1. Qualitative and quantitative PCR monitoring of minimal residual disease (MRD) in relapsed poor-risk chronic lymphocytic leukemia (CLL): early assessment can predict long-term outcome after reduced intensity allogeneic transplantation.
Farina L, Carniti C, Dodero A, Vendramin A, Raganato A, Patriarca F, Narni F, Spina F, Benedetti F, Olivieri A, Corradini P.

2. BACKGROUND
The median overall survival of CLL patients is about 10 years but :
patients younger than 65 years with high risk disease (17p deletion)
and
poor risk relapsed patients
may become eligible to a stem cell transplantation (SCT)

3. post-trasplant molecular
MRD status influences the clinical outcome after auto-SCT in CLL and indolent lymphomas
PCR -
PCR -
Mixed-PCR
PCR +
PCR +
DFS according to the
PCR status of
stem cell harvests
DFS according to
post-trasplant molecular
status
Corradini P et al, JCO 2004

4. Outcome according to MRD status at 3-6 months after auto-SCT in CLL
Progression risk
Overall Survival
Consensus PCR
PCR+
PCR-
PCR-
PCR+
Moreno C et al, Blood 2006

5. Clinical and molecular outcome according to the VDJ mutational status after auto-SCT
Molecular event-free survival
Progression-free survival
Mutated pts
median time not reached
Mutated pts
Unmutated pts
median time: 25 months
Unmutated pts p p
Experiments were done using consensus PCR
Ritgen M et al, Blood 2003

6. Rationale for reduced intensity (RIC) allo-SCT in CLL
Allogeneic stem cells are free of tumor contamination
“Graft-versus-Leukemia effect”: donor derived immune cells are potentially capable of mediating an anti-tumor effect, either specifically or as part of an alloreactive phenomenon
RIC alloSCT can be a suitable option for CLL patients who are usually old, heavely pretreated and/or affected by comorbidities

7. AIMS OF THE STUDY
Can we achieve molecular remission (MR) after RIC allo-SCT?
What is the clinical relevance of MRD results?
Are there any clinical or biological factors that can be correlated with the MRD status?

8. PATIENT CHARACTERISTICS (1)
Molecular monitoring of MRD was carried out by PCR on BM samples of 29 relapsed CLL patients achieving complete remission after RIC alloSCT.
Donor type
n (%)
Conditioning regimen and GVHD prophylaxis
HLA identical sibling
21 (72)
Thio/Flu/Cy
MTX/CSA
Unrelated
6 (21)
Thio/Cy/ATG
Haploidentical sibling
2 (7)
Thio/Flu/Cy/TBI200 cGy/Alemtuzumab

9. PATIENT CHARACTERISTICS (2)
n° patients 29
Median age (years) 60
range: 44-69
Unmutated VDJ
22/29
76%
17p deletion
6/16
38%
Median n° previous therapy 3
range: 1-6
Previous autoSCT 8
29%
Chemorefractory 11

10. MRD MONITORING
Qualitative monitoring
n=29 L V D J L
FR1
CDR1
FR2
CDR2
FR3
CDR3
FR4
1st PCR
Probe JH
VH primers
2nd PCR
Patient-specific primers
Quantitative monitoring
n=6
ΔΔCT= (CTIgH-CTGAPDH )follow-up– (CTIgH-CTGAPDH) pre-transplant

11. RESULTS (1): MR can be achieved after RIC allo-SCT%
Status
at last
follow-up
Median follow-up
(range, months)
PCR-neg 9
31*
9 alive and CR 40
(14-97)
PCR-mixed 7 24
1 nodal relapse
1 TRM
5 alive and CR 46
(38-77)
PCR-pos 13 45
8 relapse**
2 TRM
3 alive and CR 49
(33-57)
*4 patients experienced a delayed clearance of MRD within the first year after transplant
**Relapse occurred after a median time of 9 months (range, 3-26)

12. RESULTS (2): clinical impact of MRD status after RIC allo-SCT
PCR+
n=13
PCR-/mixed
n= 16 p
Relapse after alloSCT
8 (62%)
1 (6%)
0.003
After RIC allo-SCT
PCR positivity correlates
with a high relapse risk

13. RESULTS (2): clinical impact of MRD status after RIC alloSCT
Cumulative incidence of relapse
based on PCR status at 6 mos
DFS based on PCR
status at 6 mos
p = 0.031
p = 0.012
2-yr DFS 93%
MRD-
--- MRD+
2-yr DFS 46%

14. RESULTS (3): no clinical or biological factors were predictive for the achievement of MR
PCR+
n=13
PCR-/mixed
n= 16 p
Previous treatment
(≤ 2 vs > 2)
9 (69%)
8 (50%)
0.451
Chemorefractory
4 (31%)
7 (44%)
0.702
Donor type
(HLA identical sib vs MUD/mismatched)
6 (46%)
3 (19%)
0.225
GVHD
12 (75%)
0.142
….but the incidence of GVHD was 22% and 70%
in relapsed and not relapsed patients, respectively (p= 0.040).

15. RESULTS (4): the GVL effect may prevent clinical relapse in PCR –positive patients
Quantitative monitoring in PCR-pos. patients n=6
Decreasing or stable tumor load
with extensive cGVHD
n=4
Increasing tumor load
without GVHD
n=2 CR
Relapse

16. Qualitative MRD monitoring: PCR pos.
UPN 16
UPN 17
UPN 28
UPN 29
UPN 179
UPN 1u
UPN 4u
UPN 1a
UPN 2a
UPN 33
UPN 123
UPN 7u
UPN 111
Rel
Rel
Rel
Death
Rel
Death
PCR +
PCR -
DLI
CT or mAb
Relapse
acute and chronic GVHD
Rel
Death
Rel
Rel
Rel
17pdel
TRM
17pdel
TRM
Rel
17pdel
UPN # identical sibling
UPN #u unrelated
UPN #a haploidentical
Months

17. Qualitative MRD monitoring: PCRneg/mixed
UPN 9
UPN 26
UPN 31
UPN 142
UPN 178
UPN 6
UPN 2u
UPN 3u
UPN 6u
UPN 11
UPN 27
UPN 36
UPN 37
UPN 30
UPN 34
UPN 50
Qualitative MRD monitoring: PCRneg/mixed
17pdel
17pdel
Rel
Death
17pdel
TRM
Months

18. CONCLUSIONS
In poor risk relapsed CLL, molecular remission can be achieved after RIC allo-SCT.
Persistent PCR positivity correlates with a high incidence of relapse, while a mixed-PCR pattern can be observed without clinical relapse.
The postulated GVL effect may contribute to prevent clinical relapse in patients with detectable MRD, including chemorefractory and 17p deletion positive patients.
Qualitative and quantitative PCR patterns can suggest the timing for early immune therapy intervention.

19. Acknowledgments Carniti C. Raganato A. Dodero A. Vendramin A. Spina F.
Corradini P.
Hematology dept.
Istituto Nazionale Tumori
University of Milano
Patriarca F.
University of Udine
Benedetti F.
University of Verona
Narni F.
Hematology dept.
University of Modena
Olivieri A.
AOS San Carlo, Potenza