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

Key words: Neuroblastoma (NB), Circulating tumour cell (CTC), Disseminated tumour cell

(DTC), Disialoganglioside (GD2), Imagestream Imaging flow cytometer (ISx).

Financial support

This work was funded by a Newcastle University Overseas Research Studentship Award (S.

Merugu), the Children’s Cancer & Leukaemia Group (CCLG) & Little Princess Trust

(CCLGA 2016 08)(S. Merugu, D. Tweddle, L. Chen, D. Jamieson), Children with Cancer

UK (S. Merugu), the Newcastle upon Tyne Hospitals NHS Charity (E. Gavens, D. Tweddle,

L. Chen), the DUBOIS Childhood Cancer Fund, SPARKS (11NCL05) (L. Chen, D.

Tweddle), Neuroblastoma UK & Niamh’s Next Step (L. Chen, D. Tweddle) the North of

England Children’s Cancer Research Fund (D. Tweddle), Santander Universities &

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Association of Physicians intercalated scholarship (J. Wright), Mrs Anna Upjohn (M.

Robinson), Cancer Research UK (CRUK-C9380/A25138) (D. Jamieson, E. Gavens), Action

Medical Research/Great Ormond Street Hospital Children’s Charity (GN2390)(A. Gabriel, D.

Tweddle) and the Sir Bobby Robson Foundation (for purchase of the Image Stream flow

cytometer). The authors acknowledge the support of the National Institute for Health

Research Clinical Research Network: Cancer (UKCRNID16957) and the Experimental

Cancer Medicines Centre (ECMC) Paediatric network (G. Makin, D. Murphy, A. Ng, D.

Tweddle).

*Corresponding author: Deborah A. Tweddle, Wolfson Childhood Cancer Research

Centre, Northern Institute for Cancer Research, Newcastle University, Level 6, Herschel

Building, Brewery Lane, Newcastle upon Tyne, NE1 7RU, U.K. Tel: +44 (0)191 208 2230,

Fax: +44 (0) 191 208 4301; Email: deborah.tweddle@newcastle.ac.uk

Conflict of Interest

The authors declare no potential conflicts of interest

Word Count = 5, 095

Total number of Figures & Tables: = 6

Statement of translational relevance

This the first study to show that circulating tumour cell (CTCs) and disseminated tumour

cells (DTCs) are detectable in neuroblastoma (NB) patient samples at diagnosis and relapse

using the Imagestream imaging flow cytometer. Increased p53 and p21 expression in

CTCs/DTCs following MDM2 antagonist treatment may be a useful pharmacodynamic

proof-of-mechanism biomarker for early phase clinical trials. Enumeration of CTCs at

diagnosis in high-risk NB patients with bone marrow infiltration should be further

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investigated as a predictive biomarker of bone marrow response to first line induction

chemotherapy.

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Abstract

Purpose: Circulating tumour cells (CTCs) serve as non-invasive tumour biomarkers in many

types of cancer. Our aim was to detect CTCs from patients with neuroblastoma for use as

predictive and pharmacodynamic biomarkers. Experimental Design: We collected matched

blood and bone marrow samples from 40 neuroblastoma patients to detect GD2+ve/ CD45-ve

neuroblastoma CTCs from blood and disseminated tumour cells (DTCs) from bone marrow

(BM) using the Imagestream Imaging flow cytometer (ISx). In 6 cases circulating free DNA

(cfDNA) extracted from plasma isolated from the CTC sample was analysed by high-density

single nucleotide polymorphism (SNP) arrays. Results: CTCs were detected in 26/42 blood

samples (1-264/ml) and DTCs in 25/35 BM samples (57-291544/ml). Higher numbers of

CTCs in newly diagnosed, high-risk neuroblastoma patients correlated with failure to obtain a

complete BM metastatic response after induction chemotherapy (p< 0.01). Ex-vivo Nutlin-3

(MDM2 inhibitor) treatment of blood and BM increased p53 and p21 expression in CTCs and

DTCs compared with DMSO controls. In 5/6 cases cfDNA analysed by SNP arrays revealed

copy number abnormalities associated with neuroblastoma. Conclusion: This is the first

study to show that CTCs and DTCs are detectable in NB using the ISx, with concurrently

extracted cfDNA used for copy number profiling, and may be useful as pharmacodynamic

biomarkers in early phase clinical trials. Further investigation is required to determine if CTC

numbers are predictive biomarkers of BM response to first line induction chemotherapy.

