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Dose estimation after a mixed field exposure: Radium-223 and intensity modulated radiotherapy

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Introduction Radium-223 dichloride ([²²³Ra]RaCl2), a radiopharmaceutical that delivers α-particles to regions of bone metastatic disease, has been proven to improve overall survival of men with metastatic castration resistant prostate cancer (mCRPC). mCRPC patients enrolled on the ADRRAD clinical trial are treated with a mixed field exposure comprising radium-223 (²²³Ra) and intensity modulated radiotherapy (IMRT). While absorbed dose estimation is an important step in the characterisation of wider systemic radiation risks in nuclear medicine, uncertainties remain for novel radiopharmaceuticals such as ²²³Ra. Methods 24-Colour karyotyping was used to quantify the spectrum of chromosome aberrations in peripheral blood lymphocytes of ADRRAD patients at incremental times during their treatment. Dicentric equivalent frequencies were used in standard models for estimation of absorbed blood dose. To account for the mixed field nature of the treatment, existing models were used to determine the ratio of the component radiation types. Additionally, a new approach (M-FISHLET), based on the ratio of cells containing damage consistent with high-LET exposure (complex chromosomal exchanges) and low-LET exposure (simple exchanges), was used as a pseudo ratio for ²²³Ra:IMRT dose. Results Total IMRT estimated doses delivered to the blood after completion of mixed radiotherapy (after 37 IMRT fractions and two [²²³Ra]RaCl2 injections) were in the range of 1.167 ± 0.092 and 2.148 ± 0.096 Gy (dose range across all models applied). By the last treatment cycle analysed in this study (four [²²³Ra]RaCl2 injections), the total absorbed ²²³Ra dose to the blood was estimated to be between 0.024 ± 0.027 and 0.665 ± 0.080 Gy, depending on the model used. Differences between the models were observed, with the observed dose variance coming from inter-model as opposed to inter-patient differences. The M-FISHLET model potentially overestimates the ²²³Ra absorbed blood dose by accounting for further PBL exposure in the vicinity of metastatic sites. Conclusions The models presented provide initial estimations of cumulative dose received during incremental IMRT fractions and [²²³Ra]RaCl2 injections, which will enable improved understanding of the doses received by individual patients. While the M-FISHLET method builds on a well-established technique for external exposures, further consideration is needed to evaluate this method and its use in assessing non-targeted exposure by ²²³Ra after its localization at bone metastatic sites.
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Dose estimation after a mixed eld exposure: Radium-223 and intensity
modulated radiotherapy
Isabella Bastiani
a,
, Stephen J. McMahon
b
, Philip Turner
b,c
, Kelly M. Redmond
c
, Conor K. McGarry
b,c
,
Aidan Cole
b,c
, Joe M. O'Sullivan
b,c
, Kevin M. Prise
b
,LizAinsbury
d
, Rhona Anderson
a
a
Centre forHealth Effects of Radiologicaland Chemical Agents,College of Health,Medicine and Life Sciences, Brunel University London,Kingston Lane, Uxbridge, London UB8 3PH,United Kingdom
of Great Britain and Northern Ireland
b
Patrick G. Johnston Centre for Cancer Research, Queen's University Belfast, Belfast BT9 7AE,United Kingdom of Great Britain and Northern Ireland
c
Northern Ireland Cancer Centre, Belfast Health and Social Care Trust, Belfast, United Kingdom of Great Britain and Northern Ireland
d
Centre for Radiation, Chemical & Environmental Hazards,Public Health England, Didcot OX11 0RQ, United Kingdom of Great Britain and Northern Ireland
abstractarticle info
Article history:
Received 17 February 2021
Received in revised form 4 November 2021
Accepted 9 December 2021
Available online xxxx
Keywords:
Radium-223
Biodosimetry
Chromosome exchanges
Targeted alpha-particle therapy
Prostate cancer
Introduction: Radium-223 dichloride ([
223
Ra]RaCl
2
), a radiopharmaceutical that delivers α-particles to regions of
bone metastatic disease, has been proven to improve overall survival of men with metastatic castration resistant
prostate cancer (mCRPC).mCRPC patients enrolled on the ADRRAD clinical trialare treated with a mixed eld ex-
posure comprising radium-223 (
223
Ra) and intensity modulated radiotherapy (IMRT). While absorbeddose esti-
mation is an important step in the characterisation of widersystemic radiation risks in nuclear medicine, uncer-
tainties remain for novel radiopharmaceuticals such as
223
Ra.
Methods: 24-Colour karyotyping was used to quantify the spectrum of chromosome aberrations in peripheral
blood lymphocytes of ADRRAD patients at incremental times during their treatment. Dicentric equivalent
frequencies were used in standard models for estimation of absorbed blood dose. To account for the mixed
eld nature of the treatment, existing models were used to determine the ratio of the component radiation
types. Additionally, a new approach (M-FISH
LET
), based on the ratio of cells containing damage consistent with
high-LET exposure (complex chromosomal exchanges) and low-LET exposure (simple exchanges), was used as
a pseudo ratio for
223
Ra:IMRT dose.
Results: Total IMRT estimated doses delivered to the bloodafter completion of mixed radiotherapy (after 37 IMRT
fractions and two [
223
Ra]RaCl
2
injections) were in the range of 1.167 ± 0.092 and 2.148 ± 0.096 Gy (dose range
across all models applied). By the last treatment cycle analysed in this study (four [
223
Ra]RaCl
2
injections), the
total absorbed
223
Ra dose to the blood was estimated to be between 0.024 ± 0.027 and 0.665 ± 0.080 Gy,
depending on the model used. Differences between the models were observed, with the observed dose
variance coming from inter-model as opposed to inter-patient differences. The M-FISH
LET
model potentially
overestimates the
223
Ra absorbed blood dose by accounting for further PBL exposure in the vicinity of metastatic
sites.
Conclusions: The models presented provide initial estimations of cumulative dose received during incremental
IMRT fractions and [
223
Ra]RaCl
2
injections, which will enable improved understanding of the doses received by
individual patients. While the M-FISH
LET
method builds on a well-established technique for external exposures,
further consideration is needed to evaluate this method and its use in assessing non-targeted exposure by
223
Ra after its localization at bone metastatic sites.
Crown Copyright © 2021 Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Metastatic castration resistant prostate cancer (mCRPC) is an incur-
able condition. Prostate cancer has a strong predisposition to forming
bone metastases, with upwards of 90% of patients with advanced dis-
ease being affected, often with bone as the only site of metastasis [1].
As the general standard of care in the UK, patients with mCRPC are of-
fered a number of life-prolonging therapies including chemotherapy,
Nuclear Medicine and Biology 106107 (2022) 1020
Corresponding author.
E-mail addresses: isabella.bastiani@brunel.ac.uk (I. Bastiani),
stephen.mcmahon@qub.ac.uk (S.J. McMahon), philip.turner@cantab.net (P. Turner),
kelly.redmond@qub.ac.uk (K.M. Redmond), conor.mcgarry@belfasttrust.hscni.net
(C.K. McGarry), a.cole@qub.ac.uk (A. Cole), joe.osullivan@qub.ac.uk (J.M. O'Sullivan),
k.prise@qub.ac.uk (K.M. Prise), liz.ainsbury@phe.gov.uk (L. Ainsbury),
rhona.anderson@brunel.ac.uk (R. Anderson).
https://doi.org/10.1016/j.nucmedbio.2021.12.002
0969-8051/Crown Copyright © 2021 Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Contents lists available at ScienceDirect
Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio
novel anti-hormonals and Radium-223 dichloride ([
223
Ra]RaCl
2
).
Recent advances in molecular radiotherapy have generated a great
deal of interest, particularly the use of [
223
Ra]RaCl
2
after the landmark
phase III trial ALSYMPCA [24] showed for the rsttimeanoverallsur-
vival advantage associated with [
223
Ra]RaCl
2
treatment. Along with
survival prolongation, this trial also demonstrated signicant
improvement in the quality of life by delaying the onset of
symptomatic skeletal related events and alleviating pain. This led to
FDA and EMA approvals and the widespread use of [
223
Ra]RaCl
2
for
symptomatic mCRPC [5,6].
223
Ra has a half-life of 11.43 days, decaying
through creation of a succession of short-lived nuclides to stable
207
Pb.
During its decay chain,
223
Ra emits four αand two βparticles, with
94% of its decay energy released as high LET α-particles over a short
track length of <100 μm[7,8]. When living tissue is exposed to
223
Ra,
this results in the localized induction of clustered DSB lesions which
are difcult to repair, effectively leading to cell death [911].
