and 3 (n = 1, 4, 4, and 2, respectively), and the highest gastrointestinal grades were 0, 1, and 2 (n = 9, 1, and 1, respectively). At 12 months, incontinence was the only grade ≥2 toxicity. One and 2 of 9 patients had grade 2 and 3 incontinence, respectively. On the Expanded Prostate Cancer Index Composite (26-item version), the mean/median changes in scores from baseline to 12 months were –32.8/–31.1 for urinary incontinence, –1.6/–6.2 for urinary irritative/obstructive, –2.1/0 for bowel, –34.4/–37.5 for sexual function, and –10.6/–2.5 for hormonal. The mean/median change in International Prostate Symptom Score from baseline to 12 months was 0.5/0.5. Conclusions RP after neoadjuvant SBRT appears to be feasible and safe at the dose tested. The severity of urinary incontinence may be higher than RP alone.

The limited impact of treatments for COVID-19 has stimulated several phase 1 clinical trials of whole-lung low-dose radiation therapy (LDRT; 0.3-1.5 Gy) that are now progressing to phase 2 randomized trials worldwide. This novel but unconventional use of radiation to treat COVID-19 prompted the National Cancer Institute, National Council on Radiation Protection and Measurements and National Institute of Allergy and Infectious Diseases to convene a workshop involving a diverse group of experts in radiation oncology, radiobiology, virology, immunology, radiation protection and public health policy. The workshop was held to discuss the mechanistic underpinnings, rationale, and preclinical and emerging clinical studies, and to develop a general framework for use in clinical studies. Without refuting or endorsing LDRT as a treatment for COVID-19, the purpose of the workshop and this review is to provide guidance to clinicians and researchers who plan to conduct preclinical and clinical studies, given the limited available evidence on its safety and efficacy.

The discovery of X-rays in 1895 had a dramatic impact on many aspects of society. One was the hope that it would be possible to treat some of the numerous infections and inflammatory conditions for...

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-β/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident.

The purpose of this study was to determine the dynamic contributions of different immune cell subsets to primary and abscopal tumor regression after hypofractionated radiation therapy (hRT) and the impact of anti-PD-1 therapy. A bilateral syngeneic FSA1 fibrosarcoma model was used in immunocompetent C3H mice, with delayed inoculation to mimic primary and microscopic disease. The effect of tumor burden on intratumoral and splenic immune cell content was delineated as a prelude to hRT on macroscopic T1 tumors with 3 fractions of 8 Gy while microscopic T2 tumors were left untreated. This was performed with and without systemic anti-PD-1. Immune profiles within T1 and T2 tumors and in spleen changed drastically with tumor burden in untreated mice with infiltrating CD4+ content declining, while the proportion of CD4+ Tregs rose. Myeloid cell representation escalated in larger tumors, resulting in major decreases in the lymphoid:myeloid ratios. In general, activation of Tregs and myeloid-derived suppressor cells allow immunogenic tumors to grow, although their relative contributions change with time. The evidence suggests that primary T1 tumors self-regulate their immune content depending on their size and this can influence the lymphoid compartment of T2 tumors, especially with respect to Tregs. Tumor burden is a major confounding factor in immune analysis that has to be taken into consideration in experimental models and in the clinic. hRT caused complete local regression of primary tumors, which was accompanied by heavy infiltration of CD8+ T cells activated to express IFN-&gamma, and PD-1, while certain myeloid populations diminished. In spite of this active infiltrate, primary hRT failed to generate the systemic conditions required to cause abscopal regression of unirradiated microscopic tumors unless PD-1 blockade, which on its own was ineffective, was added to the RT regimen. The combination further increased local and systemically activated CD8+ T cells, but few other changes. This study emphasizes the subtle interplay between the immune system and tumors as they grow and how difficult it is for local RT, which can generate a local immune response that may help with primary tumor regression, to overcome the systemic barriers that are generated so as to effect immune regression of even small abscopal lesions.

