Traditional Pathology Meets Next-Generation in Acute Myeloid Leukemia …and Challenges our Definition of “Acute” Leukemia !!! PATH 430 MOLECULAR BASIS OF DISEASE MICHAEL RAUH, MD, PHD JANUARY 19, 2015 OBJECTIVES • Provide an overview of acute myeloid leukemia (AML) pathophysiology, current diagnosis, classification, and clinical management • Describe the emerging role of next-generation sequencing in AML and the detection of occult malignancy • Provide a foundation for the discussion of today’s papers: • Shlush et al. (Nature, 2014) • Jaiswal et al. (NEJM, 2014) The Stem Cell Concept Stem Cells: 2-20 cell divisions per year • Capable of self-renewal (although this is a rare event and stem cells are mainly quiescent) Differentiation • Are multipotent (i.e. can give rise to a remarkable number of daughter cells by committing to successive differentiation steps, culminating in terminally-differentiated, mature cells) http://www.biochemj.org/bj/404/0169/bj4040169.htm Hematopoietic Stem Cells • Hematopoietic stem cells (HSC) are found in the bone marrow, cord blood, and in smaller numbers in the peripheral blood • Long-lived cells that give rise to all blood cells • Comprise approx. 1 in 10,000 bone marrow cells • It is estimated that approx. 1,000 to 10,000 HSC contribute to the production of 1011 – 1012 new blood cells throughout the body each day Hematopoiesis • The production of mature blood cells by HSC • In adults, primarily occurs in the bone marrow http://en.wikipedia.org/wiki/Haematopoiesis http://www.allthingsstemcell. com/wpcontent/uploads/2009/02/he matopoiesis_simple1.png Hematopoiesis Myeloid Cells Lymphoid Cells Our Stem Cells Accrue Damage http://www.hematology.org/Publications/Hematologist/2013/9947.aspx Number of mutations per HSC HSC mutations increase with age Increasing age of human subjects HSC mutations increase with age • Like other cells in our body, HSC have a fidelity rate of about 0.78 × 10−9 mutations per genomic base pair per cell division • Therefore, mutations randomly appear at a rate of about 0.13 coding mutations per year of life (i.e. approx. one mutation every 7-8 years) • Mutations accumulate with age, and generally do not impact HSC function (i.e. they do not normally cause AML) • However, in some people, will these mutations occur in genes that predispose to leukemia? Classification of myeloid disorders (Blast) Bone Marrow Failure TET2, ASXL1 Blood Cytopenia(s) JAK2 Mature cells Dysplasia Blasts AML transformation Mutations JAK2, MPL BCR/ABL, CBL Myeloproliferative Neoplasms Myelodysplastic Syndromes Acute Myeloid Leukemia MPN MDS AML ↑ ↓ ↓ rare common sometimes Norm (<5%) <5% or 5-19% ≥20% rare common n/a TK pathways self-renewal, epigen Two hits Corey et al. Nature Reviews Cancer 7, 118–129 Classification of myeloid disorders Core binding factors, PML-RARA, NPM1, CEBPA FLT3, RAS Mature cells Dysplasia Blasts AML transformation Mutations MPN MDS AML ↑ ↓ ↓ rare common sometimes Norm (<5%) <5% or 5-19% ≥20% rare common n/a TK pathways self-renewal, epigen Two hits Corey et al. Nature Reviews Cancer 7, 118–129 AML diagnosis: bone marrow studies BM Aspirate: BM Biopsy: •Morphology •Immunohistochemistry AML: morphologic features Granulopoiesis Myeloblast with Auer Rod AML diagnosis requires ≥ 20% blasts in blood or bone marrow http://www.tau.ac.il/~inter05/g-all.gif AML: French-American-British(FAB) Classification M0: with minimal differentiation M1: without maturation M2: with maturation M3: promyelocytic M4: myelomonocytic M5: monoblastic /monocytic M6: erythroid M7: megakaryoblastic AML: flow cytometric analysis Blasts: express CD45 at dim levels on their surface AML: flow cytometric analysis • CD34 is a blast marker, but can be expressed by both lymphoid & myeloid blasts • Myeloid blasts express other myeloid markers (i.e. CD13, 33, 117), and this