For symptomatic mCRPC patients,
223
Ra is administered intrave-
nously as [
223
Ra]RaCl
2
under the trade name of Xogo [5].Once
administered, [
223
Ra]RaCl
2
immediately solubilises in the blood
resulting in free
223
Ra, which as a calcium mimetic, is cleared from the
blood within 24 h (h) [12] localising to areas of freshly mineralized
bone. The uptake of
223
Ra into high-turnover areas of bone is not depen-
dent on a particular malignant signalling process. Thus, [
223
Ra]RaCl
2
couldbeofbenet in a range of other malignancies which have a
predisposition to forming bony metastases such as breast, lung, kidney
and myeloma, including those with favourable long-term survival prob-
ability. Due to the short range of α-particles, this effective target cell kill
also minimises direct α-particle exposure to non-target normal cells [7,
1316]. There is also evidence, however, that
223
Ra can lead to high ab-
sorbed doses in sites adjacent to target bone metastases, including oste-
ogenic cells and the red bone marrow [1720]. It has also been reported
that additional biologicalresponses via bystander effects may also occur
[21].
Currently, the number of studies which seek to understand the bio-
logical action of
223
Ra in vivo in humans is limited [22], indeed, there re-
mains a lack of scientic rationale to underpin current dosing strategies
and there remains a great deal of uncertainty about the heterogeneous
distribution of dose at the cellular and tissue levels and the role of direct
and indirect effects such as bystander responses. Exposure of non-
cancerous cells and tissues surrounding the tumours is of concern in
all radiotherapeutic treatments, due to acute toxicity and the potential
for delayed late effects, sometimes many years after treatment
[2330]. Quantifying the absorbed dose to non-target tissues is an im-
portant step in evaluating the potential short- and longer-term second-
ary radiation risks for patients [31]. This is of particular importance if
[
223
Ra]RaCl
2
is to be used earlier in the treatment schedule. A number
of dose calculation models have been developed, for example, for calcu-
lation of the absorbed dose to blood from radiotherapy [32], however
the actual or estimated consequences of these are still being quantied
[33].
Cytogenetic analysis of chromosome aberrations in blood lympho-
cytes is widely used to estimate the dose of ionising radiation received
by an individual following a real or suspected radiation overexposure,
to help inform assessment of future health risks [34,35]. The dicentric
assay is the most common method of biological dosimetry, not least
due to the high radiation specicity and low interindividual variation
between yields of dicentric chromosome aberrations [34]. In so called
criticality situations of mixed eld neutron and gamma exposures
after nuclear reactor incidents, where individuals are irradiated by
both high-LET and low-LET sources, a model for emergency exposure
situations has been designed to estimate external doses for the individ-
ual exposure components [35]. Although originally designed for mixed
neutron:gamma exposures, for medical uses of radiation, the ratios of
delivered doses from internal and externally applied radiations can be
readily derived from patient treatment plans. Meaning that this ap-
proach can be applied to calculate alpha and X-ray doses in cases of
mixed exposures. Complex chromosome aberrations are useful bio-
markers of LET [36], and their identication in exposed individuals
could provide another approach for estimating the doses from mixed
exposures and, thus, to further rene dose estimation methods in
mixed exposure scenarios.
ADDRAD is an approved (NHS REC 15/NI/0074) phase I/II clinical
trial which seeks to address the potential benet of treating metastatic
hormone sensitive prostate cancer patients with androgen deprivation
therapy in addition to 6 cycles of [
223
Ra]RaCl
2
and intensity modulated
radiotherapy (IMRT) to the prostate and pelvic lymph nodes which
has recently reported its rst clinical data showing good response and
minimal toxicity [37]. This patient group therefore presents a unique
opportunity to address important radiobiological research questions
pertaining to the doses and cellular and tissue level damage associated
with internal
223
Ra exposure combined with IMRT. In this study, we
describe the use of 24-colour whole chromosome painting (multiplex
uorescence in situ hybridisation, M-FISH) applied to blood samples
taken from the ADRRAD cohort of patients, to quantify the patterns of
chromosome exchange complexity, with the aimof discriminating dam-
aged cells from each component radiation type and estimating absorbed
radiation dose. In addition, patient specic information is used to calcu-
late absorbed doses to blood on the basis of three different established
or adapted models, and the dose estimates are compared, with the over-
all aim to further understand the uncertainties involved in dose estima-
tion in this mixed radiation exposure scenario.
2. Methods
2.1. Physically derived blood dose estimates
The absorbed blood dose per fraction (D
b
) was estimated using data
from 13 recruited mCRPC patients. [
223
Ra]RaCl
2
was administered over
6 cycles (C1C6), comprised of one intravenous injection every
4 weeks containing 55 kBq kg
1
of
223
Ra [38] (Fig. 1). As part of the
treatment, IMRT is received during the rst 7.5 weeks, coinciding with
two [
223
Ra]RaCl
2
cycles. The IMRT treatment plan for ADRRAD targets
the prostate with a dose of 74 Gy, with 60 Gy delivered to lymph
nodes. IMRT was delivered in daily fractions of 2 Gy to the prostate
and 1.6 Gy to the lymph nodes, the latter as a concomitant boost.
Individualised patient treatment schedules are outlined in supplemen-
tary Table 1. The absorbed dose to the blood was estimated for
223
Ra
based on its pharmacokinetic properties, and the IMRT dose by two
C1 C2 C3 C4 C5 C6
[223Ra]RaCl2
IMRT
Weeks
048121620
Treatment schedule
Blood sampling
Fig. 1. Treatment time line. IMRT daily fractions fo r 7.5 weeks. [
223
Ra]RaCl
2
was
administered every 4 weeks for a total of 6 injections. Blood samples were collected at
each cycle, wit h a control sample collected at w eek 0 prior to treatment start (C 1).
Subsequent blood samples were taken every 4 weeks just prior to the next [
223
Ra]RaCl
2
administration, up to C6. Hence bl ood samples from C2 and C3 are representative of
mixed eld exposure while C4C6 are
223
Ra only. For this study only samples up to C5
were analysed.
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
11
simple blood dose models originally derived by Moquet et al. [32] with
modications as described.
2.1.1. Pharmacokinetic derived absorbed blood dose of
223
Ra
In the ADDRAD trial, [
223
Ra]RaCl
2
was administered at an activity of
55 kBq kg
1
of total body weight. Following this, the radionuclide will
clear from the blood by 24 h. Previous studies identied the amount of
223
Ra in the whole blood volume at varying time points within 24 h of
administration [12,3941]. The median percentage of circulating
223
Ra
was averaged at timepoints of 15 min, 4 h and 24 h between all
studies reporting median values with upper and lower ranges [3941].
This gave estimates for the percentage of radium in the blood of 22%
after 15 min (928%), 3% (1.34.95%) after 4 h and 0.8% (0.371%) at
24 h. This information was used to estimate the physical absorbed
dose to the blood by circulating
223
Ra.
Firstly, the total activity of
223
Ra present in the blood was estimated
at each timepoint. This was determined by multiplying the initial
injectedactivity of radium by the estimated percentage of
223
Ra remain-
ing in the blood and the decay constant of
223
Ra, 7.02 × 10
7
s
1
[42].
The rate of energy deposition was then calculated using the energy re-
lease for each decay of
223
Ra and its short-lived daughter products
(
219
Rn,
215
Po and
211
Bi) [43], with the total α-particle energy release es-
timated to be 4.30 × 10
12
J. Finally, the dose rate to blood was calcu-
lated by dividing this rate of energy deposition by the mass of blood of
the patient, which for this work was estimated using 75 ml of blood
per kg body weight.
The absorbed dose rates were then plotted as a function of time fol-
lowing injection, and the area under the curve was calculated
(GraphPad Prism 9; GraphPad Software; computed using trapezoid
rule) to estimate the patient specic dose delivered to the blood for
the rst 24 h of each treatment cycle. As less than 1% of the activity re-
mains at 24 h post-irradiation, it was assumed that the additional dose
deposited from 24 h to t = was negligible. The uncertainty on the
223
Ra absorbed blood dose estimates was dominated by the uncertainty
in the amount of circulating
223
Ra at each time period, which, as dis-
cussed below, could be up to 50% [3941].
2.1.2. IMRT blood ow model
For this part of the work, data was assessed from 13 ADDRAD pa-
tients, for whom treatment p lan and patient specic data wereavailable.
The blood ow model (BF) enabled estimation of dose within the high
dose organ area. The absorbed blood dose per fraction (D
B
)was
estimated as follows:
DB¼DfV95 VB
ðÞ ð1Þ
where D
B
was absorbed dose to blood per fraction ,D
f
was the prescribed
dose per fraction, V
95
represents the high dose volume and V
B
was the
total blood volume.