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Introduction

Neuroblastoma (NB) is a heterogeneous tumour occurring in 10.2 per million children. It has

one of the lowest survival rates of all childhood cancers, with only 67 percent of patients

surviving to five years (1). Treatment of NB depends on the International Neuroblastoma

Risk Group (INRG) classification which is determined by patient age, stage and genetics

including MYCN gene amplification, classifying patients as low, intermediate or high-risk (2).

For high-risk patients defined in Europe as metastatic disease over 1 year of age or MYCN

amplified, intensive multimodality approaches are used including induction chemotherapy,

surgical resection of the primary tumour, consolidation with myeloablative therapy and

autologous stem cell rescue and local radiotherapy followed by immunotherapy and

differentiation therapy. Despite an improved initial response to treatment survival remains

poor (<50% at 5 years) due to eventual drug resistant relapse (3).

To better understand tumour evolution and drug resistance, it is important to consider intra-

tumoural heterogeneity (4), but performing multiple biopsies from different tumour areas is

not feasible in most patients. Studying circulating tumour cells (CTCs) from blood may

inform intra-tumour heterogeneity and tumour evolution (5,6), and such studies are gaining

importance in the clinic as their detection is a non-invasive procedure involving only the

collection of peripheral blood (7). However, as CTCs are very rare, present in around one in

106 leucocytes in prostate cancer patients (8), it is important to increase sensitivity in

detection assays by including an enrichment step.

Technologies recently developed for CTC analysis include microfluidic devices with

antibody-coated microspots (CTC chip), high-throughput microfluidic mixing devices

(Herringbone- Chip) (9), or ultrasound-based isolation in microfluidic devices (10). A

dielectrophoretic array method has been used to isolate disseminated tumour cells (DTCs)

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from bone marrow (BM) from NB patients for tumour genetic analysis (11), and magnetic

bead-based enrichment has been developed to isolate DTCs from BM samples using anti-

GD2 and NCAM antibodies (12). The Amnis Image Stream Imaging flow Cytometer (ISx)

combines the features of classical flow cytometry, including an impartial analysis of a large

number of cells in a short period of time with essential features of fluorescence microscopy

allowing multiparameter cell analysis (13).

Detection of circulating free DNA and circulating tumour DNA (ctDNA) in plasma or serum

of cancer patient blood is another type of liquid biopsy with higher levels detected in patients

with metastatic disease (14). Recent studies have shown the feasibility of detecting ctDNA in

cell free DNA (cfDNA) extracted from plasma to determine genetic aberrations including in

NB (15-19).

In the present study, we detected CTCs and DTCs from NB patient blood and BM samples

identified by GD2 expression and the absence of CD45 expression using the ISx. We show

that higher numbers of CTCs in newly diagnosed patients with high-risk NB are associated

with a failure to achieve a complete BM metastatic response after first line induction therapy

and that CTCs and DTCs can be used as pharmacodynamic biomarkers of novel targeted

treatments e.g. MDM2 inhibitors in early phase clinical trials. To our knowledge this is the

first study to report use of the ISx to detect NB CTCs and DTCs in NB clinical samples.

