Voxel-Level Dosimetry for Combined Iodine 131 Radiopharmaceutical Therapy and External Beam Radiation Therapy Treatment Paradigms for Head and Neck Cancer.

PURPOSE: Targeted radiopharmaceutical therapy (RPT) in combination with external beam radiation therapy (EBRT) shows promise as a method to increase tumor control and mitigate potential high-grade toxicities associated with re-treatment for patients with recurrent head and neck cancer. This work establishes a patient-specific dosimetry framework that combines Monte Carlo-based dosimetry from the 2 radiation modalities at the voxel level using deformable image registration (DIR) and radiobiological constructs for patients enrolled in a phase 1 clinical trial combining EBRT and RPT. METHODS AND MATERIALS: Serial single-photon emission computed tomography (SPECT)/computed tomography (CT) patient scans were performed at approximately 24, 48, 72, and 168 hours postinjection of 577.2 MBq/m2 (15.6 mCi/m2) CLR 131, an iodine 131-containing RPT agent. Using RayStation, clinical EBRT treatment plans were created with a treatment planning CT (TPCT). SPECT/CT images were deformably registered to the TPCT using the Elastix DIR module in 3D Slicer software and assessed by measuring mean activity concentrations and absorbed doses. Monte Carlo EBRT dosimetry was computed using EGSnrc. RPT dosimetry was conducted using RAPID, a GEANT4-based RPT dosimetry platform. Radiobiological metrics (biologically effective dose and equivalent dose in 2-Gy fractions) were used to combine the 2 radiation modalities. RESULTS: The DIR method provided good agreement for the activity concentrations and calculated absorbed dose in the tumor volumes for the SPECT/CT and TPCT images, with a maximum mean absorbed dose difference of -11.2%. Based on the RPT absorbed dose calculations, 2 to 4 EBRT fractions were removed from patient EBRT treatments. For the combined treatment, the absorbed dose to target volumes ranged from 57.14 to 75.02 Gy. When partial volume corrections were included, the mean equivalent dose in 2-Gy fractions to the planning target volume from EBRT + RPT differed -3.11% to 1.40% compared with EBRT alone. CONCLUSIONS: This work demonstrates the clinical feasibility of performing combined EBRT + RPT dosimetry on TPCT scans. Dosimetry guides treatment decisions for EBRT, and this work provides a bridge for the same paradigm to be implemented within the rapidly emerging clinical RPT space.

Targeting of Head and Neck Cancer by Radioiodinated CLR1404 in Murine Xenograft Tumor Models with Partial Volume Corrected Theranostic Dosimetry.

Background: Delivery of radiotherapeutic dose to recurrent head and neck cancer (HNC) is primarily limited by locoregional toxicity in conventional radiotherapy. As such, HNC patients stand to benefit from the conformal targeting of primary and remnant disease achievable with radiopharmaceutical therapies. In this study, the authors investigated the tumor targeting capacity of 131I-CLR1404 (iopofosine I-131) in various HNC xenograft mouse models and the impact of partial volume correction (PVC) on theranostic dosimetry based on 124I-CLR1404 (CLR 124) positron emission tomography (PET)/computed tomography (CT) imaging. Methods: Mice bearing flank tumor xenograft models of HNC (six murine cell line and six human patient derived) were intravenously administered 6.5-9.1 MBq of CLR 124 and imaged five times over the course of 6 d using microPET/CT. In vivo tumor uptake of CLR 124 was assessed and PVC for 124I was applied using a novel preclinical phantom. Using subject-specific theranostic dosimetry estimations for iopofosine I-131 based on CLR 124 imaging, a discrete radiation dose escalation study (2, 4, 6, and 8 Gy) was performed to evaluate tumor growth response to iopofosine I-131 relative to a single fraction of external beam radiation therapy (6 Gy). Results: PET imaging demonstrated consistent tumor selective uptake and retention of CLR 124 across all HNC xenograft models. Peak uptake of 4.4% ± 0.8% and 4.2% ± 0.4% was observed in squamous cell carcinoma-22B and UW-13, respectively. PVC application increased uptake measures by 47%-188% and reduced absolute differences between in vivo and ex vivo uptake measurements from 3.3% to 1.0 percent injected activity per gram. Tumor dosimetry averaged over all HNC models was 0.85 ± 0.27 Gy/MBq (1.58 ± 0.46 Gy/MBq with PVC). Therapeutic iopofosine I-131 studies demonstrated a variable, but linear relationship between iopofosine I-131 radiation dose and tumor growth delay (p < 0.05). Conclusions: Iopofosine I-131 demonstrated tumoricidal capacity in preclinical HNC tumor models and the theranostic pairing with CLR 124 presents a promising new treatment approach for personalizing administration of iopofosine I-131.