Two variates of the model were used: BF
1,
which was applied as per
Moquet et al. [32] which estimates V
B
by assuming75 ml of blood per kg
and, BF
2
which uses thestatic volume of blood in the prostate and lymph
nodes for V
B
, estimated by calculating a scaling factor between the
whole-body volume and the area irradiated (prostate and/or lymph
nodes) on the basis of treatment plan information. This scaling factor
was then applied to the whole-body blood volume estimates to achieve
a static blood volume for prostate and lymph nodes. In both cases, D
F
and V
95
were taken from the treatment plans. The uncertainty on V
B
and V
95
was estimated to be on the order of 10%, and the uncertainty
on D
f
can be estimated on the basis of Moquet and colleagues to be up
to 20%. Hence a conservative estimate of uncertainty on D
B
would be
approximately ±25%.
2.1.3. IMRT CT plan model
For this part of the work, data from 13 ADDRAD patients was
assessed, for whom treatment plan and patient specic data were
available. The CT planned volume (CTPV) absorbed blood dose model
enables dose estimation of both high and low dose areas. The CT volume
mapped for each patient was utilized to calculate a scaling factor for
each patient relative to whole-body volume, with whole-body volume
being calculated based on the average patient weight (kg) over the
course of treatment [44]. For the following dose models, the whole CT
volume was considered within the CTPV
1
along with a high dose
volume only CTPV
2
. Utilizing the CTPV
1
model, the whole-body mean
dose was estimated, and it was assumed the blood volume had also re-
ceived this. Firstly, a scalingfactor, S, was estimated as the ratio between
the CT plan volume and the whole-body volume. The following was
then applied to estimate the absorbed blood dose per fraction:
DB¼DPB NF
ðÞSð2Þ
where D
B
was the absorbed dose to blood per fraction; D
PB
was the
mean dose in Gy to the body volume covered by the CT scan (specic
to each patient, taken from the treatment plans), N
F
was the number
of fractions of radiotherapy and S was the patient specic scaling factor.
A further estimate, CTPV
2
, was performed in the same manner but in
this case rstly calculating the average dose per fraction for prostate
and lymph nodes, and then calculating a scaling factor based on their
volume compared to the whole-body volume.
The error associated with the plan volume was considered to be on
the order of 11.5%. The clinical upper acceptable limit in dose delivery
to the treatment plan was within 3%, as anything greater than this
would trigger re-calibration of dose planned area. The scaling factor
was estimated from whole body volume which was estimated by as-
suming that 1.01 g of human body mass ts within 1 cm
3
and that the
tissue density within the target volume was consistent with this for
the scalingfactor estimation,with an error within 1%.The total error as-
sociated with CTPV estimations was considered to be within ±6%.
2.2. M-FISH dicentric quantication
2.2.1. Sample collection
Whole blood was received from 5 male patients recruited onto the
ADRADD trial (EudraCT 2014-00273-39) with full informed consent
(NHS REC 15/NI/0074) at The Belfast Health and Social Care Trust.
Blood samples were drawn into lithium heparinised anticoagulant
tubes, once every 4 weeks, immediately prior to the rst (C1) and
then within 24 h prior to each subsequent (C2C5) [
223
Ra]RaCl
2
administration (Fig. 1). The samples were then shipped at room
temperature for nextday delivery to Brunel University London. Upon ar-
rival, the samples were processed and whole blood stimulated to divide
to enable the collectionof 1st in vitro cell division metaphase cells for cy-
togenetic assessment, as described below.
2.2.2. Cell culture
For each sample, 0.4 ml of whole blood was used to inoculate 2.6 ml
of freshly prepared media (PBMAX Karyotyping Medium)
(ThermoFisher, Cat. Number 12557021) supplemented with 0.5 μg/ml
puried phytohaemagglutinin (PHA) (ThermoFisher, Cat. Number
R30852801), 10 μM 5-bromo-2-deoxyuridine (BrdU, Sigma-Aldrich
Cat. Number 19-160), 10 μl/ml heparin (Sigma-Aldrich Cat. Number
9041-08-1) and cultured in a humidied incubator at 37 °C (95% air/
5% CO
2
), at a 45° angle, and with the cap left slightly open to allow
gaseous exchange. Cultures were set up to maximise the yield of 1st
cell division of peripheral blood lymphocytes (PBLs) and harvested
using standard cytogenetic techniques after a total of 5060 h. To arrest
cells at the metaphase stage of the cell cycle, 50 μl/ml of Colcemid
KaryoMAX (ThermoFisher, Cat. Number 1521012), a tubulin inhibitor,
was added 4 h prior to harvest. After this time,the cultures were centri-
fuged at 200gfor 10 min and the cell pellet re-suspended before the ad-
dition of 0.075 M KCl hypotonic solution (Fisher ScienticCat
Number10575090) for 8 min at 37 °C. Cells were then centrifuged at
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
12
200gfor 10 min and xed in 3:1 methanol (Thermo Fisher Catalogue
Number 15654570) acetic acid (Thermo Fisher Catalogue Number
1743468) on ice. The xation process was repeated until the samples
appeared clear (~5 times), and these were then stored in the freezer at
20 °C.
2.2.3. Harlequin stain
Fixed chromosome preparations were dropped onto clean, grease-
free slides and assessed for metaphase quality. Harlequin staining was
used to assess the number of 1st division metaphase cells. For this, slides
were aged on a hot plate for 45 min at 90 °C and immersed in Hoescht
(Thermo Fisher Scientic Cat Number 62249) for 10 min, then trans-
ferred to a at tray and covered in 2× Saline-Sodium Citrate (SSC)
(Thermo Fisher Cat Number 15557036), before being exposed in UV
box to 1.0 J/cm
2
for 1 h. After exposure slideswere washed with distilled
water twice and air dried. Treated slides were stained in 5% Giemsa
(VWR Cat Number 350864) for 4 min,removed and rinsed with distilled
water. Once dry, the slides were mounted with coverslips with 4 drops
of DPX (Fisher Scientic Cat Number 15538321). Slides were scored
using brighteld microscopy with oil immersion at ×100 magnication.
The fraction of 1st/2nd/3rd division cells was determined based upon
the chromatid staining patterns [45,46]. Samples with 5% 2nd division
cells were assayed by M-FISH.
2.2.4. Multiplex uorescence in situ hybridisation (M-FISH)
M-FISH was carried out utilizing 24XCyte staining probe
(Metasystems Probe Cat NumD-0125-600-DI) as per manufacturer pro-
tocol. Patient slides were selected from 5 patients according to meta-
phase spread quality and whether samples containing 5% 2nd division
cells, a minimum of 3 patient slides were painted per cycle. In brief,
slides were incubated in 2xSSC at 70 °C (±1 °C) for 30 min. After this
time, the cooled slide was transferred into 0.1xSSC at RT for 1 min. Chro-
mosomes were then denatured in 0.07 NaOH at RTfor 1 min followed by
1 min incubation in 0.1xSSC, followed by 2xSSC at 4 °C, and then
dehydrated through immersion in a series of alcohol solutions of as-
cending strength (70%, 95% and 100%). The 24Xcyte probe was dena-
tured by incubating at 75 °C (±1 °C) for 5 min, placed on ice briey
and then incubated at 37 °C for 30 min. The probe was overlaid on to
the slide and left to hybridize in a humidied chamber at 37 °C (±
1 °C) for 23 days. Slides were washed in 0.4xSSC preheated to 72 °C
(±1 °C) for 2 min then incubated in 2xSSCT (containing 0.05%
Tween20) for 30 s. For counterstaining, the slide was rinsed in double
distilled water and left to air dry before application of DAPI/antifade
and sealing.
Slides were visualised utilizing 8-position Zeiss Axioplan 2 micro-
scope containing individual lter sets for 24XCyte probe cocktail plus
DAPI (FITC, Spectrum Orange, Texas red, Cy5, DEAC and DAPI).
Metaphase cells were imaged under x63 oil immersion and captured
by Cool Cube driven by Metafer4 version 3.14.191 software. The image
les were exported and karyotyped in ISIS version 5.8.11.
