LymphoDose: a lymphocyte dose estimation framework - application to brain radiotherapy.

OBJECTIVE: Severe radiation-induced lymphopenia occurs in 40% of patients treated for primary brain tumors and is an independent risk factor of poor survival outcomes. We developed an in-silico framework that estimates the radiation doses received by lymphocytes during VMAT brain irradiation. Approach: We implemented a simulation consisting of two interconnected compartmental models describing the slow recirculation of lymphocytes between lymphoid organs (M_1) and the bloodstream (M_2). We used dosimetry data from 33 patients treated with chemo-radiation for glioblastoma to compare three cases of the model, corresponding to different physical and biological scenarios: H1) lymphocytes circulation only in the bloodstream i.e., circulation in M_2 only; H2) lymphocytes recirculation between lymphoid organs i.e. circulation in M_1 and M_2 interconnected; H3) lymphocytes recirculation between lymphoid organs and deep-leaning computed out-of-field dose to head and neck lymphoid structures. A sensitivity analysis of the model's parameters was also performed. Main results: For H1, H2 and H3 cases respectively, the irradiated fraction of lymphocytes was 99.8±0.7%, 40.4±10.2% et 97.6±2.5%, and the average dose to irradiated pool was 309.9±74.7mGy, 52.6±21.1mGy and 265.6±48.5mGy. The recirculation process considered in the H2 case implied that irradiated lymphocytes were irradiated in the field only 1.58±0.91 times on average after treatment. The out-of-field irradiation of head and neck lymphoid structures considered in H3 was an important contribution to lymphocytes dose. In all cases, the estimated doses are low compared with lymphocytes radiosensitivity, and other mechanisms could explain high prevalence of RIL in patients with brain tumors. Significance: Our framework is the first to take into account out-of-field doses and recirculation in lymphocyte dose assessment during brain irradiation. Our results demonstrate the need to clarify the indirect effects of irradiation on lymphopenia, in order to potentiate the combination of radio-immunotherapy or the abscopal effect. .

An international phase II trial and immune profiling of SBRT and atezolizumab in advanced pretreated colorectal cancer.

BACKGROUND: Immuno-radiotherapy may improve outcomes for patients with advanced solid tumors, although optimized combination modalities remain unclear. Here, we report the colorectal (CRC) cohort analysis from the SABR-PDL1 trial that evaluated the PD-L1 inhibitor atezolizumab in combination with stereotactic body radiation therapy (SBRT) in advanced cancer patients. METHODS: Eligible patients received atezolizumab 1200 mg every 3 weeks until progression or unmanageable toxicity, together with ablative SBRT delivered concurrently with the 2nd cycle (recommended dose of 45 Gy in 3 fractions, adapted upon normal tissue tolerance constraint). SBRT was delivered to at least one tumor site, with at least one additional measurable lesion being kept from the radiation field. The primary efficacy endpoint was one-year progression-free survival (PFS) rate from the start of atezolizumab. Sequential tumor biopsies were collected for deep multi-feature immune profiling. RESULTS: Sixty pretreated (median of 2 prior lines) advanced CRC patients (38 men [63%]; median age, 59 years [range, 20-81 years]; 77% with liver metastases) were enrolled in five centers (France: n = 4, Spain: n = 1) from 11/2016 to 04/2019. All but one (98%) received atezolizumab and 54/60 (90%) received SBRT. The most frequently irradiated site was lung (n = 30/54; 56.3%). Treatment-related G3 (no G4-5) toxicity was observed in 3 (5%) patients. Median OS and PFS were respectively 8.4 [95%CI:5.9-11.6] and 1.4 months [95%CI:1.2-2.6], including five (9%) patients with PFS > 1 year (median time to progression: 19.2 months, including 2/5 MMR-proficient). Best overall responses consisted of stable disease (n = 38; 64%), partial (n = 3; 5%) and complete response (n = 1; 2%). Immune-centric multiplex IHC and RNAseq showed that SBRT redirected immune cells towards tumor lesions, even in the case of radio-induced lymphopenia. Baseline tumor PD-L1 and IRF1 nuclear expression (both in CD3 + T cells and in CD68 + cells) were higher in responding patients. Upregulation of genes that encode for proteins known to increase T and B cell trafficking to tumors (CCL19, CXCL9), migration (MACF1) and tumor cell killing (GZMB) correlated with responses. CONCLUSIONS: This study provides new data on the feasibility, efficacy, and immune context of tumors that may help identifying advanced CRC patients most likely to respond to immuno-radiotherapy. TRIAL REGISTRATION: EudraCT N°: 2015-005464-42; Clinicaltrial.gov number: NCT02992912.

