Mary Sue O'DOrisio

Introduction: Small cell lung cancer (SCLC) has a 7% 5-year overall survival. C-X-C chemokine receptor 4 (CXCR4), an attractive target for theranostic agents, is highly expressed in SCLCs, and can be targeted with pentixather using the theranostic pair ²¹² Pb/ ²⁰³ Pb. The hypothesis that [ ²¹² Pb/ ²⁰³ Pb]-pentixather can be used safely and effectively for imaging and therapy in SCLC in xenograft models was tested. Results: SPECT/CT imaging and biodistribution studies of tumor bearing mice injected with [ ²⁰³ Pb]-pentixather demonstrated CXCR4-dependent uptake in tumors and accumulation of radioligand in kidneys and livers. Dosimetry calculations estimated [ ²¹² Pb]-pentixather uptake in tumor and normal tissue. [ ²¹² Pb]-pentixather treatment (37-111 kBq/g) of SCLC xenografts (DMS273 and H69AR) significantly prolonged survival and delayed tumor growth. When NSG mice grafted with human hCD34+ bone marrow were treated with [ ²¹² Pb]-pentixather (37-111 kBq/g), significant cytopenias were observed in peripheral blood complete blood counts (CBCs) at 13-18 days post treatment which resolved by day 28-31. Flow cytometry of bone marrow hematopeotic stem cells in these animals at day 28-31 demonstrated a significantly reduced frequency of the human hematopoietic marker CD45 (hCD45+) and reconstitution of the bone marrow with murine CD45+ (mCD45+) lineages. Conclusions: [ ²⁰³ Pb]-pentixather can be used to image CXCR4 expressing SCLC xenografts and treatment with alpha emitter [ ²¹² Pb]-pentixather significantly prolongs SCLC xenograft median overall survival. Significantly greater mCD45+ bone marrow repopulation was detected in NSG mice engrafted with human bone marrow 28-31 days following [ ²¹² Pb]-pentixather treatment, relative to hCD45+ bone marrow.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for the study of GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of 6 mice developed tumors (NEC913 and NEC1452). Over 90% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2) whereas NEC1452 PDX tumors do not express SSTR2. Exome sequencing revealed loss of p53 and RB1 in both tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as valuable resource for GEP NEN therapy testing.

Peptide receptor radionuclide therapy (PRRT) is a well-established treatment in somatostatin receptor-expressing neuroendocrine tumours (NETs). The safety and efficacy of PRRT in >79 years old patients (EP) have not been systematically investigated. All patients with inoperable/metastatic/progressive G1/G2 NET, >79 years (EP), treated with PRRT at the University Hospital of Basel between 2006 and 2018, were enrolled in this retrospective matched cohort study. Each patient was manually matched with ≥1 younger patient (YP = 60–70 years). The primary endpoint was toxicity. Toxicity (subacute, long-term) was graded according to the criteria for adverse events (CTCAE) v5.0. All toxicity grades ≥ 3, or whose delta (Δ) to baseline were ≥2, were considered significant. The odds ratio (OR) for developing toxicity was tested for non-inferiority of EP vs. YP. Clinical response to PRRT and overall survival (OS) were assessed as secondary outcome measures. Forty-eight EP and 68 YP were enrolled. Both cohorts were balanced regarding median time since diagnosis, tumour location, grading, treatment scheme, and baseline biochemical parameters, except for eGFR (EP: 61 ± 16 vs. YP: 78 ± 19; mL/min/1.73 m²). Twenty-two grade ≥ 3 or Δ ≥ 2 subacute hematotoxicities occurred in 10 EP (10.3% of cycles) and 37 in 19 YP (11.6% of cycles; p = NS). Long-term grade ≥ 3 renal toxicity occurred in 7 EP and 2 YP (p = NS). The median OS was 3.4 years (EP) vs. 6.0 years (YP), HR: 1.50 [0.75, 2.98], p = NS. PRRT is a valid therapeutic option in elderly NET patients with similar toxicity and non-inferior survival compared to matched younger patients.

