E’  u%le  o)enere  la  risposta  molecolare  nella  

LLC?        

Imma  A&olico  U.O.  di  Ematologia  e    

Trapianto  di  Cellule  Staminali  POTENZA  

CLL: epidemiology •  >70% CLL pts >65 yrs at diagnosis; •  < 2%< 45 yrs •  9% 45-54yrs •  19% 55–64yrs; •  26.5% 65–74yrs; •  30% 75-84 yrs; •  13% >85 yrs. •  Incidence : 4.1/100.000 /year •  Mild increase of incidence from 1975 to 2006.

Gribben- Blood 2009

11% 30%

 From  pallia%ve  care  to  eradica%on  of  the  disease  

•  Response  to  therapy  is  the  most  important  prognos%c  factor  for  survival.    

 •  Some  of  the  novel  biological  treatments  appear  to  be  ac%ve  irrespec%ve  of  the  presence  of  nega%ve  prognos%c  features.  

       Different  treatment  modali%es  aimed  at  more  profound  remissions  with  long-­‐term  control  of  the  disease  (even  cure?).  

Possibility  to  create  more  efficacious  associa%ons  with  standard  chemotherapy  without  increasing  hematologic  toxici%es  and/or  to  prolong  drug  administra%on  in  consolida%on  or  maintenance  strategies  

CR  and  MRD    aRer  1st  line  treatment  

• Complete  eradica%on:  obvious  desired  end  point  in  clinical  trials    • Assessment  of  CR  at  the  clinical  and  morphological  level  is  not  sufficient  

The decision for relapse treatment was determined by CT scan or ultrasound results in only 2 of 176

patients (1%)!  

Meta-analysis performed with the dataset of 3 German

CLL Study Group phase 3 trials (CLL4, CLL5, and CLL8) including 1372 patients receiving 1rst line TX.

PFS  (A)  and  OS  (B)  for  pa%ents  with  versus  without  bulky  lymphadenopathy  

detected  by  pretherapeu%c  imaging  methods    (CT  scan  or  ultrasound).  

MRD  status  (interna%onal  guidelines*):    <1  CLL  cell  in  10  000  leukocytes  (0.01%  or  10-­‐4)  

PCR-­‐based  techniques  •  Low-­‐sensi*vity:            -­‐  consensus  PCR  (10-­‐3)  :  IGHV-­‐IGHD-­‐IGHJ  rearrangement  amplified  by  PCR  ;  

qualita%ve  approach  

•  High-­‐sensi*vity:            -­‐  clone-­‐specific  PCR  (10-­‐6):  clonal  IGHV-­‐IGHD-­‐IGHJ  gene  rearrangement  sequencing,  

design  of  a  primer  in  the  VH  CDR3  region  ),  “nested”  PCR  aRer  the  first-­‐step  consensus  PCR.  Quan%fica%on  of  MRD  levels  not  allowed  

           -­‐  real-­‐2me  quan2ta2ve  PCR  (RQ-­‐PCR)  (10-­‐4-­‐10-­‐5):  combina%on  of  use  of  clone-­‐

specific  sequences  with  quan%fica%on  of  the  PCR  copy  numbers;  allele-­‐specific  oligonucleo%des)  designed  within  the  VH  CDR3  sequence;  labor  intensive  

           -­‐  high-­‐throughput  approach  (10-­‐5):    no  need  for  pa%ent  customiza%on;  very  

expensive;  significant  bioinforma%cs  support  required  for  interpreta%on  of  the  results  

*Blood.  2008;111(12):5446-­‐5456  

Flow  cytometric  protocols  •  Low-­‐sensi*vity:                      -­‐  2  color  flow  cytometry:  CD19  or  CD20  in  combina%on  with  CD5.  The  

presence  of  more  than  10%  of  CD20CD5  cells/total  lymphocytes  or  more  than  25%  of  CD19CD5  cells/total  CD19  cells  in  the  BM  considered  as  posi%ve  for  residual  disease;    not  specific.  

