Detection of circulating and disseminated neuroblastoma cells using the Imagestream Flow Cytometer for use as predictive and pharmacodynamic biomarkers Swathi Merugu 1 , Lindi Chen 1 , Elizabeth Gavens 1,2 , Hany Gabra 2 , Mark Brougham 3 , Guy Makin 4,5 , Antony Ng 6 , Dermot Murphy 7 , Alem S. Gabriel 1 , Michael L. Robinson 1 , Jennifer H. Wright 1 , Susan A. Burchill 8 , Angharad Humphreys 9 , Nick Bown 9 , David Jamieson 10 and Deborah A. Tweddle 1,2* 1 Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University; 2 Great North Children’s Hospital, Newcastle; 3 Royal Hospital for Sick Children, Edinburgh; 4 Royal Manchester Children’s Hospital; 5 Manchester Academic Health Sciences Centre, University of Manchester; 6 Royal Hospital for Sick Children, Bristol; 7 Royal Hospital for Sick Children, Glasgow, 8 Leeds Institute of Medical Research, St James’s University Hospital, Leeds, LS9 7TF, 9 Northern Genetics Service, Newcastle upon Tyne Hospitals NHS Trust, 10 Northern Institute for Cancer Research, Newcastle University, U.K. 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1
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Detection of circulating and disseminated neuroblastoma cells using the Imagestream
Flow Cytometer for use as predictive and pharmacodynamic biomarkers
Swathi Merugu1, Lindi Chen1, Elizabeth Gavens1,2, Hany Gabra2, Mark Brougham3, Guy
Makin4,5, Antony Ng6, Dermot Murphy7, Alem S. Gabriel1, Michael L. Robinson1, Jennifer H.
Wright1, Susan A. Burchill8, Angharad Humphreys9, Nick Bown9, David Jamieson10 and
Deborah A. Tweddle1,2*
1Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research,
Newcastle University; 2Great North Children’s Hospital, Newcastle; 3Royal Hospital for Sick
Children, Edinburgh; 4Royal Manchester Children’s Hospital; 5Manchester Academic Health
Sciences Centre, University of Manchester; 6Royal Hospital for Sick Children, Bristol; 7Royal
Hospital for Sick Children, Glasgow, 8Leeds Institute of Medical Research, St James’s
University Hospital, Leeds, LS9 7TF, 9Northern Genetics Service, Newcastle upon Tyne
Hospitals NHS Trust, 10Northern Institute for Cancer Research, Newcastle University, U.K.
Running title: Circulating and disseminated tumour cells in neuroblastoma
Supplementary Fig. S4f). This led to an increase in G1 population after Nutlin-3 treatment but
no change in G1/S phase ratio i.e., 9.1 after Nutlin-3 treatment compared to DMSO control
(8.7) in WBCs from Case-No-12 BM (Fig. 5f & Supplementary Fig. S4d).
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Discussion
The aim of the present study was to detect NB CTCs from blood and DTCs from BM using
the high-resolution ISx to use as biomarkers in NB. Recently we reported CTC detection and
characterisation from patients with oesophageal, hepatocellular, thyroid and ovarian cancers
using the ISx (28,37). Various techniques have been used to detect CTCs such as DEPArray
and CTC-iChip. DEPArray has been previously used for NB cell lines and patient BM
samples (11) but the current study is, to our knowledge, the first to detect NB CTCs and
DTCs using the ISx. Previously we reported between 0-118 CTCs /ml in thyroid cancer and
0-20 CTCs/ml in hepatocellular carcinoma (28) using similar methods on the ISx. This
compares with 0-264 NB CTCs/ml and a mean of 12 CTCs/ml in the current study.
Seeger et al reported >104 NB cells per 105 nucleated cells in BM samples from 103/267
patients and >104 NB cells per 105 nucleated cells in 2/174 patient blood samples from high-
risk metastatic NB patients at diagnosis using anti-GD2 immunocytology. They concluded
that quantifying NB cells in BM and peripheral blood at diagnosis and during induction
therapy provides an important poor prognostic marker for patients with stage IV NB (38). In
our study higher number of CTCs were detected at diagnosis (1-264/ml) compared to relapse
(1-39/ml) due to clearance of CTCs from the blood by chemotherapy and likely earlier
detection of relapsed disease. It is not possible to compare the sensitivity of our method for
detection of CTCs with other published studies as CD45+ve cells were depleted prior to
analysis. In 5/17 cases where DTCs were detected without bone marrow involvement this
may have been due to sampling variation, the assessment of bone marrow on the basis of
morphology alone or NB cells passing through the bone marrow rather than homing there.
