TT65

Development	  of	  a	  Comprehensive	  IGH	  NGS	  Assay	  for	  DetecQng	  Suspected	  B-­‐Cell	  Clonality	  Ying  Huang  Ph.D,  Aus0n  Jacobsen,  Jeﬀ  Panganiban,  Kasey  Hu9  Ph.D.,  Duy  Duong,  Jordan  Thornes,  Jeﬀrey  E.  Miller  Ph.D,  and  Tim  Stenzel  M.D.,  Ph.D
Invivoscribe,  Inc.,  San  Diego,  USA

Abstracts	  Results:	  AnalyQcal	  ValidaQons	  •LoD	  and	  linearity	  valida1on	  •	  Clinical	  samples	  tested	  with	  the	  Iden1Clone	  IGH	  Assay	  and	  the	  LymphoTrack	  IGH	  (FR1/FR2/FR3)	  Assay	  Sample	  Single:	  	  	  	  	  	  R2=0.9971	  3	  combo:	  R2=0.9974	  	  	  IGH	  FR1:	  Single	  versus	  3	  Combo	  (FR1+FR2+FR3)	  	  	  PB	  Clonal	  Clonal	  Clonal	  Clonal	  FFPE	  Clonal	  Clonal	  Clonal	  Clonal	  3	  P224	  PB	  Clonal	  Clonal	  Clonal	  Clonal	  4	  P228	  PB	  Clonal	  Clonal	  Clonal	  Clonal	  5	  P232	  FFPE	  Clonal	  Clonal	  Clonal	  Clonal	  6	  P235	  PB	  Clonal	  Clonal	  Clonal	  Clonal	  7	  P236	  PB	  Clonal	  Clonal	  Clonal	  Clonal	  8	  P253	  FFPE	  Clonal	  Clonal	  Non-­‐Clonal	  Non-­‐Clonal	  9	  P262	  FFPE	  Non-­‐Clonal	  Non-­‐Clonal	  Non-­‐Clonal	  Non-­‐Clonal	  10	  CE-­‐0035	  PB	  Non-­‐Clonal	  Non-­‐Clonal	  Non-­‐Clonal	  Clonal	  •	  	  Sample	  CE-­‐0035	  is	  Clonal	  by	  LymphoTrack	  IGH	  FR3	  and	  Non-­‐Clonal	  by	  IGH	  FR1	  and	  IGH	  FR2	  LymphoTrack IGH FR1 Assay - V – J Sequence Frequencies

IGH	  FR2:	  Single	  versus	  3	  Combo	  (FR1+FR2+FR3)	  120100Materials	  and	  Methods	  6040VH7-J1VH7-J2

VH7-J3VH7-J4VH7-J5

VH7-J6VH7-nonenone-J1

none-J2

none-J3

none-J4

none-J5

none-J6

none-none

VH7-J3VH7-J4VH7-J5

VH7-J6VH7-nonenone-J1

none-J2

none-J3

none-J4

none-J5

none-J6

none-none

VH7-J3VH7-J4VH7-J5

VH7-J6VH7-nonenone-J1

none-J2

none-J3

none-J4

none-J5

none-J6

none-none

VH6-J6VH7-J2
VH7-J2

VH6-J5

VH7-J1VH7-J1

VH6-J4VH6-none

VH6-J3VH6-J2

VH6-J1VH5-J6VH5-J5VH5-J4VH5-none

VH5-J3VH5-J2

VH5-J1VH4-J6

VH4-J5

VH4-J4VH4-none

VH4-J3VH4-J2

VH4-J1VH3-J6

VH3-J5

VH3-J4VH3-none

VH3-J3VH3-J2VH3-J1VH2-J6

VH2-J5

VH2-J4VH2-none

VH2-J3VH2-J2VH2-J1VH1-J60

VH1-J5

20Single:	  	  	  	  	  	  R2=0.9973	  3	  combo:	  R2=0.9979	  	  VH1-J4• The	   LymphoTrack®	   IGH	   (FR1/FR2/FR3)	   Assay	   targets	   the	   FR1/J,	   FR2/J	   and	   FR3/J	   regions.	   The	   proprietary	   V	  and	   J	   consensus	   primers	   were	   designed	   and	   adapted	   to	   enable	   the	   PCR	   products	   to	   be	   sequenced	   on	   the	  MiSeq®	  plaKorm.	  	  

