Rianne Biemans

TH-302 in Combination with Radiotherapy Enhances the Therapeutic Outcome and Is Associated with Pretreatment [18F]HX4 Hypoxia PET Imaging

Purpose: Conventional anticancer treatments are often impaired by the presence of hypoxia. TH-302 selectively targets hypoxic tumor regions, where it is converted into a cytotoxic agent. This study assessed the efficacy of the combination treatment of TH-302 and radiotherapy in two preclinical tumor models. The effect of oxygen modification on the combination treatment was evaluated and the effect of TH-302 on the hypoxic fraction (HF) was monitored using [18F]HX4-PET imaging and pimonidazole IHC stainings. Experimental Design: Rhabdomyosarcoma R1 and H460 NSCLC tumor-bearing animals were treated with TH-302 and radiotherapy (8 Gy, single dose). The tumor oxygenation status was altered by exposing animals to carbogen (95% oxygen) and nicotinamide, 21% or 7% oxygen breathing during the course of the treatment. Tumor growth and treatment toxicity were monitored until the tumor reached four times its start volume (T4×SV). Results: Both tumor models showed a growth delay after TH-302 treatment, which further increased when combined with radiotherapy (enhancement ratio rhabdomyosarcoma 1.23; H460 1.49). TH-302 decreases the HF in both models, consistent with its hypoxia-targeting mechanism of action. Treatment efficacy was dependent on tumor oxygenation; increasing the tumor oxygen status abolished the effect of TH-302, whereas enhancing the HF enlarged TH-302′s therapeutic effect. An association was observed in rhabdomyosarcoma tumors between the pretreatment HF as measured by [18F]HX4-PET imaging and the T4×SV. Conclusions: The combination of TH-302 and radiotherapy is promising and warrants clinical testing, preferably guided by the companion biomarker [18F]HX4 hypoxia PET imaging for patient selection. Clin Cancer Res; 21(13); 2984–92. ©2015 AACR.

New ways to image and target tumour hypoxia and its molecular responses

Tumour hypoxia and its molecular responses have been shown to be associated with poor prognosis. Detection of hypoxia, preferably in a non-invasive manner, could therefore predict treatment outcome and serve as a tool to individualize treatment. This review gives an overview of recent literature on hypoxia imaging markers currently used in clinical trials. Furthermore, recent progress made in targeting hypoxia (hypoxia-activated prodrugs) or hypoxia response (carbonic anhydrase IX inhibitors) is summarized. Last, window-of-opportunity trials implementing non-invasive imaging are proposed as an important tool to prove anti-tumour efficacy of experimental drugs early during drug development.

Combination of radiotherapy with the immunocytokine L19-IL2: Additive effect in a NK cell dependent tumour model.

BACKGROUND AND PURPOSE Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses. MATERIAL AND METHODS In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry. RESULTS Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression. CONCLUSION An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression.

Synthesis and in Vivo Biological Evaluation of 68Ga-Labeled Carbonic Anhydrase IX Targeting Small Molecules for Positron Emission Tomography

Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [(68)Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing (68)Ga for preclinical CA IX imaging are scarce, and this is one of the first effective (68)Ga compounds reported for PET imaging of CA IX.

Preclinical assessment of efficacy of radiation dose painting based on intratumoral FDG-PET uptake

Purpose: We tested therapeutic efficacy of two dose painting strategies of applying higher radiation dose to tumor subvolumes with high FDG uptake (biologic target volume, BTV): dose escalation and dose redistribution. We also investigated whether tumor response was determined by the highest dose in BTV or the lowest dose in gross tumor volume (GTV). Experimental Design: FDG uptake was evaluated in rat rhabdomyosarcomas prior to irradiation. BTV was defined as 30% of GTV with the highest (BTVhot) or lowest (BTVcold) uptake. To test efficacy of dose escalation, tumor response (time to reach two times starting tumor volume, TGTV2) to Hot Boost irradiation (40% higher dose to BTVhot) was compared with Cold Boost (40% higher dose to BTVcold), while mean dose to GTV remained 12 Gy. To test efficacy of dose redistribution, TGTV2 after Hot Boost was compared with uniform irradiation with the same mean dose (8 or 12 Gy). Results: TGTV2 after 12 Gy delivered heterogeneously (Hot and Cold Boost) or uniformly were not significantly different: 20.2, 19.5, and 20.6 days, respectively. Dose redistribution (Hot Boost) with 8 Gy resulted in faster tumor regrowth as compared with uniform irradiation (13.3 vs. 17.1 days; P = 0.026). Further increase in dose gradient to 60% led to a more pronounced decrease in TGTV2 (10.9 days; P < 0.0001). Conclusions: Dose escalation effect was independent of FDG uptake in target tumor volume, while dose redistribution was detrimental in this tumor model for dose levels applied here. Our data are consistent with the hypothesis that tumor response depends on the minimum intratumoral dose. Clin Cancer Res; 21(24); 5511–8. ©2015 AACR.