Background: Pheochromocytoma and paraganglioma (PHEO/PGL) are rare neuroendocrine tumors which may cause potentially life-threatening complications, with about a third of cases found to harbor specific gene mutations. Thus, early diagnosis, treatment, and meticulous monitoring are of utmost importance. Because of low incidence of succinate dehydrogenase complex subunit A (SDHA)-related metastatic PHEO/PGL, currently there exists insufficient clinical information, especially its diagnostic and treatment characteristics. Methods: Ten patients with SDHA-related metastatic PHEO/PGL were followed-up prospectively and/or retrospectively between January 2010 to July 2018. They underwent biochemical tests (n=10), 123I-MIBG (n=9) scintigraphy, and multiple whole-body positron emission tomography/computed tomography (PET/CT) scans with 68Ga-DOTATATE (n=10), 18F-FDG (n=10), and 18F-FDOPA (n=6) PET/CT. Results: Our findings suggest that these tumors can occur early and at extra-adrenal locations, behave aggressively, and have a tendency to develop metastatic disease within a short period of time. None of our patients had a family history of PHEO/PGL making them appear sporadic. Nine (n=9/10, 90%) patients showed abnormal PHEO/PGL-specific biochemical markers with predominantly noradrenergic and/or dopaminergic phenotype, suggesting their utility in diagnosing and monitoring the disease. Per patient detection rates of 68Ga-DOTATATE (n=10/10), 18F-FDG (n=10/10), 18F-FDOPA (n=5/6) PET/CT, and 123I-MIBG (n=7/9) scintigraphy were 100%, 100%, 83.33%, and 77.77%, respectively. Five of 7 (n=5/7; 71.43%) 123I-MIBG positive patients had minimal 123I-MIBG avidity or detected very few lesions compared to widespread metastatic disease on 18F-FDG PET/CT, implying that diagnosis and treatment with 123/131I-MIBG is not a good option. 68Ga-DOTATATE PET/CT was found to be superior or equal to 18F-FDG PET/CT in 7 (n=7/10, 70.0%) patients and hence is recommended for evaluation and follow-up of these patients. All 7 (n=7/7, 100%) patients who received conventional therapies (chemotherapy, somatostatin analog therapy, radiation therapy, 131I-MIBG, peptide receptor radionuclide therapy) in addition to surgery showed disease progression. Conclusion: In our cohort of patients, SDHA-related metastatic PHEO/PGL followed a disease-course similar to that of SDHB-related metastatic PHEO/PGL showing highly aggressive behavior, similar imaging and biochemical phenotypes, and suboptimal response to conventional therapies. Therefore, we recommend careful surveillance of the affected patients and a search for effective therapies.

The ability to deliver DNA to target cells creating therapeutic effects remains an important goal in the field of gene therapy. A majority of clinical trials to overcome this issue have utilized viral vectors due to their efficiency at DNA delivery and ability to create high levels of gene expression. However, their inherent toxicity and a several clinical trials leading to patients contracting new diseases from the treatment have greatly hindered the progress of viral gene therapy. Non-viral gene delivery agents have a much better safety profile, but are also much less efficient at delivering DNA, leading to low gene expression. The reason for this low expression is the numerous barriers that must be overcome to achieve gene expression: circulation, tissue specific accumulation, internalization, release of DNA cargo, and nuclear localization. While peptides are currently being improved upon, enhancing binding and the ability to protect DNA, they are still deficient when it comes to tissue specificity. Numerous targeting methods, including the use of lectins, antibodies, aptamers, and peptides, have been designed to deliver molecules to a specific research. Research to incorporate targeting ligands onto non-viral gene delivery vectors is abundant in the literature; however, successful site specific gene delivery has not been achieved. The somatostatin receptor 2 (SSTR2) ligand, octreotide, is a well-researched eight amino acid peptide that has extensive SAR data available. Also, the receptors have been well characterized and octreotide is used clinically in the radioscintigraphy imaging of brain tumors. While well researched, there are unexplored opportunities to utilize octreotide to enhance non-viral gene delivery vectors. The overall scope of this thesis is to develop and synthesize non-viral gene delivery peptides conjugated to octreotide creating receptor mediated targeting of DNA polyplexes to create tissue specific accumulation. Initial experiments indicated that attachment of octreotide to the polycationic peptide WK18 does not inhibit affinity for the SSTR2 receptor. Therefore, peptides were designed and synthesized to attach octreotide onto polyacridine peptide (Acr-Lys)6. Polyplex characteristics were unchanged by the incorporation of octreotide, and exhibited very low genotoxic effects compared to the in vitro gene delivery agent PEI. Competitive binding assays suggested a stoichiometric, ligand, and temperature dependent accumulation of polyplex on SSTR2 expressing cells, but gene expression could not be achieved. The success of (Acr-Lys)6octreotide, led to the synthesis of a di-maleimide-PEG attached to each end by (Acr-Lys4)3Acr-Lys-Cys or Cys-Gly5octreotide in attempts to create distance, and better ligand availability for the receptor, by expressing octreotide away from the polyplex. Testing of this peptide in PEGylated polyplex ad-mixtures verified that separating the DNA binding peptide from octreotide did lead to better inhibition of binding to DAOY cells in a competitive binding study. However, transfection assays with this compound showed background levels of gene expression. Although gene expression was not achieved, the synthetic strategy to create a molecule incorporating a DNA binding peptide, ligand, and PEG to create better ligand presentation to its receptor when incorporated into PEGylated polyplexes is an important step in the design of gene delivery vectors.