80VH1-none

Conclusions:	   A	   comprehensive	   NGS-­‐based	   IGH	   assay	   has	   been	   developed	   for	   the	   Illumina®	   MiSeq®	   plaKorm	  that	  idenQﬁes	  clonal	  IGH	  V-­‐J	  rearrangements	  and	  DNA	  sequences.	  This	  LymphoTrack	  Assay	  has	  demonstrated	  that	   combining	   FR1,	   FR2	   and	   FR3	   helps	   to	   decrease	   the	   false-­‐negaQve	   rate	   due	   to	   somaQc	   hypermutaQon	   in	  primer	  binding	  sites	  of	  the	  involved	  VH	  gene	  segments.	  	  	  P221	  P222	  VH1-J3	  Single:	  	  	  	  	  	  R2=0.9939	  3	  combo:	  R2=0.9969	  	  1	  2	  % Total Reads

Results:	   The	   ability	   to	   detect	   FR1,	   FR2	   and	   FR3	   regions	   was	   veriﬁed	   by	   tesQng	   diﬀerent	   known	   V-­‐J	  rearrangement	   B	   cell	   line	   DNA.	   	   Cell	   line	   DNA,	   serially	   diluted	   into	   tonsil	   DNA,	   demonstrated	   the	   limit	   of	  detecQon	   (LoD)	   at	   5%	   diluQon	   using	   50	   ng	   DNA	   input.	   The	   assay	   achieved	   excellent	   linearity	   (R2	   >	   0.95)	   and	  reproducibility	  (<25%	  CV),	  demonstrated	  by	  2	  operators,	  2	  MiSeqs	  and	  2	  lots	  of	  PCR	  master	  mixes.	  Preliminary	  tesQng	  of	  genomic	  DNA	  from	  peripheral	  blood	  (PB),	  bone	  marrow	  (BM)	  aspirates,	  and	  formalin-­‐ﬁxed	  paraﬃn-­‐embedded	   Qssue	   (FFPE)	   demonstrated	   that	   the	   clonality	   detecQon	   missed	   by	   one	   FR	   could	   be	   detected	   by	  another	  FR,	  thus	  increasing	  the	  overall	  detecQon	  rate.	  	  Iden1Clone	  IGH	  LymphoTrack	  	   LymphoTrack	  	   LymphoTrack	  	  (Capillary	  IGH	  FR1	  IGH	  FR2	  IGH	  FR3	  Electrophoresis)	  Sample	  Type	  VH1-J2

Methods:	   The	   LymphoTrack®	   IGH	   (FR1/FR2/FR3)	   Assay	   targets	   the	   FR1/J,	   FR2/J	   and	   FR3/J	   regions.	   The	  proprietary	  V	  and	  J	  consensus	  primers	  were	  designed	  and	  adapted	  to	  enable	  the	  PCR	  products	  to	  be	  sequenced	  on	  the	  MiSeq®	  plaKorm.	  	  For	  each	  IGH	  FR,	  a	  set	  of	  24	  master	  mixes	  are	  provided,	  each	  designed	  with	  a	  diﬀerent	  index,	  allowing	  analysis	  of	  22	  paQent	  samples	  plus	  posiQve	  and	  negaQve	  controls.	  Single-­‐step	  mulQplexed	  PCR	  was	   followed	   by	   amplicon	   puriﬁcaQon	   using	   the	   AMPure®	   XP	   PCR	   system.	   Puriﬁed	   equimolar	   amounts	   of	  amplicons	  from	  diﬀerent	  samples	  and	  FRs	  were	  pooled	  to	  form	  a	  library.	  The	  harmonized	  and	  quanQﬁed	  library	  was	   sequenced	   using	   the	   MiSeq®	   v2	   Reagent	   kit	   (500	   cycles).	   LymphoTrack	   product	   data	   output	   from	   the	  MiSeq®	   were	   analyzed	   using	   Invivoscribe’s	   proprietary	   bioinformaQcs	   soYware,	   which	   analyzes	   and	   sorts	  sequences	  by	  both	  index	  and	  FR,	  to	  generate	  frequency	  distribuQons	  of	  V-­‐J	  rearrangements	  and	  to	  determine	  the	  somaQc	  hypermutaQon	  (SHM)	  rate	  of	  the	  IGHV	  region	  based	  on	  the	  sequence	  of	  FR1/J	  amplicons.	  