•  High-­‐sensi*vity:                            -­‐  4-­‐color  or  more  flow  cytometry  (10-­‐4-­‐10-­‐5);  more  an%gens  in  

combina%on  with  CD19  and  CD5):  (A)  CD5/CD19/CD20/CD38;  (B)  CDCD19/CD81/CD22;  and  (C)  CD5/CD19/CD79b/CD43;  either  peripheral  blood  or  BM  regardless  of  the  type  of  therapy;    with  the  notable  excep%on  of  pa%ents  treated  with  Ab-­‐containing  regimens  in  which  BM  aspirate  is  necessary  to  assess  MRD  in  the  first  3  months  aRer  comple%on  of  therapy.    6  colours  (A)  CD19/CD5/CD20/CD3/CD38/CD79b  and  (B)  CD19/CD5/CD20/CD81/CD22/CD43  

MRD  status  (interna%onal  guidelines*):    <1  CLL  cell  in  10  000  leukocytes  (0.01%  or  10-­‐4)  

*Blood.  2008;111(12):5446-­‐5456  

   FLUDARABINE/PREDNISOLONE  

2  colours  Flow  Cytometry  

           OS  and  RESPONSE                                      TTF  and  MRD  

CR  

nCR  

PR  

NR  

MRD  -­‐  

MRD  +  

p<0.001  p<0.001  

2  colours  flow  cytometry  

4  colour  FCM  

Consensus  PCR  ASO  qPCR  

3  colours  FCM  

•  97.4%,  89%,  and  100%  pts  could  be  studied  by  consensus  PCR,  qPCR,  and  flow  cytometry.  

•  164  of  248  samples  were  negaHve  for  MRD  by  consensus  PCR.    

•  Among  those,  CLL  cells  were  detected  by  qPCR  and  by  flow  cytometry  in  77  (47%)  and  39  (23%)  of  the  164  samples,  respecHvely.    

•  All  84  samples  posiHve  on  PCR  had  detectable  CLL  cells  by  qPCR  and  flow  cytometry.  

•  A  good  correlaHon  was  seen  betweenMRD  levels  by  flow  cytometry  and  by  qPCR  (n    254;  r    0.826;  P  <  .001).  

PFS   OS  Autologous  Stem  Cell  Transplanta%on  

CONSENSUS  PCR  

Is  MRD  an  independent  prognos%c  factor?  

•  Role  of  therapy  •  Role  of  biological  prognos%c  factors  

J  Clin  Oncol  30:980-­‐988  

Pa%ents   from   the   two   treatment  arms   who   presented   with   the   same  MRD   levels   had   no   significantly  different  risks  for  disease  progression  

PB  final  restaging  

BM  final  restaging  

PB  follow  up  

PB  interim  staging  

MRD  might  guide  maintenance  

and  consolidaHon  strategies,  thus  making  a  step  forward  toward  

tailored  treatment  strategies.  

BM rest  

PB interim staging PB restaging

PB F-U

2008,  144,  95–98  

postremission  intervenHons  should  be  targeted  

toward  paHents  with  unmutated  IgVH  status  (?)  

NB:  complete  responders  with  negaHve  marrow  flow  cytometry  (B).  

complete  responders  (A)  

complete  responders  with    pos  marrow  flow  cytometry  (A)    

M

M

U

U

The case of Transplantation in CLL……….

RELAPSE INCIDENCE

ASCT  

•  Three  pa)erns  of  minimal  residual  disease  observed:  nega%ve  (31%),  mixed  (24%),  and  always  posi%ve  (45%).    

•  Cumula%ve  incidence  of  relapse  according  to  the  MRD  status  at  6  and  12  months  aRer  transplanta%on  significantly  different  (p=0.031  and  p=0.04,respec%vely).  

•  Two-­‐year  DFS:  93%  and  46%  for  PCR–  and  PCR+,  respec%vely  (p=0.012).    

