Imaging PARP with [18F]rucaparib in pancreatic cancer models.

PURPOSE: Rucaparib, an FDA-approved PARP inhibitor, is used as a single agent in maintenance therapy to provide promising treatment efficacy with an acceptable safety profile in various types of BRCA-mutated cancers. However, not all patients receive the same benefit from rucaparib-maintenance therapy. A predictive biomarker to help with patient selection for rucaparib treatment and predict clinical benefit is therefore warranted. With this aim, we developed [18F]rucaparib, an 18F-labelled isotopologue of rucaparib, and employed it as a PARP-targeting agent for cancer imaging with PET. Here, we report the in vitro and in vivo evaluation of [18F]rucaparib in human pancreatic cancer models. METHOD: We incorporated the positron-emitting 18F isotope into rucaparib, enabling its use as a PET imaging agent. [18F]rucaparib binds to the DNA damage repair enzyme, PARP, allowing direct visualisation and measurement of PARP in cancerous models before and after PARP inhibition or other genotoxic cancer therapies, providing critical information for cancer diagnosis and therapy. Proof-of-concept evaluations were determined in pancreatic cancer models. RESULTS: Uptake of [18F]rucaparib was found to be mainly dependent on PARP1 expression. Induction of DNA damage increased PARP expression, thereby increasing uptake of [18F]rucaparib. In vivo studies revealed relatively fast blood clearance of [18F]rucaparib in PSN1 tumour-bearing mice, with a tumour uptake of 5.5 ± 0.5%ID/g (1 h after i.v. administration). In vitro and in vivo studies showed significant reduction of [18F]rucaparib uptake by addition of different PARP inhibitors, indicating PARP-selective binding. CONCLUSION: Taken together, we demonstrate the potential of [18F]rucaparib as a non-invasive PARP-targeting imaging agent for pancreatic cancers.

Differences in service utilization at an urban tribal health organization before and after Alzheimer's disease or related dementia diagnosis: A cohort study.

INTRODUCTION: The prevalence, mortality, and healthcare impact of Alaska Native and American Indian (ANAI) people with Alzheimer's disease and related dementias (ADRD) are unknown. METHODS: We conducted a cohort study of electronic health record data that compared healthcare service utilization in patients with and without an ADRD diagnosis. Zero-inflated negative binomial regression with robust standard errors was used to estimate utilization rates. RESULTS: Compared with patients without ADRD, utilization rates were similar before but higher after ADRD diagnosis. For those with diagnosed ADRD, utilization increased gradually over time with sharp upward change during the year of diagnosis. DISCUSSION: This is the only study quantifying changes in healthcare service utilization before and after ADRD diagnosis among ANAI people, which is crucial for tailoring geriatric care for ANAI populations.

In Vivo Pretargeted Imaging of HER2 and TAG-72 Expression Using the HaloTag Enzyme.

A novel pretargeted SPECT imaging strategy based on the HaloTag enzyme has been evaluated for the first time in a living system. To determine the efficacy of this approach, two clinically relevant cancer biomarkers, HER2 and TAG-72, were selected to represent models of internalizing and noninternalizing antigens, respectively. In MDA-MB-231/H2N (HER2-expressing) and LS174T (TAG-72-expressing) xenograft tumors in mice, pretargeting experiments were performed in which HaloTag-conjugated derivatives of the antibodies trastuzumab (anti-HER2) or CC49 (anti-TAG-72) were utilized as primary agents, and the small molecule HaloTag ligands 111In-HTL-1, -2, and -3 were evaluated as secondary agents. While this approach was not sufficiently sensitive to detect the internalizing HER2 antigen, pretargeting experiments involving the most optimal secondary agent, 111In-HTL-3, were successful in detecting the noninternalizing antigen TAG-72 and provided high-contrast SPECT images at 4 and 24 h postinjection.

Meal frequency and timing in health and disease.

