NGS immunogenetic analysis in vitro: clonality feasibility study

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1 NGS immunogenetic analysis in vitro: clonality feasibility study ERIC EuroClonality-NGS 1-day workshop Rotterdam, NL, November 24, 2017 Anton W. Langerak, Laboratory for Medical Immunology, Dept. Immunology Erasmus MC, Rotterdam

2 NGS 2nd generation workflow

DNA (10 ng) needed - enables sequence analysis of >200 amplicons of 200 nt /run; ideal for

3 Ion Torrent PGM (ThermoFisher) Semiconductor sequencing Simple biochemistry: DNA polymerase/ nt Particularly attractive for pathology labs: - suboptimal DNA specimens - small amount of DNA (10 ng) needed - enables sequence analysis of >200 amplicons of 200 nt /run; ideal for simultaneous detection of somatic mutations in multiple genes ( >450x coverage of 95% of the reads)

4 B-cell clonality feasibility study Technical validation of the primers / protocols Reproducibility How low can you go? Input / sensitivity Samples: FFPE DNA Suboptimal DNA quality (FFPE), Low tumor loads Performance NGS - spectratyping Defining SOPs, including for ARReST / Interrogate

5 B-cell clonality feasibility study Technical validation of the primers / protocols Reproducibility How low can you go? Input / sensitivity Samples: FFPE DNA Suboptimal DNA quality (FFPE), Low tumor loads Performance NGS - spectratyping Defining SOPs, including for ARReST / Interrogate

6 Experimental approach feasibility study Each lab used its own primers and Taq Gold Scheijen, Meijers et al., ms. in preparation

7 Sample overview Laboratories performing the NGS IG-clonality experiments Sample Sample types (Lab of origin; original sample number) Sample number FS Nijmegen Rotterdam Berlin Tübingen 1 Polyclonal samples Peripheral blood mononuclear cells (Nijmegen; PBMC#3) PBMC1 X X X X 2 Peripheral blood mononuclear cells (Rotterdam; PBMC) PBMC2 X X X X 3 Reactive tonsil tissue-frozen (Nijmegen;Ton19367) Tonsil1 X X X X 4 Reactive tonsil tissue-ffpe (Nijmegen;Ton19367) Tonsil2 X X X X 5 Reactive tonsil tissue-frozen (Nijmegen;Ton10615) Tonsil3 X X X X 6 Reactive tonsil tissue-ffpe (Nijmegen; Ton10615) Tonsil4 X X X X 7 Monoclonal patient samples DLBCL abdomen FFPE (Nijmegen; DPA ) Blym1 X X 8 EBV-positive B-cell lymphoma FFPE (Nijmegen; DPA ) Blym2 X X 9 DLBCL skin (Nijmegen; DPA ) Blym3 X X 10 Low-grade gastrointestinal MZL FFPE (Berlin; ) Blym4 X X 11 Paranasal sinus B-cell lymphoma FFPE (Berlin; ) Blym5 X X 12 EBV-positive PTLD (Nijmegen; DPA ) Blym6 X X 13 Paired diagnosis-relapse samples DLBCL Uterine cervix FFPE (Nijmegen; DPA ) Diag1 X X 14 DLBCL Vermiform appendix FFPE (Nijmegen; DPA ) Relap1 X X 15 DLBCL testis FFPE (Nijmegen; DPA ) Diag2 X X 16 CLL/SLL bone marrow FFPE (Nijmegen; DPA ) Relap2 X X 17 MZL, partial involvement FFPE (Nijmegen; DPA ) Diag3 X X 18 MZL FFPE (Nijmegen; DPA ) Relap3 X X 19 Glandula parotis DLBCL FFPE (Nijmegen; DPA ) Diag4 X X 20 Extranodal MZL FFPE (Nijmegen; DPA ) Relap4 X X 21 Technical samples Dilution of Blym3 with Tonsil2 (1%Blym-99% Tonsil) Dilut1 X X 22 Dilution of Blym3 with Tonsil2 (2,5%Blym-97,5% Tonsil) Dilut2 X X 23 Dilution of Blym3 with Tonsil2 (5%Blym-95% Tonsil) Dilut3 X X 24 Dilution of Blym3 with Tonsil2 (10%Blym-90% Tonsil) Dilut4 X X 25 Dilution of Blym3 with Tonsil2 (100%Blym-0% Tonsil) Dilut5 (= sample 9) X X 26 Input range Blym4 (2,5ng) InpBlym1 X X 27 Input range Blym4 (5ng) InpBlym2 X X 28 Input range Blym4 (10ng) InpBlym3 X X 29 Input range Blym4 (20ng) InpBlym4 X X 30 Input range Blym4 (40ng) InpBlym5 (= sample 10) X X 31 Input range Tonsil2 (2,5 ng) InpTonsil1 X X 32 Input range Tonsil2 (5 ng) InpTonsil2 X X 33 Input range Tonsil2 (10 ng) InpTonsil3 X X 34 Input range Tonsil2 (20 ng) InpTonsil4 X X 35 Input range Tonsil2 (40 ng) InpTonsil5 (= sample 4) X X 36 Input range Tonsil4 (2,5 ng) InpTonsil6 X X 37 Input range Tonsil4 (5 ng) InpTonsil7 X X 38 Input range Tonsil4 (10 ng) InpTonsil8 X X 39 Input range Tonsil4 (20 ng) InpTonsil9 X X 40 Input range Tonsil4 (40 ng) InpTonsil10 (= sample 6) X X

