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.

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.

In the original publication [...]

Heart disease is a significant adverse event caused by radiotherapy for some cancers. Identifying the origins of radiogenic heart disease will allow therapies to be developed. Previous studies showed non-targeted effects manifest as fibrosis in the non-irradiated heart after 120 days following targeted X-irradiation of the kidneys with 10 Gy in WAG/RijCmcr rats. To demonstrate the involvement of T cells in driving pathophysiological responses in the out-of-field heart, and to characterize the timing of immune cell engagement, we created and validated a T cell knock downrat on the WAG genetic backgrou nd. Irradiation of the kidneys with 10 Gy of X-rays in wild-type rats resulted in infiltration of T cells, natural killer cells, and macrophages after 120 days, and none of these after 40 days, suggesting immune cell engagement is a late response. The radiation nephropathy and cardiac fibrosis that resulted in these animals after 120 days was significantly decreased in irradiated T cell depleted rats. We conclude that T cells function as an effector cell in communicating signals from the irradiated kidneys which cause pathologic remodeling of non-targeted heart.

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.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 “hits.” Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability.

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...

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.

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).

The body senses "danger" from "damaged self" molecules through members of the same receptor superfamily it uses for microbial "non-self", triggering canonical signaling pathways that lead to the generation of acute inflammatory responses. For this reason, the biology of normal tissue responses to moderate and clinically relevant doses of radiation is inextricably connected to innate immunity. The complex sequence of inflammatory events that ensues causes further cell and tissue damage to eliminate potential invaders but also leads to cytoprotective responses that limit the spread of damage and to wound healing through tissue regeneration or replacement. These sequential processes are orchestrated through multiple feedback control mechanisms involving cyclical production of free radicals and cytokines that are common to both radiation and immune signaling. This requires a concerted effort by resident tissue and inflammatory cell types, with macrophages apparently leading the way. The initial response to moderate doses of radiation therefore feeds into a pro-inflammatory paradigm whose eventual outcome is critically dependent upon the properties of the immune cells that are involved in tissue damage, regeneration and repair and that are in part under genetic influence. Importantly, these canonical pathways provide targets for interventions aimed at modifying normal tissue radiation responses. In this review, we examine areas of intersection between innate immunity and normal tissue radiobiology.

Radiation therapy affects the immune system. In addition to killing radiosensitive immune cells, it can induce functional changes in those cells that survive. Our recent studies showed that the exposure of dendritic cells (DCs) to radiation in vitro influences their ability to present tumour antigen in vivo. Here we show that local radiation therapy of B16 melanoma tumours inhibits the development of systemic immunity to the melanoma antigen MART-1. This inhibition could not be overcome by intratumoral injection of DCs expressing human MART-1 after radiation therapy, suggesting that a form of immune suppression might have developed. On the other hand, injection of MART-expressing DCs prior to tumour irradiation was able to prevent inhibition from developing. These results suggest that local radiation therapy may block the generation of immunity under some circumstances and that strategies may be required to prevent this and allow radiation-induced cell death to translate fully into the development of systemic immunity.

Introduction We determined whether pretreatment immunologic parameters could predict the outcomes and toxicity in early-stage non–small-cell lung cancer (NSCLC) patients treated with stereotactic body radiation therapy (SBRT). Patients and Methods The pretreatment leukocyte, lymphocyte, and neutrophil counts, serum albumin levels, neutrophil-to-lymphocyte ratio (NLR,) and platelet-to-lymphocyte ratio (PLR) were evaluated to determine the association with locoregional control, distant metastasis-free survival (DMFS), disease-specific survival (DSS), overall survival (OS), and treatment-related toxicity. The survival rates were estimated with Kaplan-Meier analysis and multivariate analysis using the Cox proportional hazards model. Results The data from 118 patients with a median follow-up period of 28.9 months were assessed. The 3-year local control, regional control, and DMFS rates were 97%, 87%, and 92%, respectively. The 3-year OS and DSS rates were 77% and 85%, respectively. On univariate analysis, none of the pretreatment immune parameters predicted for disease control. A higher NLR ( P = .008), PLR ( P = .002), neutrophil count ( P = .059), and the presence of lymphocytopenia ( P = .032) independently prognosticated for poor OS. Receiver operating characteristic curve analysis found NLRs > 2.18 and PLRs > 187.27 optimally predicted for poor 3-year OS ( P = .0262 and P = .0089, respectively). A higher NLR predicted against the development of any symptomatic toxicity and against the development of symptomatic (grade ≥ 2) radiation pneumonitis on univariate analysis, and a higher serum albumin level independently predicted for the development of symptomatic radiation pneumonitis ( P = .0491). Conclusion In the setting of SBRT, an elevated pretreatment NLR, PLR, and neutrophil count and the presence of lymphocytopenia independently predicted for poor OS. Patients who presented with higher NLRs and lower serum albumin levels experienced less treatment-related symptomatic toxicity.