Although GD2 is expressed on > 90% of neuroblastoma bone marrow metastases at diagnosis
and relapse (39), it is possible that using this technique we are missing a small proportion of
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tumour cells which do not express GD2. A future study should compare the sensitivity of the
ISx to detect DTCs with bone marrow examination using immunocytology and also compare
with NB specific mRNA expression by qRT-PCR. In untreated high-risk NB patients there
was a weak correlation between numbers of CTCs with the level of the NB- specific mRNA
PHOX2B detected by qRT-PCR in BM but only small numbers of patients were studied (n
=22).
Higher numbers of CTCs were detected in untreated patients with high-risk NB who did not
achieve a BM CR after first line induction therapy versus those who did, suggesting that CTC
enumeration may prove useful to guide the length of induction chemotherapy in patients with
high-risk NB in the future. However, this was not the case for DTCs, which could be due to
much higher numbers of DTCs which were not visually confirmed. These observations now
need to be extended to a larger, prospective study of high-risk NB. Due to our sample size of
40 patients including 16 relapse cases, it was not possible to evaluate the prognostic
significance of CTC/DTC numbers. DNA ploidy of tumour cells was not useful for
distinguishing CTCs from WBCs due to the presence of diploidy in the majority of NB CTCs
studied, but ploidy of CTCs/DTCs did reflect the ploidy status of the primary tumour.
We also evaluated the use of cfDNA for detection of circulating nucleic acids from blood and
BM plasma collected for CTC/DTC studies in Cell Save tubes. In NB the genomic profile of
the tumour is necessary for treatment stratification. Various methodologies have been
reported for detecting NB genomic profiles with SNP arrays now frequently used in national
reference laboratories (40). Chicard et al reported ctDNA copy number analysis using
Oncoscan arrays in 66/70 patients with copy number profiles obtained in 74% of patients
(19).
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In 5/6 blood or bone marrow plasma samples collected at the same time as CTCs/DTCs,
CNA were detected in ctDNA using Oncoscan FFPE arrays. In one case the paired blood and
bone marrow plasma showed the same CNA confirming the usefulness of BM plasma for
detecting CNA in NB as previously reported (41), and in another case the CNA from plasma
cfDNA were identical to those in a concurrently biopsied metastatic disease site. cfDNA
extracted from blood and BM plasma isolated from samples collected in Cell Save tubes for
CTCs/DTCs could be used to detect metastatic NB tumour specific genomic alterations and
should be evaluated prospectively in future clinical trials.
We are developing MDM2 inhibitors as a novel therapy for NB (42-44) and MDM2
inhibitors are currently being evaluated in adult and paediatric early phase clinical trials
therefore we sought to establish if CTCs could be used as a circulating PD biomarker . The
optimum PD biomarker for MDM2 inhibitor activity is activation of the p53 pathway in
tumour cells detected by increased expression of a p53 induced gene. Macrophage Inhibitory
cytokine (MIC1) has been used as a surrogate marker in plasma samples (45), but elevation
of MIC1 levels are not specific for tumour cells highlighting the importance of developing
less invasive, but tumour specific pharmacodynamic proof-of-mechanism biomarkers. In the
current study detection of increased p53 and p21 expression in SHSY5Y cells following
Nutlin 3 treatment but not mutant p53 Be2C cells is consistent with our previous studies of
MDM2 inhibitors in these cell lines (46,47).
In healthy volunteer blood samples exposed to MDM2 inhibitors ex-vivo, blood spiking
studies with NB cells and then patient blood and BM samples, we demonstrated increased
p53 and p21 protein expression and an increase in G1 population in CTCs, DTCs and WBCs
after Nutlin-3 consistent with our previous studies testing p53 function in diagnostic NB
biopsies showing induction of the p53 pathway following ex-vivo irradiation (48). The
current study demonstrates proof-of-concept to use p21 as a sensitive and specific PD 21
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biomarker of MDM2 inhibitor activity for CTCs and DTCs detected by the ISx, but its
usefulness for CTCs may be limited by small numbers of CTCs present in relapsed blood.
p21 expression and the G1 population also increased in WBC following Nutlin 3 highlighting
the importance of studying tumour cells rather than surrogate WBC to study proof-of-concept
PD biomarkers of MDM2 inhibitor activity.