Purpose: This study examined the feasibility, efficacy (abscopal effect), and immune effects of TGFβ blockade during radiotherapy in metastatic breast cancer patients. Experimental Design: Prospective randomized trial comparing two doses of TGFβ blocking antibody fresolimumab. Metastatic breast cancer patients with at least three distinct metastatic sites whose tumor had progressed after at least one line of therapy were randomized to receive 1 or 10 mg/kg of fresolimumab, every 3 weeks for five cycles, with focal radiotherapy to a metastatic site at week 1 (three doses of 7.5 Gy), that could be repeated to a second lesion at week 7. Research bloods were drawn at baseline, week 2, 5, and 15 to isolate PBMCs, plasma, and serum. Results: Twenty-three patients were randomized, median age 57 (range 35–77). Seven grade 3/4 adverse events occurred in 5 of 11 patients in the 1 mg/kg arm and in 2 of 12 patients in the 10 mg/kg arm, respectively. Response was limited to three stable disease. At a median follow up of 12 months, 20 of 23 patients are deceased. Patients receiving the 10 mg/kg had a significantly higher median overall survival than those receiving 1 mg/kg fresolimumab dose [hazard ratio: 2.73 with 95% confidence interval (CI), 1.02–7.30; P = 0.039]. The higher dose correlated with improved peripheral blood mononuclear cell counts and a striking boost in the CD8 central memory pool. Conclusions: TGFβ blockade during radiotherapy was feasible and well tolerated. Patients receiving the higher fresolimumab dose had a favorable systemic immune response and experienced longer median overall survival than the lower dose group. Clin Cancer Res; 24(11); 2493–504. ©2018 AACR.

In radiotherapy, the treatment is adapted to each individual to protect healthy tissues but delivers most of time a standard dose according to the tumor histology and site. The only biomarkers studied to individualize the treatment are the HPV status with radiation dose de-escalation strategies, and tumor hypoxia with dose escalation to hypoxic subvolumes using FMISO- or FAZA-PET imaging. In the last decades, evidence has grown about the contribution of the immune system to radiation tumor response. Many preclinical studies have identified some of the mechanisms involved. In this context, we have realised a systematic review to highlight potential inflammatory and immune biomarkers of radiotherapy response. Some are inside the tumor microenvironment, as lymphocyte infiltration or PD-L1 expression, others are circulating biomarkers, including different types of hematological cells, cytokines and chemokines.

Innovation and progress in radiation oncology depend on discovery and insights realized through research in radiation biology. Radiobiology research has led to fundamental scientific insights, from the discovery of stem/progenitor cells to the definition of signal transduction pathways activated by ionizing radiation that are now recognized as integral to the DNA damage response (DDR). Radiobiological discoveries are guiding clinical trials that test radiation therapy combined with inhibitors of the DDR kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and immune or cell cycle checkpoint inhibitors. To maintain scientific and clinical relevance, the field of radiation biology must overcome challenges in research workforce, training, and funding. The National Cancer Institute convened a workshop to discuss the role of radiobiology research and radiation biologists in the future scientific enterprise. Here, we review the discussions of current radiation oncology research approaches and areas of scientific focus considered important for rapid progress in radiation sciences and the continued contribution of radiobiology to radiation oncology and the broader biomedical research community.

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non–small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen–specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222). Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 106, 5 × 106, 1 × 107, or 3 × 107 DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen–specific peripheral blood lymphocyte induction of IFNγ in ELISPOT assays. Tumor biopsies were evaluated for CD8+ T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR). Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8+ T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8+ T cells per mm2). Patients with increased CD8+ T cells following vaccination showed significantly increased PD-L1 mRNA expression. Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen–specific immune responses; (ii) enhanced tumor CD8+ T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556–68. ©2017 AACR.

Background Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. Methods We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5′ upstream region of the human IL32 gene into a luciferase reporter vector. Results A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32. Cis-acting elements within this 5′ upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. Conclusions Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5′ upstream region. Electronic supplementary material The online version of this article (10.1186/s12967-019-1862-y) contains supplementary material, which is available to authorized users.

Aim: We determined if pre-treatment systemic inflammation would predict poor outcomes in the setting of stereotactic radiosurgery (SRS) for brain metastases. Patients and Methods: The pretreatment albumin concentration, neutrophil and lymphocyte counts, and the platelet-to-lymphocyte ratio (PLR) were evaluated to determine association with intracranial control, local control (LC), initial MRI response (MR), and overall survival (OS) among 70 patients with 152 separate brain metastases treated with SRS alone from 2008-2015. Results: On multivariate analysis, a higher neutrophil percentage predicted for poor LC, poor initial MR, poor OS and poor intracranial control (p=0.01, p=0.01, p=0.02 and p=0.03, respectively). A lower percentage of lymphocytes predicted for poor LC and poor MR (p=0.01 and p=0.02), and an elevated PLR predicted for poor OS and poor LC (p=0.05 and p=0.04). Additionally, a lower pretreatment albumin concentration predicted for poor LC and OS (p=0.01 and p=0.03). Conclusion: Pretreatment systemic inflammation is associated with poor outcomes post-SRS.