Objective: Although personalized dosimetry may be desirable for radionuclide therapy treatments, the multiple time samples required to determine the total integrated activity puts a burden on patients and clinic resources. The aim of this paper is to demonstrate that when some prior knowledge is known about the tracer kinetic parameters, the total integrated activity (and thus radiation dose) can be estimated from a single time sample.Methods: Mathematical derivations have been performed to generate equations for the total integrated activity in terms of a single time sample of activity for monoexponential and biexponential clearance. Simulations were performed using both exponential models where the rate constants and associated parameters were randomly sampled from distributions with a known mean. The actual total integrated activity for each random sample was compared with the estimated total integrated activity using the mean value of the parameters. Retrospective analysis of ⁹⁰Y DOTATOC data from a clinical trial provided a comparison of actual kidney dose with the estimated kidney dose using the single time point approach.Results: The optimal sampling time for the single point approach was found to be equal to the mean time of the rate constant. The simulation results for the monoexponential and biexpoential models were similar. Regressions comparing the actual and estimated total integrated activity had very high correlations (r² > 0.95) along with acceptable standard errors of estimate, especially at the optimal sampling point. The retrospective analysis of the ⁹⁰Y DOTATOC data also yielded similar results with an r² = 0.95 and a standard error of estimate of 61 cGy.Conclusions: In situations where there is prior knowledge about the population averages of kinetic parameters, these results suggest that the single time point approach can be used to estimate the total integrated activity and dose with sufficient accuracy to manage radionuclide therapy. This will make personalized dosimetry much easier to perform and more available to the community.

Pretherapy PET with ⁸⁶Y-DOTATOC is considered the ideal dosimetry protocol for ⁹⁰Y-DOTATOC therapy; however, its cost, limited availability, and need for infusion of amino acids to mimic the therapy administration limit its use in the clinical setting. The goal of this study was to develop a dosimetric method for ⁹⁰Y-DOTATOC using ⁹⁰Y-DOTATOC PET/CT and bremsstrahlung SPECT/CT and to determine whether dosimetry-based administered activities differ significantly from standard administered activities. Methods: This was a prospective phase 2 trial of ⁹⁰Y-DOTATOC therapy in patients with somatostatin receptor–positive tumors. ⁹⁰Y-DOTATOC was given in 3 cycles 6–8 wk apart. In the first cycle of therapy, adults received 4.4 GBq and children received 1.85 GBq/m²; the subsequent administered activities were adjusted according to the dosimetry of the preceding cycle so as not to exceed a total kidney dose of 23 Gy and bone marrow dose of 2 Gy. The radiation dose to the kidneys was determined from serial imaging sessions consisting of time-of-flight ⁹⁰Y-DOTATOC PET/CT at 5 h after therapy and ⁹⁰Y-DOTATOC bremsstrahlung SPECT/CT at 6, 24, 48, and 72 h. The PET/CT data were used to measure the absolute concentration of ⁹⁰Y-DOTATOC and to calibrate the bremsstrahlung SPECT kidney clearance data. The radiation dose to the kidneys was determined by multiplying the time-integrated activity (from the fitted biexponential curve of renal clearance of ⁹⁰Y-DOTATOC) with the energy emitted per decay, divided by the mass of the kidneys. Results: The radiation dose to the kidneys per cycle of ⁹⁰Y-DOTATOC therapy was highly variable among patients, ranging from 0.32 to 3.0 mGy/MBq. In 17 (85%) of the 20 adult patients who received the second and the third treatment cycles of ⁹⁰Y-DOTATOC, the administered activity was modified by at least 20% from the starting administered activity. Conclusion: Renal dosimetry of ⁹⁰Y-DOTATOC is feasible using ⁹⁰Y-DOTATOC time-of-flight PET/CT and bremsstrahlung SPECT/CT and has a significant impact on the administered activity in treatment cycles.

Localization of the site of the unknown primary tumor is critical for surgical treatment of patients presenting with neuroendocrine tumor (NET) with metastases. Methods: Forty patients with metastatic NET and unknown primary site underwent ⁶⁸Ga-DOTATOC PET/CT in a single-site prospective study. The ⁶⁸Ga-DOTATOC PET/CT was considered true-positive if the positive primary site was confirmed by histology or follow-up imaging. The scan was considered false-positive if no primary lesion was found corresponding to the ⁶⁸Ga-DOTATOC–positive site. All negative scans for primary tumor were considered false-negative. A scan was classified unconfirmed if ⁶⁸Ga-DOTATOC PET/CT suggested a primary, however, no histology was obtained and imaging follow-up was not confirmatory. Results: The true-positive, false-positive, false-negative, and unconfirmed rates for unknown primary tumor were 38%, 7%, 50%, and 5%, respectively. Conclusion: ⁶⁸Ga-DOTATOC PET/CT is an effective modality in the localization of unknown primary in patients with metastatic NET.