In conclusion, this is the first study to demonstrate the clinical utility of NB CTCs detected
by the ISx as non-invasive PD biomarkers of novel therapies in early phase clinical trials. A
future, larger study is needed to investigate whether they are predictive biomarkers of
response and also to determine if they are potential prognostic biomarkers to further refine
risk stratification in high-risk NB.
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Acknowledgements
We are very grateful to all patients and families for donating samples, to research nurses at all
study sites (Great North Children’s Hospital Newcastle (GNCH), Royal Manchester
Children’s Hospital, Royal Hospitals for Sick Children in Edinburgh, Glasgow and Bristol)
for collecting samples and to Geoff Bell (GNCH) for coordinating this national, multi-centre
study.
We are thankful to the following for providing cell lines Sue Cohn (NBLW), Barbara
Spengler (Be2C), Rogier Versteeg (NGP), Jean Bernard (SKNAS) and Penny Lovat
(SHSY5Y, SHEP). We are also grateful to Lina Hamadeh for statistical support and the
Newcastle University Flow Cytometry Core Facility.
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Table 1: Summary of patients in study according to risk group, MYCN status and bone marrow involvement (n=42)a
N =42 a
TotalNumber of patients at Diagnosis
n= 26
Number of patients at Relapse
n=16
Number of patients with CTCsd Number of patients with DTCse
Diagnosisn= 20
Relapsen= 6
Diagnosisn= 20
Relapsen= 5
Risk GroupN= 42
Low n=6
3 3 1 0 0 1
Intermediaten=2
0 2 0 0 0 0
Highn=34
23 11 19 6 20 4
MYCN statusN= 42b
MNAn=16
8 8 8 5 7 4
Non-MNAn=25
17 8 12 1 13 1
BM involvedN= 42c
Yesn=24
21 3 17 2 18 2
Non=17
5 12 3 3 2 3
a2 patients studied at diagnosis and relapse, b1 case MYCN not studied, c1 case BM not examined at relapse, d42 samples studied for CTCs (1 fail), e35 samples studied for DTCs. MNA = MYCN amplified
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Figure 1: CTCs and bone marrow response. a Scatter plot of Intensity_MC GD2 against
Intensity_MC CD45 of patient blood sample (Case-No-22) generated using IDEAS software.
Potential CTCs were gated by visual inspection of images. b Immunofluorescence image of a
CTC (GD2+ve/CD45-ve cell) with an intact nucleus (DAPI) by analysis of a single dot (cell)
from the GD2 +ve region of the scatter plot. c Immunofluorescence image showing three cells
in a single image: a CTC (GD2+ve/CD45-ve) and WBCs in doublets (CD45+ve/GD2-ve) to
show compensation of fluorochromes. d Immunofluorescence image of a CD45 +ve region
showing a CD45+ve/GD2-ve WBC. e Scatter plot of Intensity_MC GD2 against Intensity_MC
CD45 of BM sample (Case-No-22) showing DTCs. BM cells in higher intensity GD2 gated
regions were GD2+ve/CD45-ve DTCs and the scatter plot shows very few CD45+ve cells
towards the X-axis, compared with the paired blood sample which had more CD45+ve cells. f
Immunofluorescence image showing a GD2+ve/CD45-ve DTC. g-h Scatter plots showing
numbers of CTCs in 41 patient blood samples (2 cases at both diagnosis and relapse, 1 fail)
and numbers of DTCs in 35 patient BM samples. In 26 cases ≥1 CTC was detected and in 25
cases ≥1 DTC was detected. Horizontal line represents median with range. The mean number
of CTCs and DTCs detected was 12 and 5431 per ml of blood and BM respectively. i Scatter
plot showing the association between numbers of CTCs in 41 blood samples in relation to
BM involvement (P<0.0001- Mann-Whitney-test). j Scatter plot showing the association
between numbers of DTCs in 35 BM samples and BM involvement (P<0.0001- Mann-
Whitney-test). k Scatter plot showing the number of CTCs/ml blood in 19 newly diagnosed
high-risk NB patients with BM involvement at diagnosis comparing those who achieved a
BM complete response (CR) after first line induction therapy versus patients whose BM was
not in CR. l Scatter plot showing the number of DTCs/ml in BM aspirates from 18 patients
with BM involvement who achieved a CR v those who did not after first line induction
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therapy (mean ± SD), BM +ve = BM involvement, BM –ve = Bone marrow not involved,
Horizontal lines = median with range.
