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Angiogenesis is an integral part of tumor growth and invasion. This has led to the emergence of several antiangiogenic therapies and stimulated efforts to accurately evaluate the extent of angiogenesis before and in response to anticancer treatment. The most commonly used approach has been the assessment of new vessel formation in histological samples. However, it is becoming apparent that this is insufficient for a full understanding of tumor physiology and for in vivo guidance of cancer management. Imaging has the potential to provide noninvasive and repeatable assessment of the angiogenic process. Imaging approaches use a variety of modalities and are aimed at either assessment of the functional integrity of tumor vasculature or assessment of its molecular status. This review summarizes the aims and methods of clinical tumor angiogenesis imaging, including present technologies and ones that will be developed within the next 5-10 years. [ABSTRACT FROM AUTHOR] more...

Despite improvements in neuroblastoma treatment, survival figures lag behind those of many other childhood malignancies. New treatments, and better use of existing treatments, are essential to reduce mortality. Neuroblastoma expresses several molecular targets for radionuclide imaging and therapy, of which the most widely exploited is the norepinephrine transporter. [ 123 I]metaiodobenzylguanidine (MIBG) imaging and [ 131 I]MIBG treatment, which target this physiologic pathway, have been in clinical practice for 40 y. Although therapy outcomes have been favorable, [ 131 I]MIBG use has not yet been optimized. Somatostatin receptors and the disialoganglioside are alternative targets, but their use remains experimental. The charity Children's Cancer Research Fund organized a workshop bringing together a broad range of scientists including radiochemists, radiobiologists, radiation physicists, clinical researchers including pediatric oncologists and nuclear medicine physicians, and patient advocates from the United Kingdom, United States, and continental Europe to share their experiences with molecular imaging and radiotherapy of neuroblastoma and discuss potential ways of improving treatment outcomes and access. These include development of alternative vectors targeting somatostatin receptors and disialoganglioside, isotopes such as α-particle and Auger electron emitters with different radiation characteristics, and combinations with external-beam radiotherapy, immunotherapy, and DNA damage repair inhibitors. Barriers to progress discussed included the unpredictable radioisotope supply, production of novel radiopharmaceuticals, lack of data regarding which are the best combination therapies, and insufficient clinical facilities. The aim was to stimulate the development and assessment of more effective treatments., (© 2025 by the Society of Nuclear Medicine and Molecular Imaging.) more...

Background: Radiopharmaceutical therapy (RPT) has gained notable attention for its potential in treating difficult cancers, with [ 177 Lu]Lu-DOTATATE being a notable example. However, the radiobiology of RPT is less understood compared to external beam radiotherapy (EBRT), and dosimetry protocols are not standardized. Organ dose limits and tumor dose-response correlations are often based on radiobiologically motivated equieffective doses (EQDX). On top of absorbed dose, these measures are also functions of the absorbed dose rate and radiobiological parameters that quantify tissue radiosensitivity and damage repair rate. Typically, the absorbed dose and repair rates are assumed to follow a monoexponential pattern, although describing the dose rate function often requires two or more phases to describe the data., Purpose: Here we present novel expressions for calculating the equieffective dose in 2 Gy fractions (EQD2) for RPT, considering various absorbed dose rate scenarios and the rate of sublethal DNA damage repair. We aimed to establish an approach that is scalable, robust, and can be used alongside various absorbed dose integration methods., Methods: By assuming a simple exponential decay for DNA damage repair and employing a biexponential function for absorbed dose rate decay, we have re-established the solutions for EQDX in a concise analytical form. Additionally, we have devised a novel hybrid solution applicable to piecewise-defined absorbed dose-rate functions, leveraging both numerical and analytical methodologies. To validate these expressions, simulated measurements were utilized, and comparisons were made with a fully numerical approach. We also investigated the reliability of three methodologies-fully numerical, fully analytical, and a hybrid approach-when simplifying comprehensive dosimetry protocols. Utilizing publicly available clinical data from two patients undergoing [ 177 Lu]Lu-DOTATATE therapy, we defined the baseline absorbed dose rate model based on the best biexponential fit to four post-injection SPECT measurements at the organ level. We then explored variations in EQD2 values resulting from the omission of the final measurement., Results: The proposed expressions were found to be accurate and scalable, providing a reliable alternative to fully numerical methods. The results of the fully numerical method converged to our solutions with increasing accuracy as the extrapolation time after injection was increased. However, we found that to achieve an accuracy in EQD2 to within 2%, the numerical method had to extrapolate for up to 890 h in some cases, at which point overflow errors are likely to occur. Our hybrid method also achieved a significant decrease in computation time compared to the fully numerical method.Using data from two patients, we found that the numerical, hybrid, and analytical approaches underestimated the baseline EQD2 to tumors by 15.6 ± 9.4 %, 5.0 ± 4.2 %, and 1.5 ± 2.9 %, respectively., Conclusions: Comprehensive dosimetric studies are often preferred in RPT when increased measurement accuracy is desired. Correspondingly, it is vital for radiobiological models to maintain a level of accuracy commensurate with comprehensive studies. Our proposed methods are accurate, scalable, and suitable for radiobiologically motivated RPT dosimetry., (© 2025 The Author(s). Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.) more...

Purpose: Esophageal cancer carries a poor prognosis with a 5-year overall survival of less than 20%. Barrett's esophagus increases the risk of esophageal adenocarcinoma. The aim of this study was to investigate the ability of EMI-137, a mesenchymal-epithelial transition factor (c-MET)-targeting optical imaging tracer, to detect dysplasia in Barrett's esophagus., Experimental Design: c-MET expression in human esophageal tissue was investigated using Gene Expression Omnibus datasets, tissue microarrays, and Barrett's esophagus biopsies. EMI-137 was tested in a dual xenograft mouse model bearing OE33 (c-MET high expression) and FLO-1 (c-MET low expression) tumors. Fluorescence molecular endoscopy was performed in a mouse model of Barrett's-like metaplasia and dysplasia (L2-IL1β). Tumors and organs of interest were evaluated through ex vivo fluorescence imaging., Results: MET mRNA expression analyses and c-MET immunostaining confirmed upregulation of c-MET in Barrett's esophagus and esophageal adenocarcinoma compared with normal epithelium. There was strong accumulation of EMI-137 in OE33 xenografts 3 hours after injection, decreasing by more than 50% on coinjection of a 10-fold molar excess of unlabeled EMI-137. The target-to-background ratio at 3 hours after injection for OE33 and FLO-1 tumors was 10.08 and 1.42, respectively. Fluorescence molecular endoscopy of L2-IL1β mice showed uptake of EMI-137 in dysplastic lesions within Barrett's esophagus with a target-to-background ratio of 1.9 in vivo and greater than 2 in ex vivo fluorescence imaging., Conclusions: EMI-137 accumulates in dysplastic lesions within Barrett's esophagus and also in c-MET-positive esophageal adenocarcinoma. EMI-137 imaging has potential as a screening and surveillance tool for patients with Barrett's esophagus and as a means to detecting dysplasia and esophageal adenocarcinoma., (©2024 The Authors; Published by the American Association for Cancer Research.) more...