2.2.5. Chromosome aberration classication
A cell was classied as being apparently normal if all 46 chromo-
somes were present and contained the appropriate uorophore combi-
nation along their entire length. Only metaphase cells with good uoro-
chrome staining were selected for analysis in cells containing 43 chro-
mosomes. Chromosomal aberrations were identied by colour junctions
along the length of each individual chromosome and/or by the presence
of chromosomal fragments (Fig. 2b). A chromosome interchange involv-
ing 2 breaks in 2 chromosomes was categorised as a simple exchange,
and further classied as a reciprocal translocation or dicentric. Ring
chromosomes, which involve 2 breaks in one chromosome were also
classed as simple [47]. Exchanges involving 3 or more breaks in 2 or
more chromosomes were classed as complex and assigned theminimal
number of breaks, arms and breaks involved (CAB) [48].Chromosomes
having breaks only, not involving any additional chromosomes, were
classed as chromosome breaks.When classifying cells with multiple ab-
errations, all aberrations were recorded as independent events and the
chromosomes involved identied. Where homologous chromosomes
were involved, efforts were made to establish whether the homologues
were in the same event or in different independent events, mainly by
consideration of chromosome length [49]. All exchanges were recorded
as either complete (all break-ends re-joined), true incomplete (where
one or more break-ends fail to nd an exchange partner) or one-way
(where one or more elements appear to be missing) [50,51]. The poten-
tial transmissibility of exchanges was also recorded, where a stable
(transmissible) exchange was dened as complete and with no evi-
dence of unstable elements e.g.dicentric or acentric fragments. Each in-
dependent complex event was also determined to be transmissible or
non-transmissible and the presence of insertion-type rearrangements
were noted [49]. Metaphase spreads were categorised as stable only if
all the exchange events detected within that spread were classied as
stable. Unstable complex chromosomal exchanges containing polycen-
tric chromosomes were broken down in to their dicentric equivalents
whereby each additional centromere within a chromosome structure
constituted a dicentric equivalent (dicentric equivalent event = n cen-
tromere 1) (Fig. 2).
2.3. M-FISH dicentric assay dose estimation
2.3.1. Mixed eld absorbed blood dose ratio
For this part of the work, M-FISH analysis was carried out n=5in-
dividuals with a minimum of n= 3 patient samples processed for C2
C5. The
223
Ra:IMRT ratio was estimated in two ways. Firstly, by physical
Fig. 2. Dicentric equivalent scoring. a) 1st division Giemsa stained metaphase imaged under bright eld microscopy ×100 magnication under oil. Chromosome with two centromeres
highlighted along with acentric fragment b) Pseudo colour processed image painted by M-FISH, captured at ×63 magnication under oil. Complex chromosomal exchange between
three chromosomes, dicentric chromosome highlighted by red arrow along with other components in yellow. c) DAPI channel of same cell (b) highlighting same aberration as a simple
dicentric exchange.
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
13
dose estimation and secondly, from categorising cells based on the com-
plexity of chromosome exchange observed. For the physical dose ratio,
the absorbed blood dose was calculated independently for [
223
Ra]
RaCl
2
and IMRT (Section 2.1) for each exposure and was summed over
the 4-week time period prior to each sample being taken. The average
absorbed blood dose per cycle was estimated by averaging theabsorbed
blood dose across all 13 patients for all treatment cycles. For the dose
ratio the overall treatment plan was used (Fig. 1), not the patient spe-
cic plan adaptation. Therefore, C2 relates to the response to one injec-
tion of [
223
Ra]RaCl
2
and 20 IMRT fractions, C3 relates to two injections of
[
223
Ra]RaCl
2
and 37 IMRT fractions, with C4 and C5 each including the
additional [
223
Ra]RaCl
2
only administrations. Summing these gives the
total absorbed blood dose of [
223
Ra]RaCl
2
and IMRT delivered at each
time point from C2 to C5. For the M-FISH derived ratio (M-FISH
LET
),
cells containing at least one complex chromosome exchange were
classed as being damaged by the traversal of high-LET α-particles
from the
223
Ra while cells containing only simple chromosome ex-
changes, as from IMRT (Section 2.2.5). The ratio of cells containing at
least one complex chromosome exchange to cells containing simple ex-
changes only was therefore a pseudo ratio for
223
Ra:IMRT absorbed
blood dose at each cycle point.
2.3.2. Dicentric absorbed blood dose estimation
The dicentric assay traditionally utilizes Giemsa staining where
chromosomes are evenly stained in one colour, (Fig. 2a), thus enabling
the identication of chromosomes containing more than one centro-
mere along with any associated acentric fragment. In this study, dicen-
trics were quantied from M-FISH painted metaphase cells (Fig. 2b
and c).
Dose estimation was rst carried out utilizing the dicentric equiva-
lent frequency as determined by M-FISH. For the IMRT, the
60
Co calibra-
tion curve of Lloyd and colleagues, 1986, was used [52] where whole
blood was irradiated in vitro utilizing a
60
Co source with dose range of
05 Gy. The dicentric yield was entered into Dose Estimate V5.2 [53]
with the following coefcients α= 0.0756 ± 0.0031, β=0.0149±0.
0060 and C = 0.0004 ± 0.0009 [52]. This being a well-established cali-
bration curve, it has been utilized in many exposure scenarios for γ-ray
and X-ray dose estimation by Public Heath England and was judged to
be the most comparable curve in terms of type and energy of radiation
exposure. As there is currently no
223
Ra calibration data, a calibration
curve based on a
239
Pu, which emits α-particles of a similar energy to
223
Ra (5.16 MeV per α-particle), was selected. The
239
Pu calibration
curve of Purrott et al. [54] was used, the curve coefcients were: β=
0.3696 ± 0.0322, C = 0.0019 ± 0.0126 [54].Thedecaychainof
223
Ra
and
239
Pu does differ, however, due to the perceived lack of risk of
a radiation accident involving alpha exposures and the complexity
of the experiments, alpha curves for biodosimetry are rare. This
curve was established by irradiating whole blood with
239
Pu in the
range of 01.6 Gy. It is important to note, however, that in this
study, the likely non-homogeneous, partial body, nature of both the
223
Ra and IMRT exposures beyond the treatment plan details, was
not further considered, neither was the microdosimetric heterogene-
ity of radium in areas of high bone turnover, and thus this represents
a key source of unquantied uncertainty. This aspect will need to be
incorporated into further development of absorbed blood dose
models. To calculate the absorbed blood dose in this mixed exposure
scenario, the criticalitymodel was used [35]. In brief, all aberra-
tions were rstly assumed to be attributed to
223
Ra and from the di-
centric equivalent yield the dose was estimated. The absorbed blood
dose ratio (
223
Ra:IMRT) calculated according to Section 2.3.1.was
then used to estimate the IMRT dose and then the gamma calibration
curve used to estimate the dicentric equivalent yield. This IMRT yield
was then subtracted from the total yield to give a new
223
Ra dicen-
tric equivalent yield. This iterative process was repeated until self-
consistent estimates were obtained.
2.4. Other statistical analysis
Statistical analysis was performed using GraphPad Prism 9
(GraphPad Software). Descriptive statistics are presented as mean ±
SE for pooled data. Standard propagation of errors was applied to esti-
mate the uncertainty in the derived dose estimates. Normality testing
indicated ANOVA was appropriate in order to test for differences in ab-
erration frequencies between treatment rounds and for differences in
estimated doses.
3. Results
3.1. Estimating the blood dose from the treatment schedule
Patient data related to the planned treatment for 13 patients was
available for physical dose estimation. Table 1 gives the absorbed
blood dose per fraction predicted by the BF model, estimated for each
patient for the prostate, lymph nodes, and total high dose region com-
bined. The majority of the absorbed blood dose was estimated as
being from lymph node exposure, due to the larger volume of irradia-
tion for this tissue (BF
1
). The total prostate-only absorbed blood dose
was found to be in the range of 0.8801.962 Gy by the end of IMRT treat-
ment (3037 fractions; 37 fractions assumed for dose ratio calculations
in Section 3.3), comparable to the 0.381.92 Gy reported by Moquet
et al. To consider the differences in lymphatic uid shift, the static vol-
ume was calculated (BF
2
) and, by combining the BF models (BF
1P
+BF
2-
LN
), the cumulative absorbed blood dose was estimated to be between 1.
131 and 2.717 Gy.
The absorbed blood dose was estimated for the whole CT plan area,
including high dose and low dose regions (CTPV
1
) and for high dose
regions only (CTPV
2
) to enable comparison with the BF model.
PreviousCTPV approaches haveused a xed scaling factorof 2.5 with
a reported uncertainty of 20%. In this work we instead estimated a pa-
tient specic scaling factor between 2.08 and 2.64, with the largest devi-
ation from the scaling factor for Patient 10 being 16% smaller than the
published 2.5 scaling factor.The increased absorbed blooddose CTPV es-
timates compared to the BF estimates observed here are likely to be re-
ective of the large lymph node irradiation volume [32].Tocomparethe
BF and CTPV models, CTPV was also calculated using the high dose vol-
umes of prostate and lymph nodes, termed as CTPV
2
. To do so, a scaling
factor for each component as detailed for BF
2
, was used. The resulting
absorbed blood doses for CTPV
2
were found to be comparable to that
of BF
2
within the uncertainty estimates (Table 2). The average
Table 1
BF model. V
B
blood volume, D
B
blood dose per fr action. BF
1P
from BF
1
prostate dose
combined with BF
2
LN dose (BF
2LN
). Patients for whom treatment plan and patien t
specic data were available n= 13. Reported estimated unce rtainty of 25% for al l BF
model variants.