8 B-cell clonality feasibility study Technical validation of the primers / protocols Reproducibility How low can you go? Input / sensitivity Samples: FFPE DNA Suboptimal DNA quality (FFPE), Low tumor loads Performance NGS - spectratyping Defining SOPs, including for ARReST / Interrogate

9 PBMC1 IGHV FR3 raw total w junction IGHV-FR3 LAB Sample count % count % count % Berlin PBMC % % % Nijmegen PBMC % % % Rotterdam PBMC % % % Tübingen PBMC % % % Left right: BERLIN-NMGN-RDAM-TÜB feature type A: 5 gene feature type B: 3 gene

10 PBMC1 IGK raw total w junction IGK V-J Intron-KDE LAB Sample count % count % count % count % Berlin PBMC % % % % Nijmegen PBMC % % % % Rotterdam PBMC % % % % Tübingen PBMC % % % % Left right: BERLIN-NMGN-RDAM-TÜB feature type A: 5 gene feature type B: 3 gene

11 Tonsil 1 (fresh frozen vs. FFPE) IGHV FR3 raw total w junction IGHV-FR3 LAB Sample count % count % count % Nijmegen Frozen % % % Nijmegen FFPE % % % Left: frozen Right: FFPE feature type A: 5 gene feature type B: 3 gene

12 Tonsil 1 (fresh frozen vs. FFPE) - IGHD raw total w junction IGHD LAB Sample count % count % count % Nijmegen Frozen % % % Nijmegen FFPE % % % Left: frozen Right: FFPE feature type A: 5 gene feature type B: 3 gene

13 B-cell clonality feasibility study Technical validation of the primers / protocols Reproducibility How low can you go? Input / sensitivity Samples: FFPE DNA Suboptimal DNA quality (FFPE), Low tumor loads Performance NGS - spectratyping Defining SOPs, including for ARReST / Interrogate

14 Tonsil 2 IGHV FR3 raw total w junction IGHV-FR3 IGHD IgK LAB Sample count % count % count % count % count % Rotterdam % % % % % Rotterdam % % % % % Rotterdam % % % % % Rotterdam % % % % % Rotterdam % % % % % Left -> Right: ng feature type A: 5 gene feature type B: 3 gene

15 Tonsil 2 IGHD raw total w junction IGHV-FR3 IGHD IgK LAB Sample count % count % count % count % count % Rotterdam % % % % % Rotterdam % % % % % Rotterdam % % % % % Rotterdam % % % % % Rotterdam % % % % % Left -> Right: ng feature type A: 5 gene feature type B: 3 gene

16 Tonsil 4 IGK raw total w junction IGHV-FR3 IGHD / LAB Sample count % count % count % count % count % Berlin % % % % % Berlin % % % % % Berlin % % % % % Berlin % % % % % Berlin % % % % % Left -> Right: ng feature type A: 5 gene feature type B: 3 gene

17 Blym4 input range IGHV IGHJ3 feature type A: 5 gene feature type B: 3 gene 1: 2.5 ng 2: 5 ng 3: 10 ng 4: 20 ng 5: 40 ng feature type A: aa length feature type B: clon. type

18 Dilution of Blym-3 with Tonsil-2 feature type A: aa length feature type B: clon. type Blym3 (100%) IGHD2-2 + IGHJ6 24 1: 0% Blym 100% Tonsil 2: 1% Blym 99% Tonsil 3: 2.5% Blym 97.5% Tonsil 4: 5% Blym 95% Tonsil 5: 10% Blym 90% Tonsil