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Materials and Methods

Cell Culture

A panel of human NB cell lines, N-type (SHSY5Y (20-22), NGP (23), and NBLW-N

(24,25)) and S- type (SHEP, SKNAS & NBLW-S (20,21)) were used to develop a protocol

for detection of NB cells using the ISx. Cell lines were routinely maintained in RPMI

medium supplemented with 10% v/v Fetal Bovine Serum (FBS, Gibco) and 1% v/v

penicillin-streptomycin (Sigma) in 5% CO2 in air in a humidified incubator at 37oC, regularly

tested and found to be free from Mycoplasma using MycoAlert™ (Lonza, Basel,

Switzerland). Authentication of the NBLW cell line was performed as described previously

(26). NGP and SHSY5Y cell lines were authenticated using STR genotyping (New Gene

Limited, International Centre for Life, Newcastle, U.K). SKNAS, SHEP and SKNBE(2c)

(Be2C) cells were authenticated using cytogenetic analysis (27). All cell lines were used

within 6 passages or within a period of 6 months.

Detection of NB cells using the ISx

A GD2 antibody conjugated with PerCP Cy5.5 (Peridinin chlorophyll protein, BD

Pharmingen, UK, 563438, 14.G2a) and anti-neural cell adhesion molecule (NCAM)

conjugated with PE CF594 antibodies (BD Pharmingen, UK, 562328, B159) were used as

NB markers in different cell lines alongside DAPI (BD Pharmingen, UK, 564907) nuclear

staining. Imagestream data was analysed using IDEAS image stream analysis software using

methods described previously (28).

Patient samples and Clinical Study design

This study was undertaken in accordance with the ethical principles of the Declaration of

Helsinki. Following institutional review board approval (ethics reference number

14/NW/0154), local institutional approval and written informed consent from the patient or

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carer, clinical samples (4-8ml blood and 1-3ml BM) were obtained from patients with newly

diagnosed or relapsed NB from 5 UK Paediatric Oncology Principal Treatment Centres

(Supplementary Fig. S1b-c). Samples were collected in Cell Save® (Veridex®, Menarine

diagnostics, UK) tubes, sent by post at room temperature within 72 h of collection, and

processed within 96 h of collection. In the clinical study 40 NB patients were recruited (32

high, 6 low and 2 intermediate risk), 24 were studied solely at diagnosis, 14 solely at relapse

and 2 at both diagnosis and relapse (Supplementary Fig. S1a & d). In total 42 blood samples

were studied for CTCs (1 fail) and 35 paired bone marrow samples. In 7 cases only a blood

sample was obtained (Supplementary Fig. S1c). International Neuroblastoma Risk Group

(INRG) and other clinical characteristics of patients are shown in Table 1 and Supplementary

Table T2. For ex-vivo Nutlin-3 treatment 4 blood and 2 bone marrow samples were collected

in EDTA tubes from 4 patients. Clinical information is correct up to 31/5/2018. All newly

diagnosed high-risk patients studied were treated on the European High Risk Neuroblastoma

trial (HR-NBL1) (29).

Analysis of patient samples using the ISx

Samples (blood and BM) were blocked to prevent non-specific antigen binding, red cell lysed

and enriched for non-haematopoietic cells using previously reported methods (30,31). Cells

were then permeabilised in perm-wash buffer (BD Pharmingen) and stained with

immunofluorescent antibodies including GD2-PerCp, NCAM-PE, and CD45- PE-Cy 7 (Bio

Legend, UK, 560915, H130) and nuclei stained with DAPI. Following incubation for 1h,

stained cells were washed, resuspended in PBS and processed on the ISx according to the

manufacturer’s protocol, and the presence of CTCs and DTCs detected using IDEAS

software (See Supplementary Methods- & Supplementary Fig. S2).

CTCs and DTCs were detected based on brightfield morphology, GD2 expression, a nuclear

signal and the absence of CD45 expression. Potential CTCs were gated in GD 2 and CD45

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scatter plots and visually confirmed from immunofluorescence images, tagged and counted

manually. The numbers of DTCs were counted using IDEAS software as there were too

many to count manually. The diameter of CTCs and DTCs in NB patient blood and BM

samples was calculated as previously described (28). The mean diameter of CTCs/DTCs was

compared with the mean diameter of white blood cells (WBCs; n=5000). The number of

CTCs used to calculate diameter ranged from 4-100 and for DTCs from 4-2000. DNA ploidy

was determined in all blood and BM samples with ≥ 4 cells based on WBC DNA and

CTC/DTC DNA ratio from a DNA histogram using IDEAS software using the formula CTC

DNA Ploidy = Mean CTC DNA/ Mean WBC DNA. If the ratio of CTC DNA was >1.25 x

WBC DNA ploidy then it was considered hyperdiploid, if not diploid (32).

