From the patient to the sequence : Primers, PCR, Detection of clonality, Sequencing

Transcription

1 6th ERIC Educational workshop on IG gene analysis in CLL, Uppsala, SE, Sept 23, 2016 From the patient to the sequence : Primers, PCR, Detection of clonality, Sequencing Anton W. Langerak Dept. of Immunology Erasmus MC, Rotterdam a.langerak@erasmusmc.nl

2 CD79b CD79a IGH : V(D)J recombination VH DH JH s Cm D J joining V D-J joining V D J IgH IgH V D J V IgL J C C C C C C J V IgL precursor IGH mrna transcription C C C C translation V DJ Cm RNA splicing mature IGH mrna

3 Potential human IG repertoire IgH Ig Ig Number of genes - V genes - D genes - J genes Combinatorial diversity > > 50 > > 5 x 10 6 Junctional region diversity ++ Estimation of total primary receptor repertoire > Further diversification into SHM-shaped repertoire >> 10 12

4 Considerations on protocol optimization

5 From patient to sequence: parameters Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing

6 Material - 1 Parameters peripheral blood bone marrow (idem) lymph node other Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Survey prev. workshop (n =63) Source of cells 60 other frozen, paraffin, biopsies PB BM LN other

7 Material - 2 Parameters EDTA tubes CPT tubes heparinized tubes other Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Survey prev. workshop (n =63) Anticoagulant EDTA CPT heparinized other

8 Material - 3 Parameters Ficoll gradient (PB / BM) cell suspension of biopsy (e.g. for flow analysis) other Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Survey prev. workshop (n =63) other FFPE (2), whole blood (4), red cell lysis (2), osmotic analysis (1) W o rkup o f cells Ficoll cell susp other

9 Material - 4 Parameters genomic DNA RNA / complementary DNA Survey prev. workshop (n =63) Nucleic acid Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Quantity of gdna / RNA -mostly ng gdna (range 1 ng 1 ug) gdna only RNA / cdna only both -mostly 1 ug RNA for cdna reaction (range 400 ng 2 ug)

10 Material - 4 advantage disadvantage gdna -more optimal for long-distance -non-productive transport -use of archival material rearrangement can also be amplified RNA/cDNA -identifies mostly only -reverse transcription productive rearrangement -allows isotype identification step required no scientific rationale for choosing gdna or RNA/cDNA advisable to use similar type of nucleic acid in multi-center trials

11 Material - 4

12 PCR methodology - 1 Parameters PCR protocol existing protocols optimized for applied PCR primer sets Survey prev. workshop (n =63) Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing PCR strategy multiplex single both

13 PCR methodology - 2 Parameters Taq polymerase no need for Taq enzyme with proofreading capacity Number of independent reactions Taq polymerase errors are very rare 1 or 2 Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Survey prev. workshop (n =63) -AmpliTaq Gold (Applied Biosystems) (23) -Platinum Taq (Invitrogen) (5) -HotStar Taq (QIAGEN) (7) -FastStart Taq (Roche) (4) -other (10)

14 PCR methodology - 3 Parameters L exon VDJ exon C exon Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing LVDJC transcript Figure from Giudicelli and Lefranc, Ig gene analysis in CLL

15 PCR methodology - 3 Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing

16 PCR methodology - 3 Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Survey prev. workshop (n =63) 5' leader 5' FR1 both 3' IGHC 3' IGHJ both

17 PCR methodology - 3 Primer sets IGHV leader primers LH (family-specific) CH (Saohota, Blood 1996) LH & VH (family-specific) CH (Fais, J Clin Invest 1998) IGHV FR1 primers FR1 consensus JH (Aubin, Leukemia 1995) Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing FR1 multiplex JH (BIOMED-2) (Van Dongen, Leukemia 2003) IGHV FR2 primers : short IGHV sequences DISCOURAGED (only recommended upon negative leader / FR1 results, due to SHM) IGHV FR3 primers : too short IGHV sequences DISCOURAGED

18 PCR methodology - 3 Primer sets IGHV leader primers LH (family-specific) CH (Saohota, Blood 1996) LH & VH (family-specific) CH (Fais, J Clin Invest 1998) IGHV FR1 primers FR1 consensus JH (Aubin, Leukemia 1995) Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing FR1 multiplex JH (BIOMED-2) (Van Dongen, Leukemia 2003) Primer sets for DNA Primers for RNA BIOMED-2 Fais ERIC guideline IVS Aubin other BIOMED-2 Fais in-house other IVS Survey prev. workshop (n =63) Survey prev. workshop (n =63)

19 PCR methodology - 3 advantage disadvantage IGHV leader -accurate SHM level -slightly lower based on whole IGHV gene detection rate IGHV FR1 -slightly higher detection rate -estimation of SHM -widely used in clonality testing level (lack of 5 V) scientific viewpoint: IGHV leader primers more optimal than FR1 primers comprehensive approach would include both strategies advisable to standardize primer choice in multi-center trials