KRASG12C inhibition using MRTX1257: a novel radio-sensitizing partner.

BACKGROUND: KRAS activating mutations are considered the most frequent oncogenic drivers and are correlated with radio-resistance in multiple cancers including non-small cell lung cancer (NSCLC) and colorectal cancer. Although KRAS was considered undruggable until recently, several KRAS inhibitors have recently reached clinical development. Among them, MRTX849 (Mirati Therapeutics) showed encouraging clinical outcomes for the treatment of selected patients with KRASG12C mutated NSCLC and colorectal cancers. In this work, we explore the ability of MRTX1257, a KRASG12C inhibitor analogous to MRTX849, to radio-sensitize KRASG12C+/+ mutated cell lines and tumors. METHODS: Both in vitro and in vivo models of radiotherapy (RT) in association with MRTX1257 were used, with different RAS mutational profiles. We assessed in vitro the radio-sensitizing effect of MRTX1257 in CT26 KRASG12C+/+, CT26 WT, LL2 WT and LL2 NRAS KO (LL2 NRAS-/-) cell lines. In vivo, we used syngeneic models of subcutaneous CT26 KRASG12C+/+ tumors in BALB/c mice and T cell deficient athymic nu/nu mice to assess both the radio-sensitizing effect of MRTX1257 and its immunological features. RESULTS: MRTX1257 was able to radio-sensitize CT26 KRASG12C+/+ cells in vitro in a time and dose dependent manner. Moreover, RT in association with MRTX1257 in BALB/c mice bearing CT26 KRASG12C+/+ subcutaneous tumors resulted in an observable cure rate of 20%. However, no durable response was observed with similar treatment in athymic nude mice. The analysis of the immune microenvironment of CT26 KRASG12C+/+ tumors following RT and MRTX1257 showed an increase in the proportion of various cell subtypes including conventional CD4 + T cells, dendritic cells type 2 (cDC2) and inflammatory monocytes. Furthermore, the expression of PD-L1 was dramatically down-regulated within both tumor and myeloid cells, thus illustrating the polarization of the tumor microenvironment towards a pro-inflammatory and anti-tumor phenotype following the combined treatment. CONCLUSION: This work is the first to demonstrate in vitro as in vivo the radio-sensitizing effect of MRTX1257, a potent KRASG12C inhibitor compatible with oral administration, in CT26 KRASG12C mutated cell lines and tumors. This is a first step towards the use of new combinatorial strategies using KRAS inhibitors and RT in KRASG12C mutated tumors, which are the most represented in NSCLC with 14% of patients harboring this mutational profile.

Radio-induced lymphopenia in the era of anti-cancer immunotherapy.

Radiation-induced lymphopenia (RIL) is characterized by a significant decrease in the absolute number of lymphocytes circulating in the blood after radiotherapy. With the major shift in cancer management initiated by cancer immunotherapy (IT), the reduction of incidence of RIL appears today as an extremely promising way of potentiating the synergy between radiotherapy and immunotherapy. However, the causes of RIL and mechanisms involved are still poorly understood. Improving our knowledge on RIL is therefore essential to limit it and thus improve the quality of care delivered to patients. The objective of this review is to provide a global view of RIL from a clinical point of view, with particular emphasis on recent knowledge and avenues explored to explain RIL and especially its depletion and remission kinetics. An opening on treatment concepts to be rethought is conducted in the context of combined RT/IT treatments.