Low-dose radiotherapy (RT) has often been used effectively for the treatment of a variety of benign diseases, particularly those with acute inflammatory features. Here we report findings on radiation treatment of acute inflammation using a murine carrageenin air pouch model.Air pouches raised on the dorsal surface of mice were injected with lambda carrageenin and were irradiated 6 h later with doses ranging from 0-5 Gy. Treatment success was evaluated at various times thereafter by volume of exudate and number of inflammatory cells, and levels of inflammation-related cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and transforming growth factor beta-1 (TGFbeta-1), and expression of inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX-2) and inducible heat shock protein 70 (HSP70) as determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively.Crude inflammatory parameters such as the amount of exudates and number of inflammatory cells remained largely unaffected by radiation or were even a slightly and transiently increased. However, the expression of iNOS was attenuated by radiation concomitant with an increase in the levels of HO-1 and HSP70. Cytokine levels varied with the radiation dose and the time point.Ionizing radiation, even at low doses, functionally modulates inflammatory cells. Our findings indicate possible mechanisms as to how low-dose radiation may exert anti-inflammatory effects and provide the first evidence that heat shock proteins may be involved in this response.

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

Importance There is a significant need to find biomarkers of response to radiotherapy and cetuximab in locally advanced head and neck squamous cell carcinoma (HNSCC) and biomarkers that predict altered immunity, thereby enabling personalized treatment. Objectives To examine whether the Kirsten rat sarcoma viral oncogene homolog ( KRAS ) – variant, a germline mutation in a microRNA-binding site in KRAS, is a predictive biomarker of cetuximab response and altered immunity in the setting of radiotherapy and cisplatin treatment and to evaluate the interaction of the KRAS -variant with p16 status and blood-based transforming growth factor β1 (TGF-β1). Design, Setting, and Participants A total of 891 patients with advanced HNSCC from a phase 3 trial of cisplatin plus radiotherapy with or without cetuximab (NRG Oncology RTOG 0522) were included in this study, and 413 patients with available samples were genotyped for the KRAS -variant. Genomic DNA was tested for the KRAS -variant in a CLIA-certified laboratory. Correlation of the KRAS -variant, p16 positivity, outcome, and TGF-β1 levels was evaluated. Hazard ratios (HRs) were estimated with the Cox proportional hazards model. Main Outcomes and Measures The correlation of KRAS -variant status with cetuximab response and outcome, p16 status, and plasma TGF-β1 levels was tested. Results Of 891 patients eligible for protocol analyses (786 male [88.2%], 105 [11.2%] female, 810 white [90.9%], 81 nonwhite [9.1%]), 413 had biological samples for KRAS -variant testing, and 376 had plasma samples for TGF-β1 measurement. Seventy patients (16.9%) had the KRAS -variant. Overall, for patients with the KRAS -variant, cetuximab improved both progression-free survival (PFS) for the first year (HR, 0.31; 95% CI, 0.10-0.94; P = .04) and overall survival (OS) in years 1 to 2 (HR, 0.19; 95% CI, 0.04-0.86; P = .03). There was a significant interaction of the KRAS -variant with p16 status for PFS in patients treated without cetuximab. The p16-positive patients with the KRAS -variant treated without cetuximab had worse PFS than patients without the KRAS -variant (HR, 2.59; 95% CI, 0.91-7.33; P = .07). There was a significant 3-way interaction among the KRAS -variant, p16 status, and treatment for OS (HR, for KRAS -variant, cetuximab and p16 positive, 0.22; 95% CI, 0.03-1.66; HR for KRAS -variant, cetuximab and p16 negative, 1.43; 95% CI, 0.48-4.26; HR for KRAS -variant, no cetuximab and p16 positive, 2.48; 95% CI, 0.64-9.65; and HR for KRAS -variant, no cetuximab and p16 negative, 0.61; 95% CI, 0.23-1.59; P = .02). Patients with the KRAS -variant had significantly elevated TGF-β1 plasma levels (median, 23 376.49 vs 18 476.52 pg/mL; P = .03) and worse treatment-related toxic effects. Conclusions and Relevance Patients with the KRAS- variant with HNSCC significantly benefit from the addition of cetuximab to radiotherapy and cisplatin, and there is a significant interaction between the KRAS -variant and p16 status. Elevated TGF-β1 levels in patients with the KRAS -variant suggests that cetuximab may help these patients by overcoming TGF-β1–induced suppression of antitumor immunity. Trial Registration clinicaltrials.gov Identifier:NCT00265941

