Absorbed Dose-Response Relationship in Patients with Gastroenteropancreatic Neuroendocrine Tumors Treated with [177Lu]Lu-DOTATATE: One Step Closer to Personalized Medicine.

[177Lu]Lu-DOTATATE has been approved for progressive and inoperable gastroenteropancreatic neuroendocrine tumors (GEP-NETs) that overexpress somatostatin receptors. The absorbed doses by limiting organs and tumors can be quantified by serial postinfusion scintigraphy measurements of the γ-emissions from 177Lu. The objective of this work was to explore how postinfusion [177Lu]Lu-DOTATATE dosimetry could influence clinical management by predicting treatment efficacy (tumor shrinkage and survival) and toxicity. Methods: Patients with GEP-NETs treated with [177Lu]Lu-DOTATATE between 2016 and 2022 and who underwent dosimetry were included. Absorbed doses were calculated for healthy organs (liver, kidneys, bone marrow, and spleen) and tumors using PLANET Dose and the local energy deposition method based on serial posttreatment SPECT/CT. Up to 5 lesions per site were selected and measured on images collected at baseline and 3 mo after treatment end (measurement masked to the somatostatin receptor imaging uptake). For toxicity assessment, laboratory parameters were regularly monitored. Clinical data, including time to death or progression, were collected from the patients' health records. Correlations between absorbed doses by organs and toxicity and between absorbed doses by lesions and tumor volume variation were studied using regression models. Results: In total, 35 dosimetric studies were performed in patients with mostly grade 2 (77%) tumors and metastases in liver (89%), lymph nodes (77%), and bone (34%), and 146 lesions were analyzed: 1-9 lesions per patient, mostly liver metastases (65%) and lymph nodes (25%). The median total absorbed dose by tumors was 94.4 Gy. The absorbed doses by tumors significantly decreased between cycles. The absorbed dose by tumors was significantly associated with tumor volume variation (P < 0.001) 3 mo after treatment end, and it was a significant prognostic factor for survival. Toxicity analysis showed a correlation between the decrease of hematologic parameters such as lymphocytes or platelet concentrations and the absorbed doses by the spleen or bone marrow. The mean absorbed dose by the kidneys was not correlated with nephrotoxicity during the studied period. Conclusion: In patients treated with [177Lu]Lu-DOTATATE for GEP-NETs, tumor and healthy organ dosimetry can predict survival and toxicities, thus influencing clinical management.

Gadolinium-Based Nanoparticles Sensitize Ovarian Peritoneal Carcinomatosis to Targeted Radionuclide Therapy.

Ovarian cancer (OC) is the most lethal gynecologic malignancy (5-y overall survival rate, 46%). OC is generally detected when it has already spread to the peritoneal cavity (peritoneal carcinomatosis). This study investigated whether gadolinium-based nanoparticles (Gd-NPs) increase the efficacy of targeted radionuclide therapy using [177Lu]Lu-DOTA-trastuzumab (an antibody against human epidermal growth factor receptor 2). Gd-NPs have radiosensitizing effects in conventional external-beam radiotherapy and have been tested in clinical phase II trials. Methods: First, the optimal activity of [177Lu]Lu-DOTA-trastuzumab (10, 5, or 2.5 MBq) combined or not with 10 mg of Gd-NPs (single injection) was investigated in athymic mice bearing intraperitoneal OC cell (human epidermal growth factor receptor 2-positive) tumor xenografts. Next, the therapeutic efficacy and toxicity of 5 MBq of [177Lu]Lu-DOTA-trastuzumab with Gd-NPs (3 administration regimens) were evaluated. NaCl, trastuzumab plus Gd-NPs, and [177Lu]Lu-DOTA-trastuzumab alone were used as controls. Biodistribution and dosimetry were determined, and Monte Carlo simulation of energy deposits was performed. Lastly, Gd-NPs' subcellular localization and uptake, and the cytotoxic effects of the combination, were investigated in 3 cancer cell lines to obtain insights into the involved mechanisms. Results: The optimal [177Lu]Lu-DOTA-trastuzumab activity when combined with Gd-NPs was 5 MBq. Moreover, compared with [177Lu]Lu-DOTA-trastuzumab alone, the strongest therapeutic efficacy (tumor mass reduction) was obtained with 2 injections of 5 mg of Gd-NPs/d (separated by 6 h) at 24 and 72 h after injection of 5 MBq of [177Lu]Lu-DOTA-trastuzumab. In vitro experiments showed that Gd-NPs colocalized with lysosomes and that their radiosensitizing effect was mediated by oxidative stress and inhibited by deferiprone, an iron chelator. Exposure of Gd-NPs to 177Lu increased the Auger electron yield but not the absorbed dose. Conclusion: Targeted radionuclide therapy can be combined with Gd-NPs to increase the therapeutic effect and reduce the injected activities. As Gd-NPs are already used in the clinic, this combination could be a new therapeutic approach for patients with ovarian peritoneal carcinomatosis.

