liquid biopsies in neuroblastoma stefan fiedler marie

LIQUID BIOPSIES IN NEUROBLASTOMATHERAPY RESPONSE MONITORING AND EARLYRELAPSE DETECTION BY ctDNA ANALYSIS INHIGH-RISK PATIENTS

TERESA GERBER1

STEFAN FIEDLER1

SOPHIA HÜTTER1

MARIE BERNKOPF1

ULRIKE PÖTSCHGER1FLORIAN HALBRIT-

ANNA BUDER2

RUTH LADENSTEIN1,3

INGE M. AMBROS1

SABINE TASCHNER-MANDL1

1CCRI, CHILDREN’S CANCER RESEARCH INSTITUTE, ST. ANNA KINDERKREBSFORSCHUNG VIENNA, AUSTRIA

2INSTITUT OF CANCER RESEARCH, MEDICAL UNIVER-SITY OF VIENNA, VIENNA, AUSTRIA;

3DEPARTMENT OF PEDIATRICS, MEDICAL UNIVERSITY OF VIENNA, VIENNA, AUSTRIA

25% DTCs

20% DTCs

15% DTCs

10% DTCs

5% DTCs

1% DTCs

RELAPSE THERAPYINDUCTION THERAPY

PRESENCE OF DTCs IN THE BONE MARROW

PRIMARY TUMOR

BONE MARROW

MAT IMMUNOTHERAPY

CLINICALRELAPSE

DETECTIONOF MRDSCTSURGERYDIAGNOSIS

TUMOR CELLS

DISSEMINATED TUMOR CELLS (DTCs)

BONE MATRIX

Pt1

Pt2

CIRCULATINGTUMOR CELL

CIRCULATING TUMOR DNA

GENOMIC CELLFFREE DNA

PERIPHERAL BLOOD

More than 95% of high-risk neuroblastoma patients show disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis. Thus, DTC detection in BM samples from these patients can be used for sensitive MRD detection. However, in case of relapses outside the BM, disease detection in this compartment may fail. Minimally invasive peripheral blood (PB) plasma based liquid biopsy samples can be taken repeatedly during the course of dis-ease, enabling a precise monitoring of the disease evolution.

We investigated the potential of combined ctDNA and DTC analysis for therapy response monitoring and enhanced MRD detection allowing an early identi� cation of disease progression or relapse.

The combination of DTC and ctDNA analysis enhances the identi� cation of patients with incomplete response to therapy and inferior outcome. Also, ctDNA analysis allows the identi� cation of disease progressions early.

BACKGROUND

AIM

CONCLUSION

RESPONSE TO INDUCTION THERAPY CAN BE MONITORED BY ctDNA AND DTC ANALYSIS

MINIMAL RESIDUAL DISEASE DETECTED BY COMBINED ctDNA AND DTC ANALYSIS INDICATES RELAPSE EARLY

This work was supported by: St. Anna Kinderkrebsforschung, Oesterreichischer Nationalbank (OeNB) and Austrian Science Fund (FWF), Grant No. I 2799-B28.

Feel free to contact teresa.gerber@ccri.at or sabine.taschner@ccri.at regarding any question to this study.We have no con� lct of interest to declare.

PETER F. AMBROS1

Response Evaluation Time Points

DXTP1TP2aTP2bTP3TP4SXTP5TP6TP7REL/PD1REL/PD2REL/PD3

Pat18Pat17Pat16Pat15Pat14Pat13Pat12Pat11Pat10Pat9Pat8Pat7Pat6Pat5Pat4Pat3Pat2Pat1

0 500 1000 1500 2000Time since diagnosis (days)

Induction Phase MAT Phase MRD Phase

DX TP1 TP2a TP2b (TP3)

SX (SX)

TP4 TP5 TP6 TP7

(SX)

DX...DiagnosisSX...Surgery

Induction Phase: TP1 ...mid of induction chemotherapyTP2a...end of induction chemotherapyTP2b...after TVD chemotherpy

MAT Phase:TP3...if surgery is performed, previous MAT therapyTP4...post MAT therapy

