Absolute  Quan-fica-on  of  Gra3  derived  cell-­‐free  DNA  (GcfDNA)    early  a3er  Liver  Transplanta-on  (LTx)  using  droplet  Digital  PCR  

 

J.  Beck1,  P.  Kanzow2,  J.  Schmitz2,  O.  Kollmar3,  M.  Oellerich2,  E.  Schütz1.    1)  Chronix  Biomedical,  Gö5ngen,  Germany,  2)  Dept.  Clinical  Chemistry,  University  Medicine,  Gö5ngen,  Germany,  3)  TransplantaCon  Surgery,  University  Medicine,  Gö5ngen,  Germany  

 

Background:  The  diagnos,c  value  of  GcfDNA  as  measure  of  gra8  integrity  a8er  LTx  has  been  recently  proven   [1,2].   The   yin   and   yang   of   using   percentage   values   vs.   absolute   GcfDNA   quan,fica,on   is,  nevertheless,  under  discussion  [3].  Where  the  ra,o  of  gra8  to  host  cfDNA  has  analy,cal  advantages  by  elimina,ng  disturbing  variables,  such  as  DNA  extrac,on  efficiency,  variabli,es  in  host  cfDNA  may  obfuscate   the   view   on   the   engra8ed   organ.   The   early   phase   a8er   LTx   was   used   as   model   to  interrogate  whether  the  percentage  or  absolute  plasma  concentra,on  of  GcfDNA  is  a  more  valuable  gra8  integrity  measure.  Methods:  GcfDNA  percentage  was  determined  by  droplet  dPCR  (BioRad)  as  described  [1].  A  synthe,c  sequence   of   non-­‐human   origin   (average   length   of   cfDNA   )   was   spiked   into   1mL   plasma,   and  quan,fied   with   ddPCR   a8er   DNA   extrac,on   in   one   fluorescent   channel.   The   total   cfDNA   was  quan,fied  using  two  combined  human  genomic  dPCRs  in  the  second  channel  as  copies/mL  (cp/mL).  Total  cfDNA  was  calculated  using  the  spike-­‐in  without  being  extracted  to  assess  the  DNA  extrac,on  efficiency  in  each  batch.  GcfDNA  concentra,on  was  defined  as  of  the  total  cfDNA(cp/mL)  x  GcfDNA%.  Plasma  obtained  during  the  first  10  days  a8er  LTx  from  15  pa,ents  (one  split-­‐LTx)  was  inves,gated.  Results:   Ten   repeated   extrac,ons   of   the   same   plasma   pool   from   healthy   volunteers   yielded   an  average  of  1069  diploid  genomic  cp/mL  plasma  with  a  CV  of  7.5%.  Of  185  samples  six  showed  a  low  (<50%)  extrac,on  efficiency;  the  remainders  had  an  average  of  67%+9%.  The  total  cfDNA  was  highly  variable  peaking  at  6hr  a8er  reperfusion  (3.9x105±2.0x105cp/mL)  weaning  to  1.3x105±0.9x105  cp/mL  at   day   10.   The   respec,ve   GcfDNA   was   3.1x105±1.8x105   cp/mL   (6hr)   and   1.5x103±0.9x103   cp/mL  (day10).  The  correla,on  between  GcfDNA%  and  GcfDNA(cp/mL)  values  was  weak  (r=0.61;p<0.05).  A  comparison  of  the  AUC  (d1-­‐d5)  of  AST  with  GcfDNA  percentage  and  concentra,on  showed  a  beeer  associa,on   with   absolute   GcfDNA   (r=0.65;p<0.05)   compared   to   percentages   (r=0.31;p=0.27).   The  ini,al  half   life  was  1.3d±0.6  for  GcfDNA(cp/mL)  and  2.9d±1.6  GcfDNA(%),  compared  to  2.6d±1.2  for  AST.  Conclusions:   A   robust   and   precise   ddPCR   method   for   absolute   quan,fica,on   of   GcfDNA,   was  developed,   combining   the   analy,cal   advantages   of   gra8/host   ra,o   (e.   g.   elimina,ng   possible   bias  from   interferences),  with   a   robust   quan,fica,on  of   total   cfDNA.   The  GcfDNA   concentra,on   seems  beeer  associated  with  AST-­‐values  early  a8er  LTX  and  showed  a  more  rapid  dynamics  than  %GcfDNA.  Even   though   the   ini,al   post   Tx   phase,  with   highly   variable   amounts   of   total   cfDNA,   is   par,cularly  complicated,  this  method  may  also  provide  a  beeer  view  on  gra8  integrity  in  other  situa,ons,  where  the   host   cfDNA   is   increased   due   to   non-­‐transplanta,on   related   causes.   As   to  whether   the   clinical  u,lity   is   improved   compared   to   percentage   values   for   stable   pa,ents   as  well,   is   subject   to   further  inves,ga,ons.  