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.

Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation.

BACKGROUND: Irradiation (IR) and immune checkpoint inhibitor (ICI) combination is a promising treatment modality. However, local and distance treatment failure and resistance can occur. To counteract this resistance, several studies propose CD73, an ectoenzyme, as a potential target to improve the antitumor efficiency of IR and ICI. Although CD73 targeting in combination with IR and ICI has shown attractive antitumor effects in preclinical models, the rationale for CD73 targeting based on CD73 tumor expression level deserves further investigations. METHODS: Here we evaluated for the first time the efficacy of two administration regimens of CD73 neutralizing antibody (one dose vs four doses) in combination with IR according to the expression level of CD73 in two subcutaneous tumor models expressing different levels of CD73. RESULTS: We showed that CD73 is weakly expressed by MC38 tumors even after IR, when compared with the TS/A model that highly expressed CD73. Treatment with four doses of anti-CD73 improved the TS/A tumor response to IR, while it was ineffective against the CD73 low-expressing MC38 tumors. Surprisingly, a single dose of anti-CD73 exerted a significant antitumor activity against MC38 tumors. On CD73 overexpression in MC38 cells, four doses of anti-CD73 were required to improve the efficacy of IR. Mechanistically, a correlation between a downregulation of iCOS expression in CD4+ T cells and an improved response to IR after anti-CD73 treatment was observed and iCOS targeting could restore an impaired benefit from anti-CD73 treatment. CONCLUSIONS: These data emphasize the importance of the dosing regimen for anti-CD73 treatment to improve tumor response to IR and identify iCOS as part of the underlying molecular mechanisms. Our data suggest that the selection of appropriate dosing regimen is required to optimize the therapeutic efficacy of immunotherapy-radiotherapy combinations.

Clinical transfer of AGuIX®-based radiation treatments for locally advanced cervical cancer: MR quantification and in vitro insights in the NANOCOL clinical trial framework.

Clinical trials incorporating metallic nanoparticles (NPs) have recently begun. Radiotherapy planning does not take into account NPs concentrations observed in the patients' target volumes. In the framework of the NANOCOL clinical trial including patients treated for locally advanced cervical cancers, this study proposes a complete method to evaluate the radiation-induced biological effects of NPs. For this, calibration phantom was developed and MRI sequences with variable flip angles were acquired. This process allowed the quantification of NPs in the tumor of 4 patients, which was compared to the results of mass spectrometry obtained from 3 patient biopsies. The concentration of the NPs was reproduced in 3D cell models. Based on clonogenic assays, the radio-enhancement effects were quantified for radiotherapy and brachytherapy, and the impact in terms of local control was evaluated. T1 signal change in GTVs revealed NPs accumulation ∼12.4 µmol/L, in agreement with mass spectrometry. Radio-enhancement effects of about 15 % at 2 Gy were found for both modalities, with a positive impact on local tumor control. Even if further follow-up of patients in this and subsequent clinical trials will be necessary to assess the reliability of this proof of concept, this study opens the way to the integration of a dose modulation factor to better take into account the impact of NPs in radiotherapy treatment.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Cytofluorometric characterization of the myeloid compartment of irradiated mouse tumors.

The use of ionizing radiation (IR) is a cornerstone for the treatment of cancer and radiotherapy (RT) is used in roughly 50% of cancer patients. It is now well established that RT exerts widespread effects on the tumor stroma, including the immune environment. Together with its deeply characterized effects on the lymphoid compartment, RT also deeply affects the myeloid cell compartment. Fluorescence-activated flow cytometry is one of the most widely used technologies in immunology, allowing the multiparametric analysis of cells on a cell-by-cell basis. Here, we provide a detailed flow cytometry protocol to analyze the myeloid cell populations of human papillomavirus (HPV)-positive TC1/Luc tumors engrafted in the oral mucosa of immunocompetent mice, and to evaluate their modulations in response to RT. The same method, with slight modifications, can be used to study the tumor myeloid cells from a variety of other mouse tumors.