Although major research efforts have focused on how specific components of foodstuffs affect health, relatively little is known about a more fundamental aspect of diet, the frequency and circadian timing of meals, and potential benefits of intermittent periods with no or very low energy intakes. The most common eating pattern in modern societies, three meals plus snacks every day, is abnormal from an evolutionary perspective. Emerging findings from studies of animal models and human subjects suggest that intermittent energy restriction periods of as little as 16 h can improve health indicators and counteract disease processes. The mechanisms involve a metabolic shift to fat metabolism and ketone production, and stimulation of adaptive cellular stress responses that prevent and repair molecular damage. As data on the optimal frequency and timing of meals crystalizes, it will be critical to develop strategies to incorporate those eating patterns into health care policy and practice, and the lifestyles of the population.

PET imaging of DNA damage using (89)Zr-labelled anti-γH2AX-TAT immunoconjugates.

PURPOSE: The efficacy of most anticancer treatments, including radiotherapy, depends on an ability to cause DNA double-strand breaks (DSBs). Very early during the DNA damage signalling process, the histone isoform H2AX is phosphorylated to form γH2AX. With the aim of positron emission tomography (PET) imaging of DSBs, we synthesized a (89)Zr-labelled anti-γH2AX antibody, modified with the cell-penetrating peptide, TAT, which includes a nuclear localization sequence. METHODS: (89)Zr-anti-γH2AX-TAT was synthesized using EDC/NHS chemistry for TAT peptide linkage. Desferrioxamine conjugation allowed labelling with (89)Zr. Uptake and retention of (89)Zr-anti-γH2AX-TAT was evaluated in the breast adenocarcinoma cell line MDA-MB-468 in vitro or as xenografts in athymic mice. External beam irradiation was used to induce DSBs and expression of γH2AX. Since (89)Zr emits ionizing radiation, detailed radiobiological measurements were included to ensure (89)Zr-anti-γH2AX-TAT itself does not cause any additional DSBs. RESULTS: Uptake of (89)Zr-anti-γH2AX-TAT was similar to previous results using (111)In-anti-γH2AX-TAT. Retention of (89)Zr-anti-γH2AX-TAT was eightfold higher at 1 h post irradiation, in cells expressing γH2AX, compared to non-irradiated cells or to non-specific IgG control. PET imaging of mice showed higher uptake of (89)Zr-anti-γH2AX-TAT in irradiated xenografts, compared to non-irradiated or non-specific controls (12.1 ± 1.6 vs 5.2 ± 1.9 and 5.1 ± 0.8%ID/g, respectively; p < 0.0001). The mean absorbed dose to the nucleus of cells taking up (89)Zr-anti-γH2AX-TAT was twofold lower compared to (111)In-anti-γH2AX-TAT. Additional exposure of neither irradiated nor non-irradiated cells nor tissues to (89)Zr-anti-γH2AX-TAT resulted in any significant changes in the number of observable DNA DSBs, γH2AX foci or clonogenic survival. CONCLUSION: (89)Zr-anti-γH2AX-TAT allows PET imaging of DNA DSBs in a tumour xenograft mouse model.

Imaging DNA damage response by γH2AX in vivo predicts treatment response to Lutetium-177 radioligand therapy and suggests senescence as a therapeutically desirable outcome.

Rationale: An effective absorbed dose response relationship is yet to be established for Lutetium-177 based radionuclide therapies such as 177Lu-DOTATATE and 177Lu-PSMA. The inherent biological heterogeneity of neuroendocrine and prostate cancers may make the prospect of establishing cohort-based dose-response relationships unobtainable. Instead, an individual-based approach, monitoring the dose-response within each tumor could provide the necessary metric to monitor treatment efficacy. Methods: We developed a dual isotope SPECT imaging strategy to monitor the change over time in the relationship between 177Lu-DOTATATE and 111In-anti-γH2AX-TAT, a modified radiolabelled antibody that allows imaging of DNA double strand breaks, in mice bearing rat pancreatic cancer xenografts. The dynamics of γH2AX foci, apoptosis and senescence following exposure to 177Lu-DOTATATE was further investigated in vitro and in ex vivo tumor sections. Results: The change in slope of the 111In-anti-γH2AX-TAT to 177Lu signal between days 5 and 7 was found to be highly predictive of survival (r = 0.955, P < 0.0001). This pivotal timeframe was investigated further in vitro: clonogenic survival correlated with the number of γH2AX foci at day 6 (r = -0.995, P < 0.0005). While there was evidence of continuously low levels of apoptosis, delayed induction of senescence in vitro appeared to better account for the γH2AX response to 177Lu. The induction of senescence was further investigated by ex vivo analysis and corresponded with sustained retention of 177Lu within tumor regions. Conclusions: Dual isotope SPECT imaging can provide individualized tumor dose-responses that can be used to predict lutetium-177 treatment efficacy. This bio-dosimeter metric appears to be dependent upon the extent of senescence induction and suggests an integral role that senescence plays in lutetium-177 treatment efficacy.