Radiopharmaceuticals as combinatorial partners for immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of multiple cancer types. However, only a fraction of patients with cancer responds to ICIs employed as stand-alone therapeutics, calling for the development of safe and effective combinatorial regimens to extend the benefits of ICIs to a larger patient population. In addition to exhibiting a good safety and efficacy profile, targeted radionuclide therapy (TRT) with radiopharmaceuticals that specifically accumulate in the tumor microenvironment has been associated with promising immunostimulatory effects that (at least in preclinical cancer models) provide a robust platform for the development of TRT/ICI combinations. We discuss preclinical and clinical findings suggesting that TRT stands out as a promising partner for the development of safe and efficient combinatorial regimens involving ICIs.

Clonogenic assay to measure bystander cytotoxicity of targeted alpha-particle therapy.

Radiation therapy induces targeted effects in the cells that are irradiated and also non-targeted effects (i.e. bystander effects) in non-irradiated cells that are close to or at short distance (<∼1 mm) from irradiated cells. Bystander effects are mediated by intercellular communications and may result in cytotoxic and genotoxic modifications. Their occurrence and relative contribution to the irradiation outcome are influenced by several parameters among which the particle linear energy transfer seems to be prominent. Bystander effects were first observed after external radiation therapy, but have been described also following targeted radionuclide therapy. Therefore, we propose a method to investigate their occurrence in experimental conditions where cells are exposed to radiopharmaceuticals. In this approach, clonogenic cell death is the biological endpoint of the bystander effects caused by irradiation with alpha particles (a potent inducer of the bystander response).

Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy.

PURPOSE: Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts. MATERIALS AND METHODS: B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice. RESULTS: In vitro analysis of bystander cytotoxic effects showed that CM killed about 30-40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects. CONCLUSIONS: This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

A Recanalized Umbilical Vein Hypermetabolic Thrombosis Mimicked an Hepatocellular Carcinoma Recurrence: A New Pitfall?

ABSTRACT: A transarterial left hepatic artery radioembolization involving 90Y microspheres was performed on a cirrhotic man with hypermetabolic 18F-FDG segment III hepatocellular carcinoma. During the 18F-FDG PET/CT follow-up, the disappearance of the hypermetabolic lesion was initially observed. Then, a focal segment III hypermetabolism reappeared mimicking a recurrence before disappearing without any treatment. Finally, the hepatic MRI demonstrated that the transitory segment III hypermetabolism matched a thrombus of the dilated recanalized umbilical vein.

Immunostimulatory effects of radioimmunotherapy.

Radiation therapy (RT) is known for its ability to kill cancer cells in an immunogenic manner. Recent preclinical data demonstrate that targeted alpha-particle therapy shares with RT the capacity to elicit immunostimulatory effects, standing out as a promising strategy to circumvent immune checkpoint inhibitor resistance in immunologically 'cold' tumors.

From the target cell theory to a more integrated view of radiobiology in Targeted radionuclide therapy: The Montpellier group's experience.

