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

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

Radiation-induced lymphopenia does not impact treatment efficacy in a mouse tumor model.

Radiation-induced lymphopenia is a common occurrence in radiation oncology and an established negative prognostic factor, however the mechanisms underlying the relationship between lymphopenia and inferior survival remain elusive. The relevance of lymphocyte co-irradiation as critical normal tissue component at risk is an emerging topic of high clinical relevance, even more so in the context of potentially synergistic radiotherapy-immunotherapy combinations. The impact of the radiotherapy treatment volume on the lymphocytes of healthy and tumor-bearing mice was investigated in a novel mouse model of radiation-induced lymphopenia. Using an image-guided small-animal radiotherapy treatment platform, translationally relevant tumor-oriented volumes of irradiation with an anatomically defined increasing amount of normal tissue were irradiated, with a focus on the circulating blood and lymph nodes. In healthy mice, the influence of irradiation with increasing radiotherapy treatment volumes was quantified on the level of circulating blood cells and in the spleen. A significant decrease in the lymphocytes was observed in response to irradiation, including the minimally irradiated putative tumor area. The extent of lymphopenia correlated with the increasing volumes of irradiation. In tumor-bearing mice, differential radiotherapy treatment volumes did not influence the overall therapeutic response to radiotherapy alone. Intriguingly, an improved treatment efficacy in mice treated with draining-lymph node co-irradiation was observed in combination with an immune checkpoint inhibitor. Taken together, our study reveals compelling data on the importance of radiotherapy treatment volume in the context of lymphocytes as critical components of normal tissue co-irradiation and highlights emerging challenges at the interface of radiotherapy and immunotherapy.

Interfering with Tumor Hypoxia for Radiotherapy Optimization.

Hypoxia in solid tumors is an important predictor of treatment resistance and poor clinical outcome. The significance of hypoxia in the development of resistance to radiotherapy has been recognized for decades and the search for hypoxia-targeting, radiosensitizing agents continues. This review summarizes the main hypoxia-related processes relevant for radiotherapy on the subcellular, cellular and tissue level and discusses the significance of hypoxia in radiation oncology, especially with regard to the current shift towards hypofractionated treatment regimens. Furthermore, we discuss the strategies to interfere with hypoxia for radiotherapy optimization, and we highlight novel insights into the molecular pathways involved in hypoxia that might be utilized to increase the efficacy of radiotherapy.

Paracrine Placental Growth Factor Signaling in Response to Ionizing Radiation Is p53-Dependent and Contributes to Radioresistance.

Placental growth factor (PlGF) is a pro-angiogenic, N-glycosylated growth factor, which is secreted under pathologic situations. Here, we investigated the regulation of PlGF in response to ionizing radiation (IR) and its role for tumor angiogenesis and radiosensitivity. Secretion and expression of PlGF was induced in multiple tumor cell lines (medulloblastoma, colon and lung adenocarcinoma) in response to irradiation in a dose- and time-dependent manner. Early upregulation of PlGF expression and secretion in response to irradiation was primarily observed in p53 wild-type tumor cells, whereas tumor cells with mutated p53 only showed a minimal or delayed response. Mechanistic investigations with genetic and pharmacologic targeting of p53 corroborated regulation of PlGF by the tumor suppressor p53 in response to irradiation under normoxic and hypoxic conditions, but with so far unresolved mechanisms relevant for its minimal and delayed expression in tumor cells with a p53-mutated genetic background. Probing a paracrine role of IR-induced PlGF secretion in vitro, migration of endothelial cells was specifically increased towards irradiated PlGF wild type but not towards irradiated PlGF-knockout (PIGF-ko) medulloblastoma cells. Tumors derived from these PlGF-ko cells displayed a reduced growth rate, but similar tumor vasculature formation as in their wild-type counterparts. Interestingly though, high-dose irradiation strongly reduced microvessel density with a concomitant high rate of complete tumor regression only in the PlGF-ko tumors. IMPLICATIONS: Our study shows a strong paracrine vasculature-protective role of PlGF as part of a p53-regulated IR-induced resistance mechanism and suggest PlGF as a promising target for a combined treatment modality with RT.

