minimal residual disease in hematologic neoplasms lloyd m. stoolman, m.d. professor of pathology and...

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

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

Methods used for detection MRD

Flow cytometry Cytogenetics PCR-based techniques

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

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

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

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

MRD in B-ALL: Neoplasm or hematogones

* mean fl. Intensity (sd) CD10 CD19 CD38

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

Hematogone Ag progression/intensity

>90% ALL show aberrancies: Ag density, non-lineal Ag expression

* mean fl. Intensity (sd) CD10 CD19 CD38

HEMATOGONES (N=8) 38 (12)* 12.9 (4.9) 16 (4.5)

CASE 153 44 6

ALL event-free survival vs. MRD (FC)

End induction, BM

Multicenter (COG)2000-2005

All FC at Hopkins

4-color analysis20-10-45-19

19-34-45-19

≥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


Day-8, Blood

4-color analysis CD 20-10-45-19

CD 9-34-45-19

≥500K events



End consolidation, BM

4-color analysis CD 20-10-45-19

CD 9-34-45-19

≥500K events


MRD in AML: LAPs or LAIPs

Most recognizable aberrancies: Lineage infidelity, Ag under/overMost frequent aberrancies: Asynchronous antigen expression

MRD directed tx in AML

Compared low-dose and high dose protocols in Induction 1MRD at end Induction 1 determined timing of Induction 2

<1% MRD then counts allowed to recover>1% <25% then 2nd 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

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 7Years after enrollment

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MRD and BMT

Retrospective analysis of cumulative survival based on tumor burden (e.g. MRD <0.1%>) 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…..

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

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.

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 “1st pull” aspirates by an experienced laboratory

Cytogenetics and PCR-based methods, where applicable, are of equal or greater sensitivity

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

minimal residual disease in hematologic neoplasms lloyd m. stoolman, m.d. professor of pathology and...

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