Targeted radionuclide therapy (TRT) is used to treat disseminated or metastatic tumours in which conventional external beam radiotherapy (EBRT) would have unacceptable side effects. Unlike EBRT, TRT delivers low doses at a continuous low dose rate. In EBRT, the effect increases progressively with the dose rate, and biological effects (tumour control and normal tissue damage) are related to the dose according to a sigmoid curve model. This model is part of the so-called quantitative radiobiology that is mostly based on the target cell theory, according to which cell death is due to (lethal) radiation hits to vital cellular targets. This model was developed for EBRT, but was adapted to low dose-rate situations by including a parameter that reflects the time needed to repair tissue damage. However, a growing body of evidence indicates that the model should take into account also the biological effects, which are due to intercellular communications (bystander effects) and amplify the effects of radiation, as well as the immune system. Moreover, extranuclear targets must be considered, although induced intracellular and intercellular signalling pathways may ultimately result in DNA damage. It is likely that bystander effects and immune response always contribute to the overall response to TRT at different levels, and that dose and dose rate are key parameters in controlling their real contribution. We hypothesize that the dose rate is the key determinant in the balance between the physical and DNA-centred response on one side, and the biological response that integrates all subcellular compartments and intercellular signalling pathways on the other side.

Therapeutic antibodies - natural and pathological barriers and strategies to overcome them.

Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment.

In Silico Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach.

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as "clustered/complex DNA damage" and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.

Revisiting the Radiobiology of Targeted Alpha Therapy.

Targeted alpha therapy (TAT) using alpha particle-emitting radionuclides is in the spotlight after the approval of 223RaCl2 for patients with metastatic castration-resistant prostate cancer and the development of several alpha emitter-based radiopharmaceuticals. It is acknowledged that alpha particles are highly cytotoxic because they produce complex DNA lesions. Hence, the nucleus is considered their critical target, and many studies did not report any effect in other subcellular compartments. Moreover, their physical features, including their range in tissues (<100 µm) and their linear energy transfer (50-230 keV/µm), are well-characterized. Theoretically, TAT is indicated for very small-volume, disseminated tumors (e.g., micrometastases, circulating tumor cells). Moreover, due to their high cytotoxicity, alpha particles should be preferred to beta particles and X-rays to overcome radiation resistance. However, clinical studies showed that TAT might be efficient also in quite large tumors, and biological effects have been observed also away from irradiated cells. These distant effects are called bystander effects when occurring at short distance (<1 mm), and systemic effects when occurring at much longer distance. Systemic effects implicate the immune system. These findings showed that cells can die without receiving any radiation dose, and that a more complex and integrated view of radiobiology is required. This includes the notion that the direct, bystander and systemic responses cannot be dissociated because DNA damage is intimately linked to bystander effects and immune response. Here, we provide a brief overview of the paradigms that need to be revisited.

Targeted Radionuclide Therapy Using Auger Electron Emitters: The Quest for the Right Vector and the Right Radionuclide.

Auger electron emitters (AEEs) are attractive tools in targeted radionuclide therapy to specifically irradiate tumour cells while sparing healthy tissues. However, because of their short range, AEEs need to be brought close to sensitive targets, particularly nuclear DNA, and to a lower extent, cell membrane. Therefore, radioimmunoconjugates (RIC) have been developed for specific tumour cell targeting and transportation to the nucleus. Herein, we assessed, in A-431CEA-luc and SK-OV-31B9 cancer cells that express low and high levels of HER2 receptors, two 111In-RIC consisting of the anti-HER2 antibody trastuzumab conjugated to NLS or TAT peptides for nuclear delivery. We found that NLS and TAT peptides improved the nuclear uptake of 111In-trastuzumab conjugates, but this effect was limited and non-specific. Moreover, it did not result in a drastic decrease of clonogenic survival. Indium-111 also contributed to non-specific cytotoxicity in vitro due to conversion electrons (30% of the cell killing). Comparison with [125I]I-UdR showed that the energy released in the cell nucleus by increasing the RIC's nuclear uptake or by choosing an AEE that releases more energy per decay should be 5 to 10 times higher to observe a significant therapeutic effect. Therefore, new Auger-based radiopharmaceuticals need to be developed.

Radiation-Induced Immunity and Toxicities: The Versatility of the cGAS-STING Pathway.

In the past decade, radiation therapy (RT) entered the era of personalized medicine, following the striking improvements in radiation delivery and treatment planning optimization, and in the understanding of the cancer response, including the immunological response. The next challenge is to identify the optimal radiation regimen(s) to induce a clinically relevant anti-tumor immunity response. Organs at risks and the tumor microenvironment (e.g. endothelial cells, macrophages and fibroblasts) often limit the radiation regimen effects due to adverse toxicities. Here, we reviewed how RT can modulate the immune response involved in the tumor control and side effects associated with inflammatory processes. Moreover, we discussed the versatile roles of tumor microenvironment components during RT, how the innate immune sensing of RT-induced genotoxicity, through the cGAS-STING pathway, might link the anti-tumor immune response, radiation-induced necrosis and radiation-induced fibrosis, and how a better understanding of the switch between favorable and deleterious events might help to define innovative approaches to increase RT benefits in patients with cancer.