The ADAM17-directed Inhibitory Antibody MEDI3622 Antagonizes Radiotherapy-induced VEGF Release and Sensitizes Non-Small Cell Lung Cancer for Radiotherapy.

The cellular response to ionizing radiation (IR) depends on tumor cell and microenvironmental factors. Here, we investigated the role of IR-induced ADAM17 matrix metalloproteinase activity for the intercellular communication between tumor cells and the tumor vasculature in non-small cell lung cancer (NSCLC) tumor models. Factors shed by ADAM17 from NSCLC tumor cells (A549, H358) and relevant for endothelial cell migration were investigated using transwell migration assays, ELISA, and flow cytometry. Tumor angiogenesis-related endpoints were analyzed with the chorio-allantoic membrane assay and in murine NSCLC tumor models. Efficacy-oriented experiments were performed in a murine orthotopic NSCLC tumor model using irradiation with an image-guided small-animal radiotherapy platform alone and in combination with the novel ADAM17-directed antibody MEDI3622. In vitro, VEGF was identified as the major factor responsible for IR-induced and ADAM17-dependent endothelial cell migration toward attracting tumor cells. IR strongly enhanced tumor cell-associated ADAM17 activity, released VEGF in an ADAM17-dependent manner, and thereby coordinated the communication between tumor and endothelial cells. In vivo, tumor growth and microvessel size and density were strongly decreased in response to the combined treatment modality of IR and MEDI3622 but not by either treatment modality alone and thus suggest that the supra-additive effect of the combined treatment modality is in part due to abrogation of the ADAM17-mediated IR-induced protective effect on the tumor vasculature. Furthermore, we demonstrate that the novel ADAM17-inhibitory antibody MEDI3622 potently improves the radiotherapy response of NSCLC. Significance: The tumor response to radiotherapy is influenced by several factors of the tumor microenvironment. We demonstrate that inhibition of the sheddase ADAM17 by the novel antibody MEDI3622 reduces IR-induced VEGF release from tumor cells relevant for endothelial cell migration and vasculature protection, thereby enhancing radiotherapy treatment outcome of NSCLC.

Probing spatiotemporal fractionation on the preclinical level.

In contrast to conventional radiotherapy, spatiotemporal fractionation (STF) delivers a distinct dose distribution in each fraction. The aim is to increase the therapeutic window by simultaneously achieving partial hypofractionation in the tumour along with near uniform fractionation in normal tissues. STF has been studied in silico under the assumption that different parts of the tumour can be treated in different fractions. Here, we develop an experimental setup for testing this key assumption on the preclinical level using high-precision partial tumour irradiation in an experimental animal model. We further report on an initial proof-of-concept experiment. We consider a reductionist model of STF in which the tumour is divided in half and treated with two complementary partial irradiations separated by 24 h. Precise irradiation of both tumour halves is facilitated by the image-guided small animal radiation research platform X-RAD SmART. To assess the response of tumours to partial irradiations, tumour growth experiments are conducted using mice carrying syngeneic subcutaneous tumours derived from MC38 colorectal adenocarcinoma cells. Tumour volumes were determined daily by calliper measurements and validated by CT-volumetry. We compared the growth of conventionally treated tumours, where the whole tumour was treated in one fraction, to the reductionist model of STF. We observed no difference in growth between the two groups. Instead, a reduction in the irradiated volume (where only one half of the tumour was irradiated) resulted in an intermediate response between full irradiation and unirradiated control. The results obtained by CT-volumetry supported the findings of the calliper-derived measurements. An experimental setup for precise partial tumour irradiation in small animals was developed, which is suited to test the assumption of STF that complementary parts of the tumour can be treated in different fractions on the preclinical level. An initial experiment supports this assumption, however, further experiments with longer follow-up and varying fractionation schemes are needed to provide additional support for STF.

The relative biological effectiveness of proton irradiation in dependence of DNA damage repair.