Background and Purpose: To evaluate the impact of a deep learning (DL)-assisted interactive contouring tool on inter-observer variability and the time taken to complete tumour contouring., Materials and Methods: Nine clinicians contoured the gross tumour volume (GTV) using the PET-CT scans of 10 non-small cell lung cancer (NSCLC) patients, either using DL-assisted or manual contouring tools. After contouring a case using one contouring method, the same case was contoured one week later using the other method. The contours and time taken were compared., Results: Use of the DL-assisted tool led to a statistically significant decrease in active contouring time of 23 % relative to the standard manual segmentation method (p < 0.01). The mean observation time for all clinicians and cases made up nearly 60 % of interaction time for both contouring approaches. On average the time spent contouring per case was reduced from 22 min to 19 min when using the DL-assisted tool. Additionally, the DL-assisted tool reduced contour variability in the parts of tumour where clinicians tended to disagree the most, while the consensus contour was similar whichever of the two contouring approaches was used., Conclusions: A DL-assisted interactive contouring approach decreased active contouring time and local inter-observer variability when used to delineate lung cancer GTVs compared to a standard manual method. Integration of this tool into the clinical workflow could assist clinicians in contouring tasks and improve contouring efficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.) more...

Background and Objective: Management of metastatic prostate cancer (mPCa) presents significant challenges. In this systematic review, meta-analysis, and meta-regression, the efficacy, safety, and quality of life (QoL) outcomes of prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (PRLT) utilising lutetium-177 ([ 177 Lu]Lu-PSMA) and actinium-225 ([ 225 Ac]Ac-PSMA) were assessed., Methods: A detailed literature search across PubMed/Medline, EMBASE, Web of Science, Scopus, and Cochrane Library was conducted, culminating in the inclusion of 100 studies involving 8711 patients. Data on prostate-specific antigen (PSA) responses, toxicity profiles, and QoL and survival outcomes were analysed. Proportional meta-analyses and meta-regression analyses were performed., Key Findings and Limitations: The estimated proportion of patients with PSA decline ≥50% was 0.49 for [ 177 Lu]Lu-PSMA and 0.60 for [ 225 Ac]Ac-PSMA in mPCa, particularly metastatic castration-resistant prostate cancer. A meta-regression analysis indicated an association between the cumulative amount of administered activity and the proportion of PSA ≥50% decline. Positive PSA responses were observed alongside improved overall survival across both therapies. Our analyses also identified the key factors associated with PSA responses and survival outcomes, including baseline haemoglobin level, and the presence of visceral metastases. Although anaemia was commonly observed, with [ 177 Lu]Lu-PSMA, severe toxicities were infrequent. Improved QoL was observed following [ 177 Lu]Lu-PSMA therapy, whereas it remained stable following the second cycle of [ 225 Ac]Ac-PSMA treatment. Heterogeneity across studies for PSA responses and toxicity profiles is a limitation., Conclusions and Clinical Implications: Our findings suggest an association between PRLT and reductions in PSA levels, as well as associations with enhanced survival outcomes in mPCa. Furthermore, our analysis shows a low incidence of severe toxicity associated with this treatment. These observations highlight the important role of PRLT in the management of mPCa., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.) more...

The reversible attachment of small molecules to oligonucleotides provides versatile tools for the development of improved oligonucleotide therapeutics. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement for traceless release of the payload in vivo . Herein, we describe a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB) for the automated synthesis of oligonucleotide-small molecule conjugates. Val-Ala(NB) was protected by a photolabile 2-nitrobenzyl group to improve the stability of the peptide linker during DNA synthesis. Intracellular cathepsin B digests the dipeptide efficiently, releasing the payload-phosphate which is converted to the free payload by endogenous phosphatase enzymes. With the advantages of modular synthesis and stimuli-responsive drug release, we believe Val-Ala(NB) will be a potentially valuable cleavable linker for use in oligonucleotide-drug conjugates., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.) more...

Background: Non-toxic approaches to enhance radiotherapy outcomes are beneficial, particularly in ageing populations. Based on preclinical findings showing that high-fibre diets sensitised bladder tumours to irradiation by modifying the gut microbiota, along with clinical evidence of prebiotics enhancing anti-cancer immunity, we hypothesised that dietary fibre and its gut microbiota modification can radiosensitise tumours via secretion of metabolites and/or immunomodulation. We investigated the efficacy of high-fibre diets combined with irradiation in immunoproficient C57BL/6 mice bearing bladder cancer flank allografts., Result: Psyllium plus inulin significantly decreased tumour size and delayed tumour growth following irradiation compared to 0.2% cellulose and raised intratumoural CD8 + cells. Post-irradiation, tumour control positively correlated with Lachnospiraceae family abundance. Psyllium plus resistant starch radiosensitised the tumours, positively correlating with Bacteroides genus abundance and increased caecal isoferulic acid levels, associated with a favourable response in terms of tumour control. Psyllium plus inulin mitigated the acute radiation injury caused by 14 Gy. Psyllium plus inulin increased caecal acetate, butyrate and propionate levels, and psyllium alone and psyllium plus resistant starch increased acetate levels. Human gut microbiota profiles at the phylum level were generally more like mouse 0.2% cellulose profiles than high fibre profiles., Conclusion: These supplements may be useful in combination with radiotherapy in patients with pelvic malignancy. Video Abstract., (© 2024. The Author(s).) more...