How to Improve SBRT Outcomes in NSCLC: From Pre-Clinical Modeling to Successful Clinical Translation.

Despite major research and clinical efforts, lung cancer remains the leading cause of cancer-related death. While the delivery of conformal radiotherapy and image guidance of stereotactic body radiotherapy (SBRT) have revolutionized the treatment of early-stage non-small-cell lung cancer (NSCLC), additional research is needed to elucidate underlying mechanisms of resistance and identify novel therapeutic combinations. Clinical progress relies on the successful translation of pre-clinical work, which so far has not always yielded expected results. Improved clinical modelling involves characterizing the preclinical models and selecting appropriate experimental designs that faithfully mimic precise clinical scenarios. Here, we review the current role of SBRT and the scope of pre-clinical armamentarium at our disposal to improve successful clinical translation of pre-clinical research in the radiation oncology of NSCLC.

TGFß receptor inhibition unleashes interferon-ß production by tumor-associated macrophages and enhances radiotherapy efficacy.

BACKGROUND: Transforming growth factor-beta (TGFß) can limit the efficacy of cancer treatments, including radiotherapy (RT), by inducing an immunosuppressive tumor environment. The association of TGFß with impaired T cell infiltration and antitumor immunity is known, but the mechanisms by which TGFß participates in immune cell exclusion and limits the efficacy of antitumor therapies warrant further investigations. METHODS: We used the clinically relevant TGFß receptor 2 (TGFßR2)-neutralizing antibody MT1 and the small molecule TGFßR1 inhibitor LY3200882 and evaluated their efficacy in combination with RT against murine orthotopic models of head and neck and lung cancer. RESULTS: We demonstrated that TGFß pathway inhibition strongly increased the efficacy of RT. TGFßR2 antibody upregulated interferon beta expression in tumor-associated macrophages within the irradiated tumors and favored T cell infiltration at the periphery and within the core of the tumor lesions. We highlighted that both the antitumor efficacy and the increased lymphocyte infiltration observed with the combination of MT1 and RT were dependent on type I interferon signaling. CONCLUSIONS: These data shed new light on the role of TGFß in limiting the efficacy of RT, identifying a novel mechanism involving the inhibition of macrophage-derived type I interferon production, and fostering the use of TGFßR inhibition in combination with RT in therapeutic strategies for the management of head and neck and lung cancer.

Deciphering the Dynamic Molecular Program of Radiation-Induced Endothelial Senescence.

PURPOSE: Radiation-induced cellular senescence is a double-edged sword, acting as both a tumor suppression process limiting tumor proliferation, and a crucial process contributing to normal tissue injury. Endothelial cells play a role in normal tissue injury after radiation therapy. Recently, a study observed an accumulation of senescent endothelial cells (ECs) around radiation-induced lung focal lesions following stereotactic radiation injury in mice. However, the effect of radiation on EC senescence remains unclear because it depends on dose and fractionation, and because the senescent phenotype is heterogeneous and dynamic. METHODS AND MATERIALS: Using a systems biology approach in vitro, we deciphered the dynamic senescence-associated transcriptional program induced by irradiation. RESULTS: Flow cytometry and single-cell RNA sequencing experiments revealed the heterogeneous senescent status of irradiated ECs and allowed to deciphered the molecular program involved in this status. We identified the Interleukin-1 signaling pathway as a key player in the radiation-induced premature senescence of ECs, as well as the endothelial-to-mesenchymal transition process, which shares strong hallmarks of senescence. CONCLUSIONS: Our work provides crucial information on the dynamics of the radiation-induced premature senescence process, the effect of the radiation dose, as well as the molecular program involved in the heterogeneous senescent status of ECs.

Metabolic features of cancer cells impact immunosurveillance.