Purpose: This is a pilot study combining focal irradiation and systemic TGFβ blockade in metastatic breast cancer. The rationale for using TGFβ blockade was to limit tumor growth and further spread as well as to curb systemic immune suppression. Combining this systemic approach with hypofractionated radiation of selective tumor metastasis aimed at vaccinating each patient in vivo with her own, relevant tumor antigens by local radiation damage and allow T cells to reach their full potential while escaping TGFβ's grip. Experimental Design: Serial blood samples from 22 patients undergoing treatment with 1mg or 10mg Fresolimumab and Radiation at the New York University School of Medicine (n=15) and at the David Geffen School of Medicine, University of California, Los Angeles (n=7) were immunophenotyped based on flow cytometric analysis of 21 surface antigens. Results: There were significant differences between the 1mg and 10mg groups with respect to several immune parameters, especially the rise in circulating central memory CD8s at the higher dose level (relative increase at 2 weeks 10mg vs 1mg, p=0.027). Regulatory networks responded to the 10mg treatment regime with a consistent expansion in CD4 Tregs while mMDSCs declined (ratio Tregs/mMDSC rising in 10mg vs 1mg, p=0.026). An overall survival benefit was seen in the 10mg Fresolimumab arm (median OS 64.1 weeks versus 20 weeks, p=0.015 log rank test) albeit not striking. CART analysis allowed accurate survival classification based on 2-week changes in CD8/mMDSC ratios and plasma Tryptophan (ROC AUC 0.979). Conclusion: Inhibiting TGFβ in the context of focal irradiation seems to create a favorable systemic immune landscape that drives T cell memory differentiation while limiting myeloid suppression. Citation Format: Dörthe Schaue, Michael W. Xie, Josephine A. Ratikan, Ewa D. Micewicz, Lin Hwang, Kym F. Faull, James W. Sayre, Percy P. Lee, John A. Glaspy, Sandra Demaria, Silvia C. Formenti, William H. McBride. Radiation and TGFβ blockade bring back memories in metastatic breast cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B86.

The immune system has the power to modulate the expression of radiation-induced normal and tumor tissue damage. On the one hand, it can contribute to cancer cure, and on the other hand, it can influence acute and late radiation side effects, which in many ways resemble acute and chronic inflammatory disease states. The way radiation-induced inflammation feeds into adaptive antigen-specific immune responses adds another dimension to the tumor-host cross talk during radiation therapy and to possible radiation-driven autoimmune responses. Understanding how radiation affects inflammation and immunity is therefore critical if we are to effectively manipulate these forces for benefit in radiation oncology treatments.

This study aimed to evaluate the therapeutic efficacy of a novel polymer platform delivering cisplatin and cytokines in the treatment of head and neck squamous cell carcinoma (HNSCC).In vivo study.Academic research laboratory.Mice were randomized to receive implantation of (1) no polymer, (2) plain polymer, (3) plain polymer with local cisplatin injection, or (4) cisplatin polymer. The 2 groups of mice implanted with cisplatin polymer or no polymer were further randomized to receive (1) 4 Grays external beam radiation for 4 days or (2) no radiation. For cytokine studies, mice were grouped into (1) no polymer, (2) plain polymer, (3) plain polymer with intratumoral injection of recombinant CCL21 twice a week, (4) polymer containing parental dendritic cells, or (5) polymer containing dendritic cells secreting CCL21 (DC-CCL21).The cisplatin-secreting polymer effectively reduced tumors in the mice by more than 16-fold (P.01). We also observed a statistically significant lower tumor weight among mice treated with cisplatin polymer and concomitant radiation compared to control groups. The DC-CCL21 polymer reduced SCCVII/SF tumors in the C3H/HeJ mice by more than 41% (P.01).Herein, we demonstrate the efficacy of a novel polymer platform in delivering cisplatin and cytokines. We also demonstrate that we can effectively grow dendritic cells in the polymer that can actively secrete CCL21 for a minimum of 5 days. This polymer may represent a new therapeutic modality for patients with HNSCC. Once this polymer platform is optimized, we will plan to pursue prospective trials in patients with HNSCC.

The power of ionizing radiation as a therapeutic modality is based in large part on two basic principles. One is the use of sophisticated delivery systems that minimize the dose to normal tissues and maximize the dose to the tumor, an approach epitomized by the advent of intensity-modulated

Purpose This study used chloroquine to direct radiation-induced tumor cell death pathways to harness the antitumor activity of the immune system. Methods and Materials Chloroquine given immediately after tumor irradiation increased the cure rate of MCaK breast cancer in C3H mice. Chloroquine blocked radiation-induced autophagy and drove MCaK cells into a more rapid apoptotic and more immunogenic form of cell death. Results Chloroquine treatment made irradiated tumor vaccines superior at inducing strong interferon gamma-associated immune responses in vivo and protecting mice from further tumor challenge. In vitro, chloroquine slowed antigen uptake and degradation by dendritic cells, although T-cell stimulation was unaffected. Conclusions This study illustrates a novel approach to improve the efficacy of breast cancer radiation therapy by blocking endosomal pathways, which enhances radiation-induced cell death within the field and drives antitumor immunity to assist therapeutic cure. The study illuminates and merges seemingly disparate concepts regarding the importance of autophagy in cancer therapy.