Over the past decade, immunotherapy has emerged as a major modality in cancer medicine. However, despite its unprecedented success, immunotherapy currently benefits only a subgroup of patients, may induce responses of limited duration and is associated with potentially treatment-limiting side effects. In addition, responses to immunotherapeutics are sometimes diminished by the emergence of a complex array of resistance mechanisms. The efficacy of immunotherapy depends on dynamic interactions between tumour cells and the immune landscape in the tumour microenvironment. Ultrasound, especially in conjunction with cavitation-promoting agents such as microbubbles, can assist in the uptake and/or local release of immunotherapeutic agents at specific target sites, thereby increasing treatment efficacy and reducing systemic toxicity. There is also increasing evidence that ultrasound and/or cavitation may themselves directly stimulate a beneficial immune response. In this review, we summarize the latest developments in the use of ultrasound and cavitation agents to promote checkpoint inhibitor immunotherapy., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.) more...

In August 2022, the Cancer Informatics for Cancer Centers brought together cancer informatics leaders for its biannual symposium, Precision Medicine Applications in Radiation Oncology, co-chaired by Quynh-Thu Le, MD (Stanford University), and Walter J. Curran, MD (GenesisCare). Over the course of 3 days, presenters discussed a range of topics relevant to radiation oncology and the cancer informatics community more broadly, including biomarker development, decision support algorithms, novel imaging tools, theranostics, and artificial intelligence (AI) for the radiotherapy workflow. Since the symposium, there has been an impressive shift in the promise and potential for integration of AI in clinical care, accelerated in large part by major advances in generative AI. AI is now poised more than ever to revolutionize cancer care. Radiation oncology is a field that uses and generates a large amount of digital data and is therefore likely to be one of the first fields to be transformed by AI. As experts in the collection, management, and analysis of these data, the informatics community will take a leading role in ensuring that radiation oncology is prepared to take full advantage of these technological advances. In this report, we provide highlights from the symposium, which took place in Santa Barbara, California, from August 29 to 31, 2022. We discuss lessons learned from the symposium for data acquisition, management, representation, and sharing, and put these themes into context to prepare radiation oncology for the successful and safe integration of AI and informatics technologies. more...

Toxicity induced by inorganic arsenic as AsO 3 3- ( iAs III ) is of global concern. Reliable detection of the maximum allowed contaminant level for arsenic in drinking water and in the cellular system remains a challenge for the water quality management and assessment of toxicity in the cellular milieu, respectively. A new Ir(III)-based phosphorescent molecule ( AS-1 ; λ Ext = 415 nm and λ Ems = 600 nm, Φ = 0.3) is synthesized for the selective detection of iAs III in an aqueous solution with a ratiometric luminescence response even in the presence of iAs V and all other common inorganic cations and anions. The relatively higher affinity of the thioimidazole ligand (HPBT) toward iAs III led to the formation of a fluorescent molecule iAs V -HPBT (λ Ext = 415 nm and λ Ems = 466 nm, Φ = 0.28) for the reaction of iAs III and AS-1 . An improved limit of quantitation (LOQ) down to 0.2 ppb is achieved when AS-1 is used in the CTAB micellar system. Presumably, the cationic surfactants favor the localization of AS-1 @CTAB Micelle in mitochondria of MCF7 cells, and this is confirmed from the images of the confocal laser fluorescence scanning microscopic studies. Importantly, cell viability assay studies confirm that AS-1 @CTAB Micelle induces dose-dependent detoxification of iAs III in live cells. Further, luminescence responses at 466 nm could be utilized for developing a hand-held device for the in-field application. Such a reagent that allows for ratiometric detection of iAs III with LOQ of 2.6 nM (0.5 ppb) in water, as well as helps in visualizing its distribution in mitochondria with a detoxifying effect, is rather unique in contemporary literature. more...

Semi-automatic and fully automatic contouring tools have emerged as an alternative to fully manual segmentation to reduce time spent contouring and to increase contour quality and consistency. Particularly, fully automatic segmentation has seen exceptional improvements through the use of deep learning in recent years. These fully automatic methods may not require user interactions, but the resulting contours are often not suitable to be used in clinical practice without a review by the clinician. Furthermore, they need large amounts of labelled data to be available for training. This review presents alternatives to manual or fully automatic segmentation methods along the spectrum of variable user interactivity and data availability. The challenge lies to determine how much user interaction is necessary and how this user interaction can be used most effectively. While deep learning is already widely used for fully automatic tools, interactive methods are just at the starting point to be transformed by it. Interaction between clinician and machine, via artificial intelligence, can go both ways and this review will present the avenues that are being pursued to improve medical image segmentation., (Creative Commons Attribution license.) more...

Purpose: Recent reports personalizing the administered activity (AA) of each cycle of peptide receptor radionuclide therapy based on the predicted absorbed dose (AD) to the kidneys (dose-limiting organ) have been promising. Assuming identical renal pharmacokinetics for each cycle is pragmatic, however it may lead to over- or under-estimation of the optimal AA. Here, we investigate the influence that earlier cycles of [ 177 Lu]Lu-DOTATATE had on the biokinetics and AD of subsequent cycles in a recent clinical trial that evaluated the safety and activity of [ 177 Lu]Lu-DOTATATE in pediatric neuroblastoma (NBL). We investigated whether predictions based on an assumption of unchanging AD per unit AA (Gy/GBq) prove robust to cyclical changes in biokinetics., Methods: A simulation study, based on dosimetry data from six children with NBL who received four-cycles of [ 177 Lu]Lu-DOTATATE in the LuDO trial (ISRCTN98918118), was performed to explore the effect of variable biokinetics on AD. In the LuDO trial, AA was adapted to the patient's weight and SPECT/CT-based dosimetry was performed for the kidneys and tumour after each cycle. The largest tumour mass was selected for dosimetric analysis in each case., Results: The median tumour AD per cycle was found to decrease from 15.6 Gy (range 8.12-26.4) in cycle 1 to 11.4 Gy (range 9.67-28.8), 11.3 Gy (range 2.73-32.9) and 4.3 Gy (range 0.72-20.1) in cycles 2, 3 and 4, respectively. By the fourth cycle, the median of the ratios of the delivered AD (AD D ) and the predicted (or "expected") AD (AD E ) (which was based on an assumption of stable biokinetics from the first cycle onwards) were 0.16 (range 0.02-0.92, p = 0.013) for the tumour and 1.08 (range 0.84-1.76, p > 0.05) for kidney. None of the patients had an objective response at 1 month follow up., Conclusion: This study demonstrates variability in Gy/GBq and tumour AD per cycle in children receiving four administrations of [ 177 Lu]Lu-DOTATATE treatment for NBL. NBL is deemed a radiation sensitive tumour; therefore, dose-adaptive treatment planning schemes may be appropriate for some patients to compensate for decreasing tumour uptake as treatment progresses. Trial registration ISRCTN ISRCTN98918118. Registered 20 December 2013 (retrospectively registered)., (© 2022. The Author(s).) more...