Connecting Contemporary Trauma Care to Florence Nightingale's Visionary Work.

The impact of Florence Nightingale's visionary work continues to influence the delivery of nursing care in the contemporary emergency department (ED). Her foundational work in the Crimean War resulted in data-based recommendations for using the environment to promote healing and wellness among sick and wounded British soldiers. She advocated for attention to environmental details, including ventilation, air, warmth, drainage, cleanliness, natural light, and low noise levels. These important environmental concepts play a significant role in the nursing management of trauma patients in today's ED. This article features an application of Nightingale's environmental concepts to a trauma patient case exemplar and demonstrates the enduring impact of her work for trauma patients who receive care in the ED.

A radioiodinated rucaparib analogue as an Auger electron emitter for cancer therapy.

INTRODUCTION: Radioligand therapy (RLT) is an expanding field that has shown great potential in the fight against cancer. Radionuclides that can be carried by selective ligands such as antibodies, peptides, and small molecules targeting cancerous cells have demonstrated a clear improvement in the move towards precision medicine. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage repair signalling pathway, with PARP inhibitors olaparib, talazoparib, niraparib, veliparib, and rucaparib having FDA approval for cancer therapy in routine clinical use. Based on our previous work with the radiolabelled PARP inhibitor [18F]rucaparib, we replaced the fluorine-18 moiety, used for PET imaging, with iodine-123, a radionuclide used for SPECT imaging and Auger electron therapy, resulting in 8-[123I]iodo-5-(4-((methylamino)methyl)phenyl)-2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-1-one, ([123I]GD1), as a potential radiopharmaceutical for RLT. METHODS: [123I]GD1 was synthesized via copper-mediated radioiodination from a selected boronic esters precursor. In vitro uptake, retention, blocking, and effects on clonogenic survival with [123I]GD1 treatment were tested in a panel of cancer cell lines. Enzymatic inhibition of PARP by GD1 was also tested in a cell-free system. The biodistribution of [123I]GD1 was investigated by SPECT/CT in mice following intravenous administration. RESULTS: Cell-free enzymatic inhibition and in vitro blocking experiments confirmed a modest ability of GD1 to inhibit PARP-1, IC50 = 239 nM. In vitro uptake of [123I]GD1 in different cell lines was dose dependent, and radiolabelled compound was retained in cells for >2 h. Significantly reduced clonogenic survival was observed in vitro after exposure of cells for 1 h with as low as 50 kBq of [123I]GD1. The biodistribution of [123I]GD1 was further characterized in vivo showing both renal and hepatobiliary clearance pathways with a biphasic blood clearance. CONCLUSION: We present the development of a new theragnostic agent based on the rucaparib scaffold and its evaluation in in vitro and in vivo models. The data reported show that [123I]GD1 may have potential to be used as a theragnostic agent.

[123I]CC1: A PARP-Targeting, Auger Electron-Emitting Radiopharmaceutical for Radionuclide Therapy of Cancer.