BACKGROUND: Tumors rewire their metabolism to achieve robust anabolism and resistance against therapeutic interventions like cisplatin treatment. For example, a prolonged exposure to cisplatin causes downregulation of pyridoxal kinase (PDXK), the enzyme that generates the active vitamin B6, and upregulation of poly ADP-ribose (PAR) polymerase-1 (PARP1) activity that requires a supply of nicotinamide (vitamin B3) adenine dinucleotide. We investigated the impact of the levels of PDXK and PAR on the local immunosurveillance (ie, density of the antigen presenting cells and adaptive immune response by CD8 T lymphocytes) in two different tumor types. METHODS: Tumors from patients with locally advanced cervical carcinoma (LACC) and non-small cell lung cancer (NSCLC) were stained for PAR, PDXK, dendritic cell lysosomal associated membrane glycoprotein (DC-LAMP) and CD8 T cell infiltration. Their correlations and prognostic impact were assessed. Cisplatin-resistant NSCLC cell clones isolated from Lewis-lung cancer (LLC) cells were evaluated for PAR levels by immunoblot. Parental (PARlow) and cisplatin-resistant (PARhigh) clones were subcutaneously injected into the flank of C57BL/6 mice. Tumors were harvested to evaluate their immune infiltration by flow cytometry. RESULTS: The infiltration of tumors by CD8 T and DC-LAMP+ cells was associated with a favorable overall survival in patients with LACC (p=0.006 and p=0.008, respectively) and NSCLC (p<0.001 for both CD8 T and DC-LAMP cells). We observed a positive correlation between PDXK expression and the infiltration by DC-LAMP (R=0.44, p=0.02 in LACC, R=0.14, p=0.057 in NSCLC), and a negative correlation between PAR levels and CD8 T lymphocytes (R=-0.39, p=0.034 in LACC, R=-0.18, p=0.017 in NSCLC). PARP1 is constitutively hyperactivated in cisplatin-resistant LLC cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PARhigh). Tumors formed by such cancer cells injected into immunocompetent mice were scarcely infiltrated by CD8 T (p=0.028) and antigen presenting cells (p=0.086). CONCLUSIONS: Oncometabolic features can impact local immunosurveillance, providing new functional links between cisplatin resistance and therapeutic failure.

(Chemo)Radiotherapy-Immunotherapy Combinations: Time to Get Tailored?

Chemoradiotherapy is considered an immunogenic anticancer treatment. Data obtained during the course of chemoradiotherapy treatment of patients with cervical cancer show heterogeneous changes in the tumor immune landscape, highlighting the need for patient selection to rationally design successful combined immunotherapies. Blood-based biomarkers could be valuable to perform such stratification.See related article by Chen et al., p. 3990.

Low Doses of Radiation Increase the Immunosuppressive Profile of Lung Macrophages During Viral Infection and Pneumonia.

PURPOSE: Severe pneumonia and acute respiratory distress syndrome (ARDS) have been described in patients with severe coronavirus disease 2019 (COVID-19). Recently, early clinical data reported the feasibility of low doses of radiation therapy (RT) in the treatment of ARDS in patients with severe COVID-19. However, the involved mechanisms remained unknown. METHODS AND MATERIALS: Here, we used airways-instilled lipopolysaccharide (LPS) and influenza virus (H1N1) as murine models of pneumonia, and toll-like receptor (TLR)-3 stimulation in human lung macrophages. RESULTS: Low doses of RT (0.5-1 Gray) decreased LPS-induced pneumonia, and increased the percentage of nerve- and airway-associated macrophages producing interleukin (IL) 10. During H1N1 viral infection, we observed decreased lung tissue damage and immune cell infiltration in irradiated animals. Low doses of RT increased IL-10 production by infiltrating immune cells into the lung. Irradiation of TLR-3 ligand-stimulated human lung macrophages ex vivo increased IL-10 secretion and decreased interferon γ production in the culture supernatant. The percentage of human lung macrophages producing IL-6 was also decreased. CONCLUSIONS: Our data highlight a mechanism by which low doses of RT regulate lung inflammation and skew lung macrophages toward an anti-inflammatory profile. These data provide a preclinical mechanistic support to clinical trials evaluating low doses of RT, such as COVID-19-induced ARDS.

