Targeted radiotherapy:

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Targeted radiotherapy   microGray doses
and the bystander effect Robert J.
Mairs,1 Natasha E. Fullerton1, Michael R.
Zalutsky2 and Marie Boyd1 1Targeted Therapy
Group, CRUK Beatson Labs, Glasgow, Scotland
2Dept of Radiology, Duke University, North
Carolina, USA
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• Neuroblastoma
• 2nd commonest solid
• tumour in children
• peak age lt 2 years
• mostly disseminated at
• 1st presentation poor
• prognosis
• rapid growth
• chemo/radiosensitive

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Catecholamines, adrenergic neurone blockers and
MIBG
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Preoperative 131IMIBG treatment3 year old
neuroblastoma stage IV patient(PA Voute, 2001)
For mets but..

• 3 consecutive treatments interval 4 weeks
• 1st scan bone and bone marrow invasion large
  retroperitoneal tumour
• 2nd scan decreased uptake in bone, bone marrow
  and primary tumour
• 3rd scan bone and bone marrow cleared primary
  tumour resectable

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Bystander Effects

• Killing/damage of un-irradiated cells due to
  irradiation of adjacent cells
• Physical radiation cross-fire, i.e., direct
  traversal
• Biological direct traversal not required

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Biological effects of ionising radiation the
traditional perspective
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Manifestation of radiation-induced biological
bystander effects (RIBBE)
Transfer of medium
cell death chromosomal aberrations mutations g
enomic instability
unirradiated cells
Transfer of serum
Esp low dose low dose rate
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Are RIBBEs significant in targeted radiotherapy?
How do we investigate RIBBE in our gene
therapy/targeted radiotherapy scheme?

• - transfectant mosaic spheroids
• - media transfer experiments

p53
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Non-mosaic multicellular spheroids
100 GFP-expressing cells
100 GFP-non-expressing cells
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Transfected mosaic spheroids derived from the
human glioma cell line UVW
The spheroids, ranging in size from 100 to 500 ?m
diameter, are composed of mixtures of cells
transfected with the GFP gene (green) and
cellstransfected with the NAT gene (red).
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3 types of therapeutic decay particle
Radionuclide decay particle range
Use 123I Auger electrons
10 nm imaging 131I
beta particles 800 ?m
therapy 211At alpha
particles 60 ?m therapy
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211Atastatine
NH
NH
NH2
131I
211AtMABG meta-astatobenzylguanidin
e
131IMIBG meta-iodobenzylguanidine
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Survival of NAT gene-transfected UVW spheroids
after treatment with 211AtMABG
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NAT expressing cells
surviving fraction
0.1
0
1
2
3
0.01
4
5
0.001
5
10
15
20
0
211AtMABG conc. (kBq/ml)
Greater TMS killing than attributable to physical
cross fire, implicating RIBBE
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Media transfer protocol
After 1hr incubation, gamma count the transferred
medium and add activity, equivalent to that of
radiopharmaceutical which leaked from cells, to
a third flask - Activity control
Transfer of medium
ACTIVITY CONTROL
RECIPIENT
DONOR
Remove targeted radionuclide Replace medium
and Incubate for 1hr
Non-irradiated cells
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Groups evaluated

• Direct Indirect incubate with MIBG 2h wash
  incubate to allow generation of bystander
  poisons (direct bystander effect)
• Donor as Direct Indirect but remove medium
  then replace with fresh medium ( direct effect
  only)
• Recipient receive medium from Donor (
  bystander effect only)
• Activity control to correct for small amount of
  IMIBG leaked from Donor cells and transferred
  to Recipients
• Untransfected wild-type UVW cells (do not
  express NAT) recipients of medium from
  MIBG-treated wild-type UVW donors

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RIBBE after ?-irradiation of UVW/NAT cells
1.1
1.0
1.0
0.9
0.8
surviving fraction
0.7
0.1
surviving fraction
0.6
0.5
Direct indirect
0.4
0.01
Donor
0.3
0
1
2
3
4
5
6
7
8
9
0.2
Recipient
dose(Gy)
0.1
0
7
8
9
0
1
2
3
4
5
6
dose (Gy)