Collection of plasma from NB patient blood and BM samples

Plasma was separated from blood (n=3 cases) and BM (n=3 cases) samples in Cell Save tubes

by two sequential centrifugations (2,000 g, 10 min) and stored at -80°C in 1 ml aliquots.

cfDNA was isolated from aliquots of double spun plasma using the QIAamp DNA Blood

Maxi Kit (Qiagen). Following isolation the cfDNA yield was quantified using the Qubit®

Fluorometer (Thermo Fisher Scientific) as per the manufacturer’s instructions. cfDNA

fragment size was determined using 1% agarose gel electrophoresis.

Nutlin-3 treatment of NB cell lines and patient samples

Two NB cell lines, p53 wt (SHSY5Y) and p53 mutant (Be2C) (27), and blood and BM

samples were exposed to Nutlin-3 an MDM2 inhibitor, and upregulation of p53 and p21

detected by immunofluorescence using the ISx and compared with DMSO (dimethyl

sulfoxide) controls (Sigma-Aldrich, UK, D2650). Nutlin-3 (Selleckchem, UK) was dissolved

in DMSO to a stock concentration of 100µM. Cells were treated with 10µM Nutlin-3 or an

equal volume of DMSO for 24 h prior to fixation, followed by incubation with GD2 PerCp

(BD Pharmingen, UK), p53 Alexa Fluor-647 (Cell Signaling technologies, UK, 2533S, 1C12)

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at 1:50, p21 Alexa Fluor-488 (Cell Signaling technologies, UK, 5487, 12D1) at 1:50) and

DAPI (BD Pharmingen, UK) and run on the ISx as described above. Cell cycle analysis was

performed using IDEAS software with round single in focussed cells gated on scatter plots.

Using the intensity of the DAPI histogram, mitotic cells and cells in G1, S & G2/M were gated

to observe the effect of Nutlin-3 treatment and DMSO in NB cell lines, healthy volunteer

blood samples and NB patient blood and BM samples. (See supplementary methods for more

details).

Cytogenetic analysis of NB tumours and cfDNA

Using single nucleotide polymorphism arrays (SNP) (n=11 cases), array comparative

genomic hybridisation (CGH) (n=21 cases) and multiplex ligation PCR dependent

amplification (MLPA) (n=6 cases), cytogenetic analysis was undertaken on primary tumours

or bone marrow metastases from all except 4 cases (Supplementary Table T2). For SNP

arrays DNA samples were hybridised to Infinium CytoSNP-850K v1.1 BeadChip (Illumina,

Inc) according to the manufacturer's instructions. Illumina IDAT files were analysed using

BlueFuse Multi software. Using targeted next generation sequencing (NGS) (26) p53 gene

mutational status was determined for the cases treated with Nutlin 3 (Supplementary Table

T3). Affymetrix Oncoscan arrays (OncoScan FFPE CNV) performed by Eurofins Genomics

(Ebersberg, Germany) were used for detecting copy number abnormalities from cfDNA from

plasma with CEL files analysed using Nexus (Biodiscovery) software.

Statistical analysis

Statistical tests were performed using Graph Pad Prism (version 6.04) and IDEAS® software

(Amnis-Imagestream Imaging Flow cytometer). The number of cases with BM involvement

and the presence or absence of CTCs and DTCs was assessed using a Fisher’s exact test. A

Mann Whitney U-test was used to calculate the relationship between numbers of CTCs and

DTCs with BM involvement and BM response to induction chemotherapy. To determine the

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correlation between numbers of CTCs/DTCs detected by qRT-PCR for the NB mRNAs

tyrosine hydroxylase (TH) and PHOX2B) (33) a Spearman rank correlation test was used

(SPSS). All p values reported were two-tailed and considered significant if p≤0.05. Data is

presented as mean ± SEM.