Antitumor efficacy of 90Y-NM600 targeted radionuclide therapy and PD-1 blockade is limited by regulatory T cells in murine prostate tumors.

BACKGROUND: Systemic radiation treatments that preferentially irradiate cancer cells over normal tissue, known as targeted radionuclide therapy (TRT), have shown significant potential for treating metastatic prostate cancer. Preclinical studies have demonstrated the ability of external beam radiation therapy (EBRT) to sensitize tumors to T cell checkpoint blockade. Combining TRT approaches with immunotherapy may be more feasible than combining with EBRT to treat widely metastatic disease, however the effects of TRT on the prostate tumor microenvironment alone and in combinfation with checkpoint blockade have not yet been studied. METHODS: C57BL/6 mice-bearing TRAMP-C1 tumors and FVB/NJ mice-bearing Myc-CaP tumors were treated with a single intravenous administration of either low-dose or high-dose 90Y-NM600 TRT, and with or without anti-PD-1 therapy. Groups of mice were followed for tumor growth while others were used for tissue collection and immunophenotyping of the tumors via flow cytometry. RESULTS: 90Y-NM600 TRT was safe at doses that elicited a moderate antitumor response. TRT had multiple effects on the tumor microenvironment including increasing CD8 +T cell infiltration, increasing checkpoint molecule expression on CD8 +T cells, and increasing PD-L1 expression on myeloid cells. However, PD-1 blockade with TRT treatment did not improve antitumor efficacy. Tregs remained functional up to 1 week following TRT, but CD8 +T cells were not, and the suppressive function of Tregs increased when anti-PD-1 was present in in vitro studies. The combination of anti-PD-1 and TRT was only effective in vivo when Tregs were depleted. CONCLUSIONS: Our data suggest that the combination of 90Y-NM600 TRT and PD-1 blockade therapy is ineffective in these prostate cancer models due to the activating effect of anti-PD-1 on Tregs. This finding underscores the importance of thorough understanding of the effects of TRT and immunotherapy combinations on the tumor immune microenvironment prior to clinical investigation.

Validation of Monte Carlo 131 I radiopharmaceutical dosimetry workflow using a 3D-printed anthropomorphic head and neck phantom.