Oligonucleotides containing cleavable linkers have emerged as versatile tools to achieve stimulus-responsive and site-specific cleavage of DNA. However, the limitations of previously reported cleavable linkers including photolabile and disulfide linkers have restricted their applications in vivo. Inspired by the cathepsin B-sensitive dipeptide linkers in antibody-drug conjugates (ADCs) such as Adcetris, we have developed Val-Ala-02 and Val-Ala-Chalcone phosphoramidites for the automated synthesis of enzyme-cleavable oligonucleotides. Cathepsin B digests Val-Ala-02 and Val-Ala-Chalcone linkers efficiently, enabling cleavage of oligonucleotides into two components or release of small-molecule payloads. Based on the prior success of dipeptide linkers in ADCs, we believe that these dipeptide linker phosphoramidites will promote new clinical applications of therapeutic oligonucleotides., (© 2021 The Authors. Published by Wiley-VCH GmbH.) more...

The intracellular environment hosts a large number of cancer- and other disease-relevant human proteins. Targeting these with internalized antibodies would allow therapeutic modulation of hitherto undruggable pathways, such as those mediated by protein-protein interactions. However, one of the major obstacles in intracellular targeting is the entrapment of biomacromolecules in the endosome. Here we report an approach to delivering antibodies and antibody fragments into the cytosol and nucleus of cells using trimeric cell-penetrating peptides (CPPs). Four trimers, based on linear and cyclic sequences of the archetypal CPP Tat, are significantly more potent than monomers and can be tuned to function by direct interaction with the plasma membrane or escape from vesicle-like bodies. These studies identify a tricyclic Tat construct that enables intracellular delivery of functional immunoglobulin-G antibodies and Fab fragments that bind intracellular targets in the cytosol and nuclei of live cells at effective concentrations as low as 1 μM., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.) more...

The emergence of effective radionuclide therapeutics, such as radium-223 dichloride, [ 177 Lu]Lu-DOTA-TATE and [ 177 Lu]Lu-PSMA ligands, over the last 10 years is driving a rapid expansion in molecular radiotherapy (MRT) research. Clinical trials that are underway will help to define optimal dosing protocols and identify groups of patients who are likely to benefit from this form of treatment. Clinical investigations are also being conducted to combine new MRT agents with other anticancer drugs, with particular emphasis on DNA repair inhibitors and immunotherapeutics. In this review, the case is presented for combining MRT with external beam radiotherapy (EBRT). The technical and dosimetric challenges of combining two radiotherapeutic modalities have impeded progress in the past. However, the need for research into the specific radiobiological effects of radionuclide therapy, which has lagged behind that for EBRT, has been recognised. This, together with innovations in imaging technology, MRT dosimetry tools and EBRT hardware, will facilitate the future use of this important combination of treatments., (Copyright © 2021 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.) more...

Background: Triple negative breast cancer (TNBC) poses a serious clinical challenge as it is an aggressive form of the disease that lacks estrogen receptor, progesterone receptor, and ERBB2 (formerly HER2) gene amplification, which limits the treatment options. The Warburg phenotype of upregulated glycolysis in the presence of oxygen has been shown to be prevalent in TNBC. Elevated glycolysis satisfies the energy requirements of cancer cells, contributes to resistance to treatment by maintaining redox homeostasis and generating nucleotide precursors required for cell proliferation and DNA repair. Expression of the monocarboxylate transporter 1 (MCT1), which is responsible for the bidirectional transport of lactate, correlates with an aggressive phenotype and poor outcome in several cancer types, including breast cancer. In this study, 3-bromopyruvate (3BP), a lactate/pyruvate analog, was used to selectively target TNBC cells that express MCT1., Methods: The cytotoxicity of 3BP was tested in MTT assays using human TNBC cell lines: BT20 (MCT1 + /MCT4 - ), MDA-MB-23 (MCT1 - /MCT4 + ), and BT20 in which MCT1 was knocked down (siMCT1-BT20). The metabolite profile of 3BP-treated and 3BP-untreated cells was investigated using LC-MS/MS. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BT20 and MDA-MB-231 cells treated with 3BP were measured using a Seahorse XF96 extracellular flux analyzer. The impact of ionizing radiation on cell survival, alone or in combination with 3BP pre-treatment, was evaluated using clonogenic assays., Results: Metabolomic analyses showed that 3BP causes inhibition of glycolysis, disturbance of redox homeostasis, decreased nucleotide synthesis, and was accompanied by a reduction in medium acidification. In addition, 3BP potentiated the cytotoxic effect of ionizing radiation, a treatment that is frequently used in the management of TNBC., Conclusions: Overall, MCT1-mediated metabolic perturbation in combination with radiotherapy is shown to be a promising strategy for the treatment of glycolytic tumors such as TNBC, overcoming the selectivity challenges of targeting glycolysis with glucose analogs., (© 2021. The Author(s).) more...