19 Dilution of Blym-3 with Tonsil-2 IGHD2-2 + IGHJ6 feature type A: 5 gene feature type B: 3 gene feature type A: aa length feature type B: clonotype 5% 2.5%

20 Summary on polyclonal and technical samples Reproducible testing of polyclonal samples by multiple centers? General overlap between four centers for IGHV-FR3, IGH D-J, and IGK Are all functional genes (V/J) detected by the primers? All the low represented genes are ORFs, except for IGHV Reproducibility and performance of primers and protocols in polyclonal samples (including FFPE fresh frozen DNA comparison)? Equal detection of clonotypes in frozen tissue and FFPE Minimal input DNA for robust performance? Minimal input DNA is around 5-10 ng Sensitivity of detection? Clone can be detected at 5% in polyclonal background

21 B-cell clonality feasibility study Technical validation of the primers / protocols Reproducibility How low can you go? Input / sensitivity Samples: FFPE DNA Suboptimal DNA quality (FFPE), Low tumor loads Performance NGS - spectratyping Defining SOPs, including for ARReST / Interrogate

22 Monoclonal and paired Dx-Re samples Laboratories performing the NGS IG-clonality experiments Sample Sample types (Lab of origin; original sample number) Sample number FS Nijmegen Rotterdam Berlin Tuebingen 7 Monoclonal patient samples DLBCL abdomen FFPE (Nijmegen; DPA ) Blym1 X X 8 EBV-positive B-cell lymphoma FFPE (Nijmegen; DPA ) Blym2 X X 9 DLBCL skin (Nijmegen; DPA ) Blym3 X X 10 Low-grade gastrointestinal MZL FFPE (Berlin; ) Blym4 X X 11 Paranasal sinus B-cell lymphoma FFPE (Berlin; ) Blym5 X X 12 EBV-positive PTLD (Nijmegen; DPA ) Blym6 X X 13 Paired diagnosis-relapse samples DLBCL Uterine cervix FFPE (Nijmegen; DPA ) Diag1 X X 14 DLBCL Vermiform appendix FFPE (Nijmegen; DPA ) Relap1 X X 15 DLBCL testis FFPE (Nijmegen; DPA ) Diag2 X X 16 CLL/SLL bone marrow FFPE (Nijmegen; DPA ) Relap2 X X 17 MZL, partial involvement FFPE (Nijmegen; DPA ) Diag3 X X 18 MZL FFPE (Nijmegen; DPA ) Relap3 X X 19 Glandula parotis DLBCL FFPE (Nijmegen; DPA ) Diag4 X X 20 Extranodal MZL FFPE (Nijmegen; DPA ) Relap4 X X

23 Summary NGS-GS comparison

24 Concordance between centers and NGS-GS BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Blym1 (Uterine DLBCL ) C128 C263 C148 C285+C371 IGK VJ Clonotype/ junction aa length IGK VJ IGKV1(D)-39 & IGKJ4 IGKV/intron-KDE Clonotype/ junction aa length IGKV/intron-KDE IGKV2(D)-30 & KDE Intron & KDE

25 Discordance between centers BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Blym1 (Uterine DLBCL ) C128 C263 C148 C285+C371 IGH DJ Clonotype/ junction aa length IGH DJ Low reads: inefficient annealing primers? IGHD5-5=5-18 & IGHJ6 IGHD2-2 & IGHJ6 1= Lab 1 (Reads: 4133) 2= Lab 2 (Reads: 4162)

26 Improved performance NGS vs. GS BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Blym5 (B cell lymphoma) Oligoclonal C258 C131/C181 C241/C278 IGH FR3 IGH VJ FR3 IGHV3-7=3-11=3-21=3-48=3-69-1=3-74 & IGHJ3 The IGHV3 & IGHJ3 clone is missed due to mismatches with VH3-FR3 BIOMED-2 primer. Green=tccagagacaacgccaaaaacacagtgtatctacacatgactggtttgagagccgaggacacggctctctattacTGTGCGAGAGATG AAGTCAGTGGTTCTGATGCTCTTGACCTCTGGggccaagggacagtggtcaccgtctcctcaggtgag

27 Improved performance NGS vs. GS BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Blym2 (EBV + lymphoma) C112 +PCB C240 +PCB C197+PCB C274/C286+PCB IGK VJ Clonotype/ junction aa length IGK VJ IGKV3(D)-7 & IGKJ5 IGKV4-1 & IGKJ4 This IGKV4-J rearrangement is only detected by NGS-clonality, not by conventional BIOMED- 2; suboptimal DNA will not allow detection of IGKV4-J R ( bp) IGKV/intron-KDE IGKV/intron-KDE Intron & KDE Intron & KDE