PURPOSE: Approximately 50% of head and neck cancer (HNC) patients will experience loco-regional disease recurrence following initial courses of therapy. Retreatment with external beam radiotherapy (EBRT) is technically challenging and may be associated with a significant risk of irreversible damage to normal tissues. Radiopharmaceutical therapy (RPT) is a potential method to treat recurrent HNC in conjunction with EBRT. Phantoms are used to calibrate and add quantification to nuclear medicine images, and anthropomorphic phantoms can account for both the geometrical and material composition of the head and neck. In this study, we present the creation of an anthropomorphic, head and neck, nuclear medicine phantom, and its characterization for the validation of a Monte Carlo, SPECT image-based, 131 I RPT dosimetry workflow. METHODS: 3D-printing techniques were used to create the anthropomorphic phantom from a patient CT dataset. Three 131 I SPECT/CT imaging studies were performed using a homogeneous, Jaszczak, and an anthropomorphic phantom to quantify the SPECT images using a GE Optima NM/CT 640 with a high energy general purpose collimator. The impact of collimator detector response (CDR) modeling and volume-based partial volume corrections (PVCs) upon the absorbed dose was calculated using an image-based, Geant4 Monte Carlo RPT dosimetry workflow and compared against a ground truth scenario. Finally, uncertainties were quantified in accordance with recent EANM guidelines. RESULTS: The 3D-printed anthropomorphic phantom was an accurate re-creation of patient anatomy including bone. The extrapolated Jaszczak recovery coefficients were greater than that of the 3D-printed insert (∼22.8 ml) for both the CDR and non-CDR cases (with CDR: 0.536 vs. 0.493, non-CDR: 0.445 vs. 0.426, respectively). Utilizing Jaszczak phantom PVCs, the absorbed dose was underpredicted by 0.7% and 4.9% without and with CDR, respectively. Utilizing anthropomorphic phantom recovery coefficient overpredicted the absorbed dose by 3% both with and without CDR. All dosimetry scenarios that incorporated PVC were within the calculated uncertainty of the activity. The uncertainties in the cumulative activity ranged from 23.6% to 106.4% for Jaszczak spheres ranging in volume from 0.5 to 16 ml. CONCLUSION: The accuracy of Monte Carlo-based dosimetry for 131 I RPT in HNC was validated with an anthropomorphic phantom. In this study, it was found that Jaszczak-based PVCs were sufficient. Future applications of the phantom could involve 3D printing and characterizing patient-specific volumes for more personalized RPT dosimetry estimates.

Safety and feasibility of an in situ vaccination and immunomodulatory targeted radionuclide combination immuno-radiotherapy approach in a comparative (companion dog) setting.

RATIONALE: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. METHODS: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. RESULTS: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. CONCLUSIONS: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.

Low-dose targeted radionuclide therapy renders immunologically cold tumors responsive to immune checkpoint blockade.

Molecular and cellular effects of radiotherapy on tumor microenvironment (TME) can help prime and propagate antitumor immunity. We hypothesized that delivering radiation to all tumor sites could augment response to immunotherapies. We tested an approach to enhance response to immune checkpoint inhibitors (ICIs) by using targeted radionuclide therapy (TRT) to deliver radiation semiselectively to tumors. NM600, an alkylphosphocholine analog that preferentially accumulates in most tumor types, chelates a radioisotope and semiselectively delivers it to the TME for therapeutic or diagnostic applications. Using serial 86Y-NM600 positron emission tomography (PET) imaging, we estimated the dosimetry of 90Y-NM600 in immunologically cold syngeneic murine models that do not respond to ICIs alone. We observed strong therapeutic efficacy and reported optimal dose (2.5 to 5 gray) and sequence for 90Y-NM600 in combination with ICIs. After combined treatment, 45 to 66% of mice exhibited complete response and tumor-specific T cell memory, compared to 0% with 90Y-NM600 or ICI alone. This required expression of STING in tumor cells. Combined TRT and ICI activated production of proinflammatory cytokines in the TME, promoted tumor infiltration by and clonal expansion of CD8+ T cells, and reduced metastases. In mice bearing multiple tumors, combining TRT with moderate-dose (12 gray) external beam radiotherapy (EBRT) targeting a single tumor augmented response to ICIs compared to combination of ICIs with either TRT or EBRT alone. The safety of TRT was confirmed in a companion canine study. Low-dose TRT represents a translatable approach to promote response to ICIs for many tumor types, regardless of location.

Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy.

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.

Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain.

Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2+, immunologically "cold") melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50-150 mm3) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 µg daily, treatment days 6-10), and anti-CTLA-4 (100 µg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8+ and CD4+) and monocytes/macrophages (F4/80+) but no changes in FOXP3+ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (86Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic ß-particle-emitting 90Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies.

Preclinical Pharmacokinetics and Dosimetry Studies of 124I/131I-CLR1404 for Treatment of Pediatric Solid Tumors in Murine Xenograft Models.