It   has   been   shown   is   several   studies   that   the   quan,fica,on   of   gra8   derived   circula,ng   free   DNA  (GcfDNA)  has  the  clinical  poten,al  for  interroga,ng  the  integrity  of  the  transplanted  organ  using  this  “liquid   biopsy”   approach   [1,5].   We   have   shown   that   GcfDNA%   is   useful   to   assess   the   minimal  required  dosage  of  immunosuppressive  drugs  in  the  early  post  transplant  phase  [2].  However,  the  use  of  percentages  can  be  misleading  of  non-­‐transplant  related  circumstances  lead  to  an  increase  of  host  cfDNA,  over  the  amount  that  is  usually  observed  in  complica,on-­‐free  solid  organ  recipients.    Furthermore   the   immediate   early   post   engra8ment   phase   is   complicated   by   the   recovery   from  preserva,on-­‐reperfusion  injury,  which  can  lead  to  extreme  cfDNA  concentra,ons.    The  minute  amounts  of  cfDNA  that  can  be  extracted  from  plasma  require  careful  control  of  extrac,on  efficiency  and  analy,cal  methods  that  yield  precise  measures  with  minimum  sample  input.  The  aim  of  this  work  was  to  establish  a  robust  system  for  reliable  quan,fica,on  of  GcfDNA  expressed  as   absolute   concentra,on   [cp/mL],   controlling   the   pre-­‐analy,cal   error   and   the   comparison   of  diagnos,c  use  with  the  established  GcfDNA  percentage  determina,on.    