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Results

Detection of GD2 & NCAM expression in cell lines using the ISx

A panel of NB cell lines (n=6) comprising neuronal (N) and substrate-adherent (S) types were

used to determine expression of the NB cell surface markers (GD2 and NCAM) using the ISx.

N-type cell lines (NGP, SHSY5Y and NBLW-N) were found to express a higher percentage

of GD2 and NCAM +ve cells than S type cell lines (SHEP, NBLW-S, and SKNAS). NGP

cells were the most strongly positive N type cell line for GD2 and NCAM (Supplementary

Table T1).

Detection of CTCs and DTCs from NB samples using the ISx

CTCs and DTCs were detected from a scatter plot of cells that were GD2+ve/ CD45-ve with

an intact nucleus (Fig. 1a & e). Potential CTCs/DTCs were gated based on intensity scatter

plots of GD2 versus CD45 for one patient sample and the template saved as a standard and

used for all remaining clinical samples using IDEAS software. Dots in scatter plots were

linked to corresponding cell imagery and visualised to help define gating boundaries (Fig.1a-

f). Once a gate was defined, cell imagery of that population could be inspected and

CTCs/DTCs were visually confirmed based on a round single cell from the bright field image

(BF), with positive GD2 expression, negative CD45 expression and an intact DAPI stained

nucleus (Fig. 1a, b & c). Confirmed CTCs/DTCs were then tagged and saved for further

analysis of cell diameter and DNA ploidy.

CTCs were detected in 26/42 patient blood samples (mean=40/ml, range, 1-264/ml at

diagnosis; mean=6/ml, range, 1-39/ml at relapse, Fig. 1g). DTCs were detected in 25/35 BM

samples (mean=30,342/ml, range, 57-291, 635/ml at diagnosis; mean=2,124/ml, range, 112-

15,688/ml at relapse, Fig. 1h). Table 1 shows a summary of cases studied in relation to

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clinical risk group and MYCN status with 32/40 (80%) cases high-risk and 26/40 (65%)

studied at diagnosis (Supplementary Fig S1a, d & e).

BM involvement with NB on either 1 or 2 aspirates and/or trephines as reported by a

consultant haematologist based on morphology was present in 24/41 cases (including 1 case

at diagnosis and relapse). In patients with reported BM involvement, CTCs were detected in

19/24 cases and DTCs in 20/21 cases (in 3 cases DTCs not studied)(Supplementary Fig S1e).

In patients without reported BM involvement CTCs were detected in 6/17 and DTCs in 5/17

cases (Table 1). The presence of CTCs or DTCs was associated with BM involvement

(p<0.01 for CTCs and p<0.0001 for DTCs, Fisher’s exact test). Similarly there was an

association between the number of CTCs and DTCs and BM involvement (p<0.0001 Mann

Whitney test, Fig. 1i & j). CTCs were detected in 2/42 cases in the absence of DTCs and BM

involvement (one low-risk and one high-risk), and in one case in the absence of DTCs and

the presence of BM involvement (Supplementary Fig 1e). In 3/35 cases DTCs were detected

in the absence of CTCs and presence of BM involvement, and in two cases in the absence of

CTCs and absence of BM involvement. There was no association between the numbers of

CTCs or DTCs and the presence of MYCN amplification in the primary tumour or other

metastatic site biopsied. CTC and DTC numbers from 16 and 21 patients respectively with

untreated high-risk NB treated on the SIOPEN HR-NBL-1 trial were compared with NB-

specific mRNA detected by qRT-PCR for TH and PHOX2B in blood and BM. There was a

weak correlation between CTC numbers and level of PHOX2B mRNA expression in BM

only (r =0.45, p< 0.05) (data not shown).