•  GCHD  more  frequent  in  pa%ents  who  did  not  relapse  (p=0.04).    •  Quan*ta*ve  monitoring  of  MRD  able  to  iden%fy  PCR+  pts  with  higher  

risk  of  relapse.  •  Sugges%on  of  a  minimal  residual  disease-­‐driven  interven%on  to  

prevent  overt  hematologic  relapse.  

ROLE  OF  MRD  IN  CLL…  MRD-­‐oriented  interven%ons:  •  PCR-­‐pts  (very  low  risk  of  disease  recurrence):  slow  

decrease  of  immunosuppressive  therapy  

•  Mixed  PCR  pa)ern  pts:  outcome  similar  to  PCR-­‐  pts;  closer  PCR  monitoring  necessary;  quan%ta%ve  monitoring  advisable  when  posi%ve  results  occur  

•  PCR+  pts    (high  risk  of  overt  relapse):  withdrawal  of  immunosuppressive  therapy  followed  by  therapeu%c;  Quan%ta%ve  PCR  monitoring    strongly  indicated  to  assess  the  %ming  and  type  of  interven%on.    

MRD-­‐nega%ve  status  1  year  aRer  alloSCT:    •  achieved  in  up  to  50%  of  the  pa%ents;  •  predicts  for  long-­‐term  clinical  remission.  •  52  pa%ents  with  MRD  monitoring  had                lower  relapse  risk  without  increase  in                NRM  in  comparison  with  the  38  pa%ents                without  MRD  follow-­‐up  •  MRD  monitoring  triggered  preemp%ve                DLI  in  6  cases,  resul%ng  in  MRD-­‐nega%ve                CR  in  3  of  them.  

WAITING  FOR  ANSWERS…  •  GCLLSG:  (CLLM1)              high  risk  (MRD>10-­‐2  or  >10-­‐4  <10-­‐2)  aRer  first-­‐line  immunochemotherapy  (FC,  FCR,  or  BR)  are  randomized  to  maintenance  therapy  with  lenalidomide  versus  placebo.  MRD-­‐guided  approach  (lenalidomide  increased  if  and  when  MRD  is  s%ll  detectable  at  predefined  %me  points)  

 •  Na%onal  Cancer  Research  Ins%tute  (NCRI)  CLL  subgroup:  (CLARET)    

       pa%ents  responding  to  previous  chemotherapy  with  detectable  MRD  (  >10-­‐4)  randomized  to  receive  either  consolida%on  therapy  with  alemtuzumab  for  6  weeks  or  no  therapy.  

CONCLUSIONS  •  MRD  status  at  the  end  of  treatment  is  one  of  the  most  powerful  predictors  of  PFS  and  OS  

•  Independent  of  the  clinical  response,  the  type  or  line  of  therapy,  and  biological  markers  

•  MRD  kine%cs  evaluated  as  a  real-­‐%me  marker  of  efficacy  and/or  resistance  to  the  administered  therapies  

•  Not  used  outside  clinical  trials  •  MRD  should  be  considered  based  on  the  fitness  status.    

•  Use  of  high-­‐sensi%vity  tests,  (mul%color  flow  cytometry)  cri%cal  

TRAPIANTO ALLOGENICO VANTAGGI

•  Infusione di cellule staminali “non contaminate” •  Possibilità di indurre risposte durature anche in

pazienti refrattari alla fludarabina •  Effetto GVL •  Minore incidenza di recidive, ma non è dimostrato un

vantaggio di sopravvivenza rispetto al trapianto autologo

3.4.  Issue  4:  monitoring  the  response    (consensus-­‐based  recommendaHons)  

Minimal  residual  disease  (MRD)  assessment  performed  by  any  method  on  bone  marrow  is  not  recommended  since  the  eradicaHon  of  MRD  cannot  be  considered  a  therapeuHc  goal  for  all  paHents  

outside  clinical  trials.  

GRAZIE  PER  L’ATTENZIONE  

Grazie per l’attenzione