Poly(adenosine diphosphate ribose) polymerase (PARP) has emerged as an effective therapeutic strategy against cancer that targets the DNA damage repair enzyme. PARP-targeting compounds radiolabeled with an Auger electron-emitting radionuclide can be trapped close to damaged DNA in tumor tissue, where high ionizing potential and short range lead Auger electrons to kill cancer cells through the creation of complex DNA damage, with minimal damage to surrounding normal tissue. Here, we report on [123I]CC1, an 123I-labeled PARP inhibitor for radioligand therapy of cancer. Methods: Copper-mediated 123I iododeboronation of a boronic pinacol ester precursor afforded [123I]CC1. The level and specificity of cell uptake and the therapeutic efficacy of [123I]CC1 were determined in human breast carcinoma, pancreatic adenocarcinoma, and glioblastoma cells. Tumor uptake and tumor growth inhibition of [123I]CC1 were assessed in mice bearing human cancer xenografts (MDA-MB-231, PSN1, and U87MG). Results: In vitro and in vivo studies showed selective uptake of [123I]CC1 in all models. Significantly reduced clonogenicity, a proxy for tumor growth inhibition by ionizing radiation in vivo, was observed in vitro after treatment with as little as 10 Bq [123I]CC1. Biodistribution at 1 h after intravenous administration showed PSN1 tumor xenograft uptake of 0.9 ± 0.06 percentage injected dose per gram of tissue. Intravenous administration of a relatively low amount of [123I]CC1 (3 MBq) was able to significantly inhibit PSN1 xenograft tumor growth but was less effective in xenografts that expressed less PARP. [123I]CC1 did not cause significant toxicity to normal tissues. Conclusion: Taken together, these results show the potential of [123I]CC1 as a radioligand therapy for PARP-expressing cancers.

Patterns of Healthcare Use and Mortality After Alzheimer's Disease or Related Dementia Diagnosis Among Alaska Native Patients: Results of a Cluster Analysis in a Tribal Healthcare Setting.

Background: Alaska Native and American Indian (AN/AI) people represent a rapidly aging population with disproportionate burdens of Alzheimer's disease and related dementias (ADRD) risk factors. Objective: To characterize healthcare service use patterns and mortality in the years following ADRD diagnosis for patients in an Alaska Native Tribal health system. Methods: The study sample included all AN/AI patients aged 55 or older with an ADRD diagnosis who were seen between 2012-2018 (n = 407). We used cluster analysis to identify distinct patterns of healthcare use for primary care, emergency and urgent care, inpatient hospital stays, and selected specialty care. We compared demographic and clinical factors between clusters and used regression to compare mortality. Results: We identified five clusters of healthcare service use patterns after ADRD diagnosis: 1) people who use a low amount of all services (n = 107), 2) people who use a high amount of all services (n = 60), 3) people who use a high amount of primary and specialty care (n = 105), 4) people who use a high amount of specialty care (n = 65), and 5) people who use a high amount of emergency and urgent care (n = 70). The cluster with the highest use had the greatest proportion of comorbidities and had a 2.3-fold increased risk of mortality compared to the cluster with the lowest healthcare service use. Conclusion: Results indicate that those receiving the most services had the greatest healthcare-related needs and increased mortality. Future research could isolate factors that predict service use following ADRD diagnosis and identify other differential health risks.

Correlation between molar activity, injection mass and uptake of the PARP targeting radiotracer [18F]olaparib in mouse models of glioma.