Telomerase represents an attractive target in oncology as it is expressed in cancer but not in normal tissues. The oligonucleotide inhibitors of telomerase represent a promising anticancer strategy, although poor cellular uptake can restrict their efficacy. In this study, gold nanoparticles (AuNPs) were used to enhance oligonucleotide uptake. "match" oligonucleotides complementary to the telomerase RNA template subunit (hTR) and "scramble" (control) oligonucleotides were conjugated to diethylenetriamine pentaacetate (DTPA) for 111 In-labeling. AuNPs (15.5 nm) were decorated with a monofunctional layer of oligonucleotides (ON-AuNP) or a multifunctional layer of oligonucleotides, PEG(polethylene glycol)800-SH (to reduce AuNP aggregation) and the cell-penetrating peptide Tat (ON-AuNP-Tat). Match-AuNP enhanced the cellular uptake of radiolabeled oligonucleotides while retaining the ability to inhibit telomerase activity. The addition of Tat to AuNPs increased nuclear localization. 111 In-Match-AuNP-Tat induced DNA double-strand breaks and caused a dose-dependent reduction in clonogenic survival of telomerase-positive cells but not telomerase-negative cells. hTR inhibition has been reported to sensitize cancer cells to ionizing radiation, and 111 In-Match-AuNP-Tat therefore holds promise as a vector for delivery of radionuclides into cancer cells while simultaneously sensitizing them to the effects of the emitted radiation. more...

Treatment options for patients with pancreatic cancer are limited and survival prospects have barely changed over the past 4 decades. Chemoradiation treatment (CRT) has been used as neoadjuvant therapy in patients with borderline resectable disease to reduce tumour burden and increase the proportion of patients eligible for surgery. Antimetabolite drugs such as gemcitabine and 5-fluorouracil are known to sensitise pancreatic tumours to radiation treatment. Likewise, photodynamic therapy (PDT) has also been shown to enhance the effect of radiation therapy. However, PDT is limited to treating superficial lesions due to the attenuation of light by tissue. The ability of the related technique, sonodynamic therapy (SDT), to enhance CRT was investigated in two murine models of pancreatic cancer (PSN-1 and BxPC-3) in this study. SDT uses low intensity ultrasound to activate an otherwise non-toxic sensitiser, generating toxic levels of reactive oxygen species (ROS) locally. It is applicable to greater target depths than PDT due to the ability of ultrasound to propagate further than light in tissue. Both CRT and the combination of CRT plus SDT delayed tumour growth in the two tumour models. In the PSN-1 model, but not the BxPC-3 model, the combination treatment caused an increase in survival relative to CRT alone (p = 0.038). The improvement in survival conferred by the addition of SDT in this model may be related to differences in tumour architecture between the two models. MRI and US images showed that PSN-1 tumours were less well perfused and vascularised than BxPC-3 tumours. This poor vascularisation may explain why PSN-1 tumours were more susceptible to the effects of vascular damage exerted by SDT treatment., (Copyright © 2021. Published by Elsevier B.V.) more...

Brain metastases (BM) are frequently detected during the follow-up of patients with malignant tumors, particularly in those with advanced disease. Despite a major progress in systemic anti-cancer treatments, the average overall survival of these patients remains limited (6 months from diagnosis). Also, cognitive decline is regularly reported especially in patients treated with whole brain external beam radiotherapy (WBRT), due to the absorbed radiation dose in healthy brain tissue. New targeted therapies, for an earlier and/or more specific treatment of the tumor and its microenvironment, are needed. Radioimmunotherapy (RIT), a combination of a radionuclide to a specific antibody, appears to be a promising tool. Inflammation, which is involved in multiple steps, including the early phase, of BM development is attractive as a relevant target for RIT. This review will focus on the (1) early biomarkers of inflammation in BM pertinent for RIT, (2) state of the art studies on RIT for BM, and (3) the importance of dosimetry to RIT in BM. These two last points will be addressed in comparison to the conventional EBRT treatment, particularly with respect to the balance between tumor control and healthy tissue complications. Finally, because new diagnostic imaging techniques show a potential for the detection of BM at an early stage of the disease, we focus particularly on this therapeutic window., Competing Interests: Author NF was employed by GenesisCare. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Corroyer-Dulmont, Jaudet, Frelin, Fantin, Weyts, Vallis, Falzone, Sibson, Chérel, Kraeber-Bodéré, Batalla, Bardet, Bernaudin and Valable.) more...

Purpose: To investigate a deep learning approach that enables three-dimensional (3D) segmentation of an arbitrary structure of interest given a user provided two-dimensional (2D) contour for context. Such an approach could decrease delineation times and improve contouring consistency, particularly for anatomical structures for which no automatic segmentation tools exist., Methods: A series of deep learning segmentation models using a Recurrent Residual U-Net with attention gates was trained with a successively expanding training set. Contextual information was provided to the models, using a previously contoured slice as an input, in addition to the slice to be contoured. In total, 6 models were developed, and 19 different anatomical structures were used for training and testing. Each of the models was evaluated for all 19 structures, even if they were excluded from the training set, in order to assess the model's ability to segment unseen structures of interest. Each model's performance was evaluated using the Dice similarity coefficient (DSC), Hausdorff distance, and relative added path length (APL)., Results: The segmentation performance for seen and unseen structures improved when the training set was expanded by addition of structures previously excluded from the training set. A model trained exclusively on heart structures achieved a DSC of 0.33, HD of 44 mm, and relative APL of 0.85 when segmenting the spleen, whereas a model trained on a diverse set of structures, but still excluding the spleen, achieved a DSC of 0.80, HD of 13 mm, and relative APL of 0.35. Iterative prediction performed better compared to direct prediction when considering unseen structures., Conclusions: Training a contextual deep learning model on a diverse set of structures increases the segmentation performance for the structures in the training set, but importantly enables the model to generalize and make predictions even for unseen structures that were not represented in the training set. This shows that user-provided context can be incorporated into deep learning contouring to facilitate semi-automatic segmentation of CT images for any given structure. Such an approach can enable faster de-novo contouring in clinical practice., (© 2021 Mirada Medical Ltd. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.) more...