Sample	  ID	  VH1-J1

Introduc1on:	   	  PCR-­‐based	  methods	  targeQng	  immunoglobulin	  heavy	  chain	  (IGH)	  frameworks	  1,	  2,	  and	  3	  (FR1,	  FR2	   and	   FR3)	   are	   the	   current	   gold	   standard	   for	   ﬁrst-­‐line	   clonality	   tesQng	   in	   suspected	   B-­‐cell	   proliferaQons.	  
Recently,	   next-­‐generaQon	   sequencing	   (NGS)	   based	   approaches	   for	   tesQng	   immune	   receptor	   genes	   have	   been	  suggested	   to	   improve	   sensiQvity	   and	   to	   idenQfy	   the	   speciﬁc	   V-­‐J	   DNA	   sequence	   required	   to	   track	   clones	   in	  follow-­‐up	   tesQng.	   Here	   we	   report	   the	   development	   of	   a	   comprehensive	   NGS-­‐based	   IGH	   assay	   which	   allows	  detecQon	  of	  all	  three	  FRs,	  simultaneously,	  in	  a	  single	  Illumina®	  MiSeq®	  run.	  

Results:	  Clinical	  Study	  LymphoTrack IGH FR2 Assay - V – J Sequence Frequencies

IGH	  FR3:	  Single	  versus	  3	  Combo	  (FR1+FR2+FR3)	  120

9.2	  104	  3.50	  8.0	  • Precision	   and	   reproducibility	   were	   validated	   for	   each	   FR.	   A	   total	   of	   combined	   96	   data	   points	   were	   generated	  from	  8	  MiSeq®	  runs	  (2	  operators	  x	  2	  runs	  x	  2	  days)	  for	  each	  FR.	  

1	  48	  295	  0.50	  29.3	  243	  1.52	  12.5	  104	  1.43	  16.1	  • DNA	  from	  diﬀerent	  clinical	  samples	  was	  tested	  by	  both	  the	  capillary-­‐based	  IdenQClone®	  IGH	  Clonality	  Assay	  and	  the	  NGS-­‐based	  LymphoTrack®	  IGH	  (FR1/FR2/FR3)	  Assay.	  

Tonsil	  16	  varies	  0.04	  34.8	  varies	  0.06	  26.5	  104	  0.07	  28.6	  VH6-none

VH6-J6VH6-J5VH6-J4VH6-J3VH6-J2

VH6-J1

VH5-none

VH5-J6VH5-J5VH5-J4VH5-J3VH5-J2

VH5-J1

VH4-none

VH4-J6

VH4-J5

VH4-J4VH4-J3VH4-J2

VH4-J1VH3-none

VH3-J6

VH3-J5

VH3-J4VH3-J3VH3-J2VH3-J1VH2-none

VH2-J6

VH2-J5

VH2-J4VH2-J3VH2-J2VH2-J1VH1-none

VH1-J6

VH1-J5200

VH6-none

3.93	  

VH6-J6243	  VH6-J514.1	  

VH6-J41.28	  

VH6-J3295	  VH6-J248	  VH6-J12.5	  

VH5-none• For	  each	  FR,	  LoD	  and	  linearity	  were	  validated	  in	  both	  single	  FR	  and	  3	  combined	  FRs	  MiSeq®	  runs.	  