A novel concept for tumour targeting with radiation: Inverse dose-painting or targeting the “Low Drug Uptake Volume”

BACKGROUND AND PURPOSE We tested a novel treatment approach combining (1) targeting radioresistant hypoxic tumour cells with the hypoxia-activated prodrug TH-302 and (2) inverse radiation dose-painting to boost selectively non-hypoxic tumour sub-volumes having no/low drug uptake. MATERIAL AND METHODS 18F-HX4 hypoxia tracer uptake measured with a clinical PET/CT scanner was used as a surrogate of TH-302 activity in rhabdomyosarcomas growing in immunocompetent rats. Low or high drug uptake volume (LDUV/HDUV) was defined as 40% of the GTV with the lowest or highest 18F-HX4 uptake, respectively. Two hours post TH-302/saline administration, animals received either single dose radiotherapy (RT) uniformly (15 or 18.5Gy) or a dose-painted non-uniform radiation (15Gy) with 50% higher dose to LDUV or HDUV (18.5Gy). Treatment plans were created using Eclipse treatment planning system and radiation was delivered using VMAT. Tumour response was quantified as time to reach 3 times starting tumour volume. RESULTS Non-uniform RT boosting tumour sub-volume with low TH-302 uptake (LDUV) was superior to the same dose escalation to HDUV (p<0.0001) and uniform RT with the same mean dose 15Gy (p=0.0077). Noteworthy, dose escalation to LDUV required on average 3.5Gy lower dose to the GTV to achieve similar tumour response as uniform dose escalation. CONCLUSIONS The results support targeted dose escalation to non-hypoxic tumour sub-volume with no/low activity of hypoxia-activated prodrugs. This strategy applies on average a lower radiation dose and is as effective as uniform dose escalation to the entire tumour. It could be applied to other type of drugs provided that their distribution can be imaged.

In vivo optical imaging of MMP2 immuno protein antibody: tumor uptake is associated with MMP2 activity

Matrix metalloproteinase-2 (MMP2) is important in tumorigenesis, angiogenesis and tumor invasion. In this study, we investigated if the Cy5-tagged small immuno protein targeting the catalytic domain of human MMP2 (aMMP2-SIP) detects MMP2 in tumors non-invasively. For this purpose, we generated MMP2 expressing (empty vector EV) and knock-down (KD) HT1080, U373 and U87 cells, which were injected subcutaneously in the lateral flank of NMRI-nu mice. Optical imaging (Optix MX2) performed at 0.5, 2, 4, 8, 24 and 48 hour post injection (h.p.i.) of Cy5 tagged aMMP2-SIP, indicated significantly lower tumor to background ratios at both 24 (P = 0.0090) and 48 h.p.i. (P < 0.0001) for the U87 MMP2-KD compared to control tumors. No differences were found for HT1080 and U373 models. U87 MMP2-KD tumors had significantly lower MMP2 activity (P < 0.0001) than EV tumors as determined by gelatin zymography in tumor sections and lysates, while no differences were observed between EV and MMP2-KD in HT1080 and U373. In line with these data, only U87 MMP2-KD tumors had a reduced tumor growth compared to control tumors (P = 0.0053). aMMP2-SIP uptake correlates with MMP2 activity and might therefore be a potential non-invasive imaging biomarker for the evaluation of MMP2 activity in tumors.