Differential therapeutic effects of PARP and ATR inhibition combined with radiotherapy in the treatment of subcutaneous versus orthotopic lung tumour models.

BACKGROUND: Subcutaneous mouse tumour models are widely used for the screening of novel antitumour treatments, although these models are poor surrogate models of human cancers. METHODS: We compared the antitumour efficacy of the combination of ionising radiation (IR) with two DNA damage response inhibitors, the PARP inhibitor olaparib and the ATR inhibitor AZD6738 (ceralasertib), in subcutaneous versus orthotopic cancer models. RESULTS: Olaparib delayed the growth of irradiated Lewis lung carcinoma (LL2) subcutaneous tumours, in agreement with previous reports in human cell lines. However, the olaparib plus IR combination showed a very narrow therapeutic window against LL2 lung orthotopic tumours, with nearly no additional antitumour effect compared with that of IR alone, and tolerability issues emerged at high doses. The addition of AZD6738 greatly enhanced the efficacy of the olaparib plus IR combination treatment against subcutaneous but not orthotopic LL2 tumours. Moreover, olaparib plus AZD6738 administration concomitant with IR even worsened the response to radiation of head and neck orthotopic tumours and induced mucositis. CONCLUSIONS: These major differences in the responses to treatments between subcutaneous and orthotopic models highlight the importance of using more pathologically relevant models, such as syngeneic orthotopic models, to determine the most appropriate therapeutic approaches for translation to the clinic.

Dual oxidase 1 limits the IFNγ-associated antitumor effect of macrophages.

BACKGROUND: Macrophages play pivotal roles in tumor progression and the response to anticancer therapies, including radiotherapy (RT). Dual oxidase (DUOX) 1 is a transmembrane enzyme that plays a critical role in oxidant generation. METHODS: Since we found DUOX1 expression in macrophages from human lung samples exposed to ionizing radiation, we aimed to assess the involvement of DUOX1 in macrophage activation and the role of these macrophages in tumor development. RESULTS: Using Duox1-/- mice, we demonstrated that the lack of DUOX1 in proinflammatory macrophages improved the antitumor effect of these cells. Furthermore, intratumoral injection of Duox1-/- proinflammatory macrophages significantly enhanced the antitumor effect of RT. Mechanistically, DUOX1 deficiency increased the production of proinflammatory cytokines (IFNγ, CXCL9, CCL3 and TNFα) by activated macrophages in vitro and the expression of major histocompatibility complex class II in the membranes of macrophages. We also demonstrated that DUOX1 was involved in the phagocytotic function of macrophages in vitro and in vivo. The antitumor effect of Duox1-/- macrophages was associated with a significant increase in IFNγ production by both lymphoid and myeloid immune cells. CONCLUSIONS: Our data indicate that DUOX1 is a new target for macrophage reprogramming and suggest that DUOX1 inhibition in macrophages combined with RT is a new therapeutic strategy for the management of cancers.

Radiotherapy-immunotherapy combinations - perspectives and challenges.

