Next-Generation Sequencing of FLT3/ITD for Minimal Residual Disease Monitoring inLeukemia Patients

H66Zhiyi  Xie  Ph.D1,  Andrew  R.  Carson  Ph.D.  ,  Valerie  McClain1,  Selena  Zheng1,  Wenli  Huang1  ,  Jeﬀrey  E.  Miller  Ph.D1,  Mark  J.  Levis  M.D.,  Ph.D2,  Tim  Stenzel  M.D.,  Ph.D1  1Invivoscribe  Inc.,  San  Diego,  CA,  2  Sidney  Kimmel  Comprehensive  Cancer  Center  at  Johns  Hopkins,  BalQmore,  MD

Introduc*on	  	  Minimal	  residual	  disease	  (MRD)	  detec*on	  in	  pa*ents	  with	  leukemia	  has	  proven	  to	   be	   useful	   in	   the	   clinical	   management	   of	   disease	   and	   can	   facilitate	   the	  development	  of	  new	  therapies.	  Muta*ons	  in	  fms	  related	  tyrosine	  kinase	  3	  (FLT3)	  gene	   are	   the	   most	   common	   muta*ons	   found	   in	   acute	   myeloid	   leukemia	   (AML)	  and	   are	   characterized	   by	   an	   aggressive	   phenotype	   with	   a	   high	   prevalence	   of	  	  relapse.	  Internal	  tandem	  duplica*on	  (ITD)	  muta*ons	  within	  the	  juxtamembrane	  domain	  are	  the	  most	  common	  muta*ons	  in	  the	  FLT3	  gene.	  The	  development	  of	  a	   sensi*ve	   and	   speciﬁc	   assay	   for	   FLT3/ITD	   muta*ons	   represents	   a	   signiﬁcant	  advancement	  in	  guiding	  treatment	  decisions.	  	  Results:	  LOD	  LOB	  and	  Linearity	  Results:	  Clinical	  Sample	  Tes*ng	  As	   shown	   in	   Table	   2,	   the	   limit	   of	   detec*on	   (LOD)	   for	   cell	   line	   A	   (30bp	   ITD)	   and	  cell	   line	   B	   (126	   bp	   ITD)	   were	   determined	   to	   be	   at	   2.5x10-­‐5	   and	   1.0x10-­‐4,	  respec*vely.	  This	  was	  concordant	  with	  the	  fact	  that	  ITDs	  of	  a	  certain	  sequence	  and	   at	   longer	   lengths	   are	   harder	   to	   detect	   by	   PCR-­‐based	   assays.	   There	   was	   no	  ITD	   detected	   in	   the	   nega*ve	   cell	   line	   (cell	   line	   C)	   indica*ng	   that	   the	   limit	   of	  blank	  (LOB)	  is	  zero.	  There	  was	  no	  amplicon	  (ITD	  posi*ve	  or	  nega*ve)	  detected	  in	  the	  no	  template	  control	  (NTC)	  samples.	  