 Pa-ents,  Material  and  Methods  Blood  samples  from  pa,ents  a8er  liver  (LTx),  heart  (HTx)  and  kidney  (KTx)  were  drawn  according  to  IRB   approved   protocols.   288   samples   from   23   Ltx   were   included.   For   the   cfDNA   extrac,on  inves,ga,ons  pools  from  normal  volunteers  are  used.  EDTA-­‐whole  blood  was  drawn  and  processed  within  4  hours  and  stored  frozen  at  -­‐80°C  un,l  extrac,on.  For  LTx  pa,ents  cfDNA  tubes  (9mL)  Streck  Inc.  were  used  for  a  subset  of  draws.  cfDNA  Extrac-ons  Before   extrac,on   all   plasma   samples  were   centrifuged   at   4000   x   g   for   20  min   at   4°C.   Two   cfDNA  extrac,on  methods:  QIAamp  Circula,ng  Nucleic  Acid  Extrac,on  Kit  (QIAGEN),  Roches  High  Pure  Viral  Extrac,on  Large  Volume  (LV)  Kit  (ROCHE  LV)  were  used  to  the  manufacturers  recommenda,ons.  The  LV   Kit   was   also   used   with   1/3   and   without   the   recommended   carrier   RNA.   Extrac,ons   were  performed  on  the  same  plasma  pool  on  three  different  days.  Two  different  volumes  were  extracted  with  each  kit:  2.5  mL  and  5  mL  for  QIAGEN,  1mL  and  2.5  mL  for  ROCHE  LV.  ddPCR  were  performed  in  triplicate   for   each   extrac,on   day.   For   the   in-­‐assay   assessment   of   the   extrac,on   yield,   an   ar,ficial  spike  added  to  the  plasma  immediately  before  adding  the  protease/binding  buffer.  The  spike  consists  of   a   non-­‐human   derived   320bp   DNA   that   is   prepared   by   PCR   on   the   vector-­‐cloned   fragment.   The  product  was  stored  in  1.4x106-­‐fold  concentra,on  and  was  diluted  freshly  prior  to  extrac,on.    ddPCR  Assays  All   primers   were   purchased   from   Sigma-­‐Aldrich   and   probes   from  MWG-­‐Biotech   Eurofins.   ddPCRs  were   performed   using   a   Bio-­‐Rad   QX100/QX200   (Bio-­‐Rad)   and   data   were   analyzed   using   the  QuantaSo8  version  1.5.38  so8ware  (Bio-­‐Rad)  and  exported  to  Excel  for  further  calcula,ons.  ddPCRs  were  performed  in  1x  ddPCR  Supermix  for  Probes  or  EvaGreen  mastermix  as  appropriate  (Bio-­‐Rad)  Absolute  cfDNA  quan-fica-on  For   the  quan,fica,on  of   absolute   cfDNA   two  assays   each   targe,ng  one   single   copy   genomic   locus  were   combined   in   one   ddPCR   (HEX   channel)   together  with   one   assay   targe,ng   the   ar,ficial   spike  product  (FAM  channel)  GcfDNA  [%]  was  measured  as  described  elsewhere  (1).  The  GcfDNA  [cp/mL]  was   the   calculated  by  mul,plying   the  GcfDNA   [%]  with   the   cfDNA   [cp/mL]   (Figure  1).   20  µl   of   the  spike-­‐dilu,on  used  for  the  respec,ve  extrac,on  were  diluted  to  a  final  volume  of  50  µL  three  ,mes  and  each  dilu,on  was  measured  in  duplicates.  ddPCRs  contained  8  µL  of  extrac,on  eluate  or  diluted  spike  control  and  4  µL  for  samples  drawn  within  the  first  48hrs  a8er  surgery.  cfDNA  length  asessment  The  absolute  cfDNA  values  were  corrected   for   the   respec,ve  spike   recovery   rate  and   the  amplicon  lengths  of  98bp  and  90bp,  either  assuming  the  size  distribu,on  as  determined  in  cfDNA  samples  from  healthy  volunteers  and  pa,ents  and  the  formula  given  in  Figure  2  or  using  the  cfDNA  length  index  as  determined   by   ddPCR   (Figure   3).   Simula,ons   were   computed   for   the   range   of   observed   size  distribu,ons,   where   the   PCR   efficiency   and   the   length   index   were   calculated   using   R   (10,000  simula,ons  per   size  distribu,on,   1.   peak  50   to  70%,   second  and   third  peak  5   to  45%   to   cover   the  observed  range  of  the  length  index  (3  to  30).  

!  Given   the  minute   amounts   of   cell-­‐free   DNA,   the   absolute   quan-fica-on   of   cfDNA   requires   at   first  careful   control   of   the   extrac-on   stage.   By   adding   and   quan-fying   a   spiked-­‐in   ar-ficial   DNA   the  extrac-on  variability  can  be  corrected.  

!  For   accurate   cfDNA  quan-fica-on   a   triplex   ddPCR   assay  was   developed   targe-ng   two   genomic   loci  and   the   spiked-­‐in  DNA.  By   combining   two  genomic   targets  higher  numbers  of   posi-ve  droplets   are  yielded,  while  keeping  the  needed  sample  volume  low.  

!  Since   PCR   efficiency   is   lowered   by   the   fragmenta-on   of   the   cfDNA,   a   length   correc-on   factor  was  implemented.   Individual   differences   in   the   cfDNA   length   distribu-on   profiles   introduce   only   small  addi-onal  bias,  which  may  not  be  clinically  relevant.    

!  The  GcfDNA  concentra-on  seems  be]er  associated  with  AST-­‐values  early  a3er  LTX.  

!  The   ini-al  post  Tx  phase   is  characterized  by  highly  variable  and  high  amounts  of  total  cfDNA,  which  declines  with  a  approximate  half  life  of    1.9  days.    