Synthesis and in vitro antitumour activity of carboplatin analogues containing functional handles compatible for conjugation to drug delivery systems.

We describe herein the synthesis of a series of carboplatin derivatives with different functional groups at position 3 of the cyclobutane ring. This pharmacomodulation approach aims at facilitating the vectorisation of these analogues, via their subsequent conjugation to a drug delivery system. Five different derivatives bearing a hydroxy, keto, iodo, azido or amino function at position 3 were synthesised. One of these compounds was coupled to a bifunctional maleimide-containing linker. All compounds were tested in vitro for their cytotoxicity on four different cell lines including two platinum-resistant colorectal cancer cell line (SK-OV-3, HCT116, D3E2, D5B7) using an MTS assay. Overall, the tested compounds were up to six times more potent than carboplatin, especially on D5B7 human colorectal cancer cells. We demonstrated that these modifications led to potent analogues which are compatible with conjugation to a drug delivery system.

Comparison of commercial dosimetric software platforms in patients treated with 177 Lu-DOTATATE for peptide receptor radionuclide therapy.

PURPOSE: The aim of this study was to quantitatively compare five commercial dosimetric software platforms based on the analysis of clinical datasets of patients who benefited from peptide receptor radionuclide therapy (PRRT) with 177 Lu-DOTATATE (LUTATHERA® ). METHODS: The dosimetric analysis was performed on two patients during two cycles of PRRT with 177 Lu. Single photon emission computed tomography/computed tomography images were acquired at 4, 24, 72, and 192 h post injection. Reconstructed images were generated using Dosimetry Toolkit® (DTK) from Xeleris™ and HybridRecon-Oncology version_1.3_Dicom (HROD) from HERMES. Reconstructed images using DTK were analyzed using the same software to calculate time-integrated activity coefficients (TIAC), and mean absorbed doses were estimated using OLINDA/EXM V1.0 with mass correction. Reconstructed images from HROD were uploaded into PLANET® OncoDose from DOSIsoft, STRATOS from Phillips, Hybrid Dosimetry Module™ from HERMES, and SurePlan™ MRT from MIM. Organ masses, TIACs, and mean absorbed doses were calculated from each application using their recommendations. RESULTS: The majority of organ mass estimates varied by <9.5% between all platforms. The highest variability for TIAC results between platforms was seen for the kidneys (28.2%) for the two patients and the two treatment cycles. Relative standard deviations in mean absorbed doses were slightly higher compared with those observed for TIAC, but remained of the same order of magnitude between all platforms. CONCLUSIONS: When applying a similar processing approach, results obtained were of the same order of magnitude regardless of the platforms used. However, the comparison of the performances of currently available platforms is still difficult as they do not all address the same parts of the dosimetric analysis workflow. In addition, the way in which data are handled in each part of the chain from data acquisition to absorbed doses may be different, which complicates the comparison exercise. Therefore, the dissemination of commercial solutions for absorbed dose calculation calls for the development of tools and standards allowing for the comparison of the performances between dosimetric software platforms.

The therapeutic effectiveness of 177Lu-lilotomab in B-cell non-Hodgkin lymphoma involves modulation of G2/M cell cycle arrest.

