LIQUID BIOPSIES IN NEUROBLASTOMA THERAPY RESPONSE MONITORING AND EARLY RELAPSE DETECTION BY ctDNA ANALYSIS IN HIGH-RISK PATIENTS TERESA GERBER 1 STEFAN FIEDLER 1 SOPHIA HÜTTER 1 MARIE BERNKOPF 1 ULRIKE PÖTSCHGER 1 FLORIAN HALBRIT- ANNA BUDER 2 RUTH LADENSTEIN 1,3 INGE M. AMBROS 1 SABINE TASCHNER-MANDL 1 1 CCRI, CHILDREN’S CANCER RESEARCH INSTITUTE, ST. ANNA KINDERKREBSFORSCHUNG VIENNA, AUSTRIA 2 INSTITUT OF CANCER RESEARCH, MEDICAL UNIVER- SITY OF VIENNA, VIENNA, AUSTRIA; 3 DEPARTMENT OF PEDIATRICS, MEDICAL UNIVERSITY OF VIENNA, VIENNA, AUSTRIA 25% DTCs 20% DTCs 15% DTCs 10% DTCs 5% DTCs 1% DTCs RELAPSE THERAPY INDUCTION THERAPY PRESENCE OF DTCs IN THE BONE MARROW PRIMARY TUMOR BONE MARROW MAT IMMUNOTHERAPY CLINICAL RELAPSE DETECTION OF MRD SCT SURGERY DIAGNOSIS 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 identification of disease progression or relapse. The combination of DTC and ctDNA analysis enhances the identification of patients with incomplete response to therapy and inferior outcome. Also, ctDNA analysis allows the identification 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 confilct of interest to declare. PETER F. AMBROS 1 Response Evaluation Time Points DX TP1 TP2a TP2b TP3 TP4 SX TP5 TP6 TP7 REL/PD1 REL/PD2 REL/PD3 Pat18 Pat17 Pat16 Pat15 Pat14 Pat13 Pat12 Pat11 Pat10 Pat9 Pat8 Pat7 Pat6 Pat5 Pat4 Pat3 Pat2 Pat1 0 500 1000 1500 2000 Time since diagnosis (days) Induction Phase MAT Phase MRD Phase DX TP1 TP2a TP2b (TP3) SX (SX) TP4 TP5 TP6 TP7 (SX) DX...Diagnosis SX...Surgery Induction Phase: TP1 ...mid of induction chemotherapy TP2a...end of induction chemotherapy TP2b...after TVD chemotherpy MAT Phase: TP3...if surgery is performed, previous MAT therapy TP4...post MAT therapy MRD PHASE: TP5...after Radiotherapy TP6...mid of immunotherapy TP7...post immunotherapy Liquid Biopsy (LB) marker pos neg LB+ LB- no CR PD PD CR CR no CR no PD no PD PD DT DOD PD AWD AWD DOD DOD ADF AWD AWD PD LB Positvity post-Induction Patient Cohort CR at any time during therapy CR no CR Patient cohort showing PD/REL PD/REL no PD/REL Last follow-up PD/REL Dead of Disease (DOD) During Therapy (DT) Alive with Disease (AWD) Alive Disease-Free (ADF) ctDNA PB amplification DTCs ctDNA BM amplification cut-off ctDNA Pat6 0 1 2 20 40 60 80 0 200 400 600 days 0 50 100 150 200 Ratio (MYCN/NAGK) DTCs/10 6 MNCs day 372 day 433 IND MAT MRD REL TH PD PD PD/REL No LB +/- DTC +/- BM +/- PB +/- Pat3 1 Pat4 1 Pat12 1 Pat17 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 pos LB marker neg negative Marker Positivity ctDNA PB pos ctDNA BM pos DTC pos no data ctDNA/DTC marker positivity at REL/PD Ratio [target/reference gene] DX TP1 TP2 DX TP1 TP2 DX TP1 TP2 ctDNA BM DTC 1 10 100 1 10 100 Pat1 Pat2 Pat3 Pat4 Pat5 Pat6 Pat7 Pat8 Pat9 Pat10 Pat11 Pat12 Pat13 Pat14 Pat15 Pat16 Pat17 Pat18 cut-off 1 100 10.000 1.000.000 0 ** ** A B C ctDNA PB DTCs/10 6 MNCs * * * * ddPCR PB 01.01.2019 Female Name: MA-MUS AIPF Microscopy Characteristics Gender Age Amplification Disease Progression/Relapse No % Male Female <18 month >18 month MYCN other 10 56% 8 44% 12 67% 5 11% 13 89% 16 89% 2 11% Total No patients 18 ddPCR ctDNA n=382 ctDNA n=133 DTC n=136 PERIPHERAL BLOOD BONE MARROW BONE MARROW PERIPHERAL BLOOD ddPCR AIPF Microscopy MIBG PRIMARY 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/progressive 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 amplified loci in ctDNA from: (A) PB or (B) BM plasma. (C) Quantification of DTC in BM aspirates by AIPF. Response to induction therapy is reflected by all three markers, showing similar kinetics.