Qualitative and quantitative PCR monitoring of minimal residual

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Qualitative and quantitative PCR monitoring of minimal residual disease (MRD) in relapsed poor-risk chronic

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.

BACKGROUND The median overall survival of CLL patients is about 10 years but :

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

MRD status influences the clinical outcome after auto-SCT in CLL and indolent lymphomas PCR

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

Outcome according to MRD status at 3 -6 months after auto-SCT in CLL Progression

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

Clinical and molecular outcome according to the VDJ mutational status after auto-SCT Molecular event-free

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 Unmutated pts median time: 25 months p 0. 0006 p 0. 0008 Experiments were done using consensus PCR Ritgen M et al, Blood 2003

Rationale for reduced intensity (RIC) allo-SCT in CLL • Allogeneic stem cells are free

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 allo. SCT can be a suitable option for CLL patients who are usually old, heavely pretreated and/or affected by comorbidities

AIMS OF THE STUDY ØCan we achieve molecular remission (MR) after RIC allo-SCT? ØWhat

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

PATIENT CHARACTERISTICS (1) Molecular monitoring of MRD was carried out by PCR on BM

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 allo. SCT. Donor type n (%) Conditioning regimen and GVHD prophylaxis HLA identical sibling 21 (72) Thio/Flu/Cy MTX/CSA Unrelated 6 (21) Thio/Cy/ATG MTX/CSA Haploidentical sibling 2 (7) Thio/Flu/Cy/TBI 200 c. Gy/Alemtuzumab

PATIENT CHARACTERISTICS (2) n° patients 29 Median age (years) 60 range: 44 -69 Unmutated

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

MRD MONITORING Qualitative monitoring n=29 L L 1 st PCR FR 1 VH primers

MRD MONITORING Qualitative monitoring n=29 L L 1 st PCR FR 1 VH primers Quantitative monitoring CDR 1 V D FR 2 CDR 2 J FR 3 CDR 3 Probe 2 nd PCR FR 4 JH Patient-specific primers n=6 ΔΔCT= (CTIg. H-CTGAPDH )follow-up– (CTIg. H-CTGAPDH) pre-transplant

RESULTS (1): MR can be achieved after RIC allo-SCT n % Status at last

RESULTS (1): MR can be achieved after RIC allo-SCT n % 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)

RESULTS (2): clinical impact of MRD status after RIC allo-SCT PCR+ n=13 Relapse after

RESULTS (2): clinical impact of MRD status after RIC allo-SCT PCR+ n=13 Relapse after 8 (62%) allo. SCT PCR/mixed n= 16 1 (6%) After RIC allo-SCT PCR positivity correlates with a high relapse risk p 0. 003

RESULTS (2): clinical impact of MRD status after RIC allo. SCT Cumulative incidence of

RESULTS (2): clinical impact of MRD status after RIC allo. SCT Cumulative incidence of relapse based on PCR status at 6 mos p = 0. 031 MRD--- MRD+ DFS based on PCR status at 6 mos 2 -yr DFS 93% p = 0. 012 2 -yr DFS 46%

RESULTS (3): no clinical or biological factors were predictive for the achievement of MR

RESULTS (3): no clinical or biological factors were predictive for the achievement of MR Previous treatment PCR+ n=13 9 (69%) PCR-/mixed n= 16 8 (50%) p 0. 451 4 (31%) 6 (46%) 7 (44%) 3 (19%) 0. 702 0. 225 6 (46%) 12 (75%) 0. 142 (≤ 2 vs > 2) Chemorefractory Donor type (HLA identical sib vs MUD/mismatched) GVHD …. but the incidence of GVHD was 22% and 70% in relapsed and not relapsed patients, respectively (p= 0. 040).

RESULTS (4): the GVL effect may prevent clinical relapse in PCR – positive patients

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 c. GVHD n=4 CR Increasing tumor load without GVHD n=2 Relapse

UPN 16 Rel UPN 17 Rel UPN 28 Qualitative MRD monitoring: PCR pos. Death

UPN 16 Rel UPN 17 Rel UPN 28 Qualitative MRD monitoring: PCR pos. Death UPN 29 Rel UPN 179 Death PCR + Death PCR DLI Rel UPN 1 u CT or m. Ab Rel UPN 4 u 17 pdel UPN 2 a Relapse Rel TRM 17 pdel UPN 1 a acute and chronic GVHD TRM UPN 33 Rel UPN 123 UPN # identical sibling UPN #u unrelated UPN #a haploidentical 17 pdel UPN 7 u UPN 111 Months 1 2 3 6 9 12 15 18 21 24 27 30 40 50 60 70

UPN 9 UPN 26 Qualitative MRD monitoring: PCRneg/mixed UPN 31 UPN 142 17 pdel

UPN 9 UPN 26 Qualitative MRD monitoring: PCRneg/mixed UPN 31 UPN 142 17 pdel UPN 178 UPN 6 UPN 2 u UPN 3 u UPN 6 u UPN 11 UPN 27 Rel 17 pdel UPN 36 Death TRM 17 pdel UPN 37 UPN 30 UPN 34 UPN 50 Months 1 2 3 6 9 12 15 18 21 24 27 30 40 50 60 70

CONCLUSIONS Ø In poor risk relapsed CLL, molecular remission can be achieved after RIC

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

Acknowledgments Carniti C. Raganato A. Dodero A. Vendramin A. Spina F. Corradini P. Hematology

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