NCAM was expressed in only 3/11 GD2+ve/CD45-ve blood samples initially examined,

whereas in GD2+ve/CD45-ve cells from BM aspirates weak NCAM+ve DTCs were observed

in 9/11 samples. From these early observations, it appears that there are differences in NCAM

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expression in NB cell lines compared with NB CTCs and DTCs. It has been reported that

polysialylated NCAM and non-polysialylated NCAM expression differ between in vitro and

in vivo conditions (34). Hence NCAM expression was not considered further to detect and

confirm CTCs/DTCs in this study.

Higher CTC numbers are associated with incomplete bone marrow response to

induction chemotherapy

To determine whether the number of CTCs or DTCs at diagnosis were associated with BM

response to chemotherapy in high-risk NB patients with BM involvement at diagnosis

(n=21), numbers of CTCs/DTCs were plotted against BM response after first line induction

therapy (COJEC or N7) (Fig. 1k & l). BM response after induction chemotherapy was

determined by the presence of neuroblastoma cell infiltration in either BM aspirate or

trephine biopsy according to the International Neuroblastoma Response Criteria Bone

Marrow Working Group classification (35). Absence of morphological evidence of NB on 2

aspirates and 2 trephines was considered a complete response (CR) i.e. BM in CR and a +ve

BM aspirate or trephine at 1 or more sites was considered an incomplete response i.e. BM not

in CR (35,36). Patients with higher numbers of CTCs at diagnosis were found to have an

incomplete BM response after first line induction therapy (Fig. 1k, p<0.01, Mann Whitney U

test). In contrast, there was no association between numbers of DTCs at diagnosis and BM

response to first line induction chemotherapy (Fig. 1l, p=0.38).

Detection of ploidy using the ISx

The DNA content of WBCs and CTCs/DTCs in the CD45 depleted blood cell population was

determined from a DNA histogram plotted using IDEAS software. On the DNA histogram of

CD45 depleted cells, visually confirmed CTCs/DTCs were overlaid to determine DNA

content of CTCs/DTCs, from which ploidy could be extrapolated (Fig. 2a & c). The ploidy 14

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status determined in this way from 24/42 CTC samples and 25/35 DTC samples is shown in

Supplementary Table T2. For CTCs 19/24 samples were diploid and 5/24 hyperdiploid and

for DTCs 17/25 diploid and 8/25 hyperdiploid.

The DNA ploidy of CTCs in five cases determined using the ISx was compared with ploidy

from a corresponding primary tumour or BM aspirate determined using high density SNP

arrays. Case-No-15 CTCs were found to be diploid using the ISx (Fig. 2a) and the

corresponding primary tumour SNP array showed diploidy with multiple segmental

chromosomal abnormalities (SCA) without MYCN amplification (Fig. 2b). Case-No-33 CTCs

detected at 1st relapse were hyperdiploid using the ISx (Fig. 2c) and a SNP array performed

15 months later at further relapse on a BM aspirate showed near-triploidy (Fig. 2d). MYCN

and ALK amplification were detected in the SNP array profile from this case as well as 1p

and 6q loss as shown in Fig. 2d. MYCN and ALK amplification were the only SCAs detected

in the SNP array from a lymph node metastasis from the same patient at diagnosis when

diploidy was present. The concordance of ploidy results from 5/5 cases with primary tumour

or BM aspirate SNP arrays with CTC ploidy results from the same patients suggests that

measurement of ploidy status using the ISx is accurate and reliable (Supplementary Table

T2). In addition the ploidy from CTCs/DTCs of 3 cases determined using the ISx was

compared with the ploidy from SNP arrays performed on cfDNA collected at the same time

and found to be concordant (Supplementary Table T2). However, the low frequency of

hyperploidy indicates that ploidy could only be used to distinguish a diploid WBC from a

hyperploid CTC/DTC in a minority of cases.

