1. Minimal Residual Disease in Hematologic Neoplasms Lloyd M. Stoolman, M.D. Professor of Pathology and Director, Clinical and Research Flow Cytometry Laboratories Department of Pathology, University of Michigan

2. What is meant by “minimal residual disease” in acute leukemia  Undetected by morphologic means but revealed by more sensitive modalities  What are standard criteria for remission ? –No clusters/sheets of blasts in core –No detectable “neoplastic” blasts in aspirate –<5% blasts on 2-500 cell differential count

3. Methods used for detection MRD  Flow cytometry  Cytogenetics  PCR-based techniques

4. Flow cytometry  Strengths –>90% of ALL, 30-80% of AML cases –Strong correlation with relapse  When clone frequency in 0.01-5% range  Sensitivity can be extended (depends solely on number of cells analyzed) –Shortest TAT, available  Limitations –Subjectivity of phenotypic criteria for neoplasia –Phenotypic instability, overlap with normals

5. Cytogenetics (including FISH)  Strengths –Most specific if abnormalities present –Culture step selects for viable tumor  Limitations –Requires large structural abnormalities –Less sensitive than other modalities –Longest TAT

6. PCR-based techniques  Strengths –Most sensitive (0.0001%), highly specific –Readily automated, less subjective –Short TAT  Limitations –Require large panel of disease-specific primers/probes with <50% coverage for AML/ALL –ALL coverage can be extended but requires synthesis of patient-specific primers –Transcript number vs. leukemia cell frequency

7. Uses supported by clinical trials  Chemotherapy recipients –Duration and intensity of induction –Timing HSCT (if donor) –Remission surveillance  Bone marrow transplant recipients –When to transplant –Intensity of conditioning –Guide for immuno tx post transplant

8. MRD in B-ALL: Neoplasm or hematogones * mean fl. Intensity (sd)CD10CD19CD38 HEMATOGONES (N=8)38 (12)*12.9 (4.9)16 (4.5) 1.Low side-scatter (at or below mature lymphs) 2.Consistent antigen progression as cells mature: 45 , 34 , 38 , Tdt , 10 , 19 , 20  3.No surface Ig (BCR), myeloid or T-antigens 4.Maintain 10-19-38 density in narrow range until mature

9. Hematogone Ag progression/intensity

10. >90% ALL show aberrancies: Ag density, non-lineal Ag expression * mean fl. Intensity (sd)CD10CD19CD38 HEMATOGONES (N=8)38 (12)*12.9 (4.9)16 (4.5) CASE153446

11. ALL event-free survival vs. MRD (FC) End induction, BM Multicenter (COG) 2000-2005 All FC at Hopkins 4-color analysis 20-10-45-1919-34-45-19 ≥500K events M.J. Borowitz et. al. Blood. 2008: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study

12. ALL event-free survival vs. MRD (FC) Day-8, Blood 4-color analysis CD 20-10-45-19 CD 9-34-45-19 ≥500K events M.J. Borowitz et. al. Blood. 2008: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study

13. ALL event-free survival vs. MRD (FC) End consolidation, BM 4-color analysis CD 20-10-45-19 CD 9-34-45-19 ≥500K events M.J. Borowitz et. al. Blood. 2008: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study

14. MRD in AML: LAPs or LAIPs Most recognizable aberrancies: Lineage infidelity, Ag under/over Most frequent aberrancies: Asynchronous antigen expression

15. MRD directed tx in AML Compared low-dose and high dose protocols in Induction 1 MRD at end Induction 1 determined timing of Induction 2 <1% MRD then counts allowed to recover >1% 1% <25% then 2 nd phase begun immediately >25% then high risk, early HSCT MRD at end Induction 2 determined subsequent tx decision >1% MRD after three cycles then HSCT Rubnitz et. Al. Lancet Oncol. 2010: Minimal residual disease- directed therapy for childhood acute myeloid leukemia: results of the AML02 multicenter trial