PURPOSE: Radiopharmaceuticals targeting poly(ADP-ribose) polymerase (PARP) have emerged as promising agents for cancer diagnosis and therapy. PARP enzymes are expressed in both cancerous and normal tissue. Hence, the injected mass, molar activity and potential pharmacological effects are important considerations for the use of radiolabelled PARP inhibitors for diagnostic and radionuclide therapeutic applications. Here, we performed a systematic evaluation by varying the molar activity of [18F]olaparib and the injected mass of [TotalF]olaparib to investigate the effects on tumour and normal tissue uptake in two subcutaneous human glioblastoma xenograft models. METHODS: [18F]Olaparib uptake was evaluated in the human glioblastoma models: in vitro on U251MG and U87MG cell lines, and in vivo on tumour xenograft-bearing mice, after administration of [TotalF]olaparib (varying injected mass: 0.04-8.0 µg, and molar activity: 1-320 GBq/µmol). RESULTS: Selective uptake of [18F]olaparib was demonstrated in both models. Tumour uptake was found to be dependent on the injected mass of [TotalF]olaparib (µg) but not the molar activity. An injected mass of 1 µg resulted in the highest tumour uptake (up to 6.9 ± 1.3%ID/g), independent of the molar activity. In comparison, both the lower and higher injected masses of [TotalF]olaparib resulted in lower relative tumour uptake (%ID/g; P < 0.05). Ex vivo analysis of U87MG xenograft sections showed that the heterogeneity in [18F]olaparib intratumoural uptake correlated with PARP1 expression. Substantial upregulation of PARP1-3 expression was observed after administration of [TotalF]olaparib (> 0.5 µg). CONCLUSION: Our findings show that the injected mass of [TotalF]olaparib has significant effects on tumour uptake. Moderate injected masses of PARP inhibitor-derived radiopharmaceuticals may lead to improved relative tumour uptake and tumour-to-background ratio for cancer diagnosis and radionuclide therapy.

Immuno-imaging of ICAM-1 in tumours by SPECT.

PURPOSE: Molecular imaging of cancer cells' reaction to radiation damage can provide a non-invasive measure of tumour response to treatment. The cell surface glycoprotein ICAM-1 (CD54) was identified as a potential radiation response marker. SPECT imaging using an 111In-radiolabelled anti-ICAM-1 antibody was explored. METHODS: PSN-1 cells were irradiated (10 Gy), and protein expression changes were investigated using an antibody array on cell lysates 24 h later. Results were confirmed by western blot, flow cytometry and immunofluorescence. We confirmed the affinity of an 111In-labelled anti-ICAM-1 antibody in vitro, and in vivo, in PSN-1-xenograft bearing mice. The xenografts were irradiated (0 or 10 Gy), and [111In]In-anti-ICAM-1 SPECT/CT images were acquired 24, 48 and 72 h after intravenous administration. RESULTS: ICAM-1 was identified as a potential marker of radiation treatment using an antibody array in PSN-1 cell lysates following irradiation, showing a significant increase in ICAM-1 signal compared to non-irradiated cells. Western blot and immunohistochemistry confirmed this upregulation, with an up to 20-fold increase in ICAM-1 signal. Radiolabelled anti-ICAM-1 bound to ICAM-1 expressing cells with good affinity (Kd = 24.0 ± 4.0 nM). [111In]In-anti-ICAM-1 uptake in tumours at 72 h post injection was approximately 3-fold higher than non-specific isotype-matched [111In]In-mIgG2a control (19.3 ± 2.5%ID/g versus 6.3 ± 2.2%ID/g, P = 0.0002). However, ICAM1 levels, and [111In]In-anti-ICAM-1 uptake in tumours was no different after irradiation (uptake 9.2%ID/g versus 14.8%ID/g). Western blots of the xenograft lysates showed no significant differences, confirming these results. CONCLUSION: Imaging of ICAM-1 is feasible in mouse models of pancreatic cancer. Although ICAM-1 is upregulated post-irradiation in in vitro models of pancreatic cancer, it shows little change in expression in an in vivo mouse xenograft model.

Radiolabeled cCPE Peptides for SPECT Imaging of Claudin-4 Overexpression in Pancreatic Cancer.