Measurement of CTC/DTC diameter

Cell diameter was also investigated as an additional feature to differentiate CTCs/DTCs from

WBCs. The diameter of all CTCs and DTCs in samples with ≥4 CTCs/DTCs was measured

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using IDEAS software in 21 and 25 samples respectively (Supplementary Fig S3a & b). A

significant difference was observed between the diameter of WBCs in blood or BM samples

versus CTCs or DTCs respectively (Wilcoxon signed rank test, p<0.0001, Supplementary Fig

S3g & h). However, after plotting the values for each patient the intra-patient variability

between CTC/DTC diameter and WBC diameter suggested cell size would not be a useful

parameter to isolate NB CTCs/DTCs from residual WBCs.

cfDNA copy number analysis using SNP arrays

cfDNA collected in Cell Save tubes (n=6 samples) was analysed by Oncoscan FFPE arrays

and copy number variations (CNV) successfully detected in 5/6 samples. In one case blood

and BM plasma were collected at the same time and the CNV found to be identifical in both

samples (Fig. 3a & b). In 3 cases MYCN amplification was detected by SNP arrays in cfDNA

(Fig 3c & d). In 2 cases the numbers of SCAs detected in the blood plasma at relapse

increased from diagnosis illustrating temporal heterogeneity (Fig. 3 c & d).

CTCs/DTCs can be used as PD biomarkers of MDM2 inhibitor activity

MDM2 inhibitors prevent binding of MDM2 to p53 so stabilising and activating p53 leading

to increased transcription of target genes including p21, which mediates a G1 cell cycle arrest.

p53 mutant Be2C cells and p53 wt SHSY5Y cells were treated with the MDM2 inhibitor

Nutlin-3 for 24h or DMSO control, immunostained with GD2, p53, p21, and DAPI and run on

the ISx. p53 and p21 expression was determined by the percentage of p53 and p21 positive

cells from intensity histograms of p53 and p21 (Fig. 4a and Supplementary Fig. S4a & b).

Mutant Be2C cells expressed a higher percentage of p53 positive cells at baseline compared

with SHSY5Y cells with no increase following Nutlin-3 treatment, whereas in SHSY5Y cells

there was a statistically significant increase in p53 expression following Nutlin -3 treatment

(Fig. 4a and Supplementary Fig. S4a & b and Fig. 5g). In p53 mutant Be2C cells there was

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low baseline expression of p21 and no increase following Nutlin-3 treatment (Supplementary

Fig. S4b and Fig. 5h), resulting in a virtually unchanged cell cycle profile (Fig. 5f), whereas

in p53 wt SHSY5Y cells there was an almost 10 fold increase in p21 expression (Fig. 4a and

Supplementary Fig. S4a) resulting in a strong G1/S cell cycle arrest and increase in G1/S ratio

from 6.4 to 33 (Supplementary Fig. S4c and Fig. 5f).

In order to validate the panel of antibodies with GD2-PerCp, PE Cy7 CD45, p53-Alexa Fluor-

647, p21 Alexa Fluor-488 and DAPI and to compensate data with all fluorochromes, healthy

volunteer blood samples (n=3) were spiked with SHSY5Y cells or Be2c cells, treated with

Nutlin-3 for 24h or DMSO control and p53 and p21 expression measured. After 24h there

was increased expression of p53 and p21 in GD2+ve/CD45-ve SHSY5Y NB cells and

residual GD2-ve, CD45+ve WBC remaining after WBC depletion (Fig. 4b & Fig. 5g & h).

The fold increase in p21 in CD45+ve cells, although statistically significant, was not as great

as for SHSY5Y cells (Fig. 5h), resulting in an increased G1 population but unchanged G1/S

ratio after Nutlin-3 treatment (Fig. 5f). In contrast there was no change in p53, p21 or cell

cycle profile after Nutlin 3 treatment of spiked p53 mutant Be2c cells compared with DMSO

control cells (Fig 5f-h).