339 Background: Stereotactic Body Radiotherapy (SBRT) delivers high dose per fraction radiotherapy to targets with high precision. Such hypofractionated RT appears to act as an immune adjuvant, altering the tumor infiltrating immune landscape and enriching it for lymphocytes as numerous preclinical investigations would suggest. Based on this hypothesis, hundreds of ongoing trials listed in clinicaltrials.gov currently test the combination of RT (largely SBRT) with various immunotherapies. However, studies directly measuring the representation of infiltrating immune cells after SBRT in patients are few and far between and none exist in the context of prostate cancer. We therefore sought to interrogate the tumor-immune interface after prostate SBRT using fresh tissue in patients. Methods: Fresh prostate tissue from patients (N=10) enrolled in a clinical trial of prostate SBRT (three fractions of 8 Gy directed to the prostate and seminal vesicles) in the neoadjuvant setting two weeks prior to radical prostatectomy was subjected to multicolor flow cytometry and compared to that of Gleason Grade and T stage matched controls who did not undergo neoadjuvant therapy. Results: With a threshold of significance level of 0.05 for unadjusted p-values, using two-sided two-sample t-test, myeloid cells and particularly CD14+/hiCD16+DR+ intermediate monocytes/macrophages were enriched, while lymphocytes, including T cells and CD56+16− NK cells were decreased in SBRT-treated prostates as compared to unirradiated controls. Conclusions: The immune infiltrates in prostates two weeks after SBRT demonstrates a significant lymphoid to myeloid shift consistent with a tumor microenvironment after SBRT that is likely immunosuppressive beyond what can be targeted through the PD-1/L1 or CTLA-4 axis alone. This may have implications for the design of immunotherapy trials, especially in prostate cancer, that test SBRT in combination with immunotherapies.

OBJECTIVE: Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer’s patches. METHODS: C3H mice were irradiated with 0 or 12 Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24 hours later. 48 hours after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer’s patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96 hours after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer’s patches were subjected to immune profiling using flow cytometry. RESULTS: Compound #5 significantly increased the number of visible Peyer’s patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer’s patch isolated from these mice demonstrated an overall increase in the total number of Peyer’s patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11b(high) in Peyer’s patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside the Peyer’s patches 24 hours after drug treatment. A single dose of Compound #5 significantly increased the number of CD45(+) cells after 12 Gy TBI. Importantly, 96 hours after 12 Gy TBI Compound #5 induced a significant rise in the number of visible Peyer’s patches and the number of Peyer’s patch-associated regenerating crypts. CONCLUSION: In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer’s patches.

The ability to recognize and respond to universal molecular patterns on invading microorganisms allows our immune system to stay on high alert, sensing danger to our self-integrity. Our own damaged cells and tissues in pathological situations activate similar warning systems as microbes. In this way, the body is able to mount a response that is appropriate to the danger. Toll-like receptors are at the heart of this pattern recognition system that initiates innate pro-oxidant, pro-inflammatory signaling cascades and ultimately bridges recognition of danger to adaptive immunity. The acute inflammatory lesions that are formed segue into resolution of inflammation, repair and healing or, more dysfunctionally, into chronic inflammation, autoimmunity, excessive tissue damage and carcinogenesis. Redox is at the nexus of this decision making process and is the point at which ionizing radiation initially intercepts to trigger similar responses to self-damage. In this review we discuss our current understanding of how radiation-damaged cells interact with Toll-like receptors and how the immune systems interprets these radiation-induced danger signals in the context of whole-body exposures and during local tumor irradiation.

The past 20 years have seen dramatic changes in the delivery of radiation therapy, but the impact of radiobiology on the clinic has been far less substantial. A major consideration in the use of radiotherapy has been on how best to exploit differences between the tumour and host tissue characteristics, which in the past has been achieved empirically by radiation-dose fractionation. New advances are uncovering some of the mechanistic processes that underlie this success story. In this Review, we focus on how these processes might be targeted to improve the outcome of radiotherapy at the individual patient level. This approach would seem a more productive avenue of treatment than simply trying to increase the radiation dose delivered to the tumour.