Patient
ID
V
B
body
(ml)
V
B
prostate
(ml)
V
B
LN
(ml)
BF
1
BF
2
BF
1P
+BF
2LN
Prostate
D
B
(Gy)
LN
D
B
(Gy)
Total
dose
D
B
(Gy)
LN
D
B
(Gy)
Total dose D
B
(Gy)
1 9030 12.9 54.5 0.038 0.132 0.170 0.007 0.045
2 7615 11.7 56.8 0.041 0.164 0.205 0.010 0.050
3 6288 9.5 49.2 0.040 0.171 0.212 0.009 0.049
4 6560 12.2 57.2 0.060 0.231 0.291 0.014 0.073
5 6461 13.8 63.9 0.056 0.209 0.265 0.015 0.071
6 6985 10.8 58.9 0.041 0.184 0.225 0.011 0.052
7 7650 9.4 52.0 0.033 0.149 0.181 0.008 0.040
8 5700 7.0 32.4 0.032 0.124 0.157 0.004 0.037
9 6290 11.5 49.1 0.050 0.154 0.204 0.007 0.058
10 6629 14.0 52.3 0.056 0.171 0.227 0.009 0.065
11 5559 9.1 48.4 0.043 0.188 0.231 0.009 0.052
12 7883 7.1 49.2 0.024 0.135 0.159 0.007 0.031
13 6486 10.2 54.8 0.042 0.161 0.203 0.009 0.051
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
14
absorbed blood dose per fraction of IMRT wasestimated as 0.101 Gy for
CTPV
1
and 0.016 Gy for CTPV
2
(average n= 13 patients), each with an
estimated uncertainty on the order of 6%.
BF
1
and CTPV
1
cannot be directly compared as they use different
irradiation volumes. The BF
1
model assumes the blood ow within
prostate and lymph node regions was identical and in doing so,
it appears to signicantly overestimate the lymph node blood volume.
On the basis of this and the simple nature of the other model
assumptions, this method likely has the largest uncertainty.
Accordingly, only CTPV
1
and CTPV
2
are considered further as these
models represent an estimate of combined absorbed blood dose for
high dose regions and low dose regions (CTPV
1
) and high dose regions
only (CTPV
2
). Supplementary Table 1 details the patient specic
injected activities and CT planning volumes. Table 2 shows the individ-
ual absorbed blood dose estimates for each
223
Ra administration, and
the IMRT absorbed blood dose estimates for each fraction. The average
223
Ra absorbed blood dose per treatment cycle was 0.012 ± 0.002 Gy.
The estimated
223
Ra absorbed blood dose per fraction was not found
to be statistically different between patients (P= 0.097). To estimate
the
223
Ra absorbed blood dose per treatment cycle, the median values
of the activity kinetics reported in the literature were utilized. Based
on the highest and lowest limits reported for each timepoint the
223
Ra
blood dose estimates per treatment cycle could vary by up to 50%. This
value being reported as a conservative estimate of uncertainty for dose
estimates, with more work needed to quantify the variations between
patient clearance.
3.2. Frequency and type of chromosome aberration in ADRADD patients
M-FISH analysis was carried out on blood samples received from 5
patients for C1C5. The control samples of all 5 patients were analysed
and following this a minimum of 3 patients were analysed per treat-
ment cycle. The background frequency of chromosomal aberrations
was found to be within the expected normal range for individuals in
the 50+ age bracket [55,56]. Specically, frequencies of 0.0, 0.024 ± 0.
011 and 0.020 ± 0.009 were observed for simple dicentrics, reciprocal
translocations and break-only aberrations, respectively (sample C1;
pooled for 5 patients). One cell containing an unstable complex rear-
rangement was found in Patient 2 (0.004 ± 0.004), for which the origin
was not clear.
Simple chromosomal exchanges were observed to signicantly in-
crease in frequency between C1 and C2 (0.024 ± 0.011 to 0.319 ± 0.
042 (P< 0.001)) and further at C3 to 0.484 ± 0.042 (P<0.031)(Supple-
mentary Table 2). No other statistical differences were noted between
treatment cycles (C34, P= 0.966 or C4C5, P= 0.996), suggesting
the majority of simple aberrations formed early in the treatment regime
persist over the period sampled, and/or the induction of new aberra-
tions was balanced by cell death of unstable (e.g. dicentric) types. The
frequency of complex exchanges increased from 0.058 ± 0.016 at C2
to 0.174 ± 0.022 at C3 (P= 0.007), rising further to 0.265 ± 0.037 at
C4 and 0.210 ± 0.030 at C5 (Supplementary Table 2).
In terms of classication of complexity, the frequency of damaged
PBLs assigned to each exposure type (
223
Ra or IMRT) was reported in
Fig. 3 and Table 3. This categorisation was based upon the presence or
absence of a complex chromosome exchange and shows that the major-
ity of damaged PBLs sampled, with combined IMRT and
223
Ra contained
mostly simple exchanges only at C2 (cells containing simple exchanges
0.204 ± 0.025 and containing at least one complex exchange 0.050 ± 0.
014, P< 0.001) and C3 (0.243 ± 0.021 and 0.150 ± 0.018, P=0.006).
After this time, when patients continue to receive
223
Ra only, the frac-
tion of damaged PBLs with at least one complex exchange increased.
In the
223
Ra only cycles, no signicant difference between cells contain-
ing a simple aberration and those containing at least one complex ex-
change was observed (C4 P=0.992,C5P=0.558).
3.3. M-FISH dicentric assay absorbed blood dose estimates
The absorbed blood dose ratios were estimated for C2C5, this was
equivalent to 4 intravenous injections of
223
Ra and completed IMRT
schedule of 37 fractions by C5. For the physical dose ratio, this was
based on the average absorbed blood dose per fraction across all 13 pa-
tients for both the IMRT and
223
Ra dose. The M-FISH
LET
ratios described
in Section 2.3.1, was obtained from blood samples received from 5
patients with a minimum of 3 patient samples analysed per cycle. The
absorbed blood dose ratios for all models can be seen in Table 3.
Applying these blood dose ratios to the dicentric assay, the absorbed
blood dose was estimated in three ways (Table 4). The CTPV methods
were derived from the physical absorbed blood dose estimates as per
method Section 2.1. with CTPV
1
estimating the absorbed blood dose
across the planned volume including both high and low dose volumes
while CTPV
2
including the high dose regions only. The M-FISH
LET
absorbed blood dose was instead estimated from the ratio of cells
consistent with high LET exposure (
223
Ra) and low LET exposure
(IMRT).
The absorbed blood dose estimates from the CTPV models suggest
the dose after 20 IMRT fractions, measured at C2 (Table 4), was between
1.327 ± 0.115 Gy and 1.395 ± 0.115 Gy. The M-FISH
LET
method was
found to be in similar range at 0.956 ± 0.114 Gy at C2. The IMRT ab-
sorbed blood dose after the end of fractionation (C3 assuming 37 com-
pleted fractions) was estimated for CTPV
1
as 2.148 ± 0.096 Gy and 2.
073 ± 0.096 Gy for CTPV
2
, with M-FISH
LET
blood dose estimates of 1.
167 ± 0.092 Gy.
Table 2
Physically derived absorbed blooddose, in Gy.
223
Ra absorbedblood dose was representa-
tive of an average dose to the blood during localization period. The absorbed blood dose
after all treatment cycles was calculatedfor n= 6 injections with theexception of Patient
5 where n= 5. IMRT absorbed blood dose estimated for whole CT plan area CTPV
1
and
high dose regions only CTPV
2
. Scaling factor est imated from patient body volume
derived from ave rage weight during treatment. Patient numbers n=13patientsfor
whom treatment plan and patient specic data were available. Uncertainties of up to 6%
for CTPV models and up to 50% considered for the
223
Ra estimates.