Overexpression of tight-junction protein claudin-4 has been detected in primary and metastatic pancreatic cancer tissue and is associated with better prognosis in patients. Noninvasive measurement of claudin-4 expression by imaging methods could provide a means for accelerating detection and stratifying patients into risk groups. Clostridium perfringens enterotoxin (CPE) is a natural ligand for claudin-4 and holds potential as a targeting vector for molecular imaging of claudin-4 overexpression. A glutathione S-transferases (GST)-tagged version of the C terminus of CPE (cCPE) was previously used to delineate claudin-4 overexpression by SPECT but showed modest binding affinity and slow blood clearance in vivo. Methods: On the basis of the crystal structure of cCPE, a series of smaller cCPE194-319 mutants with putatively improved binding affinity for claudin-4 was generated by site-directed mutagenesis. All peptides were conjugated site-specifically on a C-terminal cysteine using maleimide-diethylenetriamine pentaacetate to enable radiolabeling with 111In. The binding affinity of all radioconjugates was evaluated in claudin-4-expressing PSN-1 cells and HT1080-negative controls. The specificity of all cCPE mutants to claudin-4 was assessed in HT1080 cells stably transfected with claudin-4. SPECT/CT imaging of BALB/c nude mice bearing PSN-1 or HT1080 tumor xenografts was performed to determine the claudin-4-targeting ability of these peptides in vivo. Results: Uptake of all cCPE-based radioconjugates was significantly higher in PSN-1 cells than in HT1080-negative controls. All peptides showed a marked improvement in affinity for claudin-4 in vitro when compared with previously reported values (dissociation constant: 2.2 ± 0.8, 3 ± 0.1, 4.2 ± 0.5, 10 ± 0.9, and 9.7 ± 0.7 nM). Blood clearance of [111In]In-cCPE194-319, as measured by SPECT, was considerably faster than that of [111In]In-cCPE.GST (half-life, <1 min). All radiopeptides showed significantly higher accumulation in PSN-1 xenografts than in HT1080 tumors at 90 min after injection of the tracer ([111In]In-cCPE194-319, 2.7 ± 0.8 vs. 0.4 ± 0.1 percentage injected dose per gram [%ID/g], P < 0.001; [111In]In-S313A, 2.3 ± 0.9 vs. 0.5 ± 0.1 %ID/g, P < 0.01; [111In]In-S307A + N309A + S313A, 2 ± 0.4 vs. 0.3 ± 0.1 %ID/g, P < 0.01; [111In]In-D284A, 2 ± 0.2 vs. 0.7 ± 0.1 %ID/g, P < 0.05; [111In]In-L254F + K257D, 6.3 ± 0.9 vs. 0.7 ± 0.2 %ID/g, P < 0.001). Conclusion: These optimized cCPE-based SPECT imaging agents show great promise as claudin-4-targeting vectors for in vivo imaging of claudin-4 overexpression in pancreatic cancer.

[18F]AZD2461, an Insight on Difference in PARP Binding Profiles for DNA Damage Response PET Imaging.

BACKGROUND: Poly (ADP-ribose) polymerase (PARP) inhibitors are extensively studied and used as anti-cancer drugs, as single agents or in combination with other therapies. Most radiotracers developed to date have been chosen on the basis of strong PARP1-3 affinity. Herein, we propose to study AZD2461, a PARP inhibitor with lower affinity towards PARP3, and to investigate its potential for PARP targeting in vivo. METHODS: Using the Cu-mediated 18F-fluorodeboronation of a carefully designed radiolabelling precursor, we accessed the 18F-labelled isotopologue of the PARP inhibitor AZD2461. Cell uptake of [18F]AZD2461 in vitro was assessed in a range of pancreatic cell lines (PSN-1, PANC-1, CFPAC-1 and AsPC-1) to assess PARP expression and in vivo in xenograft-bearing mice. Blocking experiments were performed with both olaparib and AZD2461. RESULTS: [18F]AZD2461 was efficiently radiolabelled via both manual and automated procedures (9 % ± 3 % and 3 % ± 1 % activity yields non-decay corrected). [18F]AZD2461 was taken up in vivo in PARP1-expressing tumours, and the highest uptake was observed for PSN-1 cells (7.34 ± 1.16 %ID/g). In vitro blocking experiments showed a lesser ability of olaparib to reduce [18F]AZD2461 binding, indicating a difference in selectivity between olaparib and AZD2461. CONCLUSION: Taken together, we show the importance of screening the PARP selectivity profile of radiolabelled PARP inhibitors for use as PET imaging agents.

Early Detection in a Mouse Model of Pancreatic Cancer by Imaging DNA Damage Response Signaling.

