liquid biopsies in neuroblastoma stefan fiedler marie
TRANSCRIPT
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 [email protected] or [email protected] 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.