Is monitoring of chimerism predicting relapse and allowing preemptive immunotherapy? Fabio Ciceri, MD San Raffaele Scientific Institute Milano, Italy

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1 Is monitoring of chimerism predicting relapse and allowing preemptive immunotherapy? Fabio Ciceri, MD San Raffaele Scientific Institute Milano, Italy

2 Fabio Ciceri, MD San Raffaele Scientific Institute, Milano. Italy Is monitoring of chimerism predicting relapse and allowing preemptive immunotherapy? Disclosure of Interest: Nothing to Disclose

3 Background Leukemia relapse after allogeneic HSCT represents a major clinical issue Early detection of Minimal Residual Disease (MRD) may lead to preemptive therapeutic interventions, improving HSCT outcome Hematopoietic chimerism may represent a valid surrogate MRD marker for post-transplantation monitoring of many patients without disease-specific MRD markers Recently developed chimerism assays based on quantitative PCR grant improved sensitivity in detecting low percentages of host cells

4 qchim validation study Host chimerism, qpcr Marker 2 (%) Study Cohort Chimerism 403 BM samples 115 pts, 116 transplants Disease: AML 58 HD 13 ALL 12 MDS 12 MM 5 NHL 4 other 11 Donor: MRD 19 MUD 33 UCB 2 MMRD 62 Timing of analysis: median 91 days ( ) All samples analyzed in parallel for: Morphological Evaluation Short Tandem Repeat PCR chimerism (AmpFISTR kit, Applied Biosystems) qpcr chimerism, 2 markers/sample (Allele Seq TM, Celera Genomics) Host chimerism, qpcr Marker 1 (%)

5 qpcr-str Chimerism Comparison in Bone Marrow Analyzed Samples (%) qpcr > 0% > 0.5% > 1% > 2% STR 80.1% pos 32.5% pos 22.6% pos 11.9% pos 20.0% pos qpcr and STR positive qpcr positive and STR negative qpcr negative and STR positive qpcr and STR negative 0 >0 >0.5 >1 >2 qpcr Host Chimerism Threshold (%)

6 Host Chimerism (%) qpcr Chimerism and Time after HSCT Host Chimerism (%) Days after HSCT Days after HSCT Slow clearance of residual host hematopoiesis? Early relapses? Graft versus Tumor effect? Enrichment in proficient donor hematopoiesis vs stroma?

7 qpcr Chimerism and Relapse Prediction Host Chimerism (%) 297 samples, 90 pts Disease: AML 58 ALL 12 MDS 12 CML 3 CMML 3 MFI 2 9 pts relapsed (24 samples) 79 did not relapse (273 samples) > 0 > 0.5 > 1 > 2 Relapse No relapse 20 (83%) 9 (37%) 7 (29%) 4 (17%) specificity sensitivity 213 (78%) 75 (27%) 45 (16%) 20 (7%)

8 qpcr Chimerism and Relapse Prediction In 4/5 of the patients with two or more time-points of qpcr 10 analysis before relapse Q-PCR we could detect Increasing Mixed 1 Chimerism (IMC) 0.8 before overt hematologic recurrence WT1/ABL*10 HC % 4 HC % HC % HC % BL* STR-PCR Q-PCR HLA-PCR STR-PCR SSO HLA-PCR WT1 SSO WT Months after HSCT

9 Mixed Chimerism Patient Donor HLA Chimerism Assays Short Tandem Repeats (STRs) HLA typing Patient Donor Patient Mixed Chimerism Donor Highly polymorphic lengths On different chromosomes Detects donor-recipient HLA mismatches Focused on the MHC region Mazzi et al., Leukemia, 2008

10 Molecular Mechanism and Functional Consequences of Leukemia HLA Loss T cell specific proliferation Acquired Uniparental Disomy Vago et al., New Engl J Med, 2009

11 Leukemia Immune Evasion by Genomic HLA loss Further Evidences Isoda, PNAS, 2009 Villalobos, Blood, 2010 Waterhouse, BBMT, 2011 Katagiri, Blood, 2011 Stölzel, Transplantation, 2012 Toffalori, Blood, 2012

12 Patient and Transplantation Characteristics 230 Transplants at OSR ( ) High-risk Myeloid Malignancies: 26% Adverse Cytogenetic Risk 22% Hyperleucocytosis 52% Dysplasia 33% Chemorefractory or PIF 56% Active Disease or Up Front 1 HLA Mismatch in GvL Direction Evaluable for Engraftment T cell Infusion Follow-up: Standard Bone Marrow Evaluation comprising HLA Typing 12% 23% 23% Disease Diagnosis 4% 2% 1% 3% AML saml MDS MPD CML 55% BAL CMML Type of Transplant 3% MMRD MUD CBU 74%

13 Incidence of HLA Loss 21 cases of HLA Loss/83 relapses 25% of relapses Disease: AML (=19), MDS (=1) and MFI (=1) None after MUD HSCT, all after MMRD (29%) Risk factor analysis performed in the MMRD HSCT subset Median Time to Relapse HLA loss vs Classical 307 vs 86 Days, range vs (p< )