!  Absolute  GcfDNA  quan-fica-on  may  provide  a  be]er  view  on  gra3  integrity  in  situa-ons,  where  the  host   cfDNA   is   increased   due   to   non-­‐transplanta-on   related   causes.   Whether   the   clinical   u-lity   is  improved  compared  to  percentage  values  is  subject  to  further  inves-ga-ons.    

Figure   1:  Overview  of   different   ddPCR   assays   applied   for   the   exact   absolute   quan,fica,on   of   gra8  derived  cell-­‐free  DNA.  

Figure  2:  Upper  le8:  Schema,c  drawing  of  PCR  efficiency  decrease  caused  by  random  fragmenta,on  of   the   template   given   that   all   fragments   are  of   exactly   the   same   size.   Lower   le8:  Correc,on   factor  es,ma,on   for   lowered   PCR   efficiency   based   on   the   fragment   length   profile   of   cfDNA.   Upper   and  lower  right:  Experimental  data  showing  the  effect  of  PCR  efficiency  correc,on  based  on  amplicon  and  template  lengths.  

Figure  3:  Agilent  Bioanalyzer  images  (upper  le8)  were  used  to  determine  the  percentage  of  molecules  present   in   the   shortest   peak.   Upper   right:   This   percentage   is   highly   nega,vely   correlated  with   the  frac,onal  abundance  of   larger  sized  amplicons  as  determined   in  the   length   index  ddPCR  detailed   in  Figure  1.  Lower  right:  Modeling  of   the  PCR  efficiency  versus  the   length   index  and  a  mean  amplicon  size  of  94  bp  as  used  in  the  total  cfDNA  concentra,on  ddPCR.  The  model  can  be  used  to  correct  the  template   length   dependent   decrease   in   PCR   efficiency   for   any   given   experimentally   determined  length  index.  

Figure   4:   Bland-­‐Altman   Plot   showing   the  maximum   devia,on   between   the   absolute   cfDNA   values  either  corrected  by  the  samples  experimentally  determined  length  index  (n=141,  range:  3.7  to  27.5)  or   by   a   mean   length   correc,on   factor   of   0.59.   The   95%   confidence   interval   indicates   that   the  differences  in  cfDNA  length  profiles  introduce  an  error  of  only  10%.  

Figure  5:  Comparison  of  different  extrac,on  kits  with  respect  to  spike  recovery  rate  (upper  panel)  and  cfDNA  yield  (middle  panel).  All  extrac,ons  were  performed  on  the  same  plasma  pool  in  triplicate  and  on   three   consecu,ve   days,   mean   values   and   standard   devia,ons   over   all   extrac,ons   are   shown.  Lower  panel:   cfDNA  yields  corrected   for   the  spike   recovery   rate;  no  significant  differences  between  the   different   extrac,on   methods   remained,   proving   the   importance   and   feasibility   to   correct   for  extrac,on  efficiency  by  means  of  a  spiked-­‐in  control.  

Table   1:  Mul,variate   correla,on   of   GcfDNA   percentages   and   absolute   GcfDNA   concentra,on   with  clinical   parameters.   Aeributable   to   their   higher   dynamic   range   the   absolute   GcfDNA   values   are  significantly  correlated  to  the  AST  levels,  while  the  percent  GcfDNA  that  do  not  account  for  the  total  cfDNA  content  per  mL  of  plasma  are  not.  CIT  =  cold  ischemia  ,me,  WIT  =  warm  ischemia  ,me,  AST  =  aspartate  aminotransferase  

Figure  6:  Absolute  GcfDNA  concentra,ons  within  the  first  eight  days  post  surgery  of  14  pa,ents  were  used  to  calculate  the  range  demarcated  in  the  figure.  The  absolute  GcfDNA  in  the  early  post-­‐Tx  phase  are   highly   variable   spanning   over   nearly   two   powers   of   ten.   Addi,onally   shown   is   the   absolute  GcfDNA   ,mecourse   of   a   pa,ent   who   received   a   marginal   donor   organ   (HELLP-­‐liver).   The   GcfDNA  levels   of   this   pa,ent  were   low   and   the   post   opera,ve   gra8   func,on  was   excellent,   indica,on   that  early  GcfDNA  determina,ons  are  suitable  to  assess  the  gra8  quality  of  marginal  donors.  

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