The immunocytokine L19-IL2: An interplay between radiotherapy and long-lasting systemic anti-tumour immune responses

ABSTRACT Recently, we have shown that the administration of the tumour-targeted antibody-based immunocytokine L19-IL2 after radiotherapy (RT) resulted in synergistic anti-tumour effect. Here we show that RT and L19-IL2 can activate a curative abscopal effect, with a long-lasting immunological memory. Ionizing radiation (single dose of 15Gy, 5 × 2Gy or 5 × 5Gy) was delivered to primary C51 colon tumour-bearing immunocompetent mice in combination with L19-IL2 and response of secondary non-irradiated C51 or CT26 colon tumours was evaluated. 15Gy + L19-IL2 triggered a curative (20%) abscopal effect, which was T cell dependent. Moreover, 10Gy + L19-IL2 treated and cured mice were re-injected after 150 days with C51 tumour cells and tumour uptake was assessed. Age-matched controls (matrigel injected mice treated with 10Gy + L19-IL2, mice cured after treatment with surgery + L19-IL2 and mice cured after high dose RT 40Gy + vehicle) were included. Several immunological parameters in blood, tumours, lymph nodes and spleens were investigated. Treatment with 10Gy + L19-IL2 resulted in long-lasting immunological memory, associated with CD44+CD127+ expression on circulating T cells. This combination treatment can induce long-lasting curative abscopal responses, and therefore it has also great potential for treatment of metastatic disease. Preclinical findings have led to the initiation of a phase I clinical trial (NCT02086721) in our institute investigating stereotactic ablative radiotherapy with L19-IL2 in patients with oligometastatic solid tumours.

High dose rate and flattening filter free irradiation can be safely implemented in clinical practice.

Purpose: We hypothesize that flattening filter free (FFF) high dose rate irradiation will decrease cell survival in normal and cancer cells with more pronounced effects in DNA repair deficient cells. Additionally, we hypothesize that removal of the flattening filter will result in an enhanced relative biological effectiveness independent of the dose rate. Materials and methods: Clonogenic survival was assessed after exposure to dose rates of 4 or 24 Gy/min (FFF 10 megavolt [MV] photon beam) using a Varian TrueBeam accelerator. Additionally, cells were exposed to 4 Gy/min with or without flattening filter. Relative biological effectiveness estimations were performed comparing the different beam photon spectra. Results: Cell survival in tumor and normal cell lines was not influenced by high dose rate irradiation. The intrinsic radiation sensitivity of DNA repair deficient cells was not affected by high dose rate compared to normal dose rate. Furthermore, the relative biological effectiveness was not significantly different from unity in any of the cell lines for both FFF and conventional flattened beam exposures. Conclusions: High dose rate irradiation did not affect long-term survival and DNA repair for cell lines of different tissues. This suggests that high dose rate does not influence treatment outcome or treatment toxicity and could be safely implemented in clinical routine.

Distinct radiation responses after in vitro mtDNA depletion are potentially related to oxidative stress

Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ0). Overall survival after irradiation was increased (p<0.001) for BEAS-2B ρ0 cells, while decreased for both tumor ρ0 lines (p<0.05). In agreement, increased residual DNA damage was observed after mtDNA depletion for A549 and 143B cells. Intrinsic radiosensitivity (surviving fraction at 2Gy) was not influenced. We investigated whether ROS levels, oxidative stress and/or antioxidant responses were responsible for altered radiation responses. Baseline ROS formation was similar between BEAS-2B parental and ρ0 cells, while reduced in A549 and 143B ρ0 cells, compared to their parental counterparts. After irradiation, ROS levels significantly increased for all parental cell lines, while levels for ρ0 cells remained unchanged. In order to investigate the presence of oxidative stress upon irradiation reduced glutathione: oxidized glutathione (GSH:GSSG) ratios were determined. Irradiation reduced GSH:GSSG ratios for BEAS-2B parental and 143B ρ0, while for A549 this ratio remained equal. Additionally, changes in antioxidant responses were observed. Our results indicate that mtDNA depletion results in varying radiation responses potentially involving variations in cellular ROS and antioxidant defence mechanisms. We therefore suggest when mitotoxic drugs are combined with radiation, in particular at high dose per fraction, the effect of these drugs on mtDNA copy number should be explored.