20 IGH - intergenic distances of J genes tel 25 kb cen D H segments JH CmC C3 C1 C 1 C2 C4 C C 2 3 enhancer enhancer DH7-27 JH1 JH2 JH3 JH4 JH5 JH bp Adapted from IMGT database, M.P. Lefranc, Leukemia 2003; 17:

21 IGH extended PCR products VH DH JH s Cm n D J joining V D-J joining VH3-21 JH3 JH4 JH5 JH6 ~140 bp ~512 bp ~913 bp ~1529 bp Dept. of Immunology, Erasmus MC, Rotterdam

22 PCR methodology - 3 Parameters What about amplification of IG light chains? Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing

23 IGK gene complex (2p11.2) 76 Vk and 5 Jk rearrangeable gene segments 25 kb tel D-41 1D-17 1D-16 3D-15 2D-14 1D-13 ~800 kb D-20 2D-19 2D-18 1D-12 3D-11 2D-10 1D-42 1D-43 1D D D-40 1D-39 2D-38 1D-37 2D-36 1D-35 3D-34 1D-33 1D-32 3D-31 2D-30 2D-29 2D-28 1D-27 2D-26 3D-25 2D-24 2D-23 1D-22 6D-21 J C cen Kde intron enhancers J 1-5 RSS C Kde Adapted from IMGT database, M.P. Lefranc, Leukemia 2003; 17:

24 IGK (V-J) : multiplex PCR V J V 2f V 4 V 5 V 3f V 1f/6 V 7 J primers bp MwM PB-MNC tonsil ss he ho NL-6 ss ho ES-3 FR-2 IGK tube A V -J ss he ho PB-MNC V 1f/6/V 7-J nt V 3f-J nt V 2f/V 4/V 5-J nt 3000 tonsil NL ES FR BIOMED-2 report: Leukemia 2003; 17:

25 IGK gene complex additional rearrangeable elements 25 kb tel D-41 1D-17 1D-16 3D-15 2D-14 ~800 kb D-20 2D-19 2D-18 1D-13 1D-12 3D-11 2D-10 1D-42 1D-43 1D D D-40 1D-39 2D-38 1D-37 2D-36 1D-35 3D-34 1D-33 1D-32 3D-31 2D-30 2D-29 2D-28 1D-27 2D-26 3D-25 2D-24 2D-23 1D-22 6D-21 J C cen Kde intron enhancers J 1-5 RSS C Kde Adapted from IMGT database, M.P. Lefranc, Leukemia 2003; 17:

26 IGK locus : V-J and deletional R 1 Langerak, Crit Rev Immunol 2006;26:23

27 IGK locus : V-J and deletional R 2 1 Langerak, Crit Rev Immunol 2006;26:23

28 IGK locus : V-J and deletional R Langerak, Crit Rev Immunol 2006;26:23

29 IGK locus : V-J and deletional R gdna cdna Vk-Jk (productive) + + Vk-Jk (non-product.) + (+) Vk-Jk (prod/non-prod) + intron-kde + - cdna : no detection of inactivated alleles!

30 IGL gene complex (#22q11.2) 56 V and 5J rearrangeable gene segments cen kb J -C cluster tel enhancer 3 5 kb J 1C 1 J 2 C 2 J 3C 3 J 4 4 J 5 5 J 6 6J 7C 7 Adapted from IMGT database, M.P. Lefranc, Leukemia 2003; 17:

31 BIOMED-2 multiplex IGL tube: V -J V J V primers J primer bp / MwM tonsil PB-MNC GBS-7 IGL tube V -J GBS-2 ES Tonsil nt PB-MNC GBS-7 GBS-2 ES BIOMED-2 report: Leukemia 2003; 17:

32 PCR methodology - 3 Parameters Primer sets IGK primers Vk (family specific) Jk / Ck (refs. in Kostareli, Leukemia 2010) multiplex (BIOMED-2) (Van Dongen, Leukemia 2003) IGL primers Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing VL (family-specific) JL /CL (refs. in Kostareli, Leukemia 2010) multiplex (BIOMED-2) (Van Dongen, Leukemia 2003)

33 Processing of PCR products - 1 Survey prev. workshop (n =63) analysis prior to sequencing YES : 53 NO: 10 Survey prev. workshop (n =63) analytical method Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing PCR product analysis agarose PAGE HD GS other Other -dhplc (1) -capill. electr. (1)

34 Processing of PCR products - 2 Survey: purification of PCR product prior to sequencing? YES : 52 NO: 11 Survey: cloning of PCR product prior to sequencing? YES : 12 (exceptional cases; sequencing failure, NO : 51 Survey: cloning kits? intracl. heterogeneity) (once: all cases) pgem T Easy (2), TOPO cloning kit (7) Survey: prior to sequencing check for insert? YES : 10 Survey: number of clones sequenced? range 3-50, mostly around 10 Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing

35 Clonality analysis PCR product analysis agarose PAGE HD GS other Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Preferred methods for clonality testing heteroduplex analysis high resolution PAGE Gene Scan / fragment analysis / spectratyping agarose gel electrophoresis : too low resolution DISCOURAGED

36 Clonality testing strategies advantage disadvantage HD analysis / -unlabeled products -lower detection limit PAGE allow direct sequencing GS analysis -higher detection limit -labeled products less -optimal visualization optimal in sequencing

37 HD interpretation sequencing strategy Parameters Cases A, C, E, F, G monoallelic direct sequencing Case D bi-allelic Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing What is the next step? direct sequencing single PCR sequencing gel excision sequencing cloning sequencing

38 HD interpretation sequencing strategy Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Case B What are possible scenarios? a. Small clone below detection limit follow-up sample b. Clone is present, but not recognized different primer set required c. Lymphocytosis not due to CLL clone check immunophenotype

39 GS interpretation sequencing strategy Parameters IGH FR1 Case 1 Case 2 Case 3 Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Case 1 : monoallelic direct sequencing in principle possible Case 2 : monoallelic + background 1. direct sequencing? ; 2. gel excision Case 3 : polyclonal 1. no clone (no CLL?); 2. small clone below detection (follow-up sample?)

40 Sequencing strategy Survey prev. workshop (n =63) Sequencing strategy direct cloning gel excision Parameters Material Cell source Anticoagulant Work-up of cells Type of nucleic acid PCR methodology PCR protocol Taq polymerase PCR primers Processing and clonality Sequencing Primers used for sequencing Strands sequenced same nested M13 other 0 forw ard reverse both

41 Sequencing strategy Direct sequencing one product from multiplex PCR starting from IGHJ / IGHC, then reverse sequencing via IGHV family primer one product from single PCR sequencing via specific primers from both sides Sequencing after gel excision + elution bi-allelic rearrangement : physical separation of products Sequencing after subcloning final option, e.g. physical separation of biallelic rearrangements impossible

42 Background: effect on sequencing strategy background sequences

43 Question What would be your next step with the above result obtained via multiplex PCR? A. Analyze sequence via IMGT B. Check on gel, if possible separate PCR products, and sequence individually C. Clone the PCR product and sequence individual colonies

44 Question What would be your next step with the above result obtained via multiplex PCR? A. Analyze sequence via IMGT RISK OF UNRELIABLE SEQUENCE B. Check on gel, if possible separate PCR products, and sequence individually C. Clone the PCR product and sequence individual colonies TOO EARLY 2x VH3 rearrangement ( ) first VH3 rearrangement OK

45 Question 2x VH3 rearrangement ( ) first VH3 rearrangement OK second VH3 rearrangement cloning required

46 Bi-clonal CLL Ig light chain status different type same type VH-JH (prod.) ambiguous ambiguous Vk-Jk /VL-JL (prod.) + ambiguous Cave: Vk-Jk + intron-kde Use surface membrane Ig Light chain info for interpretation Sorting of cells

47 ERIC recommendations for IGHV analysis Most relevant parameters Type of nucleic acid PCR primers Clonality analysis Sequencing RNA / cdna and/or gdna Preferably leader primers (FR1 primers PAGE / HD analysis or GeneScan analysis mostly direct (w or w/o gel excision) reliable sequence from two strands Many parameters: no clear scientific rationale for one or the other option Some strategies show complementary value Clonality testing is an essential phase in the strategy!

48 Further reading on ERIC guidelines

49 IGHV mutational analysis The importance of optimization, standardization, and stereotypy Kinderdijk, NL

50 Thanks to Erasmus MC, Rotterdam Ruud Meijers Alice Muggen Martine Kallemeijn Fatemeh Saberi Hosnijeh Ellen van Gastel Ingrid Wolvers Joyce Schilperoord Kim Heezen Tamara Wabeke Michele van der Klift Irene Groen Vincent van der Velden Jacques van Dongen Simar Pal Singh Rudi Hendriks Uppsala University, Uppsala Richard Rosenquist Lesley-Ann Sutton Pitie-Salpetriere, Paris Frederic Davi San Raffaele, Milan Paolo Ghia Nikea, Athens / Piraeu Chrysoula Belessi G. Papanicolaou, Thessaloniki Kostas Stamatopoulos Anastasia Hadzidimitriou CEITEC, Brno Nikos Darzentas Sebastian Böttcher Jan Philipé Neus Villamor Juan Flores Alberto Orfao University Kiel Matthias Ritgen Monika Brüggemann HOVON CLL network Arnon Kater Mark David Levin