40

VH5-J68.4	  

VH5-J57.04	  

VH5-J4104	  

VH5-J38.2	  

VH5-J2

7.72	  

VH5-J1243	  VH4-none

19.4	  

VH4-J6

2.58	  

VH4-J5295	  VH4-J448	  VH4-J35	  VH4-J2• The	  analyQcal	  validaQon	  panel	  consists	  of	  tonsil	  DNA	  (IVS-­‐0000)	  and	  a	  diluQon	  series	  of	  10%,	  5%,	  2.5%	  and	  1.0%,	  cell	  line	  DNA	  (IVS-­‐0013)	  diluted	  into	  tonsil	  DNA.	  

60

VH4-J1

6.2	  

VH3-none

12.99	  VH3-J6104	  VH3-J59.9	  

VH3-J415.74	  VH3-J3243	  VH3-J217.5	  

VH3-J15.08	  80

VH2-none295	  

100

VH2-J6Mean	  CV%	  	  %	  Reads	  	  VH2-J5Size	  (bp)	  VH2-J4

Mean	  CV%	  	  %	  Reads	  	  VH2-J3Size	  (bp)	  VH2-J2

Mean	  	  CV%	  %	  Reads	  	  VH2-J1Size	  (bp)	  32	  Illumina®	  and	  MiSeq®	  are	  registered	  trademarks	  of	  Illumina,	  Inc.	  	  VH1-J4LymphoTrack IGH FR3 Assay - V – J Sequence Frequencies
12010	  • The	  staQsQcal	  program	  JMP	  was	  used	  to	  perform	  the	  staQsQcal	  analyses.

VH1-J3

IGH	  FR3	  N	  VH1-none

Analyze	  Data	  IGH	  FR2	  VH1-J2

IGH	  FR1	  IVS-­‐0013	  %Dilu1ons	  	  VH1-J6Run	  MiSeq	  0

VH1-J5Pool	  Library	  	  20VH1-J4

Quan1fy	  Amplicons	  40VH1-J3Purify	  Amplicons	  	  60VH1-J2

PCR	  

	  	  	  	  	  •	  	  	  Precision	  and	  reproducibility	  valida1on	  80VH1-J1• Single-­‐step	  mulQplexed	  PCR	  was	  followed	  by	  amplicon	  puriﬁcaQon	  using	  the	  AMPure	  XP	  PCR	  system.	  Puriﬁed	  equimolar	   amounts	   of	   amplicons	   from	   diﬀerent	   samples	   and	   FRs	   were	   pooled	   to	   form	   a	   library.	   The	  harmonized	  and	  quanQﬁed	  library	  generated	  with	  the	  LymphoTrack	  Assay	  was	  sequenced	  using	  the	  MiSeq®	  v2	   Reagent	   kit	   (500	   cycles).	   LymphoTrack	   Assay	   output	   fastq	   data	   were	   analyzed	   using	   Invivoscribe	  proprietary	   bioinformaQcs	   soYware,	   which	   can	   sort	   the	   sequences	   by	   both	   index	   and	   FR,	   to	   generate	  frequency	   distribuQons	   of	   V-­‐J	   rearrangements	   and	   to	   determine	   the	   somaQc	   hypermutaQon	   (SHM)	   rate	   of	  the	  IGHV	  region	  based	  on	  the	  sequence	  of	  FR1/J	  amplicons.	  

% Total Reads• For	   each	   IGH	   FR,	   a	   set	   of	   24	   master	   mixes	   are	   provided,	   each	   designed	   with	   a	   diﬀerent	   index,	   allowing	  analysis	  of	  22	  paQent	  samples	  plus	  posiQve	  and	  negaQve	  controls.	  	  	  VH1-J1	  % Total Reads

100Conclusions	  A	  comprehensive	  NGS-­‐based	  IGH	  assay	  has	  been	  developed	  for	  the	  Illumina®	  MiSeq®	  plaKorm	  that	  idenQﬁes	  clonal	  IGH	  V-­‐J	  rearrangements	  and	  DNA	  sequences.	  The	  LymphoTrack®	  Assay	  has	  demonstrated	  that	  combining	  FR1,	  FR2	  and	  FR3	  helps	  to	  decrease	  the	  false-­‐negaQve	  rate	  due	  to	  somaQc	  hypermutaQon	  in	  primer	  binding	  sites	  of	  the	  involved	  VH	  gene	  segments.