Patient ID
223
Ra IMRT
D
B
(Gy) CTPV
1
D
B
(Gy) CTPV
2
D
B
dose (Gy)
1 0.0116 0.105 0.013
2 0.0114 0.104 0.015
3 0.0116 0.101 0.016
4 0.0118 0.113 0.022
5 0.0116 0.100 0.020
6 0.0116 0.112 0.017
7 0.0117 0.092 0.014
8 0.0118 0.095 0.012
9 0.0117 0.089 0.015
10 0.0115 0.113 0.017
11 0.0114 0.108 0.018
12 0.0116 0.094 0.012
13 0.0114 0.088 0.015
Fig. 3. Frequency of cells containing either si mple () or at least one complex ()
chromosome exchange. Data pooled from 5 patients total, n= 3 pat ients/cycle (C2, 3
and 5) and n= 4 for C3. Error bars represent standard error of the mean for pooleddata.
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
15
For the
223
Ra absorbed blood dose at C2, the CTPV
1
and CTPV
2
were
found to be 0.008 ± 0.025 and 0.048 ± 0.032, both lower than the M-
FISH
LET
estimate of 0.150 ± 0.046 Gy. For C3 the
223
Ra absorbed blood
dose estimated from CTPV methods was between 0.013 ± 0.025 and
0.082 ± 0.033 Gy (CTPV
1
and CTPV
2
respectively) with the M-FISH
LET
absorbed blood dose estimated at 0.719 ± 0.073 Gy. By the nal
sample point studied here (C5), the absorbed blood dose from
223
Ra
was estimated as 0.024 ± 0.027 Gy by CTPV
1
and 0.141 ± 0.042 Gy
for CTPV
2
with the M-FISH
LET
absorbed blood dose estimate at 0.
665 ± 0.080 Gy (see Fig. 4).
4. Discussion
In the future, α-particle emitters, such as
223
Ra, alone or in combina-
tion, are likely to be adopted in the treatment of a wide range of cancers,
contributing to more targeted, personalized medicine with the potential
to impact a large number of patients [17,57,58]. To date, however, there
are only limited studies using biological endpoints which seek to under-
stand the biological action of
223
Ra in vivo in humans, to improve theop-
timal delivery of these and, to minimise the risk of adverse effects
through radiation exposure of normal tissues [5963]. Quantifying the
absorbed blood dose delivered from the treatment is an important
step in maximising clinical efcacy and evaluating radiation risks, esti-
mates of which are currently based on population studies [31]. In this
study, whole blood was sampled from patients recruited onto the
ADRRAD trial [37]. The treatment includes a planned mixed eld expo-
sure from daily IMRT fractions, 37 × 2 Gy to prostate with concomitant
boost (37 × 1.6 Gy) to lymph nodes, over a period of 7.5 weeks, together
with 6 intravenous injections of [
223
Ra]RaCl
2
over a period of 20 weeks.
Dose estimation for non-targeted tissues in mixed exposure scenarios is
challenging. To account for the mixed eld nature of the treatment,
existing models were used to determine the ratio of the component ra-
diation types. Additionally, a new approach (M-FISH
LET
), based on the
ratio of cells containing damage consistent with high-LET exposure
(complex chromosomal exchanges) and low-LET exposure (simple ex-
changes), was used as a pseudo ratio for
223
Ra:IMRT absorbed blood
dose.
223
Ra exposure was dictated by its unique pharmacokinetic proper-
ties and its ability to target calcium-dependent bone turnover. Once in-
travenously administered,
223
Ra is rapidly cleared through the gastroin-
testinal tract and the remainder through the kidneys [39,41].Previous
studies on rodents found minimal uptake of
223
Ra in non-targeted
areas such as kidneys and the spleen [13,64] with the highest absorbed
doses being observed in humans in neighbouring sites of target bone
metastases, including osteogenic cells and the red bone marrow [17,
19,20]. Here, the
223
Ra absorbed blood dose was estimated from the
injected activity using an existing clearance model, based on the quan-
tity of
223
Ra in circulation at three time points (15 min, 4 h and 24 h
post intravenous administration). The uncertainty for the
223
Ra physical
absorbed blood dose estimations per cycle was based on the errors asso-
ciated with the injected activity, patient weight and clearance models
used. The injected activity per patient was estimated from the known
activity in the syringe prior to administration and the remaining activity
after injection, the error associated with this was considered negligible.
Patient individual weights were found to uctuate during treatment by
8% (up to 6.2 kg lost by C6). Therefore, the
223
Ra absorbed blood dose
was estimated for each chosen timepoint from the data available at
the specic treatment cycle rather than an average across treatment
Table 3
Absorbed blood dose ratios. M-FISH derived ratio based on the complexity of cellular damage with
223
Ra assigned cells to include those with at least 1 complex exchange and, IMRT
assigned cells dened as containing only simple exchanges. Frequency per total cells scored pooled per cycle, with calculated SE. D
B
per cycle of patient average (n= 13), complexity
of cellular damage from pooled patient data (n=5).
Cycle Cellular classication D
B
per cycle
223
Ra: IMRT
223
Ra cells (f) IMRT cells (f)
223
Ra (Gy) IMRT CTPV
1
(Gy) IMRT CTPV
2
(Gy) CTPV
1
CTPV
2
M-FISH
LET
C2 0.050 ± 0.014 0.204 ± 0.025 0.012 ± 0.002 2.021 ± 0.050 0.318 ± 0.017 1:174 1:27 1:4
C3 0.150 ± 0.018 0.243 ± 0.021 0.023 ± 0.003 3.739 ± 0.092 0.587 ± 0.031 1:161 1:25 2:3
C4 0.198 ± 0.025 0.229 ± 0.026 0.035 ± 0.003 3.739 ± 0.092 0.587 ± 0.031 1:108 1:17 1:1
C5 0.162 ± 0.021 0.220 ± 0.024 0.046 ± 0.004 3.739 ± 0.092 0.587 ± 0.031 1:81 1:13 3:4
Table 4
Absorbed blood doses during treatment (Gy) calculated on the basis of CTPV
1
,CTPV
2
and M-FISH
LET
models. Patients sampled duringtreatment cycles were coded and identier number
(ID) assigned, n= 5 patients were assayedby M-FISH to determine the dicentric equivalent yield.The absorbed blooddose was calculatedwith use of dicentricassay for each patientwith
the dose ratios estimated from CT plan and M-FISH
LET
derived models. The SE was reported for the absorbed blood dose estimation, this being the largest uncertainty for M-FISH
LET
dose
estimation. CTPV
1
and CTPV
2
reported SE of absorbed blood dose estimation with a propagated conservative uncertainty estimated up to 50% not included.
Cycle Patient ID Cells scored Dicentric equivalents
(yield ± SE)
CTPV
1
CTPV
2
M-FISH
LET
223
Ra IMRT
223
Ra IMRT
223
Ra IMRT
2 1 55 8 (0.145 ± 0.066) 0.007 ± 0.033 1.268 ± 0.246 0.044 ± 0.054 1.201 ± 0.246 0.207 ± 0.105 0.845 ± 0.245
3 101 16 (0.158 ± 0.067) 0.008 ± 0.029 1.329 ± 0.182 0.046 ± 0.043 1.261 ± 0.182 0.221 ± 0.081 0.899 ± 0.181
4 104 21 (0.202 ± 0.048) 0.009 ± 0.029 1.516 ± 0.180 0.053 ± 0.045 1.448 ± 0.180 0.263 ± 0.086 1.070 ± 0.179
Total 260 45 (0.173 ± 0.035) 0.008 ± 0.025 1.395 ± 0.115 0.048 ± 0.032 1.327 ± 0.115 0.235 ± 0.055 0.959 ± 0.114
3 1 100 30 (0.30 ± 0.0820) 0.012 ± 0.031 1.874 ± 0.184 0.071 ± 0.051 1.799 ± 0.184 0.584 ± 0.128 0.947 ± 0.182
2 101 38 (0.376 ± 0.076) 0.013 ± 0.031 2.113 ± 0.184 0.080 ± 0.053 2.038 ± 0.184 0.701 ± 0.139 1.138 ± 0.182
4 101 45 (0.446 ± 0.111) 0.014 ± 0.032 2.310 ± 0.185 0.088 ± 0.055 2.234 ± 0.184 0.801 ± 0.149 1.300 ± 0.182
5 105 45 (0.429 ± 0.065) 0.014 ± 0.031 2.264 ± 0.181 0.086 ± 0.053 2.188 ± 0.181 0.777 ± 0.144 1.261 ± 0.179
Total 407 158 (0.388 ± 0.042) 0.013 ± 0.025 2.148 ± 0.096 0.082 ± 0.033 2.073 ± 0.096 0.719 ± 0.073 1.167 ± 0.092
4 1 48 15 (0.313 ± 0.099) 0.018 ± 0.042 1.908 ± 0.264 0.106 ± 0.082 1.797 ± 0.264 0.680 ± 0.198 0.789 ± 0.262
2 102 53 (0.520 ± 0.062) 0.023 ± 0.035 2.497 ± 0.184 0.141 ± 0.066 2.384 ± 0.184 1.048 ± 0.169 1.216 ± 0.181
3 103 32 (0.311 ± 0.116) 0.018 ± 0.033 1.902 ± 0.182 0.106 ± 0.058 1.791 ± 0.182 0.677 ± 0.136 0.785 ± 0.179
Total 253 100 (0.395 ± 0.052) 0.020 ± 0.028 2.162 ± 0.119 0.121 ± 0.043 2.050 ± 0.119 0.833 ± 0.097 0.966 ± 0.116
5 1 106 47 (0.443 ± 0.083) 0.028 ± 0.037 2.290 ± 0.180 0.169 ± 0.070 2.142 ± 0.180 0.864 ± 0.151 1.175 ± 0.178
4 101 35 (0.347 ± 0.084) 0.025 ± 0.036 2.009 ± 0.184 0.147 ± 0.068 1.862 ± 0.183 0.705 ± 0.140 0.959 ± 0.182
5 102 18 (0.176 ± 0.043) 0.017 ± 0.033 1.394 ± 0.182 0.099 ± 0.057 1.254 ± 0.181 0.392 ± 0.105 0.533 ± 0.180
Total 309 100 (0.324 ± 0.042) 0.024 ± 0.027 1.937 ± 0.108 0.141 ± 0.042 1.791 ± 0.108 0.665 ± 0.080 0.905 ± 0.105
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
16
along with the median values of
223
Ra in the blood at each timepoint
(averaged between reported studies). The average absorbed blood
dose per cycle was estimated as 0.012 ± 0.002 Gy (average of Table 2
as per methods Section 2.3.1). In order to estimate the uncertainty for
the clearance model, the reported ranges in terms of upper and lower
range limits were taken from the literature [3941] with a variation
from the median of up to 50%. The average uncertainty on the delivered
223
Ra activity was thus estimated to be as high as50%. The additional ab-
sorbed blood dose delivered after 24 h period was not considered in this
model. Although the additional absorbed blood dose is likely to be
within the 50% uncertainty described, it may not be implicitly negligible
and does form an additional contribution to the uncertainty not quanti-
ed here.