A	  total	  of	  16	  clinical	  samples	  tested	  to	  be	  nega*ve	  for	  FLT3/ITD	  by	  the	  standard	  PCR	   assay	   previously	   were	   re-­‐tested	   by	   the	   MRD	   assay.	   The	   inves*gator	  conduc*ng	  the	  MRD	  assay	  was	  blind	  to	  any	  informa*on	  regarding	  the	  presence	  or	  absence	  of	  FLT3/ITD	  muta*on,	  its	  length,	  or	  the	  mutant-­‐to-­‐wild	  type	  allelic	  ra*o.	   The	   summary	   of	   clinical	   sample	   tes*ng	   by	   standard	   PCR	   assay	   and	   the	  MRD	  assay	  is	  shown	  in	  Table	  4.	  The	  MRD	  assay	  correctly	  detected	  the	  ITD	  size	  in	   9	   clinical	   samples.	   Six	   pa*ents	   without	   detectable	   FLT3/ITD	   by	   the	   MRD	  assay	  are	  disease	  free.	  	  Table	  	  2.	  Determina/on	  of	  the	  LOD	  and	  LOB	  of	  the	  assay	  Cell	  Line	  ITD	  Size	  (bp)	  Materials	  and	  Methods	  A	  	  The	  next-­‐genera*on	  sequencing	  (NGS)	  MRD	  assay	  was	  designed	  to	  target	  exons	  14	  and	  15	  of	  the	  FLT3	  gene	  with	  a	  single	  PCR	  ampliﬁca*on.	  Amplicons	  from	  up	  to	  24	   samples	   were	   puriﬁed,	   pooled	   and	   sequenced	   before	   being	   analyzed	   using	  proprietary	   soTware	   developed	   by	   Invivoscribe.	   	   Valida*on	   was	   carried	   out	   by	  	   spiking	   in	   ﬁxed	   amounts	   of	   mutant	   DNA	   into	   wild-­‐type	   DNA	   to	   establish	   a	  sensi*vity	   equivalent	   to	   detec*on	   of	   at	   least	   one	   ITD-­‐containing	   cell	   out	   of	  10,000.	   The	   DNA	   input	   of	   the	   assay	   was	   700	   ng	   (>100,000	   cell	   equivalent).	   The	  assay	  was	  applied	  to	  bone	  marrow	  DNA	  from	  pa*ents	  with	  FLT3/ITD	  AML.	  	  	  Barcode	   crossover	   is	   deﬁned	   as	   errors	   introduced	   into	   the	   DNA	   barcode	   from	  oligo	   synthesis,	   PCR,	   or	   sequencing	   causing	   one	   barcode	   sequence	   to	   become	  another	   barcode	   sequence.	   	   These	   errors	   are	   introduced	   at	   ~1x10-­‐4	   frequency.	  
MiSeq	   instrumenta*on	   also	   has	   up	   to	   a	   ~1%	   rate	   of	   run	   to	   run	   DNA	   carryover	  which	  presents	  a	  problem	  for	  MRD	  level	  detec*on.	  We	  have	  designed	  our	  assay	  with	   a	   proprietary	   approach	   to	   reduce	   the	   background	   errors	   below	   our	   5x10-­‐5	  detec*on	  limit.	  	  Results:	  Sensi*vity	  and	  Speciﬁcity	  	  The	   sensi*vity/speciﬁcity	   and	   precision/reproducibility	   of	   the	   FLT3/ITD	   MRD	  	   assay	   were	   demonstrated	   by	   tes*ng	   DNA	   from	   two	   cell	   lines	   diluted	   into	   a	  background	   DNA	   from	   a	   wild	   type	   FLT3	   cell	   line.	  	   The	   valida*on	   was	   carried	   out	  	  with	   diﬀerent	   operators	   and	   instruments	   and	   conducted	   on	   diﬀerent	   days.	  
Valida*on	  data	   	  for	  samples	  with	  read	  frequency	  of	  1x10-­‐4	   	  and	  5x10-­‐5	   (LOD)	  of	  the	  assay	  is	  shown	  in	  Table	  1.	  The	  results	  show	  excellent	  sensi*vity/speciﬁcity	  and	  precision	  /reproducibility.	  
Table	  	  1.	  Summary	  of	  sensi/vity	  and	  speciﬁcity	  Cell	  Line	  ITD	  Size	  (bp)	  Expected	  Frequency	  Sample	  Size	  TP	  FP	  TN	  FN	  Sensi/vity	  Speciﬁcity	  A	  30	  1.0	  x	  10-­‐4	  36	  36	  0	  N/A	  0	  100.0%	  N/A	  5.0	  x	  10-­‐5	  B	  68	  68	  0	  N/A	  0	  100.0%	  N/A	  126	  1.0	  x	  10-­‐4	  36	  35	  0	  N/A	  1	  97.2%	  N/A	  C	  N/A	  0.0	  38	  N/A	  0	  38	  N/A	  N/A	  100.0%	  TP:	  true	  posi*ve;	  FP:	  false	  posi*ve;	  TN:	  true	  nega*ve;	  FN:	  false	  nega*ve	  30	  Expected	  Frequency	  Sample	  Size	  ITD	  Detec/on	  Posi/ve	  Nega/ve	  %	  Posi/ve†	  Table	  	  4.	  Summary	  of	  clinical	  sample	  tes/ng	  	  by	  standard	  PCR	  assay	  and	  the	  MRD	  assay	  	  36	  36	  0	  100.0%	  5.0	  x	  10-­‐5	  68	  68	  0	  100.0%	  2.5	  x	  10-­‐5	  36	  35	  1	  97.2%	  1	  2	  B	  126	  1.0	  x	  10-­‐5	  36	  35	  1	  97.2%	  C	  N/A	  0.0	  38	  0	  38	  0.0%	  NTC	  N/A	  0.0	  34	  0	  0	  0.0%	  †	  A	  posi*ve	  call	  requires	  the	  detec*on	  of	  a	  minimum	  of	  3	  NGS	  reads	  containing	  a	  FLT3	  ITD	  muta*on.	  