Controlled and efficient activation is the crucial aspect of designing an effective prodrug. Herein we demonstrate a proof of concept for a light activatable prodrug with desired organelle specificity. Mertansine, a benzoansamacrolide, is an efficient microtubule-targeting compound that binds at or near the vinblastine-binding site in the mitochondrial region to induce mitotic arrest and cell death through apoptosis. Despite its efficacy even in the nanomolar level, this has failed in stage 2 of human clinical trials owing to the lack of drug specificity and the deleterious systemic toxicity. To get around this problem, a recent trend is to develop an antibody-conjugatable maytansinoid with improved tumor/organelle-specificity and lesser systematic toxicity. Endogenous CO is recognized as a regulator of cellular function and for its obligatory role in cell apoptosis. CO blocks the proliferation of cancer cells and effector T cells, and the primary target is reported to be the mitochondria. We report herein a new mitochondria-specific prodrug conjugate ( Pro-DC ) that undergoes a photocleavage reaction on irradiation with a 400 nm source (1.0 mW cm -2 ) to induce a simultaneous release of the therapeutic components mertansine and CO along with a BODIPY derivative ( BODIPY(PPH3)2 ) as a luminescent marker in the mitochondrial matrix. The efficacy of the process is demonstrated using MCF-7 cells and could effectively be visualized by probing the intracellular luminescence of BODIPY(PPH3)2 . This provides a proof-of-concept for designing a prodrug for image-guided combination therapy for mainstream treatment of cancer., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.) more...

Purpose: Selective internal radiation therapy (SIRT) is administered to treat tumors of the liver and is generally well tolerated. Although widely adopted for its therapeutic benefits, SIRT is rarely combined with external beam radiation therapy (EBRT) owing to the complexity of the dosimetry resulting from the combination of treatments with distinct radiobiological effects. The purpose of this study was to establish a dosimetric framework for combining SIRT and EBRT using clinical experience derived from representative patients with hepatocellular carcinoma (HCC) who received both therapies., Methods and Materials: Treatments from 10 patients with HCC given EBRT either before or after SIRT were analyzed. The dosimetry framework used here considered differences in the radiobiological effects and fractionation schemes of SIRT versus EBRT, making use of the concepts of biological effective dose (BED) and equivalent dose (EQD). Absorbed dose from SIRT was calculated, converted to BED, and summed with BED from EBRT dose plans. Two of these patients were used in a virtual planning exercise to investigate the feasibility of combining stereotactic body radiation therapy and SIRT., Results: The combination of EBRT and SIRT in 10 patients with HCC showed no major toxicity. No Child-Pugh scores went above 8 and albumin-bilirubin scores from only 1 patient worsened to grade 3 (> -1.39) from treatment through 3-months follow-up. A framework with radiobiological modeling was developed to manage the combined treatments in terms of their sum BED. The exploratory SIRT plus SABR inverse dose plans for 2 patients, incorporating radiobiologically informed 90 Y SIRT dosimetry, achieved dose distributions comparable to SBRT alone., Conclusions: Treatment with both EBRT and SIRT can be given safely to patients with HCC. The BED and EQD concepts should be used in combined dosimetry to account for the differing radiobiological effects of EBRT and SIRT. Inverse dose planning of EBRT after SIRT could provide improved dose distributions and flexibility to the clinical workflow. Further research into combination therapy is needed through prospective trials., (© 2020 The Author(s).) more...

Purpose/objective: To use a model-based approach to identify a sub-group of patients with locally advanced lung cancer who would benefit from proton therapy compared to photon therapy for reduction of cardiac toxicity., Material/methods: Volumetric modulated arc photon therapy (VMAT) and robust-optimised intensity modulated proton therapy (IMPT) plans were generated for twenty patients with locally advanced lung cancer to give a dose of 70 Gy (relative biological effectiveness (RBE)) in 35 fractions. Cases were selected to represent a range of anatomical locations of disease. Contouring, treatment planning and organs-at-risk constraints followed RTOG-1308 protocol. Whole heart and ub-structure doses were compared. Risk estimates of grade⩾3 cardiac toxicity were calculated based on normal tissue complication probability (NTCP) models which incorporated dose metrics and patients baseline risk-factors (pre-existing heart disease (HD))., Results: There was no statistically significant difference in target coverage between VMAT and IMPT. IMPT delivered lower doses to the heart and cardiac substructures (mean, heart V5 and V30, P < .05). In VMAT plans, there were statistically significant positive correlations between heart dose and the thoracic vertebral level that corresponded to the most inferior limit of the disease. The median level at which the superior aspect of the heart contour began was the T7 vertebrae. There was a statistically significant difference in dose (mean, V5 and V30) to the heart and all substructures (except mean dose to left coronary artery and V30 to sino-atrial node) when disease overlapped with or was inferior to the T7 vertebrae. In the presence of pre-existing HD and disease overlapping with or inferior to the T7 vertebrae, the mean estimated relative risk reduction of grade⩾3 toxicities was 24-59%., Conclusion: IMPT is expected to reduce cardiac toxicity compared to VMAT by reducing dose to the heart and substructures. Patients with both pre-existing heart disease and tumour and nodal spread overlapping with or inferior to the T7 vertebrae are likely to benefit most from proton over photon therapy., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.) more...

The purpose of this study was to establish the dose-response relationship of selective internal radiation therapy (SIRT) in patients with metastatic colorectal cancer (mCRC), when informed by radiobiological sensitivity parameters derived from mCRC cell lines exposed to 90 Y. Methods: Twenty-three mCRC patients with liver metastases refractory to chemotherapy were included. 90 Y bremsstrahlung SPECT images were transformed into dose maps assuming the local dose deposition method. Baseline and follow-up CT scans were segmented to derive liver and tumor volumes. Mean, median, and D 70 (minimum dose to 70% of tumor volume) values determined from dose maps were correlated with change in tumor volume and volumetric RECIST response using linear and logistic regression, respectively. Radiosensitivity parameters determined by clonogenic assays of mCRC cell lines HT-29 and DLD-1 after exposure to 90 Y or external beam radiotherapy (EBRT; 6 MV photons) were used in biologically effective dose (BED) calculations. Results: Mean administered radioactivity was 1,469 ± 428 MBq (range, 847-2,185 MBq), achieving a mean absorbed radiation dose to tumor of 35.5 ± 9.4 Gy and mean normal liver dose of 26.4 ± 6.8 Gy. A 1.0 Gy increase in mean, median, and D 70 absorbed dose was associated with a reduction in tumor volume of 1.8%, 1.8%, and 1.5%, respectively, and an increased probability of a volumetric RECIST response (odds ratio, 1.09, 1.09, and 1.10, respectively). Threshold mean, median and D 70 doses for response were 48.3, 48.8, and 41.8 Gy, respectively. EBRT-equivalent BEDs for 90 Y are up to 50% smaller than those calculated by applying protraction-corrected radiobiological parameters derived from EBRT alone. Conclusion: Dosimetric studies have assumed equivalence between 90 Y SIRT and EBRT, leading to inflation of BED for SIRT and possible undertreatment. Radiobiological parameters for 90 Y were applied to a BED model, providing a calculation method that has the potential to improve assessment of tumor control., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.) more...

Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics. Herein we report the synthesis of a mismatch DNA binding bis-ruthenium(ii) dipyridophenazine (dppz) complex that can be radiolabelled with the Auger electron emitting radionuclide indium-111 ( 111 In). Greater stabilisation accompanied by enhanced MLCT (metal to ligand charge-transfer) luminescence of both the bis-Ru(dppz) chelator and non-radioactive indium-loaded complex was observed in the presence of a TT mismatch-containing duplex compared to matched DNA. The radioactive construct [ 111 In]In-bisRu(dppz) ([ 111 In][In- 2 ] 4+ ) targets cell nuclei and is radiotoxic towards MMR-deficient human colorectal cancer cells showing substantially less detrimental effects in a paired cell line with restored MMR function. Additional cell line studies revealed that [ 111 In][In- 2 ] 4+ is preferentially radiotoxic towards MMR-deficient colorectal cancer cells accompanied by increased DNA damage due to 111 In decay. The biodistribution of [ 111 In][In- 2 ] 4+ in live mice was demonstrated using SPECT. These results illustrate how a Ru(ii) polypyridyl complex can incorporate mismatch DNA binding and radiometal chelation in a single molecule, generating a DNA-targeting AE radiopharmaceutical that displays selective radiotoxicity towards MMR-deficient cancer cells and is compatible with whole organism SPECT imaging., (This journal is © The Royal Society of Chemistry 2020.) more...

Commercial mouse chow is designed to provide a complete, nutrient-rich diet, and it can contain upwards of 100 mg/kg manganese, an essential mineral. Manganese acts as a relaxation time-shortening contrast agent for both T 1 and T 2 , and where standard chow is hydrated in the gastrointestinal tract, bright signals are produced when using T 1 -weighted imaging (T 1 WI). As a result of peristalsis, gastrointestinal hyperintensities result in temporally unstable signals, leading to image ghosting and decreased resolution from that prescribed. To avoid the problem, various methods of gastrointestinal tract modulation, including the use of intestinal cleansing with laxatives and dietary modulation, have been reported. Here, dietary modulation has been extended to the use of a biologically innocuous, long-term change of diet. In this study, we report on the use of a commercially available manganese-free chow to improve the image quality of the gastrointestinal tract. This manganese-free chow, apart from the omitted manganese which is available in tap water, is a complete diet and readily available. We investigated the time-dependent, diet-related gastrointestinal intensities on short-TR T 1 WI magnetic resonance imaging; monitored body mass, food and water consumption and standard blood biochemistry analysis following diet change; and determined manganese concentration in blood plasma following a five-day change to manganese-free chow. We show that the manganese-free chow presents a refinement to other gastrointestinal tract modulation, as it avoids the need for invasive procedures for gut voiding and can be provided ad libitum so that animals can be maintained with no need for prescribed diet change before imaging. more...

Molecular radiotherapy using 177 Lu-DOTATATE is a most effective treatment for somatostatin receptor-expressing neuroendocrine tumors. Despite its frequent and successful use in the clinic, little or no radiobiologic considerations are made at the time of treatment planning or delivery. On positive uptake on octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiologic and DNA damage effects in the tumor. Here, we visualized and quantified the extent of DNA damage response after 177 Lu-DOTATATE therapy using SPECT imaging with 111 In-anti-γH2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with β-emitting therapeutic radiopharmaceuticals. Methods: Six cell lines were exposed to external-beam radiotherapy (EBRT) or 177 Lu-DOTATATE, after which the number of γH2AX foci and the clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor-positive tumor xenografts were treated with 177 Lu-DOTATATE or sham-treated and coinjected with 111 In-anti-γH2AX-TAT, 111 In-IgG-TAT control, or vehicle. Results: Clonogenic survival after external-beam radiotherapy was cell-line-specific, indicating varying levels of intrinsic radiosensitivity. Regarding in vitro cell lines treated with 177 Lu-DOTATATE, clonogenic survival decreased and γH2AX foci increased for cells expressing high levels of somatostatin receptor subtype 2. Ex vivo measurements revealed a partial correlation between 177 Lu-DOTATATE uptake and γH2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to 177 Lu-DOTATATE irradiation. Conclusion: 111 In-anti-γH2AX-TAT allows monitoring of DNA damage after 177 Lu-DOTATATE therapy and reveals heterogeneous damage responses., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.) more...

The purpose of this exploratory study was to investigate the combination of a radiopharmaceutical, nanoparticles and ultrasound (US) enhanced delivery to develop a clinically viable therapeutic strategy for tumours overexpressing the epidermal growth factor receptor (EGFR). Molecularly targeted radionuclides have great potential for cancer therapy but are sometimes associated with insufficient delivery resulting in sub-cytotoxic amounts of radioactivity being delivered to the tumour. Liposome formulations are currently used in the clinic to reduce the side effects and improve the pharmacokinetic profile of chemotherapeutic drugs. However, in contrast to non-radioactive agents, loading and release of radiotherapeutics from liposomes can be challenging in the clinical setting. US-activated cavitation agents such as microbubbles (MBs) have been used to release therapeutics from liposomes to enhance the distribution/delivery in a target area. In an effort to harness the benefits of these techniques, the development of a liposome loaded radiopharmaceutical construct for enhanced delivery via acoustic cavitation was studied. The liposomal formulation was loaded with peptide, human epidermal growth factor (HEGF), coupled to a chelator for subsequent radiolabelling with 111 Indium ([ 111 In]In 3+ ), in a manner designed to be compatible with preparation in a radiopharmacy. Liposomes were efficiently radiolabelled (57%) within 1 h, with release of ~12% of the radiopeptide following a 20 s exposure to US-mediated cavitation in vitro. In clonogenic studies this level of release resulted in cytotoxicity specifically in cells over-expressing the epidermal growth factor receptor (EGFR), with over 99% reduction in colony survival compared to controls. The formulation extended the circulation time and changed the biodistribution compared to the non-liposomal radiopeptide in vivo, although interestingly the biodistribution did not resemble that of liposome constructs currently used in the clinic. Cavitation of MBs co-injected with liposomes into tumours expressing high levels of EGFR resulted in a 2-fold enhancement in tumour uptake within 20 min. However, owing to the poor vascularisation of the tumour model used the same level of uptake was achieved without US after 24 h. By combining acoustic-cavitation-sensitive liposomes with radiopharmaceuticals this research represents a new concept in achieving targeted delivery of radiopharmaceuticals., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...

