1. Acute Myeloid Leukemia Diagnosis in the 21st Century Bryan L. Betz, PhD; Jay L. Hess, MD, PhD Arch Pathol Lab Med. 2010;134:1427–1433 R4 채정민 /Prof 박태성

2. INTRODUCTION  Acute leukemia classification French-American-British classification –morphology and cytochemical stains 2008 World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissue –morphology, immunophenotyping, cytogenetics, and molecular studies review basics of AML diagnosis under the 2008 WHO classification –focused on molecular studies

3. DIAGNOSIS the starting point for diagnosis of leukemia –morphologic examination > 20% blasts in bone marrow or in blood –20% < blasts and cytogenetic abnormalities –assess the degree of dysplasia in the different lineages cytochemistry –played a central role in older leukemia classification schemes –no longer required –in rare cases → helpful in the identification of monocytic differentiation immunophenotyping by multicolor flow cytometry –the current standard of care to subclasify

4. DIAGNOSIS blasts are expressed as the percentage of nucleated cells –200-cell count in peripheral blood –500-cell count in the blood marrow > 50% erythroid precursors –erythroid progenitors are also excluded from the blast count

5. DIAGNOSIS

7. THE IMPACT OF CYTOGENETIC ABNORMALITIES  3 risk group following genetic abnormalities favorable –APL with t(15;17) → trans–retinoic acid –AML with t(8;21) or inv(16)/t(16;16) → cytarabine, doxorubicin intermediate –AML with t(9;11) –gains of whole chromosomes –loss of the Y chromosome –with normal cytogenetics adverse –t(6;9) –inv(3)/t(3;3) –complex karyotype (more than 3, older patients, poor prognosis)

8. THE IMPACT OF CYTOGENETIC ABNORMALITIES AML with specific recurrent cytogenetic abnormality (blast < 20%) –t(8;21)(q22;q22) –inv(16)(p13.1q22) –t(16;16)(p13.1;q22) –t(15;17)(q22;q12) blast < 20% but other recurrent cytogenetic abnormalities –myelodysplastic disorders with a cytogenetic abnormality

9. THE IMPACT OF CYTOGENETIC ABNORMALITIES AML with complex karyotype –extremely poor prognosis, survival < 1 year monosomal karyotype –2 autosomal monosomies or an autosomal monosomy + 1 or more structural abnormalities –[monosomy 5 and monosomy 7, or monosomy 5 with del(7q)] the 4-year overall survival –3% in monosomy group vs 26% in other cases Breems DA, Van Putten WL, De Greef GE, et al. Monosomal karyotype in acute myeloid leukemia: a better indicator of poor prognosis than a complex karyotype. J Clin Oncol. 2008;26(29):4791–4797

11. THE IMPACT OF CYTOGENETIC ABNORMALITIES  Leukemogenesis the class I mutations –FLT3, KIT, NRAS/KRAS, PTPN11 –cell proliferation and survival advantage –kinase signaling pathways –occur late –AML progression the class II mutations –CEBPA, NPM1, recurrent chromosomal translocations/inversions –impair myeloid differentiation –affecting genes involved in transcriptional regulation –occur early –stable throughout the disease course –initiating mutations

12. THE IMPACT OF CYTOGENETIC ABNORMALITIES class I and class II mutations occur together –FLT3-ITD with concurrent NPM1 mutation is common –enhanced proliferation (class I) and a block in differentiation (class II) class II mutations –generally do not coexist in AML –high stability of these mutations at relapse –class II aberrations are founder mutations –associated with characteristic clinicopathologic features recent studies (specific gene-expression, microRNA, and DNA methylation) → class II subtype

15. THE IMPACT OF CYTOGENETIC ABNORMALITIES the core-binding factor leukemias (t(8;21) and inv(16)) –high frequency of KIT exon 8 and 17 gene mutations (20%–40%) KIT exon 17 mutations (especially D816V) –inferior outcome in the t(8;21) subset KITD816 mutation was present in t(8;21) AML –the median survival ↓ (1836 days → 304 days) Cairoli R, Beghini A, Grillo G, et al. Blood. 2006;107(9):3463–3468 Schnittger S, Kohl TM, Haferlach T, et al. Blood. 2006;107(5):1791–1799 KIT mutations in inv(16) AML –need further study p53 abnormalities –high-risk patients (poor prognosis)

16. FLT3-ITD Mutation: Unfavorable Risk Marker FMS-related tyrosine kinase 3 (FLT3) –membrane-bound receptor tyrosine kinase –supports the proliferation and survival of hematopoietic progenitors Internal tandem duplication mutations –occur about 30% of CN-AML cases –strongly associated with poor outcome –shorter relapse-free and overall survival –duplication and tandem insertion of a small, variably sized (3–400 nucleotides) fragment of the genes

17. NPM1 Mutation: Favorable Risk Marker nucleophosmin (NPM1) –multifunctional phosphoprotein –shuttles between nuclear compartments and the cytoplasm –predominately located in the nucleolus –ribosome assembly and regulation of ARF and p53 tumor suppressor function NPM1 mutations –first discovered in AML owing to the cytoplasmic mislocalization of the mutated NPM1 protein → referred to as NPM1c positive, associated with CD34- immunophenotype –the most common genetic lesion in AML –30% of adult de novo cases and in 50% to 60% of AMLs with normal cytogenetics –good response to induction