helps to assign their “lineage” and make the diagnosis of AML AML: G-band Karyotyping AML: recurring chromosomal translocations http://www.asco.org/ AML: Fluorescent in situ Hybridization (“FISH”) HOW DO THESE TRANSLOCATIONS CAUSE AML? Core binding factor translocations impair cellular differentiaton (i.e. maturation) Normal Progenitor Cell Maturation Programs Activated AML/RUNX1 RUNX1T1 t(8;21) Maturation Arrest inv(16) Maturation Arrest MYH11 http://www.elsevierimages .com/image/28065.htm The t(15;17) translocation also impairs cellular differentiation (i.e. maturation) Maturation Arrest: ‘M3’ Acute Promyelocytic Leukemia (APL) http://www.bioscience.org/2009/v14/af/3333 APL: using ATRA to induce blast differentiation ARE THERE ANY OTHER SUCCESSFUL TARGETED AML THERAPIES? No! (not yet…) Standard 3+7 AML “Induction” Chemotherapy An anthracycline, Daunorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription. 3 days, IV • Kills dividing cells – not particularly targeted! • After induction, if <5% blasts, considered in morphological remission. Cytosine arabinoside (Ara-C) is similar enough to human cytosine deoxyribose (deoxycytidine) to be incorporated into human DNA, but different enough that it kills the cell. MORPHOLOGY, IMMUNOPHENOTYPING, CHROMOSOMAL ANALYSIS… PUTTING IT ALL TOGETHER TO ARRIVE AT A DIAGNOSIS… AML: Current (2008) Classification WHO Acute myeloid leukemia and related neoplasms: Acute myeloid leukemia with recurrent genetic abnormalities AML with t(8;21)(q22;q22); RUNX1-RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11 APL with t(15;17)(q22;q12); PML-RARA M3 AML with t(9;11)(p22;q23); MLLT3-MLL AML with t(6;9)(p23;q34); DEK-NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1 Provisional entity: AML with mutated NPM1 Provisional entity: AML with mutated CEBPA Only 2 gene mutations! Acute myeloid leukemia with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukemia, not otherwise specified AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Acute erythroid leukemia Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Old FAB: M0 M1 M2 M4 M5 M6 M7 AML: cytogenetic risk stratification “CBF” & “PML-RARA” The problem: Traditional diagnostics and treatments are reaching their limitations Where can we turn for novel insights and approaches? AML: tradition meets next-generation Success story: Higher-throughput sequencing technologies make somatic mutation profiling more feasible enhancing diagnostic and prognostic yield • Next generation genomic sequencing • Couples pH changes during DNA synthesis to sequence data • In-house at Queen’s University Ion Torrent next-generation sequencing pH sensors below the sample wells record digital sequences Ion Torrent next-generation sequencing Bioinformatics programs align the short sequences to a reference genome and ‘variants’ are called Types of DNA Mutations (4 “Tiers”) Tier 1 (coding exons) comprise only 1.3% of the genome • Mutations in Tier 1 (coding exons) are likely very important • However, little is currently know of the function of other genomic tiers www.genome.gov/Multimedia/Slides/.../04_Wilson_Fitting.pdf The New Genetic Model of AML Blue = cooperativity Red = exclusivity Moving Towards Revised Diagnostic Categories And targeted therapeutics SUMMARY • Currently, AML is diagnosed using blast counts, immunophenotyping, chromosomal analysis, and (rarely) mutations • Apart from ATRA in t(15;17) AML, treatment is mainly onesize-fits all • Gene mutation profiling is helping to refine diagnostic risk categories and to guide rational and targeted therapeutics • Paper 1: Mutation profiling unexpectedly reveals evidence of a pre-leukemic state • Paper 2: How common is this pre-leukemic state and what are the implications? AML: Darwinian evolution of leukemia through sequential HSC mutations THANK YOU! QUESTIONS?