14 1. Disease-related Factors Disease Cytogenetic Profile Molecular Profile Hyperleucocytosis Dysplasia Chemorefractory Disease History Previous Relapses (81% vs 63%) Previous allohsct (43% vs 29%) Active disease at HSCT (76% vs 67%) No significant correlations with HLA loss

15 2. Transplant-related Factors Conditioning Regimen Median Stem Cell Dose of Graft Donor Lymphocite Infusions (DLI) GvHD Prophylaxis Median T cell Dose of Graft No correlations with HLA loss T cell repleted graft (p=0.01) Acute GvHD (agvhd) Chronic GvHD (cgvhd)

16 3. Immunogenetic Factors Sex Mismatch HLA-DPB1 Matching Status Parental Antigen Mismatches NK Alloreactivity Mismatched HLA Loci No significant correlations with HLA loss HLA A*03 [Chi 2 =3.16; p=0.07] HLA C*04 [Chi 2 =8.07; p=0.04] MMRD transplants Total (n=164) No Relapse (n=98) Classical Relapse (n=45) HLA loss Relapse (n=21) P value HLA-C *04 in mismatched haplotype, n (%) HLA-A *03 in mismatched haplotype, n (%) 16 (9.8%) 10 (10.2%) 1 (2.2%) 5 (23.8%) (14.6%) 8 (8.2%) 16 (35.5%) 3 (14.3%) 0.075

17 Summary Unmanipulated T cell-replete graft acute GvHD chronic GvHD Long Equilibrium HLA-C*04?

18 Current Diagnosis of HLA Loss Relapse Outside HLA Inside HLA Morphology Phenotype STR chimerism SSO-PCR HLA-A,B,C,DR typing D C D D CCC C D P P PP Presence of leukemic cells of patient origin, typing negative for patient-specific HLA Minimum Blast percentage 5-10%, analysis of FACS-purified blasts preferrable Diagnosis possible only in the presence of morphologically overt disease

19 Development of a new qpcr-based Assay Outside HLA Celera Allele Seq TM Chimerism Assay Inside HLA Sponsored R&D Project insertion deletion 34 indels 19 chromosomes SNPs SNPs HLA-A,B,C,DRB1, DQB1, DPB1 Low Percentage of HLA-loss blasts (0.2%) detected in a single assay Identification of informative allele groups in 4 ethnic groups and validation in 165 transplants performed in the OSR BMT Unit. Generic Forward Primer Probe SNP1 Specific Reverse Primer SNP2 Primers and probes specific for HLA allelic polymophysms (SNPs), to grant optimal specificity

20 Development of a new qpcr-based Assay Outside HLA Celera Allele Seq TM Chimerism Assay Inside HLA Sponsored R&D Project insertion deletion 34 indels 19 chromosomes SNPs SNPs HLA-A,B,C,DRB1, DQB1, DPB1 Low Percentage of HLA-loss blasts (0.2%) detected in a single assay Identification of informative allele groups in 4 ethnic groups and validation in 165 transplants performed in the OSR BMT Unit. Generic Forward Primer Probe SNP1 Specific Reverse Primer SNP2 Primers and probes specific for HLA allelic polymophysms (SNPs), to grant optimal specificity

21 Development of a new qpcr-based Assay UPN#1 Outside HLA UPN#2 CA025 Pre-transplant CA022 Pre-transplant days days Host: 17.6% Host: 79.9% HLA-A*11 Inside HLA HLA-A*68 Pre-transplant +245 days Pre-transplant +180 days Host: 17.1% Host: 0.08% Classical Relapse HLA Loss Relapse

22 Clinical Utility of Inside HLA qpcr Chimerism "Inside HLA" host chimerism (%) Classical Relapse 5 0 HLA Loss Relapse "Outside HLA" host chimerism (%)

23 Conclusions qpcr-based post-transplantation chimerism evaluation is a promising tool for relapse prediction, comparing at higher sensitivity with standard techniques and thus allowing earlier therapeutic intervention Intra-patient longitudinal evaluation and assessment in peripheral blood may improve the specificity of the assay Newly developed inside HLA reactions allow the early differential diagnosis of classical and HLA loss relapses, paving the way for targeted pre-emptive interventions

24 Acknowledgements Benedetta Mazzi Luca Vago Lorenza Chiesa Cinzia Pultrone Eleonora Toffoletti Veronica Tettamanzi Chiara Brambati Cristina Toffalori Pietro Crivello Katharina Fleischhauer Unit of Molecular and Functional Immunogenetics Monica Zanussi Laboraf Molecular Biology Raffaella Greco Maria Teresa Lupo Stanghellini Lara Crucitti Consuelo Corti Massimo Bernardi Jacopo Peccatori Chiara Bonini Hematology and BMT Unit Doug Bost CELERA