Cancer is the second leading cause of death for children between the ages of 5 and 14 y. For children diagnosed with metastatic or recurrent solid tumors, for which the utility of external-beam radiotherapy is limited, the prognosis is particularly poor. The availability of tumor-targeting radiopharmaceuticals for molecular radiotherapy (MRT) has demonstrated improved outcomes in these patient populations, but options are nonexistent or limited for most pediatric solid tumors. 18-(p-iodophenyl)octadecylphosphocholine (CLR1404) is a novel antitumor alkyl phospholipid ether analog that broadly targets cancer cells. In this study, we evaluated the in vivo pharmacokinetics of 124I-CLR1404 (CLR 124) and estimated theranostic dosimetry for 131I-CLR1404 (CLR 131) MRT in murine xenograft models of the pediatric solid tumors neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Methods: Tumor-bearing mice were imaged with small-animal PET/CT to evaluate the whole-body distribution of CLR 124 and, correcting for differences in radioactive decay, predict that of CLR 131. Image volumes representing CLR 131 provided input for Geant4 Monte Carlo simulations to calculate subject-specific tumor dosimetry for CLR 131 MRT. Pharmacokinetics for CLR 131 were extrapolated to adult and pediatric humans to estimate normal-tissue dosimetry. In neuroblastoma, a direct comparison of CLR 124 with 124I-metaiodobenzylguanidine (124I-MIBG) in an MIBG-avid model was performed. Results: In vivo pharmacokinetics of CLR 124 showed selective uptake and prolonged retention across all pediatric solid tumor models investigated. Subject-specific tumor dosimetry for CLR 131 MRT presents a correlative relationship with tumor-growth delay after CLR 131 MRT. Peak uptake of CLR 124 was, on average, 22% higher than that of 124I-MIBG in an MIBG-avid neuroblastoma model. Conclusion: CLR1404 is a suitable theranostic scaffold for dosimetry and therapy with potentially broad applicability in pediatric oncology. Given the ongoing clinical trials for CLR 131 in adults, these data support the development of pediatric clinical trials and provide detailed dosimetry that may lead to improved MRT treatment planning.

Targeted Molecular Radiotherapy of Pediatric Solid Tumors Using a Radioiodinated Alkyl-Phospholipid Ether Analog.

External-beam radiotherapy plays a critical role in the treatment of most pediatric solid tumors. Particularly in children, achieving an optimal therapeutic index to avoid damage to normal tissue is extremely important. Consequently, in metastatic disease, the utility of external-beam radiotherapy is limited. Molecular radiotherapy with tumor-targeted radionuclides may overcome some of these challenges, but to date there exists no single cancer-selective agent capable of treating various pediatric malignancies independently of their histopathologic origin. We tested the therapeutic potential of the clinical-grade alkyl-phospholipid ether analog CLR1404, 18-(p-iodophenyl)octadecyl phosphocholine, as a scaffold for tumor-targeted radiotherapy of pediatric malignancies. Methods: Uptake of CLR1404 by pediatric solid tumor cells was tested in vitro by flow cytometry and in vivo by PET/CT imaging and dosimetry. The therapeutic potential of 131I-CLR1404 was evaluated in xenograft models. Results: In vitro, fluorescent CLR1404-BODIPY showed significant selective uptake in a variety of pediatric cancer lines compared with normal controls. In vivo tumor-targeted uptake in mouse xenograft models using 124I-CLR1404 was confirmed by imaging. Single-dose intravenous injection of 131I-CLR1404 significantly delayed tumor growth in all rodent pediatric xenograft models and extended animal survival while demonstrating a favorable side effect profile. Conclusion:131I-CLR1404 has the potential to become a tumor-targeted radiotherapeutic drug with broad applicability in pediatric oncology. Because 131I-CLR1404 has entered clinical trials in adults, our data warrant the development of pediatric clinical trials for this particularly vulnerable patient population.

Optimisation of a pyrazole series of progesterone antagonists; Part 1.

The design and synthesis of a novel series of non-steroidal progesterone receptor antagonists is described. Ligand-lipophilicity efficiency (LLE) was used in the selection of a prototype agent for in vivo pharmacology studies.