targeted radiotherapy: microgray doses and the bystander effect robert j. mairs, 1 natasha e....

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Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs , 1 Natasha E. Fullerton 1 , Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted Therapy Group, CRUK Beatson Labs, Glasgow, Scotland

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Page 1: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

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

Page 2: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

NeuroblastomaNeuroblastoma

• 2nd commonest solid tumour in children

• peak age < 2 years

• mostly disseminated at 1st presentation: poor prognosis

• rapid growth

• chemo/radiosensitive

Page 3: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Meta-iodobenzylguanidine(MIBG)

NH

NH

NH2

I

HO NH2

AdrenalineHO

Noradrenaline

HO NH2

HO

OH

Guanethidine

NH2

HN

NHN

Catecholamines, adrenergic neurone blockers Catecholamines, adrenergic neurone blockers and MIBG and MIBG

Noradrenaline transporter

Page 4: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Preoperative [Preoperative [131131I]MIBG treatmentI]MIBG treatment

3 year old neuroblastoma stage IV patient(PA Voute, 2001)

• 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

For mets but..

Page 5: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

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

Page 6: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Biological effects of ionising radiation: Biological effects of ionising radiation: the traditional perspectivethe traditional perspective

Page 7: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Transfer of medium

Transfer of serum

cell death

chromosomal aberrations

mutations

genomicinstability

Manifestation of radiation-induced biological bystander effects (RIBBE)

unirradiateunirradiateddcellscells

Esp low dose & low dose rate

Page 8: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

p53

Are RIBBEs significant in targeted radiotherapy?

- transfectant mosaic spheroids

- media transfer experiments

How do we investigate RIBBE in our gene therapy/targeted radiotherapy scheme?

Page 9: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

NonNon-mosaic multicellular spheroids-mosaic multicellular spheroids

100% GFP-expressing cells 100% GFP-non-expressing cells

Page 10: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Transfected mosaic Transfected mosaic spheroids derived from spheroids derived from the human glioma cell the human glioma cell line UVWline 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).

Page 11: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

3 types of therapeutic decay particle3 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

Page 12: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

[211At]astatine

[211At]MABGmeta-astatobenzylguanidine

[131I]MIBGmeta-iodobenzylguanidine

NH

NH

NH2

131I

NH

NH

N H2

211At

Page 13: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

0.001

0.01

0.1

1

surv

ivin

g f

ract

ion

0 5 10 15 20

[211At]MABG conc. (kBq/ml)

543210

% NAT expressing cells% NAT expressing cells

Survival of NAT gene-transfected UVW spheroids after treatment with [211At]MABG

Greater TMS killing than attributable to physical cross fire, implicating RIBBE

Page 14: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Transfer of mediumTransfer of medium

Non-irradiated cellsNon-irradiated cellsRemove targeted radionuclideRemove targeted radionuclideReplace medium andReplace medium andIncubate for Incubate for 1hr1hr

After 1hr incubation, gamma count thetransferred medium and add activity,equivalent to that of radiopharmaceutical which leaked from cells, to a third flask -[= Activity control]

Clonogenic Assay

24h24h

DONOR RECIPIENT ACTIVITY CONTROL

Media transfer protocol

Page 15: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

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 [*I]MIBG 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

Groups evaluated

Page 16: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

RIBBE after RIBBE after -irradiation of UVW/NAT cells

- 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

Recipient

Donor

Direct + indirect

su

rviv

ing

fra

cti

on

00.10.20.3

0.40.50.60.70.80.91.01.1

0 1 2 3 4 5 6 7 8 9

dose (Gy)

0.01

0.1

1.0

su

rviv

ing

fra

cti

on

0 1 2 3 4 5 6 7 8 9

dose(Gy)

Page 17: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

RIBBE after treatment with RIBBE after treatment with -emitter [-emitter [131131I]MIBG I]MIBG

Untransfected cells

Activity control

Direct + indirect

Donor

Recipient

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

su

rviv

ing

fra

cti

on

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 1 2 3 4 5 6 7 8

[131I]MIBG activity conc. (MBq/ml)

Page 18: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

RIBBE elicited by treatment with Auger electron emitter ([RIBBE elicited by treatment with Auger electron emitter ([123123I]MIBG) I]MIBG) and and -emitter ([-emitter ([211211At]MABG) - At]MABG) - high LEThigh LET

- 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

su

rviv

ing

fra

cti

on

0

0.10.20.30.40.50.60.70.80.91.01.1

0 1 2 3 4 5 6 7 8

Untransfected

Activity control

Recipient

Donor

Direct + Indirect

[123I]MIBG activity conc (MBq/ml)

su

rviv

ing

fra

cti

on

[211At]MABG activity conc (kBq/ml)

35302520151050

0.10.20.30.40.50.60.70.80.91.01.1

0

Page 19: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

RIBBE in a second cell line - EJ138 bladder carcinoma

- Cell line shows RIBBE in response to external beam irradiation- Recipient cells - dose response at low doses then plateau

0.01

0.1

1.0

su

rviv

ing

fra

cti

on

0 1 2 3 4 5 6 7 8 9Dose (Gy)

00.1

0.20.3

0.40.50.6

0.70.80.91.01.1

su

rviv

ing

fra

cti

on

0 1 2 3 4 5 6 7 8 9

Dose (Gy)

RIBBE after RIBBE after -irradiation

Recipient

Donor

Direct + indirect

Page 20: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

RIBBE following exposure of EJ138 cells to -emitter -emitter [131I]MIBG

Effect similar to that observed in UVW cell line: - no U-shaped survival curve - RIBBE cell kill less than in donor cells

su

rviv

ing

fra

cti

on

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

[131I]MIBG activity conc. (MBq/ml)

0 1 2 3 4 5 6 7 8 9 10 11

Untransfected cells

Activity control

Direct + indirect

Donor

Recipient

Page 21: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

RIBBE following exposure of EJ138 cells to Auger electron emitter ([123I]MIBG) and -emitter ([211At]MABG) - high LET

Similar to effect observed in UVW cell line: - U-shaped survival curves for RIBBE-kill

su

rviv

ing

fra

cti

on

00.10.20.30.40.50.60.70.80.91.01.1

0 40

su

rviv

ing

fra

cti

on

[123I]MIBG activity conc (MBq/ml)

EJ138 parental

Activity control

Recipient

Donor

Direct + Indirect

00.10.20.30.40.50.60.70.80.91.0

1.1

0 1 2 3 4 5 6 7 8 9 10 11 453530252015105

[211At]MABG activity conc (kBq/ml)

RIBBE elicited by treatment with RIBBE elicited by treatment with -emitter ([-emitter ([211211At]MABG) At]MABG) and Auger electron emitter ([and Auger electron emitter ([123123I]MIBG) - I]MIBG) - high LEThigh LET

Page 22: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

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

Page 23: Targeted radiotherapy: microGray doses and the bystander effect Robert J. Mairs, 1 Natasha E. Fullerton 1, Michael R. Zalutsky 2 and Marie Boyd 1 1 Targeted

Acknowledgements

Glasgow UniversityMarie BoydRob MairsSusan Ross Tony McCluskeyKer Wei TanNatasha Fullerton

Duke UniversityMike ZalutskyPhil WelshGanesan VaidyanathanJennifer Dorrens

FundersCancer Research UKNIHNeuroblastoma Soc.Glasgow UniversityIan Sunter Res TrustGlasgow Cancer School

CollaboratorsHeinz BonishJohn BabichKevin PriseLez FairbairnNicol KeithSally PilmottMike Zalutsky