Despite its widespread use in oncology, the PET radiotracer 18F-FDG is ineffective for improving early detection of pancreatic ductal adenocarcinoma (PDAC). An alternative strategy for early detection of pancreatic cancer involves visualization of high-grade pancreatic intraepithelial neoplasias (PanIN-3s), generally regarded as the noninvasive precursors of PDAC. The DNA damage response is known to be hyperactivated in late-stage PanINs. Therefore, we investigated whether the SPECT imaging agent 111In-anti-γH2AX-TAT allows visualization of the DNA damage repair marker γH2AX in PanIN-3s in an engineered mouse model of PDAC, to facilitate early detection of PDAC. Methods: Genetically engineered KPC (KRasLSL.G12D/+; p53LSL.R172H/+; PdxCre) mice were imaged with 18F-FDG and 111In-anti-γH2AX-TAT. The presence of PanIN/PDAC as visualized by histologic examination was compared with autoradiography and immunofluorescence. Separately, the survival of KPC mice imaged with 111In-anti-γH2AX-TAT was evaluated. Results: In KPC mouse pancreata, γH2AX expression was increased in high-grade PanINs but not in PDAC, corroborating earlier results obtained from human pancreas sections. Uptake of 111In-anti-γH2AX-TAT, but not 111In-IgG-TAT or 18F-FDG, within the pancreas correlated positively with the age of KPC mice, which correlated with the number of high-grade PanINs. 111In-anti-γH2AX-TAT localizes preferentially in high-grade PanIN lesions but not in established PDAC. Younger, non-tumor-bearing KPC mice that show uptake of 111In-anti-γH2AX-TAT in the pancreas survive for a significantly shorter time than mice with physiologic 111In-anti-γH2AX-TAT uptake. Conclusion:111In-anti-γH2AX-TAT imaging allows noninvasive detection of DNA damage repair signaling upregulation in preinvasive PanIN lesions and is a promising new tool to aid in the early detection and staging of pancreatic cancer.

Imaging DNA Damage Repair In Vivo After 177Lu-DOTATATE Therapy.

Molecular radiotherapy using 177Lu-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 177Lu-DOTATATE therapy using SPECT imaging with 111In-anti-γH2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with ß-emitting therapeutic radiopharmaceuticals. Methods: Six cell lines were exposed to external-beam radiotherapy (EBRT) or 177Lu-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 177Lu-DOTATATE or sham-treated and coinjected with 111In-anti-γH2AX-TAT, 111In-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 177Lu-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 177Lu-DOTATATE uptake and γH2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to 177Lu-DOTATATE irradiation. Conclusion:111In-anti-γH2AX-TAT allows monitoring of DNA damage after 177Lu-DOTATATE therapy and reveals heterogeneous damage responses.

Dual-isotope imaging allows in vivo immunohistochemistry using radiolabelled antibodies in tumours.

While radiolabelled antibodies have found great utility as PET and SPECT imaging agents in oncological investigations, a notable shortcoming of these agents is their propensity to accumulate non-specifically within tumour tissue. The degree of this non-specific contribution to overall tumour uptake is highly variable and can ultimately lead to false conclusions. Therefore, in an effort to obtain a reliable measure of inter-individual differences in non-specific tumour uptake of radiolabelled antibodies, we demonstrate that the use of dual-isotope imaging overcomes this issue, enables true quantification of epitope expression levels, and allows non-invasive in vivo immunohistochemistry. The approach involves co-administration of (i) an antigen-targeting antibody labelled with zirconium-89 (89Zr), and (ii) an isotype-matched non-specific control IgG antibody labelled with indium-111 (111In). As an example, the anti-HER2 antibody trastuzumab was radiolabelled with 89Zr, and co-administered intravenously together with its 111In-labelled non-specific counterpart to mice bearing human breast cancer xenografts with differing HER2 expression levels (MDA-MB-468 [HER2-negative], MDA-MB-231 [low-HER2], MDA-MB-231/H2N [medium-HER2], and SKBR3 [high-HER2]). Simultaneous PET/SPECT imaging using a MILabs Vector4 small animal scanner revealed stark differences in the intratumoural distribution of [89Zr]Zr-trastuzumab and [111In]In-IgG, highlighting regions of HER2-mediated uptake and non-specific uptake, respectively. Normalisation of the tumour uptake values and tumour-to-blood ratios obtained with [89Zr]Zr-trastuzumab against those obtained with [111In]In-IgG yielded values which were most strongly correlated (R = 0.94; P = 0.02) with HER2 expression levels for each breast cancer type determined by Western blot and in vitro saturation binding assays, but not non-normalised uptake values. Normalised intratumoural distribution of [89Zr]Zr-trastuzumab correlated well with intratumoural heterogeneity HER2 expression.