Objective To evaluate the adverse effects and therapeutic efficacy of our biocompatible polymer platform delivering targeted local therapy of cytokine CCL21 and cisplatin in a partially resected xenograft animal model of head and neck squamous cell carcinoma. In addition, to evaluate the efficacy of cotreatment with radiotherapy and assess the biocompatibility of the cisplatin-eluting polymer in the murine neck. Study Design Experimental animal study. Setting Academic research laboratory. Subjects and Methods SCCVII/SF cell injection established head and neck squamous cell carcinoma tumors in C3H/HeJ mice. Subjects underwent surgery, and a chemokine-eluting polymer was implanted into the resected site. Subjects treated with cisplatin received radiation or no radiation, and tissue was harvested after 8 weeks to assess polymer biocompatibility. Results Our results with the polymer platform significantly ( P.05) reduced SCCVII/SF tumor size in C3H/HeJ mice with cisplatin (49% ± 8.7%, Δ3.4 ± 0.6 cm

The coming of age for immunotherapy (IT) as a genuine treatment option for cancer patients through the development of new and effective agents, in particular immune checkpoint inhibitors, has led to a huge renaissance of an old idea, namely to harness the power of the immune system to that of radiation therapy (RT). It is not an overstatement to say that the combination of RT with IT has provided a new conceptual platform that has re-energized the field of radiation oncology as a whole. One only has to look at the immense rise in sessions at professional conferences and in grant applications dealing with this topic to see its emergence as a force, while the number of published reviews on the topic is staggering. At the time of writing, over 97 clinical trials have been registered using checkpoint inhibitors with RT to treat almost 7,000 patients, driven in part by strong competition between pharmaceutical products eager to find their market niche. Yet, for the most part, this enthusiasm is based on relatively limited recent data, and on the clinical success of immune checkpoint inhibitors as single agents. A few preclinical studies on RT-IT combinations have added real value to our understanding of these complex interactions, but many assumptions remain. It seems therefore appropriate to go back in time and pull together what actually has been a long history of investigations into radiation and the immune system (Figure 1) in an effort to provide context for this interesting combination of cancer therapies.

Purpose Immunotherapy could be a useful adjunct to standard cytotoxic therapies such as radiation in patients with micrometastatic disease, although successful integration of immunotherapy into treatment protocols will require further understanding of how standard therapies affect the generation of antitumor immune responses. This study was undertaken to evaluate the impact of radiation therapy (RT) on immunosuppressive T regulatory (Treg) cells. Methods and Materials Treg cells were identified as a CD4 + CD25 hi Foxp3 + lymphocyte subset, and their fate was followed in a murine TRAMP C1 model of prostate cancer in mice with and without RT. Results CD4 + CD25 hi Foxp3 + Treg cells increased in immune organs after local leg or whole-body radiation. A large part, but not all, of this increase after leg-only irradiation could be ascribed to radiation scatter and Treg cells being intrinsically more radiation resistant than other lymphocyte subpopulations, resulting in their selection. Their functional activity on a per-cell basis was not affected by radiation exposure. Similar findings were made with mice receiving local RT to murine prostate tumors growing in the leg. The importance of the Treg cell population in the response to RT was shown by systemic elimination of Treg cells, which greatly enhanced radiation-induced tumor regression. Conclusions We conclude that Treg cells are more resistant to radiation than other lymphocytes, resulting in their preferential increase. Treg cells may form an important homeostatic mechanism for tissues injured by radiation, and in a tumor context, they may assist in immune evasion during therapy. Targeting this population may allow enhancement of radiotherapeutic benefit through immune modulation.

Many lung cancer treatment regimens include radiotherapy. We sought to improve the efficacy of such treatment by invoking the targeted delivery of a model radiosensitizer (nicotinamide) to malignant tissues. Ephrin receptors (Eph), which are often overexpressed in lung cancers, were selected as the target of our delivery system. Molecular targeting was achieved utilizing a small peptide derived from the C-terminal portion of azurin, a copper-containing redox protein (“cupredoxin”) that is capable of binding to ephrin receptors. We prepared and screened a sub-library of peptides derived from the C-terminal region of azurin and found several small analogues that bound ephrin receptors EphA2, EphB2, and EphB4. One such peptide, termed AzV36, was selected for conjugation with nicotinic acid via an amide bond to form AzV36-NicL. The resulting linear peptide contains 15 residues (including unusual and d-amino acids), is very stable in human serum, and can be easily manufactured. AzV36-NicL conjugate was tested in vivo for its ability to radiosensitize Lewis lung carcinoma (LCC) in artificial metastasis and solid tumor engraftment models. The compound increased the efficacy of radiotherapy with tumor colonies being ~2–13 fold lower than with radiation alone depending on experimental schedule. In contrast, equimolar amounts of unconjugated peptide (AzV36-L) or nicotinamide alone only marginally improved radiation efficacy. The targeted delivery of radiosensitizer(s) to ephrin receptors may enhance the efficacy of radiation treatment of lung cancer and of other cancers that overexpress ephrin receptor(s).