Systematic dose-effect studies of radionuclide and external beam therapies for cancer treatment

Student thesis: Doctoral ThesisDoctor of Philosophy


Introduction: Contrary to External Beam Radiation Therapy (EBRT), the radiobiological effects in cells and tissues once exposed to Auger Electron (AE)-emitters technetium-99m (99mTc) and iodine-123 (123I) and Beta (β)─ particle emitter rhenium-188 (188Re) and their relationship with the absorbed dose are poorly understood. Yet this knowledge is crucial to predict treatment outcome and reassess safety of radionuclides used for imaging. In this PhD thesis, I aimed to correlate in vitro and in vivo radiotherapeutic effect induced by 99mTc, and 123I and 188Re with the absorbed dose in comparison to EBRT.

Methods: Triple negative breast cancer cells (MDA-MB-231) expressing the human sodium/iodide symporter (hNIS) fused to monomeric green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used as a model to enable the controlled cellular uptake of radionuclides in the chemical forms that are substrates for hNIS. This novel model enabled the systematic comparison of the AE-emitters [99mTc]TcO4─ and [123I]I─ and the β─-emitter [188Re]ReO4─ throughout Chapters 2-4). Firstly, in Chapter 2, in vitro radiotoxicity of [99mTc]TcO4─ at 30 min and 24 hours was determined using clonogenic and γ- H2AX assays with activity concentrations up to 4 MBq/mL. Uptake, efflux, and subcellular location of [99mTc]TcO4─ were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose (MIRD) formalism and establish dose-response curves. [99mTc]TcO4─ in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging. Additionally, in Chapter 3, in vitro radiotoxicity of [99mTc]TcO4─, [123I]I─ and [188Re]ReO4─ with activity concentrations up to 4 MBq/mL following 24 and 72 hours was determined using clonogenic assays.

The nuclear-absorbed dose and the dose-effect relationships were determined as described in Chapter 2. The daughter radionuclides technetium-99, tellurium-123 or osmium-188 and EBRT served as controls in Chapter 2 and
3. Finally, in Chapter 4, in vivo longitudinal SPECT/CT imaging with ~55 and of [123I]I─ and ~55 or ~4.8 MBq [188Re]ReO4─ using MDA-MB-231.hNIS-GFP tumour-bearing mice was performed to obtain time-activity curves to estimate the absorbed dose delivered to hNIS-GFP-expressing tumours and NIS- expressing organs using the OLINDA/EXM® software. Tumour growth curves and median survival time were determined to establish dose-effect relationships. The therapeutic effect of [123I]I─ and [188Re]ReO4─ and EBRT were compared when delivering the same absorbed dose of 12.6 Gy to tumours using a precision X-ray irradiator. Moreover, absorbed dose per unit activity administered of tumours and NIS-expressing organs were extrapolated to reference adult female and male models using OLINDA/EXM® software and following the International Commission on Radiological Commission on Radiological Protection (ICRP) publication 89.

Results: In Chapter 2, [99mTc]TcO4− resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 hours incubation in hNIS- expressing cells only. We found that 24,430 decays/cell (30 mBq/cell; 95% CI= [SF0.30; SF0.44]) and a lower nuclear-absorbed dose of 0.79 Gy (95% CI= [SF0.30; SF0.44]) vs 2.59 Gy EBRT (95% CI= [SF0.35; SF0.39]) were required to achieve SF0.37. In vivo retention of [99mTc]TcO4─ after 24 hours remained high at 28.0% ± 4.5% of the administered activity/gram tissue (%AD/g) in MDA-MB- 231.hNIS-GFP tumours. In Chapter 3, [99mTc]TcO4− and [123I]I─ reduced SF in hNIS-expressing cells only, whereas [188Re]ReO4─ reduced SF in hNIS- expressing cells and to a lower extent in MDA-MB-231 cells (P<0.0001). [123I]I─ required 2.4-fold and 1.5-fold lower number of decays/cell (9,497 decays/cell; 95% CI= [SF0.42; SF0.32]) to achieve SF0.37 in comparison to [99mTc]TcO4− and [188Re]ReO4─ following 72 hours incubation. Additionally, [99mTc]TcO4−, [123I]I─ and [188Re]ReO4─ had superior therapeutic effectiveness compared to EBRT per nuclear-absorbed dose (0.68, 0.61 and 0.82 Gy, respectively, vs 2.59 Gy).