Ionizing radiation has historically been used to treat cancer by killing tumour cells, in particular by inducing DNA damage. This view of radiotherapy (RT) as a simple cytotoxic agent has dramatically changed in recent years, and it is now widely accepted that RT can deeply reshape the tumour environment by modulating the immune response. Such evidence gives a strong rationale for the use of immunomodulators to boost the therapeutic value of RT, introducing the era of 'immunoradiotherapy'. The increasing amount of preclinical and clinical data concerning the combination of RT with immunomodulators, in particular with immune checkpoint inhibitors such as anti-PD-1/PD-L1 and anti-CTLA4, reflects the interest of the scientific and medical community concerning immunoradiotherapy. The expectations are enormous since the rationale for performing such combinations is strong, with the possibility to use a local treatment such as RT to amplify a systemic antitumour response, as illustrated by the case of the abscopal effect. Nevertheless, several points remain to be addressed such as the need to find biomarkers to identify patients who will benefit from immunoradiotherapy, the identification of the best sequences/schedules for combination with immunomodulators and mechanisms to overcome resistance. Additionally, the effects of immunoradiotherapy on healthy tissues and related toxicity remain largely unexplored. To answer these critical questions and make immunoradiotherapy keep its promising qualities, large efforts are needed from both the pharmaceutical industry and academic/governmental research. Moreover, because of the work of both these entities, the arsenal of available immunomodulators is quickly expanding, thus opening the field to increasing combinations with RT. We thus forecast that the field of immunoradiotherapy will further expand in the coming years, and it needs to be supported by appropriate investment plans.

Stereotactic Lung Irradiation in Mice Promotes Long-Term Senescence and Lung Injury.

PURPOSE: Lung cancer will be treated more frequently using stereotactic body radiation therapy, and preclinical research to model long-term toxicity of ablative doses of radiation is crucial. Stereotactic lung irradiation of a small volume can induce radiation pneumonitis and fibrosis in normal tissues. METHODS AND MATERIALS: Senescence has been reported to contribute to lung fibrosis, and we investigated in vivo the effects of ablative doses of ionizing radiation on senescence-associated processes. The left lung of p16INK4a-LUC knock-in mice was exposed to a single dose or fractionated radiation doses in a millimetric volume using a small animal radiation research platform. RESULTS: Single or fractionated ablative radiation induces acute and very long-term p16INK4a activation in the irradiated lung target volume associated with lung injury. We observed a panel of heterogeneous senescent cells including pneumocytes, macrophages, and endothelial cells that accumulated around the radiation-induced lung focal lesion, suggesting that different senescent cell types may contribute to radiation injury. CONCLUSIONS: This work provides important information on the long-term effects of ablative radiation doses in the normal lung and strongly suggests that stress-induced senescence is involved in stereotactic body radiation therapy-induced late fibrosis.

TP53 Pathway Alterations Drive Radioresistance in Diffuse Intrinsic Pontine Gliomas (DIPG).

PURPOSE: Diffuse intrinsic pontine gliomas (DIPG) are the most severe pediatric brain tumors. Although accepted as the standard therapeutic, radiotherapy is only efficient transiently and not even in every patient. The goal of the study was to identify the underlying molecular determinants of response to radiotherapy in DIPG. EXPERIMENTAL DESIGN: We assessed in vitro response to ionizing radiations in 13 different DIPG cellular models derived from treatment-naïve stereotactic biopsies reflecting the genotype variability encountered in patients at diagnosis and correlated it to their principal molecular alterations. Clinical and radiologic response to radiotherapy of a large cohort of 73 DIPG was analyzed according to their genotype. Using a kinome-wide synthetic lethality RNAi screen, we further identified target genes that can sensitize DIPG cells to ionizing radiations. RESULTS: We uncover TP53 mutation as the main driver of increased radioresistance and validated this finding in four isogenic pairs of TP53WT DIPG cells with or without TP53 knockdown. In an integrated clinical, radiological, and molecular study, we show that TP53MUT DIPG patients respond less to irradiation, relapse earlier after radiotherapy, and have a worse prognosis than their TP53WT counterparts. Finally, a kinome-wide synthetic lethality RNAi screen identifies CHK1 as a potential target, whose inhibition increases response to radiation specifically in TP53MUT cells. CONCLUSIONS: Here, we demonstrate that TP53 mutations are driving DIPG radioresistance both in patients and corresponding cellular models. We suggest alternative treatment strategies to mitigate radioresistance with CHK1 inhibitors. These findings will allow to consequently refine radiotherapy schedules in DIPG.