28 Discordance between NGS and GS BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Blym5 (B cell lymphoma) Oligoclonal C258 C131/C181 C241/C278-C280 IGK VJ IGK VJ IGKV3(D)-15 & IGKJ4-p IGKV3(D)-15 & IGKJ4-up C131 clone not detected by NGS; Sequence PCR fragment

29 Comparison Dx-Re BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Diagnosis#1 (D1) C100 C250 C135 C277/C281 Relapse#1 (R1) C100 C250 C135 C277/C283 IGH FR3 IGH DJ ARResT output: 5 and 3 genes D1 R1 IGHV4-34 D1 R1 IGHD5-24 IGK D1R1 D1R1 D1R1 IGKV1-8 IGKV5-2 Intron

30 Comparison Dx-Re BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Diagnosis#2 (D2) nsp C254 C146/C195 C276/C281 Relapse#2 (R2) C121 C149 C282 C242/Cw281 IGH FR3 IGHV3-23(D) IGH DJ IGHV3-13 IGHV3-23 IGHV3-30 IGHV3-66 D2R2 D2R2 D2R2 D2R2 ARResT output: 5 and 3 genes D2R2 IGKV2-29 IGKV3-11 D2 R2 IGHD2-21 IGKV4-1 IGK D2 R2 IGHD6-6 IGKV1-12 D1R1 IGK Intron D2 R2 D2 R2 D2 R2 D2 R2 D2 R2

31 B-cell clonality feasibility study Technical validation of the primers / protocols Reproducibility How low can you go? Input / sensitivity Samples: FFPE DNA Suboptimal DNA quality (FFPE), Low tumor loads Performance NGS - spectratyping Defining SOPs, including for ARReST / Interrogate

32 Summary EuroClonality-NGS IG primers technically validated for detection of clonality (3-tube reaction/ion Torrent PGM) Increased sensitivity and specificity by NGS-based approach NGS-based clonality provides valuable clonotype sequence information Scheijen, Meijers et al., ms. in preparation

33 Future directions T-cell clonality feasibility study Illumina-based detection Biological validation IG - define threshold for clonality detection using reactive lesions - comparison GS vs NGS-based clonality in different disease entities - define procedures for interpretation - sufficient samples (reactive, defined categories, FFPE/FF) - Pathology review meeting spring 2018 Guidelines for interpretation clonality (after biological validation study) (minimal amount of reads, assignment of a minor clone, etc )

34 Acknowledgements Radboud University Medical Center, Nijmegen (NL) Blanca Scheijen, Jos Rijntjes, Michiel van den Brand, Patricia Groenen Erasmus Medical Center, Rotterdam (NL) Ruud Meijers, Michèle van der Klift, Ton Langerak Charité-University Medicine, Berlin (DE) Markus Möbs, Michael Hummel University Hospital and Comprehensive Cancer Center, Tübingen (DE) Julia Steinhilber, Falko Fend CEITEC, Brno (CZ) / University Schleswig-Holstein, Kiel (DE) Tomas Reigl, Nikos Darzentas Kiel and Paris teams for IGH FR3 and IGH D-J primer design

35 EuroClonality-NGS, Belfast, October 2017

36 PBMC1 IGHD raw total w junction IGHD LAB Sample count % count % count % Berlin PBMC % % % Nijmegen PBMC % % % Rotterdam PBMC % % % Tübingen PBMC % % % Left right: BERLIN-NMGN-RDAM-TÜB feature type A: 5 gene feature type B: 3 gene

37 Tonsil 1 (fresh frozen vs. FFPE) - IGK raw total w junction IGK V-J Intron-KDE LAB Sample count % count % count % count % Tübingen Frozen % % % % Tübingen FFPE % % % % Left: frozen Right: FFPE feature type A: 5 gene feature type B: 3 gene

38 Concordance between centers and NGS-GS BIOMED-2 IGH VJ FR3 IGH DJ IGK VJ IGK V/intron- KDE Blym4 (Low-grade MZL) C130 C135+PCB C197 C281 IGH FR3 Clonotype/ junction aa length IGH VJ FR3 IGHV3-48 & IGHJ3 IGH DJ Clonotype/ junction aa length IGH DJ IGHD3-22 & IGHJ3

bioinformatics: state of art tools for NGS immunogenetics

bioinformatics: state of art tools for NGS immunogenetics Nikos Darzentas, Ph.D. CEITEC MU, Brno, Czech Republic bat.infspire.org nikos.darzentas@gmail.com Ministry of Health of theczech Republic, grant#