Some patients with B-cell non-Hodkin lymphoma Lymphoma (NHL) become refractory to rituximab (anti-CD20 antibody) therapy associated with chemotherapy. Here, the effect of the anti-CD37 antibody-radionuclide conjugate lutetium-177 (177Lu)-lilotomab (Betalutin®) was investigated in preclinical models of NHL. In SCID mice bearing DOHH2 (transformed follicular lymphoma, FL) cell xenografts, 177Lu-lilotomab significantly delayed tumor growth, even at low activity (100 MBq/kg). In athymic mice bearing OCI-Ly8 (diffuse large B-cell lymphoma, DLBCL) or Ramos (Burkitt's lymphoma) cell xenografts, 177Lu-lilotomab activity had to be increased to 500 MBq/kg to show a significant tumor growth delay. Clonogenic and proliferation assays showed that DOHH2 cells were highly sensitive to 177Lu-lilotomab, while Ramos cells were the least sensitive, and U2932 (DLBCL), OCI-Ly8, and Rec-1 (mantle cell lymphoma) cells displayed intermediate sensitivity. The strong 177Lu-lilotomab cytotoxicity observed in DOHH2 cells correlated with reduced G2/M cell cycle arrest, lower WEE-1- and MYT-1-mediated phosphorylation of cyclin-dependent kinase-1 (CDK1), and higher apoptosis. In agreement, 177Lu-lilotomab efficacy in vitro, in vivo, and in patient samples was increased when combined with G2/M cell cycle arrest inhibitors (MK-1775 and PD-166285). These results indicate that 177Lu-lilotomab is particularly efficient in treating tumors with reduced inhibitory CDK1 phosphorylation, such as transformed FL.

Pretargeted radioimmunotherapy and SPECT imaging of peritoneal carcinomatosis using bioorthogonal click chemistry: probe selection and first proof-of-concept.

Rationale : Pretargeted radioimmunotherapy (PRIT) based upon bioorthogonal click chemistry has been investigated for the first time in the context of peritoneal carcinomatosis using a CEA-targeting 35A7 mAb bearing trans-cyclooctene (TCO) moieties and several 177Lu-labeled tetrazine (Tz) radioligands. Starting from three Tz probes containing PEG linkers of varying lengths between the DOTA and Tz groups (i.e. PEGn = 3, 7, or 11, respectively, for Tz-1, Tz-2, and Tz-3), we selected [177Lu]Lu-Tz-2 as the most appropriate for pretargeted SPECT imaging and demonstrated its efficacy in tumor growth control. Methods: An orthotopic model of peritoneal carcinomatosis (PC) was obtained following the intraperitoneal (i.p.) injection of A431-CEA-Luc cells in nude mice. Tumor growth was assessed using bioluminescence imaging. Anti-CEA 35A7 mAb was grafted with 2-3 TCO per immunoglobulin. Pretargeted SPECT imaging and biodistribution experiments were performed to quantify the activity concentrations of [177Lu]Lu-Tz-1-3 in tumors and non-target organs to determine the optimal Tz probe for the PRIT of PC. Results: The pharmacokinetic profiles of [177Lu]Lu-Tz-1-3 alone were determined using both SPECT imaging and biodistribution experiments. These data revealed that [177Lu]Lu-Tz-1 was cleared via both the renal and hepatic systems, while [177Lu]Lu-Tz-2 and [177Lu]Lu-Tz-3 were predominantly excreted via the renal system. In addition, these results illuminated that the longer the PEG linker, the more rapidly the Tz radioligand was cleared from the peritoneal cavity. The absorbed radiation dose corresponding to pretargeting with 35A7-TCO followed 24 h later by [177Lu]Lu-Tz-1-4 was higher for tumors following the administration of [177Lu]Lu-Tz-2 (i.e. 0.59 Gy/MBq) compared to either [177Lu]Lu-Tz-1 (i.e. 0.25 Gy/MBq) and [177Lu]Lu-Tz-3 (i.e. 0.18 Gy/MBq). In a longitudinal PRIT study, we showed that the i.p. injection of 40 MBq of [177Lu]Lu-Tz-2 24 hours after the systemic administration of 35A7-TCO significantly slowed tumor growth compared to control mice receiving only saline or 40 MBq of [177Lu]Lu-Tz-2 alone. Ex vivo measurement of the peritoneal carcinomatosis index (PCI) confirmed that PRIT significantly reduced tumor growth (PCI = 15.5 ± 2.3 after PRIT vs 30.0 ± 2.3 and 30.8 ± 1.4 for the NaCl and [177Lu]Lu-Tz-2 alone groups, respectively). Conclusion : Our results clearly demonstrate the impact of the length of PEG linkers upon the biodistribution profiles of 177Lu-labeled Tz radioligands. Furthermore, we demonstrated for the first time the possibility of using bioorthogonal chemistry for both the pretargeted SPECT and PRIT of peritoneal carcinomatosis.