Background: Brain metastases (BM) develop frequently in patients with breast cancer. Despite the use of external beam radiotherapy (EBRT), the average overall survival is short (6 months from diagnosis). The therapeutic challenge is to deliver molecularly targeted therapy at an early stage when relatively few metastatic tumor cells have invaded the brain. Vascular cell adhesion molecule 1 (VCAM-1), overexpressed by nearby endothelial cells during the early stages of BM development, is a promising target. The aim of this study was to investigate the therapeutic value of targeted alpha-particle radiotherapy, combining lead-212 (212Pb) with an anti-VCAM-1 antibody (212Pb-αVCAM-1)., Methods: Human breast carcinoma cells that metastasize to the brain, MDA-231-Br-GFP, were injected into the left cardiac ventricle of nude mice. Twenty-one days after injection, 212Pb-αVCAM-1 uptake in early BM was determined in a biodistribution study and systemic/brain toxicity was evaluated. Therapeutic efficacy was assessed using MR imaging and histology. Overall survival after 212Pb-αVCAM-1 treatment was compared with that observed after standard EBRT., Results: 212Pb-αVCAM-1 was taken up into early BM with a tumor/healthy brain dose deposition ratio of 6 (5.52e108 and 0.92e108) disintegrations per gram of BM and healthy tissue, respectively. MRI analyses showed a statistically significant reduction in metastatic burden after 212Pb-αVCAM-1 treatment compared with EBRT (P < 0.001), translating to an increase in overall survival of 29% at 40 days post prescription (P < 0.01). No major toxicity was observed., Conclusions: The present investigation demonstrates that 212Pb-αVCAM-1 specifically accumulates at sites of early BM causing tumor growth inhibition., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.) more...

Objective: To identify a subgroup of lung cancer plans where the analytical dose calculation (ADC) algorithm may be clinically acceptable compared to Monte Carlo (MC) dose calculation in intensity modulated proton therapy (IMPT)., Methods: Robust-optimised IMPT plans were generated for 20 patients to a dose of 70 Gy (relative biological effectiveness) in 35 fractions in Raystation. For each case, four plans were generated: three with ADC optimisation using the pencil beam (PB) algorithm followed by a final dose calculation with the following algorithms: PB (PB-PB), MC (PB-MC) and MC normalised to prescription dose (PB-MC scaled). A fourth plan was generated where MC optimisation and final dose calculation was performed (MC-MC). Dose comparison and γ analysis (PB-PB vs PB-MC) at two dose thresholds were performed: 20% (D20) and 99% (D99) with PB-PB plans as reference., Results: Overestimation of the dose to 99% and mean dose of the clinical target volume was observed in all PB-MC compared to PB-PB plans (median: 3.7 Gy(RBE) (5%) (range: 2.3 to 6.9 Gy(RBE)) and 1.8 Gy(RBE) (3%) (0.5 to 4.6 Gy(RBE))). PB-MC scaled plans resulted in significantly higher CTVD2 compared to PB-PB (median difference: -4 Gy(RBE) (-6%) (-5.3 to -2.4 Gy(RBE)), p ≤ .001). The overall median γ pass rates (3%-3 mm) at D20 and D99 were 93.2% (range:62.2-97.5%) and 71.3 (15.4-92.0%). On multivariate analysis, presence of mediastinal disease and absence of range shifters were significantly associated with high γ pass rates. Median D20 and D99 pass rates with these predictors were 96.0% (95.3-97.5%) and 85.4% (75.1-92.0%). MC-MC achieved similar target coverage and doses to OAR compared to PB-PB plans., Conclusion: In the presence of mediastinal involvement and absence of range shifters Raystation ADC may be clinically acceptable in lung IMPT. Otherwise, MC algorithm would be recommended to ensure accuracy of treatment plans., Advances in Knowledge: Although MC algorithm is more accurate compared to ADC in lung IMPT, ADC may be clinically acceptable where there is mediastinal involvement and absence of range shifters. more...

Objective: We compared the sensitivity of intensity modulated proton therapy (IMPT) and photon volumetric modulated arc therapy (VMAT) plans to setup uncertainties in locally advanced non-small cell lung cancer (NSCLC) using probabilistic scenarios., Methods: Minimax robust (MM) and planning target volume (PTV) optimised IMPT and VMAT nominal plans were created with physical dose of 70 Gy in 35 fractions in 10 representative patients. Using population data of setup errors, a fractionated treatment course was simulated, summed (D sum ) and compared to the nominal plan. Three treatment-course simulations were done for each plan. Target robustness criteria were: dose deviation of ≤5% to clinical target volume (CTV) D 98% and CTV V 95% ≥ 99.9%. Voxelwise simulation repeatability was analysed using Bland-Altman plots. Acceptable limits of agreement were 2% of the prescription dose., Results: All D sum met target robustness criteria. While fraction VMAT and MM-IMPT doses were excellent, simulated fraction doses in PTV-IMPT were suboptimal. Almost all (>99%) of VMAT and MM-IMPT fraction doses met both target robustness criteria. For PTV-IMPT, only 96.9 and 80.3% of fractions met CTVD 98% and V 95% criteria respectively. Simulation repeatability was excellent (limits of agreement range: 0.41-1.1 Gy) with strong positive correlations., Conclusion: When considering the whole treatment course, setup errors do not influence robustness irrespective of planning techniques used. However, on a fraction level, VMAT and MM-IMPT plans are superior compared to PTV-IMPT plans., Advances in Knowledge: Probabilistic analysis provides a fast and practical method for evaluating VMAT and IMPT plan sensitivity against setup uncertainty. VMAT and robust-optimised IMPT plans have comparable sensitivity to setup uncertainties in conventionally fractionated treatment for NSCLC. more...