Table	  	  3.	  Determina/on	  of	  the	  linearity	  of	  the	  assay	  DNA	   from	   two	   cell	   lines	   with	  known	   ITD	   (30	   bp	   and	   126	   bp,	  respec*vely)	   were	   serially	  diluted	   into	   a	   background	   DNA	  from	   a	   wild-­‐type	   FLT3	   cell	   line	  and	   tested	   with	   the	   FLT3/ITD	  MRD	   assay.	   Input	   DNA	   quan*ty	  was	   700	   ng	   per	   dilu*on	   point.	  
The	   experimental	   data	   was	  presented	  in	  Table	  3	  and	  plobed	  in	   the	   Figure	   1.	   	   As	   shown	   in	  Figure	   1,	   the	   linearity	   of	   the	  assay	  is	  excellent	  in	  the	  range	  of	  10-­‐2	  –	  10-­‐5.	  	  Expected	  Frequency	  1.0	  x	  10-­‐2	  1.0	  x	  10-­‐3	  1.0	  x	  10-­‐4	  5.0	  x	  10-­‐5	  2.5	  x	  10-­‐5	  Figure	  	  1.	  Linearity	  of	  the	  FLT3	  ITD	  MRD	  assay	  Detected	  Frequency	  Cell	  Line	  A	  8.2	  x	  10-­‐3	  8.1	  x	  10-­‐3	  7.8	  x	  10-­‐3	  8.3	  x	  10-­‐3	  7.5	  x	  10-­‐4	  7.8	  x	  10-­‐4	  8.2	  x	  10-­‐4	  7.4	  x	  10-­‐4	  7.8	  x	  10-­‐5	  6.3	  x	  10-­‐5	  7.3	  x	  10-­‐5	  7.2	  x	  10-­‐5	  4.8	  x	  10-­‐5	  3.3	  x	  10-­‐5	  2.4	  x	  10-­‐5	  3.3	  x	  10-­‐5	  2.5	  x	  10-­‐5	  3.4	  x	  10-­‐5	  2.1	  x	  10-­‐5	  1.8	  x	  10-­‐5	  FLT3/ITD	  MRD	  assay	  	  of	  follow-­‐up	  samples	  Standard	  FLT3/ITD	  PCR	  assay	  Sample	  Number	  1.0	  x	  10-­‐4	  Cell	  Line	  B	  3.0	  x	  10-­‐3	  2.6	  x	  10-­‐3	  2.7	  x	  10-­‐3	  2.3	  x	  10-­‐3	  2.7	  x	  10-­‐4	  2.5	  x	  10-­‐4	  3.3	  x	  10-­‐4	  2.5	  x	  10-­‐4	  7.2	  x	  10-­‐6	  2.3	  x	  10-­‐5	  2.8	  x	  10-­‐5	  2.6	  x	  10-­‐5	  1.9	  x	  10-­‐5	  1.6	  x	  10-­‐5	  1.7	  x	  10-­‐5	  0.00	  5.2	  x	  10-­‐6	  5.3	  x	  10-­‐6	  0.00	  0.00	  Diagnos/c	  Samples	  ITD	  Size	  (bp)	  Allelic	  Ra/o	  Follow-­‐up	  Samples	  33	  1490%	  Neg	  48	  245%	  Neg	  Follow-­‐up	  Samples	  Detected	  	  ITD	  Size	  (bp)	  Detected	  ITD	  Frequency	  33	  1.4	  x	  10-­‐6	  On	  treatment	  48	  1.7	  x	  10-­‐4	  Unavailable	  Died	  3	  69	  1%	  Neg	  69	  1.1	  x	  10-­‐4	  4	  24	  59%	  Neg	  24	  2.0	  x	  10-­‐4	  On	  treatment	  5	  72	  17%	  Neg	  72	  2.8	  x	  10-­‐5	  On	  treatment	  6	  21	  1%	  Neg	  21	  4.0	  x	  10-­‐6	  Relapsed	  7	  15	  11%	  39	  124%	  Neg	  8	  45	  Unavailable	  Neg	  9	  36	  Unavailable	  Neg	  10	  78	  110%	  Neg	  15	  1.4	  x	  10-­‐5	  39	  3.3	  x	  10-­‐4	  N/A	  0.0	  Disease	  free	  No	  PCR	  ampliﬁca*on	  Disease	  free	  N/A	  0.0	  On	  treatment	  Disease	  free	  11	  96	  Unavailable	  Neg	  N/A	  0.0	  Disease	  free	  12	  30	  9%	  Neg	  N/A	  0.0	  Disease	  free	  13	  30	  Neg	  N/A	  0.0	  Disease	  free	  14	  Detected*	  	  646%	  Neg	  24	  3.7	  x	  10-­‐3	  Relapsed	  and	  died	  15	  Detected*	  Neg	  	  18	  1.0	  x	  10-­‐4	  Relapsed	  and	  died	  16	  Detected*	  	  Neg	  N/A	  0.0	  Disease	  free	  *Size & ratio unavailable

Conclusions	  The	   FLT3/ITD	   MRD	   assay	   is	   a	   highly-­‐speciﬁc	   test,	   developed	   with	   the	  accompanying	   bioinforma*cs	   soTware	   under	   full	   ISO13485	   design	   control,	  which	   is	   at	   least	   two	   orders	   of	   magnitude	   more	   sensi*ve	   than	   current	  commercially	   available	   assays.	   In	   addi*on,	   the	   chemistry	   and	   bioinforma*cs	  soTware	   reliably	   picked	   up	   even	   the	   larger	   ITDs	   missed	   en*rely	   by	   other	  commercial	   assays.	   Importantly,	   the	   results	   of	   clinical	   samples	   tested	   by	   this	  MRD	   assay	   showed	   100%	   concordance	   with	   clinical	   outcomes.	   This	   assay	  provides	   a	   reliable	   tool	   to	   assess	   MRD	   in	   FLT3+	   AML	   pa*ents.	   The	   FLT3/ITD	  MRD	  test	  is	  currently	  being	  oﬀered	  through	  Invivoscribe’s	  interna*onal	  clinical	  Laboratories	  for	  Personalized	  Molecular	  Medicine	  (LabPMM).