Figure 2: Ploidy determination from CTCs in NB blood samples. a DNA histogram showing
the DNA content of residual WBCs and CTCs from a blood sample (Case-No-15) following
WBC depletion. The histogram shows an overlay of the DNA content of CTCs (red peak) and
WBCs (black peak) to determine the ploidy status of CTCs. The gatings were DNA 1 (single
CTCs/WBCs) and DNA 2 (CTC/WBC doublets), but for ploidy determination, only single
CTC gatings were used and compared with a WBC DNA ratio of 1. From the histogram,
Case-No-15 was considered diploid as CTC DNA was < 1.25× WBC DNA ploidy. bi SNP
array log R ratio track of Case-No-15 primary tumour at diagnosis showing a diploid tumour
and multiple segmental chromosomal abnormalities (SCA) (2p, 4q with hyper-rearrangement,
6p, 9p, 11q, 12pq, 17q gain and 1p, 3p, 4p, 11q loss). bii B allele frequency c DNA histogram
from Case-No-33 blood sample at first relapse with DNA ploidy of CTCs determined from
WBC DNA ratio found to be hyperdiploid with CTC DNA >1.25× WBC DNA ploidy. di
Log R ratio track of Case-No-33 BM aspirate at further relapse showing near-triploidy
(whole chromosomal abnormalities-chr7,chr12, chr18, chr20, chr22 gain and chr9, chr10,
chr11, chr14, chr16, chr19 loss, and SCA-loss of 1p and 6q). dii B allele frequency. The
profile also shows amplification of MYCN and ALK on chromosome 2p which were also
present in the primary tumour at diagnosis when the tumour was diploid. Yellow straight line
on the SNP log R ratio indicates 0, normal diploid copy number and the green data points
indicate the log R ratio of all individual SNPs. An increased or decreased log R ratio
indicates gained and deleted regions of chromosomes respectively.
Figure 3: a & b Comparison of copy number profiles from corresponding cfDNA from blood
and BM (Case-No-23). ai ) SNP array log R ratio track and (aii) B allele frequency plot log R
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ratio from Oncoscan FFPE array of cfDNA from blood and (bi) and (bii) from BM showing
the SCAs including +2p,-3q, +6p, -6q, -9p, +11p, -11q, -17p, + 17q and WCAs such as -5,
+7, -10, -19, +18 which are identical in cfDNA from both sites. The primary tumour was not
biopsied in this case at diagnosis so unavailable for comparison c & d Comparison of copy
number profiles from Case 11-diagnostic primary tumour and Case 11-R cfDNA from blood
at relapse ci & di) SNP array log R ratio track and (cii & d ii) B allele frequency plot log R
ratio c) Illumina array showing ALK and MYCN amplification together with -1p, -10q +11q
and +17q. d) Oncoscan array of Case 11-R cfDNA showing ALK and MYCN amplification -
1p and +17q and additional gains and losses including +1q, -5p, -18p, +18q. Interestingly 10q
loss and 11q gain were not detected in Case 11-R cfDNA.
Figure 4: The effect of Nutlin-3 treatment on p53 wt SHSY5Y cells, SHSY5Y cells spiked
into healthy volunteer blood, CD45+ve WBCs (blood and BM), CTCs and DTCs (Case-No-
12). ISx immunofluorescence images of (a & b) p53 wt SHSY5Y neuroblastoma cells and
spiked cells following treatment with DMSO or 10µM Nutlin-3 for 24h showing increased
p53 and p21 expression following Nutlin-3 treatment. (c & d) Case-No-12 Blood and BM
WBCs (CD45+ve/GD2-ve) showing increased nuclear p53 and p21 expression in Nutlin-3
treated cells compared with controls. (e & f) Case-No-12 CTCs and DTCs showing increased
nuclear p53 and p21 expression following Nutlin-3 treatment compared with DMSO control.
Figure 5: The effect of Nutlin-3 on p53 and p21 expression and the cell cycle in DTCs &
BM CD45+ve cells from Case No-12. a & c Histograms showing increased p53 expression in
DTCs and BM CD45+ve cells when treated with 10µM Nutlin-3 for 24 h compared with
DMSO. b & d Histograms showing increased p21 expression after Nutlin-3 treatment
compared with DMSO controls in DTCs and BM CD45+ve cells. e Histograms showing cell
cycle analysis of DTCs after Nutlin-3 treatment showing an increased G1 population. f Bar