The physical absorbed blood doses reported here are higher than
those reported by Stephan et al. using
224
Ra and in a similar range to
those reported by Schumann et al. for
223
Ra [65,66]. The decay kinetics
and energies of
224
Ra are broadly comparable to those of
223
Ra, how-
ever, as Stephan and colleagues report, there are a number of limitations
associated with the application of the ICRP 67 model [67], including the
lack of information regarding the local distribution of the activity. The
aim of the
223
Ra clearance calculations in this work was not to test the
ICRP model, rather, to provide a very simple kinetic method for valida-
tion of the newly proposed biological absorbed blood dose estimation
methods presented. Nevertheless, further work is required to assess
the most appropriate means of estimating dose, and this will include re-
assessment of more detailed kinetic models including those from ICRP.
IMRT utilizes conformal beams to accurately target a designated area
minimising exposure to non-target organs/tissues. Unlike
223
Ra, this
treatment is tailored to the disease burden of each patient with the
number of fractions, dose and area covered dependent upon patient
specic information (Supplementary Table 1). Using this, estimates
were carried out employing two models previously described by
Moquet and colleagues, which were designed to provide a relatively
simple assessment of RT doses [32].Therst model, the blood ow
model (BF), was based on the time taken for blood to ow through
the high dose planned area. Moquet et al., assumed that 6 L of whole
blood ows through the high dose region within a 1-min IMRT exposure
time [32]. For this study, individual patient weights were available,
therefore blood volume was more accurately estimated by assuming
each kg of body weight contains 75 ml of blood [68],(BF
1
). The
absorbed blood dose per fraction was then estimated to range
between 0.159 and 0.291 Gy and this was equivalent to a cumulative
dose of 5.8728.715 Gy at IMRT treatment completion (see supplemen-
tary Table1). Although lymph nodes are in proximity ofvascularized tis-
sue, they do not circulate blood, instead they circulate lymph uid. BF
1
was therefore likely to overestimate the lymph node absorbed dose.
The uid shift within lymph nodes is approximated to ~4 L per day
[69] which may be negligible during treatment. To model the lymph
node exposure, the static absorbed blood dose was calculated (BF
2
)
using similar principles, subsequently scaled down to lymph node
planned volume. Lymph node absorbed blood dose per fraction
represents 0.0040.015 Gy which was equivalent to 0.1520.508 Gy
by IMRT completion (patient individualised schedule). This BF
2
model
was likely to be a more accurate representation of static PBL irradiation
in this tissue that may then be ltered to the circulatory system. By com-
bining thetwo models, the cumulative absorbed blood dose to high dose
volume likely was between 1.131 and 2.470 Gy by completion of
individualised IMRT schedule. Neither of these models consider the
low dose regions that are also exposed during treatment; therefore, so
both will underestimate the total blood dose. The original work of
Moquet and colleagues [32] was relatively simple and requires further
validation to ascertain exposure circumstances in which such models
can be applied. In this study, patient specic data was utilized to rene
these models, with limited success. The uncertainties in CTPV absorbed
blood dose were derived by propagation of the uncertainties associated
with factors in Eqs. (1) and (2). For CTPV physical absorbed blood dose
these were the planned volume (considered minimal at 11.5% varia-
tion), the dose to planned volume and the estimated scaling factors.
The dose to the plan region was expected to have an uncertainty of
less than 3%, based on published estimates of the upper bound of
inter-treatment dosimetric uncertainty [70] and greater errors would
be detected by treatment QA and trigger re-planning and re-
validation. To estimate the whole-body volume, it was assumed that 1.
01 g of human body mass ts within 1 cm
3
and that the tissue density
within the target volume was consistent with this for the scaling factor
estimation, with an error within 1%. The total error associated with CTPV
estimations was considered to be 6%. It is important to note, however,
that the estimates of uncertainty are themselves uncertain and further
work is needed here to better understand how patient specicdata
can contribute to such estimates. In addition, more detail with respect
to beam-on times are required to improve theBF dose model, in partic-
ular, for larger dose volumes, therefore, despite its use previously, this
model was not considered further.
To enable the absorbed blood dose estimation by dicentric assay in
this mixed (
223
Ra and IMRT) exposure scenario, the absorbed blood
dose ratio between each source was estimated per treatment cycle.
This then facilitated use of the criticalitydose estimation technique,
originally designed to separate and quantify neutron and gamma expo-
sures following a nuclear accident or incident [35], but here used to as-
sess the IMRT and α-particle absorbed doses, on the basis of either the
Fig. 4. Comparison of
223
Ra dose estimates. M-FISH
LET
ratio was implemented in the dicentric assay estimates of blood dose for n= 5 patients (patient ID 15), the estimates for these
patients were compared across all other models. CTPV
1
and CTPV
2
calculated fromthe dicentric assay utilizing the physical blood dose estimates by
223
Ra and respective IMRT models.
Simple linear regression plotted for CTPV
1
Y = 0.019 X + 0.004, R
2
= 0.559 and for CTPV
2
Y = 0.115 X + 0.025, R
2
= 0.600. Physical
223
Ra dose estimated through clearance of
223
Ra from circulation 24 h post administration. Simple linear regression plotted for Y = 0.028 X + 0.012, R
2
= 0.301. The error associated with the physical
223
Ra was conservatively
estimated to 50% and propagated to CTPV
1
,CTPV
2
. The error for M-FISH
LET
dose was propagated as 30% for C2 and 14% for C3C5.
I. Bastiani, S.J. McMahon, P. Turner etal. Nuclear Medicine and Biology 106107 (2022) 1020
17
treatment planning information (CTPV ratios), or the categorisation of
complexity of aberrations observed using M-FISH. The dicentric assay
was applied to two separate models, the rst utilizing the ratio derived
from physical models,
223
Ra clearance and IMRT CTPV models, and the
second utilizing the M-FISH
LET
ratio based on the categorisation of
high LET to low LET exposed cells. The absorbed blood dose estimations
were carried out for 5 patients for which blood samples were drawn and
the dicentric equivalent frequency estimated. The CTPV derived ratios
assume that IMRT induced aberrations accumulated in the circulatory
blood pool by C3 are not cleared from the peripheral pool in the follow-
ing C4 and C5 treatment cycles and similarly for the
223
Ra, it was as-
sumed the aberrations accumulate through treatment with no clear-
ance. This results in a plateau of IMRT dose for CTPV
1
as the dose ratio
was based on a large IMRT component with a small
223
Ra dose. For
CTPV
2
, as the model was based on high dose regions areas, the IMRT es-
timated dose was lower than CTPV
1
, therefore when expressed as a ratio
with
223
Ra, this assumes a higher proportion of absorbed blood dose to
be attributed to
223
Ra, which decreases the estimated dose for C4 and
C5. The dicentric assay was estimated from the ratio of physical
223
Ra
and CTPV dose estimates with a conservative error of 50% being attrib-
uted to the
223
Ra dose, this was considered to be the largest uncertainty.