18. NPM1 Mutation: Favorable Risk Marker NPM1 mutation without FLT3-ITD (NPM1 mut /FLT3- ITD neg ) –favorable outcome –prognosis is similar to that associated with the favorable t(8;21) and inv(16)/t(16;16) core-binding factor leukemias the NPM1 WT /FLT3-ITD pos –unfavorable outcome FLT3-ITD mutation status can affect the prognostic impact of NPM1 → both should be tested for concurrently and their prognostic impact interpreted collectively

19. NPM1 Mutation: Favorable Risk Marker more than 40 different mutations within exon 12 of the NPM1 gene net insertion of 4 nucleotides immunohistochemical detection of cytoplasmic NPM1 –alternative to the genetic test –conflicting reports → genetic testing remains the preferred screening method

20. CEBPA Mutation: Favorable Risk Marker mutation of the CCAAT/enhancer binding protein a (CEBPA) gene –suppress transcription factor for the development and differentiation of granulocytes from hematopoietic precursors –promote leukemogenesis by blocking granulocytic differentiation –frequency in newly diagnosed AML: 5% to 9% –predominant at M1 and M2 FAB morphologic subtypes CEBPA mutation in CN-AML –frequency: 15% –independent prognostic marker –associated with lower relapse rates and improved overall survival –favorable prognosis is similar to that of NPM1 mut /FLT3-ITD neg AML

21. CEBPA Mutation: Favorable Risk Marker N-terminal frameshift mutations –truncation of the full-length p42 CEBPA protein C-terminal in-frame mutations –affect both the full-length p42 CEBPA protein and a shorter p30 isoform –lead to proteins with impaired dimerization and DNA binding activities testing for CEBPA mutations –requires a technology capable of detecting the wide range of nucleotide alterations (duplications, insertions,deletions, substitutions) –only available in a limited but growing number of clinical laboratories

22. CEBPA Mutation: Favorable Risk Marker positive CEBPA mutation test –typically biallelic mutations (both N- and C-terminal mutations) → complete loss of CEBPA function –up to one-third of CN-AML cases exhibit only a single mutation(monoallelic) –only biallelic CEBPA mutations are associated with a favorable clinical outcome in CN-AML silencing CEBPA –recently more than mutation of CEBPA –as a result of high levels of CpG methylation –frequently associated with mutations in the transcription factor NOTCH(common in T-acute lymphoblastic leukemia) –associated with a distinctly poor prognosis

23. EMERGING APPLICATIONS OF AML GENETICS: OPPORTUNITIES AND CHALLENGES  Prediction of Transplant Benefit genotype NPM1 mut /FLT3-ITD neg predicted a lack of benefit from allogeneic stem cell transplant at first remission transplant improved outcome in patients with the FLT3-ITD pos or NPM1 WT /FLT3-ITD neg /CEBPA WT genotypes future therapeutic decision-making algorithms in CN-AML escpecially intermediate-risk CN-AML group Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008;358(18):1909– 1918

24. EMERGING APPLICATIONS OF AML GENETICS: OPPORTUNITIES AND CHALLENGES  Molecularly Targeted Therapies all–trans-retinoic acid in acute promyelocytic leukemia –blocked PML-RARA fusion protein function –revolutionized the prognosis and treatment of patients with this previously fatal disease kinase signaling pathways –high frequency of kinase-activating gene mutations, including FLT3 and KIT –tyrosine kinase inhibitors (TKIs), including sunitinib, midostaurin, and lestaurtinib –ongoing trials are exploring combination therapy with TKIs and conventional chemotherapy for patients with FLT3 mutations

25. EMERGING APPLICATIONS OF AML GENETICS: OPPORTUNITIES AND CHALLENGES  Molecularly Targeted Therapies differential sensitivity of the particular mutations to TKIs imatinib –activity against KIT proteins with exon 8 mutations –not the D816 mutations found in exon 17 dasatinib or midostaurin –can target D816 mutations DNA methylation abnormalities, such as CEBPA silencing –may respond particularly well to demethylating agents activity of decitabine and azacitidine in AML –ongoing trials

26. EMERGING APPLICATIONS OF AML GENETICS: OPPORTUNITIES AND CHALLENGES  Disease Monitoring quantitative molecular tests –sensitivities as low as 10 -5 to 10 -6 –assess both early treatment response and low levels of posttreatment disease –minimal reduction or a persistence of the marker → relapse –opportunity for early intervention before morphologic evidence of disease –in acute promyelocytic leukemia (PML-RARA) and the core-binding factor leukemias(RUNX1-RUNX1T1 and CBFB-MYH11) have provided encouraging results

27. EMERGING APPLICATIONS OF AML GENETICS: OPPORTUNITIES AND CHALLENGES  Disease Monitoring FLT3 gene mutations –occur in leukemic subclones –the mutation status can change during treatment –limited clinical utility NPM1 mutations –highly stable and generally persist at relapse –promise for minimal residual disease –correlated decreasing mutant NPM1 copy numbers with treatment response and persistence of posttransplant NPM1 mutations with relapse –important markers for disease monitoring in patients with CN-AML mRNA expression levels –overexpressed in AML blasts –WT1 has been extensively evaluated

28. CONCLUSIONS acute myeloid leukemia –whole-genome sequencing for mutation discovery –array comparative genomic hybridization to identify submicroscopic genomic deletions and amplifications –DNA methylation arrays to detect epigenetic modifications additional molecular markers –prognostic and therapeutic significance –new challenges for molecular pathology laboratories, informatics infrastructure, hematopathologists –introduction of targeted AML therapies –better patient care