• This cell line demonstrates RIBBE after external
  beam irradiation
• Direct irradiation is more cytotoxic than RIBBE
  at high dose
• - RIBBE are most significant at low doses

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RIBBE after treatment with ?-emitter 131IMIBG
1.1
1.0
0.9
0.8
0.7
0.6
surviving fraction
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
6
7
8
131IMIBG activity conc. (MBq/ml)

• Medium from irradiated cells very cytotoxic -
  dose response
• significant effect at high doses RIBBE cell kill
  almost as potent as direct kill in cells treated
  with radiopharmaceutical

D/R
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RIBBE elicited by treatment with Auger electron
emitter (123IMIBG) and ?-emitter
(211AtMABG) - high LET
1.1
1.1
1.0
1.0
0.9
0.9
Direct Indirect
0.8
0.8
surviving fraction
0.7
0.7
Donor
surviving fraction
0.6
0.6
Recipient
0.5
0.5
Activity control
0.4
0.4
Untransfected
0.3
0.3
0.2
0.2
0.1
0.1
0
0
35
30
25
20
15
10
5
0
0
1
2
3
4
5
6
7
8
211AtMABG activity conc (kBq/ml)
123IMIBG activity conc (MBq/ml)

• U-shaped survival curves for RIBBE-kill - i.e.
  dose-related cytotoxicity at low activity
• concentration and diminishing bystander kill
  at higher activity concentration
• RIBBE elicited by high LET targeted
  radionuclides result in cell kill of magnitude
  similar
• to that caused by direct irradiation - hence
  potentially powerful therapeutic effect

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RIBBE in a second cell line - EJ138 bladder
carcinoma
RIBBE after ?-irradiation
1.0
1.1
1.0
0.9
surviving fraction
0.1
0.8
0.7
0.6
surviving fraction
0.5
0.01
0
1
2
3
4
5
6
7
8
9
0.4
Dose (Gy)
0.3
0.2
0.1
0
0
2
3
4
5
6
7
8
9
1
Dose (Gy)
- Cell line shows RIBBE in response to external
beam irradiation - Recipient cells - dose
response at low doses then plateau
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RIBBE following exposure of EJ138 cells to
?-emitter 131IMIBG
1.1
1.0
0.9
0.8
0.7
0.6
0.5
surviving fraction
0.4
0.3
0.2
0.1
0
131IMIBG activity conc. (MBq/ml)
Effect similar to that observed in UVW cell line
- no U-shaped survival curve -
RIBBE cell kill less than in donor cells
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RIBBE following exposure of EJ138 cells to Auger
electron emitter (123IMIBG) and ?-emitter
(211AtMABG) - high LET
1.1
1.1
1.0
1.0
0.9
0.9
0.8
0.8
Direct Indirect
0.7
0.7
surviving fraction
Donor
0.6
0.6
surviving fraction
0.5
Recipient
0.5
0.4
0.4
Activity control
0.3
0.3
EJ138 parental
0.2
0.2
0.1
0.1
0
0
0
40
45
35
30
25
20
15
10
5
0
1
2
3
4
5
6
7
8
9
10
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211AtMABG activity conc (kBq/ml)
123IMIBG activity conc (MBq/ml)
RIBBE elicited by treatment with ?-emitter
(211AtMABG) and Auger electron emitter
(123IMIBG) - high LET
Similar to effect observed in UVW cell line
- U-shaped survival curves for RIBBE-kill
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Conclusion

• RIBBE from targeted radionuclides appear distinct
  from external beam irradiation
• dose response
• LET dependent
• U shaped survival curves
• RIBBE from high LET radionuclides at low doses
  are more
• cytotoxic than direct irradiation
• Only cells which take up radiopharmaceutical
  produce RIBBE

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Acknowledgements
Glasgow University Marie Boyd Rob Mairs Susan
Ross Tony McCluskey Ker Wei Tan Natasha
Fullerton Duke University Mike Zalutsky Phil
Welsh Ganesan Vaidyanathan Jennifer Dorrens
Funders Cancer Research UK NIH Neuroblastoma
Soc. Glasgow University Ian Sunter Res
Trust Glasgow Cancer School
Collaborators Heinz Bonish John Babich Kevin
Prise Lez Fairbairn Nicol Keith Sally
Pilmott Mike Zalutsky