In Chapter 4, 55 MBq of [123I]I─ resulted in an average absorbed dose of 12.6 ± 0.7 Gy to tumours and a significantly increase in median survival days compared to untreated mice (43 vs 20 days; P<0.0001). However, 55 MBq of [188Re]ReO4─ led to 12.5-fold greater absorbed dose to tumours, at the cost of systemic toxicity. An absorbed dose of 15 ± 4 Gy and 12.6 ± 0.7 Gy (P=0.7816) to tumours by [188Re]ReO4─ and [123I]I─, respectively, resulted in a non- significant difference in the observed median survival and tumour growth delay (P=0.5955 and P=0.0230, respectively). However, 11-fold less administered activity of [188Re]ReO4─ (4.8 MBq ± 0.6 MBq) was required to achieve the same absorbed dose to tumours. Furthermore, 12 ± 0.05 Gy EBRT led to a greater median survival compared to [188Re]ReO4─ and [123I]I─ (54 days vs 45 and 43 days, respectively; P<0.0001 and P<0.0001). However, EBRT did not stop development of metastatic disease. Extrapolated human absorbed doses of [188Re]ReO4─ to a 1 g tumour were 13.8-fold and 11.2-fold greater than [123I]I─ in female and male models, respectively.

Discussion/Conclusion: In Chapter 2, we showed that [99mTc]TcO4− caused DNA damage and reduced clonogenicity in this model, but only when the radionuclide was taken up into the cells for long-term (24 hours). Based on the obtained in vitro and in vivo data, our data extrapolation to humans suggested that greater activities (~58 GBq) than current activities recommended by the Administration of Radioactive substances Advisory Committee (ARSAC) guidelines for diagnostic imaging of 99mTc-radiolabelled radiotracers would be required to achieve the same intracellular activities that led to long-term in vitro radiotoxicity in our studies. However, further work is required to elucidate whether 99mTc is safe as an imaging tool, especially 99mTc- radiopharmaceuticals that are known to be retained in the cells at 24 hours (i.e., [99mTc]Tc-HMPAO). Moreover, our data suggests no nuclear targeting was required to induce therapeutic efficacy by [99mTc]TcO4─ or [123I]I─ when accumulated into cells (Chapter 3). However, the lower estimated number of decays/cell of [123I]I─ to reach SF37 following 24 and 72 hours in comparison to [99mTc]TcO4─, in addition to its longer half-life (13 hours vs 6 hours, respectively), greater emission of AEs/decay (13.7 vs 4.4) and lower photon/electron ratio (6.27 vs 7.78, respectively) encourage further prospective studies with 123I for the development of AEs-radiopharmaceuticals.
Additionally, although [188Re]ReO4─ did not necessarily require cellular accumulation, its in vitro therapeutic efficacy was greater when accumulated into cells. Furthermore, the in vitro estimated cellular dose-response curves were mainly dependent on the subcellular localisation of radionuclides and there was a superior therapeutic effect at the same nuclear-absorbed dose of [99mTc]TcO4─, [123I]I─ and [188Re]ReO4─ when compared to EBRT. This was in stark contrast to the in vivo therapeutic efficacy of [123I]I─ and [188Re]ReO4─ which was less per radiation dose than the therapeutic efficacy from EBRT (Chapter 4). Despite the similar therapeutic effectiveness of [188Re]ReO4─ and [123I]I─ per radiation dose, the 11-fold less administered activity of [188Re]ReO4─ could have been attributed to the longer-range of β─ particles and cross-fire effect, highlighting a greater in vivo therapeutic effectiveness of [188Re]ReO4─ to [123I]I─ per administered activity. Therefore, the need of higher activities with 123I to achieve the same therapeutic effect as 188Re is a key factor that should be taken into consideration when choosing this AE-emitter as a therapeutic tool as limiting factors, including absorbed dose to critical organs and photon/electron ratio emission, will determine the maximum administered activity to patients. In summary, data obtained in this thesis will guide further development of new MRT radiopharmaceuticals with AEs-emitters and β─ particles at preclinical and clinical levels and can be used as a reference to predict their therapeutic efficacy and imaging safety.
Date of Award1 Aug 2023
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorSamantha Terry (Supervisor), Gilbert Fruhwirth (Supervisor) & Giuseppe Schettino (Supervisor)

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