We next treated NB patient blood (n=4) and BM samples (n=2) with Nutlin-3 ex- vivo for 24

hr or DMSO control and measured p53 and p21 expression (Supplementary Table T3). In 4/6

samples GD2+ve cells were present but a BM and blood sample from a patient with low-risk

NB at diagnosis had no detectable GD2+ve /CD45-ve CTCs or DTCs. In the 4 Nutlin-3

treated samples (3 blood and 1 BM) with CTCs/DTCs present, increased p53 and p21

expression was seen in GD2+ve/CD45-ve CTCs and DTCs compared with DMSO control

(Fig. 4e & f, Fig. 5a, b, g & h). In Case-No-12 a high number of CTCs (n=264/ml) and

DTCs (n=6383/ml) were detected (Supp Table T3), and a histogram was generated for cell

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cycle changes in DTCs and p53 and p21 expression in CTCs and DTCs, before and after

10µM Nutlin-3 treatment (Fig. 4e & f and Fig. 5a-b & e-h). It was not possible to generate

histograms from CTCs from the two blood samples taken at relapse due to low numbers of

CTCs (Supp Table T3). In CTCs and DTCs baseline nuclear and cytoplasmic p53 was

detectable with nuclear accumulation of p53 following Nutlin-3 treatment (Fig. 4e & f and

Fig. 5a & g). Consistent with p53 activation there was increased expression of p21 in CTCs

and DTCs following Nutlin-3 treatment (Fig. 4e & f, Fig. 5b and h), with a 12 fold increase in

p21 in DTCs (Fig. 5b). This led to an increase in the G1 population of Case-No-12 DTCs

(56% for DMSO treated and 70%-Nutlin-3 treated), but no reduction in S phase in DTCs

(Fig. 5e & f), so an unchanged G1/S ratio (9.8-DMSO and 9.2-Nutlin-3).

To determine the effect of Nutlin-3 on WBCs, 4 patient samples and 3 spiked healthy

volunteer blood samples were treated with Nutlin-3 or DMSO control and p53, p21 and cell

cycle arrest measured. Compared with NB cell lines, CTCs and DTCs there was very low

baseline expression of p53 in WBCs, but in all cases there was nuclear p53 accumulation

following Nutlin-3 treatment (Fig. 4c & d, Fig. 5c & g, Supplementary Fig. S4e), and

increased p21 expression compared with DMSO controls (Fig. 4c & d, Fig. 5d & h,

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

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chart showing cell cycle analysis of Be2C cells (p53 mutant), spiked (SPIK) Be2C cells,

SHSY5Y cells, spiked SHSY5Y cells (SPIK), WBC from healthy volunteer blood samples

(HV) (n=3), WBCs from Case-No-12 blood and BM samples, and DTCs from Case-No-12

following Nutlin-3 treatment, showing a G1 arrest in SHSY5Y cells and spiked SHSY5Y

cells and a partial G1 arrest compared with DMSO controls in all other samples except mutant

Be2C cells and spiked mutant Be2C cells. g and h Bar charts showing the percentage of cells

expressing p53 and p21 in Be2C cells (n=3), spiked Be2C cells (n=3), SHSY5Y cells (n=3),

spiked SHSY5Y cells (n=3), HV WBC (n=3), Case-No-12 blood and BM WBC and DTCc

(n=3 for Case-No-12; error bars represent analysed data from three different files using

IDEAS software) following Nutlin-3 treatment compared with DMSO controls. In SHSY5Y

cells, spiked SHSY5Y cells, HV WBCs, Case-No-12 blood and BM WBCs and DTCs

increased p53 and p21 expression was observed following Nutlin-3 compared with DMSO

controls, but not in p53 mutant Be2C cells and spiked mutant Be2C cells. For SHSY5Y cells,

spiked SHSY5Y cells, Be2C cells, spiked Be2C cells, healthy volunteer WBC (n=3), Case-

No-12 CD45+ve, blood and BM cells and DTCs, a minimum of = 1000 cells were analysed.

Paired t test, p<0.05*, p<0.01** and p<0.001***).

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