Clinical parameters and empirical evidence are the primary determinants for current treatment planning in radiation oncology. Personalized medicine in radiation oncology is only at the very beginning to take the genetic background of a tumor entity into consideration to define an individual treatment regimen, the total dose or the combination with a specific anticancer agent. Likewise, stratification of patients towards proton radiotherapy is linked to its physical advantageous energy deposition at the tumor site with minimal healthy tissue being co-irradiated distal to the target volume. Hence, the fact that photon and proton irradiation also induce different qualities of DNA damages, which require differential DNA damage repair mechanisms has been completely neglected so far. These subtle differences could be efficiently exploited in a personalized treatment approach and could be integrated into personalized treatment planning. A differential requirement of the two major DNA double-strand break repair pathways, homologous recombination and non-homologous end joining, was recently identified in response to proton and photon irradiation, respectively, and subsequently influence the mode of ionizing radiation-induced cell death and susceptibility of tumor cells with defects in DNA repair machineries to either quality of ionizing radiation.This review focuses on the differential DNA-damage responses and subsequent biological processes induced by photon and proton irradiation in dependence of the genetic background and discusses their impact on the unicellular level and in the tumor microenvironment and their implications for combined treatment modalities.

Effects of various factors on sleep disorders and quality of life in Parkinson's disease.

In Parkinson's disease (PD), sleep disorders (SD) occur as a result of the neurochemical changes in sleep centres, neurodegenerative changes in dopaminergic neurons, and other factors. The most common SD include excessive daytime sleepiness, insomnia, restless legs syndrome and nocturia. The aim of the study was to compare quality of sleep, as a factor that greatly impacts quality of life (QoL), between PD patients and a control group and to further examine SD in the PD group with focus on incidence and SD types as well as on effects various factors (age, sex, PD characteristics, medication usage) have on these disorders. The study included 110 patients who met the criteria for the diagnosis of PD and 110 age-matched healthy controls. We used the Pittsburgh Sleep Quality Index, PD Sleep Scale, Epworth Sleepiness Scale, PD QoL Questionnaire-8 and PD Questionnaire-39 (items 30 and 33). In the group with PD, we considered the duration of the disease, the stage of disease according to the Hoehn and Yahr scale, medications and their impact on the SD. The average duration of the disease was 6 years and the mean stage was 2.44. The result showed significant differences in the sleep quality between groups. In the PD group, SD differences were also found according to gender, duration of the disease and medication usage. The most common SD were fragmented sleep, insomnia and nocturia. To improve the QoL of PD patients, it is necessary to pay more attention to detecting and solving SD.

Quantitative electroencephalography in schizophrenia and depression.

BACKGROUND: Standard (qualitative) electroencephalography (EEG) is routinely used in the diagnostic evaluation of psychiatric patients. Quantitative EEG (qEEG) findings differ between patients with schizophrenia, patients with depression, but results are not consistent. The aim of our study was to determine the differences in qEEG parameters between patients with schizophrenia, patients with depression, and healthy subjects. SUBJECTS AND METHODS: The study included 30 patients with schizophrenia, 33 patients with depression, and 30 healthy subjects. All study participants underwent standard EEG. Artifact-free 100-second epochs were selected from the recorded material and analyzed with Fast Fourier Transformation (FFT) analysis. RESULTS: The results are presented as absolute spectral power values (µV2) of delta, theta, alpha, and beta components of the EEG spectrum. EEGs were recorded from 12 locations including Fp1, Fp2, F3, F4, F7, F8, T3, T4, P3, P4, O1, and O2. In comparison with healthy subjects, patients with schizophrenia showed increased delta, theta, and beta activity and decreased alpha activity. Similar results were obtained in patients with depression, but in fewer regions. In patients with schizophrenia, delta power over Fp1, Fp2, F4, and F8 regions was increased in comparison with those in patients with depression. Interhemispheric asymmetry was found in patients with schizophrenia and healthy subjects, but not in patients with depression. CONCLUSION: The finding that patients with schizophrenia differed from patients with depression in delta power values could be potentially used in differential diagnosis between schizophrenia and depression. The role of qEEG in clinical differentiation between these two mental disorders may be especially important in cases of negative-symptom schizophrenia.