MRD PHASE:TP5...after RadiotherapyTP6...mid of immunotherapyTP7...post immunotherapy

Liquid Biopsy (LB)marker

posneg

LB+

LB-noCR

PD

PDCR

CR

noCR

noPD

noPD

PD

DT

DOD

PD

AWD

AWDDOD

DOD

ADF

AWD

AWD

PD

LB Positvity post-Induction Patient Cohort

CR at any timeduring therapy

CRno CR

Patient cohortshowing PD/REL

PD/RELno PD/REL

Last follow-up

PD/RELDead of Disease (DOD)

During Therapy (DT)Alive with Disease (AWD)

Alive Disease-Free (ADF)

ctDNA PB amplification

DTCsctDNA BM amplification

cut-off ctDNA

Pat6

012

20406080

0 200 400 600days

0

50

100

150

200

Ratio

(MYC

N/NA

GK) DTCs/10 6 MNCs

day 372 day 433

IND MAT MRD REL THPD PD

PD/RELNo

LB+/-

DTC+/-

BM+/-

PB+/-

Pat3 1Pat4 1Pat12 1Pat17 1

Pat2 1

Pat2 2

Pat3 2

Pat3 3

Pat5 1

Pat5 2

Pat6 1

Pat6 2

Pat8 1

Pat15 1

Pat16 1

Pat16 2

Pat16 3

Pat16 4

Pat18 1

Pat18 2

LB marker posLB marker negnegative

MarkerPositivity

ctDNA PB posctDNA BM posDTC pos

no data

ctDNA/DTC marker positivity at REL/PD

Ratio

[tar

get/r

efer

ence

gen

e]

DX TP1 TP2 DX TP1 TP2 DX TP1 TP2

ctDNA BM DTC

1

10

100

1

10

100

Pat1Pat2Pat3Pat4Pat5Pat6Pat7Pat8Pat9Pat10Pat11Pat12Pat13Pat14Pat15Pat16Pat17Pat18cut-off

1

100

10.000

1.000.000

0

****

A B CctDNA PB

DTCs

/106

MNC

s

**

**

ddPCR

PB

01.01.2019Fem

aleNam

e: MA-MUS

AIPFMicroscopy

Characteristics Gender

Age

Amplification

Disease Progression/Relapse

No %

MaleFemale

<18 month>18 month

MYCNother

10 56%8 44%

12 67%

5 11%13 89%

16 89%2 11%

Total No patients 18

ddPCR

ctDNAn=382

ctDNAn=133

DTCn=136

PERIPHERALBLOOD

BONEMARROW

BONE MARROW

PERIPHERAL BLOODddPCR

AIPFMicroscopy

MIBGPRIMARY TUMOR SITE

Response evaluation time points in each patient (Pat 1-18) are indicated relative to the time of diagnosis (DX). Surgery (SX), relapse/disease progression (REL/PD) ctDNA and DTCs were investigated at TP1-7; additional PB samples were available between these time points and during follow up.

Therapy phases and response evaluation time points (TP1-7) according to the SIOPEN/HR-NBL-1 trial during induction therapy, Myeloablative therapy (MAT) and minimal-residual disease treatment (MRD).

Detection of 80% of relapse/progress ive disease events (REL/PD) using ctDNA/DTC markers in 12/18 HR-NB patients experiencing one or more events. Frequent sampling of minimal-invasive PB frequently allows the detection of progressive disease.

ctDNA/DTC liquid biopsy marker positivity in late therapy phases (TP2-TP7)(n=18).Patients positive for at least one marker after induction therapy show incomplete response to therapy and inferior outcome.

PB and BM ctDNA and DTC kinetics in longitudinally collected samples from HR-NB patient 6. Therapeutic interventions and progressive disease/relapse events (PD) are depicted on top.ctDNA analysis provides important additional information if a relapse occurs outside of the BM.

Response kinetics of ctDNA/DTC markers during induction therapy. (A,B) ddPCR levels of ampli� ed loci in ctDNA from: (A) PB or (B) BM plasma. (C) Quanti� cation of DTC in BM aspirates by AIPF.Response to induction therapy is re� ected by all three markers, showing similar kinetics.