Future studies will aim to reduce this uncertainty by increasing the
number of patients in the study and by including later timepoints.
The M-FISH
LET
model was based on the chromosomal aberration
spectrum in PBL sampled 4 weeks after each [
223
Ra]RaCl
2
administration. Following IMRT completion, the dose was estimated at
1.167 ± 0.092 Gy. This was based on the assumption that all IMRT in-
duced aberrations would be of simple type while
223
Ra aberrations
were of complex type. As a larger proportion of cells containing complex
aberrations than simple chromosomal aberrations was observed, this
was reected in the dose ratio. Accordingly, the dose attributed to
223
Ra was proportionally larger than that attributed to IMRT. The result-
ing IMRT absorbed blood dose estimated by M-FISH
LET
was therefore
lower than both CTPV dose estimates. The physically derived
223
Ra esti-
mates were representative of the period taken for
223
Ra to clear from the
blood. Due to the sampling schedule being every 4 weeks, it cannot be
excluded that absorbed dose from
223
Ra was also received by circulating
PBLs in the vicinity of metastatic sites, especially as metastatic sites tend
to be highly vascularized [7173]. The M-FISH
LET
absorbed blood dose
estimates may better account for this, with the
223
Ra dose by C5
estimated to be 0.665 ± 0.080 Gy. This estimate was signicantly
larger than CTPV
1
0.024 ± 0.027 Gy and CTPV
2
0.141 ± 0.042 Gy. The
largest uncertainty in the M-FISH
LET
absorbed blood dose estimation
was found to be also in C2 whereby the variation in the frequency of
cells consistent with IMRT exposure(cells containing simple aberrations
only) was up to 13% and for those consistent with
223
Ra exposure of up
to 28%. The error from the calibration curve used was estimated to be12
and 23% for IMRTand
223
Ra absorbed blood doseestimates, respectively.
The total propagated error on absorbed dose was within 30% for the M-
FISH
LET
derived estimates.
In this study, the complexity of chromosome exchange observed in
PBLs was used as a biomarker of radiation quality [36] from which to
make estimates of absorbed blood dose ratio, termed the M-FISH
LET
.
Based upon principles of radiation track structure and PBL cell geometry
[36,74], all cells which contained at least one complex chromosome ex-
change [75,76] were categorised as having been traversed by high LET
α-particles emitted from the
223
Ra, while damaged cells containing
only simple chromosome exchanges (reciprocal translocation, dicen-
trics and rings) were categorised as being exposed to low LET radiation
from IMRT. Although in vitro studies do show the majority of high-LET
induced damage to result in complex chromosome aberrations largely
independent of dose [7781] it is also the case that the simple ex-
changes can be directly induced after α-particles of lower incident LET
[74] and, exposure to low-LET radiation will result in the formation of
complex exchanges, in a manner strongly dependent upon dose [50,
82,83]. For instance, an increasing fraction of complex exchanges of up
to 2040% have previously been attributed to exposure of a large target
eld in IMRT treated prostate cancer patients [84,85]. Therefore, it is
likely that IMRT absorbed blood dose may be underestimated using
the M-FISH
LET
reported here. Given the potential usefulness of this
ratio in cases of unknown exposures where physical information is not
available, further work to determine frequencies of complex exchange
occurrence in IMRT only and
223
Ra only treated patients is required.
As the M-FISH
LET
absorbed dose ratio and the dicentric
quantication was based on cytogenetic observations of sampled PBL,
the resulting doses estimated will be directly affected by haematopoietic
cell death and repopulation dynamics. The IMRT dose estimates were
found to plateau between C3C5 suggesting cells containing unstable
aberrations remain over the time course studied. However, IMRT has
been shown to signicantly decrease the number of PBLs in circulation,
and therefore the clearance and repopulation dynamics should also be
taken into consideration for the CTPV models [25,86,87]. White blood
cell counts have been found to increase within 68 weeks of therapy
completion with a signicant increase in lymphocyte population after
3 months [26,88]. The 4-week period between C3C4 (after end of
IMRT) could provide a sufcient break for haematopoietic cells to
boost PBL repopulation, which if the case, would have the effect of dilut-
ing the frequency of persisting unstable chromosomal events. Newly in-
duced aberrations by subsequent
223
Ra treatment cycles would then
add to this aberrant cell pool. There is also the potential of bystander re-
sponses playing a role both in cell turnover and aberration formation
[89]. Further work on blood samples representative of later [
223
Ra]
RaCl
2
administrations (C6) and follow up samples (up to 1 year post
start of treatment), together with patient hematological counts, will
help elucidate the cellular dynamics of damaged PBL. An assessment of
the occurrence and type of stable chromosome exchange from these
samples will also offer the potential to make estimates of absorbed
dose delivered to the bone marrow.
In conclusion, in this study we have evaluated a number of absorbed
blood dose methods for mixed eld exposure, in a unique population of
patients receiving external beam photons and a systemically delivered
α-emitter
223
Ra. We highlight key observations and limitations to estab-
lish an approach from which we can make dose assessments to better
understand mixed eld exposures. The models presented provide an
initial estimation of cumulative absorbed dose received to the blood
during incremental IMRT fractions and [
223
Ra]RaCl
2
injections, all of
which move towards assessing patient specic dose information for
mixed eld treatment to help optimise treatment outcomes and
minimise patient risk in the future.
Acknowledgements
This project received funding from Public Health England, as part of
the PHE PhD studentship scheme. This work was supported by the
Movember Prostate Cancer UK Centre of Excellence (CEO13_2-004)
and the Research and Development Division of the Public Health Agency
of NI (COM/4965/14).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.nucmedbio.2021.12.002.
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... The research papers cover studies in cytogenetics after radium-223 treatment [9], the role of antioxidants in the radiosensitivity to [ 177 Lu]Lu-DOTATATE [10], relating the biological effects of radionuclides such as Auger electron-emitters gallium-67 and indium-111 to that of EBRT [11] and determining the DNA damaging capacity of Auger electron-emitter thallium-201 [12]. The future of Auger electron-emitting radiopharmaceuticals was also discussed in a commentaries [13]. ...
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Background: Radionuclide radium-223 improves survival in men with metastatic castrate-resistant prostate cancer. The United States (US) Food and Drug Administration Adverse Events Reporting System (FAERS) is a post-market pharmacovigilance database valuable for adverse event (AE) assessments. We analyzed FAERS to identify disproportionate AE signals related to radium-223, and to explore radium-223's international utilization. Materials and methods: We identified 2182 radium-223 cases associated with AE(s) from 2013 to 2018. The duration of radium-223 therapy was calculated. Reporting odds ratio (ROR) and proportional reporting ratio (PRR), with 95% confidence intervals (CIs), were calculated for AEs of interest. ROR shows disproportionate signals if the lower limit of the 95% CI > 1. PRR shows disproportionate signals if PRR ≥ 2, χ2 statistic ≥ 4, and ≥ 3 AEs were reported. We identified any US Food and Drug Administration enforcement actions for radium-223. Results: A majority (60.8%) of events occurred outside the US. Among patients with radium-223-associated AEs, the median therapy duration was only 56 days (corresponding to 2-3 treatment cycles). Disproportionate signals were detected for general health deterioration (ROR, 5.03; 95% CI, 4.23-5.98 and PRR, 4.94; 95% CI, 4.16-5.87), bone pain (ROR, 4.53; 95% CI, 3.67-5.59 and PRR, 4.48; 95% CI, 3.63-5.53), and hematologic AEs including anemia (ROR, 2.89; 95% CI, 2.55-3.27 and PRR, 2.80; 95% CI, 2.48-3.17), thrombocytopenia (ROR, 3.22; 95% CI, 2.77-3.74 and PRR, 3.16; 95% CI, 2.72-3.67), and pancytopenia/bone marrow failure (ROR, 4.83; 95% CI, 4.11-5.67 and PRR, 4.73; 95% CI, 4.03-5.55). There were no enforcement actions for radium-223. Conclusions: Patients with metastatic castrate-resistant prostate cancer experiencing AEs are only receiving one-half the prescription dose of radium-223 required for survival benefit. Radium-223 is associated with health deterioration, bone pain, and hematologic AEs. Real-world analyses are important for ongoing radium-223 risk-benefit assessments.