Tumor Imaging Using Radiolabeled Matrix Metalloproteinase-Activated Anthrax Proteins.

Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111In-radiolabeled PA-L1, 111In-PA-WTK563C, or 111In-LFE687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1-mediated delivery of 111In-LFE687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LFE687A Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.

PET Imaging of PARP Expression Using 18F-Olaparib.

Poly(ADP-ribose) polymerase (PARP) inhibitors are increasingly being studied as cancer drugs, as single agents, or as a part of combination therapies. Imaging of PARP using a radiolabeled inhibitor has been proposed for patient selection, outcome prediction, dose optimization, genotoxic therapy evaluation, and target engagement imaging of novel PARP-targeting agents. Methods: Here, via the copper-mediated 18F-radiofluorination of aryl boronic esters, we accessed, for the first time (to our knowledge), the 18F-radiolabeled isotopolog of the Food and Drug Administration-approved PARP inhibitor olaparib. The use of the 18F-labeled equivalent of olaparib allows direct prediction of the distribution of olaparib, given its exact structural likeness to the native, nonradiolabeled drug. Results:18F-olaparib was taken up selectively in vitro in PARP-1-expressing cells. Irradiation increased PARP-1 expression and 18F-olaparib uptake in a radiation-dose-dependent fashion. PET imaging in mice showed specific uptake of 18F-olaparib in tumors expressing PARP-1 (3.2% ± 0.36% of the injected dose per gram of tissue in PSN-1 xenografts), correlating linearly with PARP-1 expression. Two hours after irradiation of the tumor (10 Gy), uptake of 18F-olaparib increased by 70% (P = 0.025). Conclusion: Taken together, we show that 18F-olaparib has great potential for noninvasive tumor imaging and monitoring of radiation damage.

Imaging of Claudin-4 in Pancreatic Ductal Adenocarcinoma Using a Radiolabelled Anti-Claudin-4 Monoclonal Antibody.

PURPOSE: Despite its widespread use, the positron emission tomography (PET) radiotracer 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been shown in clinical settings to be ineffective for improving early diagnosis of pancreatic ductal adenocarcinoma (PDAC). A promising biomarker for PDAC detection is the tight junction protein claudin-4. The purpose of this study was to evaluate a new single-photon emission computed tomography (SPECT) imaging agent, [111In]anti-claudin-4 mAb, with regard to its ability to allow visualisation of claudin-4 in a xenograft and a genetically engineered mouse model of PDAC. PROCEDURES: The ability of [111In]anti-claudin-4 mAb to selectively target claudin-4 was assessed using two human xenograft tumour models with differential claudin-4 status in mice. [111In]anti-claudin-4 mAb was also used to detect PDAC development in genetically engineered KPC mice. The PDAC status of these mice was confirmed with [18F]FDG-PET, magnetic resonance imaging (MRI), histology, and immunofluorescence microscopy. RESULTS: High uptake of [111In]anti-claudin-4 mAb was observed in PDAC xenografts in mice, reaching 16.9 ± 4.5 % of injected dose per gram (% ID/g) at 72 h post-injection. This uptake was mediated specifically by the expression of claudin-4. Uptake of [111In]anti-claudin-4 mAb also enabled clear visualisation of spontaneous PDAC formation in KPC mice. CONCLUSIONS: [111In]anti-claudin-4 mAb allows non-invasive detection of claudin-4 upregulation during development of PDAC and could potentially be used to aid in the early detection and characterisation of this malignancy.