16. AML event-free survival vs. MRD (FC) End induction (d22), BM 3-year event free 63% and overall survival 71% Superior to prior trials and approaching ALL MRD-directed timing for induction 2 considered major factor in success Rubnitz et. Al. Lancet Oncol. 2010: Minimal residual disease-directed therapy for shildhood acute myeloid leukemia: results of the AML02 multicenter trial 0 1 2 3 4 5 6 7 Years after enrollment Cumulative incidence of relapse or induction failure (%) 0 20 40 60 80 100

17. MRD and BMT  Retrospective analysis of cumulative survival based on tumor burden (e.g. MRD ) from chimerism detection  Chimerism generally considered less sensitive that FC/PCR detection of MRD and less specific since also positive when graft fails M.A. Pulsipher et. al. Biol. Blood Marrow Transplant. 2009: Allogeneic Transplantation for Pediatric ALL: The Emerging role of Peritransplantation Minimal Residual Disease/Chimerism Monitoring…..

18. Relapse detection method and survival after donor lymphocyte infusion in SCT  Retrospective analysis 118 patients with hematologic malignancies  Used lineage-specific chimerism (reported sensitivity of 0.02-0.04%) or quantitative PCR for CML patients (reported sensitivity of ~0.0001%)  Compared survival of patient who received DLI after (1) positive cytogenetics (CML only), (2) positive chimerism or PCR or (3) positive morphologic relapse D. Sairafi et. al. Biol. Blood Marrow Transplant. 2010: Leukemia Lineage- Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

19. MRD at time SCT and survival  Prospective analysis 13 ALL patients with MRD by FC at time of SCT  Used flow cytometry (reported sensitivity of 0.01%) to assess MRD  All patients in morphologic remission at time of SCT  Compared survivals of patient with MRD <0.01% (MRD negative) to those with MRD 0.01-3.3% within 10d SCT I. Elorza et. al. Haematologica 2010: Relationship between minimal residual disease measured by multiparametric flow cytometry prior to allogeneic hematopoietic stem cell transplantation and outcome in children with acute lymphoblastic leukemia.

20. Conclusions  A “morphologic” remission in acute leukemia is no longer the best therapeutic endpoint  Minimal residual disease evaluation by one or more methods is desirable  Multiparameter flow cytometry is a rapid, sensitive and specific method in ALL and many AML cases when performed on “1 st pull” aspirates by an experienced laboratory  Cytogenetics and PCR-based methods, where applicable, are of equal or greater sensitivity

21. References  References for figures – –M.J. Borowitz et. al. Blood. 2008. 111, 5477-5485: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study – –Rubnitz et. Al. Lancet Oncol. 2010. 11, 543-552: Minimal residual disease-directed therapy for childhood acute myeloid leukemia: results of the AML02 multicenter trial – –M.A. Pulsipher et. al. Biol. Blood Marrow Transplant. 2009. 15, 62-71: Allogeneic Transplantation for Pediatric ALL: The Emerging role of Peritransplantation Minimal Residual Disease/Chimerism Monitoring….. – –D. Sairafi et. al. Biol. Blood Marrow Transplant. 2010 (in press, available online): Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions –. –I. Elorza et. al. Haematologica 2010. 95, 936-941: Relationship between minimal residual disease measured by multiparametric flow cytometry prior to allogeneic hematopoietic stem cell transplantation and outcome in children with acute lymphoblastic leukemia.  Reviews – –D. Campana. Seminars Hematol. 2009. 46(1), 100-106: Minimal Residual disease in Acute Lymphoblastic Leukemia – –D. Shook et. al. Clinical Lymphoma, Myeloma and Leukemia Supplement. Sept. 2009, S281-S285. Minimal Residual Disease Quantitation in Acute Myeloid Leukemia. – –A. Al-Mawali, D. Gillis and I. Lewis. Am. J. Clin. Pathol. 2009. 131, 16-26: The Role of Multiparameter Flow Cytometry for Detection of Minimal Residual Disease in Acute Myeloid Leukemia – –M.C. Bene and J.S. Kaeda. Haematologica. 2009. 94(8), 1135-1150: how and why minimal residual disease studies are necessary in leukemia: a review from WP10 and WP12 of the European LeukemiaNet