Acute Myeloid Leukemia With Recurrent Genetic Abnormalities Other Than Translocations

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1 AJCP / Review Article Acute Myeloid Leukemia With Recurrent Genetic Abnormalities Other Than Translocations Pei Lin, MD, 1 and Brunangelo Falini, MD 2 From the 1 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston; and the 2 Department of Hematology, Institute Hematology at University of Perugia, Perugia, Italy. Key Words: Hematopathology; Immunopathology; Flow cytometry Am J Clin Pathol July 2015;144:19-28 ABSTRACT Objectives: Session 2 of the workshop focused on cases of acute myeloid leukemia (AML) with gene mutations in the setting of a normal karyotype. Methods: Among 22 AML cases submitted, 14 had the NPM1 mutation, most also accompanied by mutations of other genes such as FLT3-ITD, DNMT3A, or, rarely, TP53; three cases had the heterozygous CEBPA mutation; and two cases had MYC amplification. Results: We explored prognostic implications of gene mutations such as DNMT3A, issues related to the classification of AML cases with the NPM1 mutation, and myelodysplasia-related changes arising from chronic myelomonocytic leukemia after a short latency interval. Disparate patterns of treatment response to targeted therapy using an FLT3 inhibitor, designated as cytotoxic or differentiation, and their genetic underpinnings were described. Finally, a minimal screening panel for gene mutations and the optimal approach for monitoring minimal residual disease were discussed. Conclusions: In aggregate, this session highlighted the need for a refined molecular classification of AML as well as improved risk stratification based on systematic assessment for genetic alterations and their evolution over time. Morphologic, cytogenetic, and molecular genetic alterations are the basis of disease classification and risk stratification in acute myeloid leukemia (AML). Conventional cytogenetic (CG) and fluorescence in situ hybridization (FISH) may identity abnormalities in about 50% to 60% of cases that can be further stratified into favorable-, intermediate-, or poor-risk subgroups. The remaining 40% to 50% cases with a normal karyotype (cytogenetically normal; CN) are heterogeneous and carry a variety of molecular aberrations. In the 2008, World Health Organization (WHO) classification, mutations of NPM1, FLT3-ITD, and CEBPA are listed among the most common recurrently mutated genes, and those with the NPM1 or CEBPA mutation are designated as provisional entities with distinct biological and clinical features. 1 According to the 2010 European LeukemiaNet (ELN) proposal Table 1, 2 AML with the NPM1 mutation and FLT3-ITD wild-type or CEBPA double mutations belong to the low-risk group, with a survival rate similar to that of AML with core binding factor abnormalities. By contrast, AML with the FLT3-ITD mutation, with or without the NPM1 mutation, belongs to the intermediate-risk group. Low-risk patients are generally managed with chemotherapy, whereas intermediate- and high-risk patients may require allogeneic stem cell transplantation (SCT). Since the publication of the WHO classification and the initial ELN proposal, additional novel recurrent gene mutations have been identified. 3 DMNT3A, TET2, IDH1/2, ASXL1, RUNX1, and PTPN11 are among the genes most commonly mutated, some of which are considered initiating or founder mutations, whereas others are secondary events involved in disease progression. Mapping of the genomic landscape of AML has revealed clonal hierarchy and clonal Am J Clin Pathol 2015;144:

2 Lin and Falini / AML With Recurrent Genetic Abnormalities Table 1 European LeukemiaNet Risk Stratification Model Risk Favorable t(8;21), inv(16), t(15;17) CN: NPM1+ FLT3-ITD CN: CEBPA+ (double) Intermediate I NK: Mutated NPM1 + and FLT3-ITD+ NK: Wild-type NPM1 with or without FLT3-ITD+ Intermediate II t(9;11)(p22;q23); others, excluding favorable/adverse Adverse inv(3)/t(3;3) t(6;9)(p23;q34) t(v;11)(v;q23) 5 or del(5q), 7, abnl(17p) Complex karyotype ( 3) CN, cytogenetically normal; NK, normal karyotype. evolution, providing a new framework for disease classification, risk stratification, and targeted therapy. 4-6 Updated risk models incorporating newly identified gene mutations have been introduced, although these models need further clinical validation based on large cohorts of similarly treated patients This session of the workshop focused mainly on cases of CN-AML or related diseases with recurrent molecular aberrations. Of the 22 cases included in the session Table 2, 14 had the NPM1 mutation, with most accompanied by other gene mutations. Rare cases with additional karyotypic abnormalities were included to illustrate their prognostic impact in the context of otherwise well-characterized cases of CN-AML with the NPM1 mutation. These cases included three AMLs with the heterozygous CEBPA mutation, alone or concomitant with other gene mutations, and two cases of AML with MYC amplification with unusual morphologic features. In view of the newly identified mutations and their therapeutic implications, a practical approach to refined molecular subtypes of AML was emphasized. Specific issues related to clinical practice were discussed and are summarized in this report, including (1) significance of the DNMT3A mutation in NPM1-mutated AML; (2) classification of cases of AML with the NPM1 mutation that also meet WHO classification criteria for AML with myelodysplasia-related changes (MRCs) based on morphologic evidence of dysplasia, cytogenetic aberrations, or a history of myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML); (3) prognostic significance of the heterozygous vs homozygous CEBPA mutation; (4) recommendations for a minimal screening panel of genes mutated in CN-AML in routine practice; (5) monitoring of minimal residual disease (MRD) by molecular methods; and (6) two distinct patterns of treatment response (cytotoxic vs differentiation) and FLT3-ITD mutant burden in the bone marrow of patients with AML treated with an FLT3 inhibitor. AML With the NPM1 Mutation What Is the Prognostic Impact of DNMT3A in NPM1-Mutated AML? Based on a unique gene expression signature and distinctive clinical features, NPM1-mutated AML is designated as a provisional entity in the WHO classification. 12 This mutation is seen in approximately 30% of all AMLs but occurs in 40% to 60% of CN-AML and results in abnormal cytoplasmic localization of NPM1 that can be assessed by immunohistochemistry (IHC). Most cases of AML with the NPM1 mutation have myelomonocytic or monocytic morphologic features and an immunophenotype without CD34 expression, but exceptional cases expressing CD34 have been shown and are reported to correlate with clinically more aggressive disease. 13 FLT3-ITD and DNMT3A mutations are also common recurrent mutations in CN-AML, in about 40% and 30% of cases, respectively, and often coexist with the NPM1 mutation. AML cases with all three gene mutations have distinct microrna and messenger RNA expression patterns. 3 The FLT3-ITD mutation negatively affects prognosis, and affected patients are typically treated with SCT. The impact of FLT3-ITD, however, is dependent on allelic burden, with a low burden (defined as mutant to wild-type ratio <0.5%) still predicting a favorable outcome. 14 The DNMT3A mutation usually involves locus 2644C>T, R882C. Like the NPM1 mutation, the DNMT3A mutation is considered a founder event, likely arising in leukemic stem cells, and affected patients require high-dose daunorubicin therapy. 11 Case 91 was a 56-year-old man with an aggressive CN-AML carrying mutations of FLT3-ITD, DNMT3A, and NPM1 but with a low FLT3-ITD allelic burden (mutant/ wild-type ratio of 0.45) Image 1 and no evidence of abnormalities involving MLL (11q23), 5p15.2, or 5q31. The immunophenotype was characterized by partial expression of CD34 and aberrant CD5 and CD7 expression. Although the patient readily achieved complete remission after induction, the disease quickly relapsed and became refractory, even after SCT. The prognostic significance of the DNMT3A mutation in AML has been explored mostly in retrospective studies. Most studies suggest that this mutation confers inferior survival in patients with CN-AML. However, existing data are inconsistent and controversial regarding which risk groups are significantly affected Gaidzik et al 18 and Ribeiro et al 19 found no impact of the DNMT3A mutation on patients with CN-AML but a negative impact in patients with unfavorable-risk AML. Key points: This case suggests that DNMT3A may confer an aggressive clinical course in patients with otherwise 20 Am J Clin Pathol 2015;144:19-28

3 AJCP / Review Article Table 2 Cases of AML With Recurrent Genetic Abnormalities in Session 2 Case No. Diagnosis Interesting Features 91 AML with NPM1, FLT3-ITD and DNMT3A mutations Aggressive AML in cases with a low FLT3-ITD mutant/wild-type ratio 398 AML with NPM1 mutation and trisomy 4 Trisomy 4 in AML with NPM1 mutation 255 AML with NPM1 and FLT3-ITD mutations Presenting as CMML with rapid progression to AML 428 AML with NPM1, FLT3-ITD and RAS mutations, Presenting as CMML with rapid progression to AML trisomy AML with NPM1 mutation Typical phenotype (CD34-HLA-DR ), but with AML-M1 morphology 207 AML with NPM1 and FLT3-ITD mutations Cup-like morphology 52 AML with NPM1 and FLT3-ITD mutations, CD25+ CD25 expression and clinically aggressive 218 AML with NPM1 mutation and GSTM1 Prognosis of low-risk AML with an adverse epigenetic marker hypermethylation 299 AML with MRC, NPM1, FLT3-ITD and CEBPA Presenting with myeloid sarcoma in the mandible, MRC mutations 355 AML with NPM1, FLT3-ITD, DNMT3A and TP53 Preceded by a history of MDS for more than 4 y mutations 186 AML with NPM1 mutation Additional FLT3-ITD mutation at relapse with rapid progression 318 AML with NPM1 and FLT3-ITD mutations Relapse with t(2;16),t(11;14) 360 AML with NPM1 and FLT3-ITD mutations Relapse with t(12;13)(etv6/flt3) 254 AML with FLT3-ITD mutation Two disparate response patterns to quizartinib (an FLT3 inhibitor) treatment 381 AML with NPM1 and IDH2 mutations Responded to a single-agent PR-104 treatment (hypoxia-inducing agent) 409 AML with CEBPA heterozygous mutation Issues related to heterozygous vs homozygous CEBPA mutation 399 AML with CEBPA heterozygous mutation Possible preceding history of MDS 129 AML with MRC and MYC amplification Complex karyotype with MYC amplification as double minutes 183 AML with MYC amplification Resemble APL with large intracellular inclusions 95 AML with MRC Dysplastic megakaryocytes 216 Acute leukemia, probably myeloid Positive for stem cell markers (CD34, CD117, CD38, HLA-DR) only 227 Acute leukemia, not further classifiable Positive for stem cell markers AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; MRC, myelodysplasiarelated changes. low-risk AML and underscores the need for routine testing of DNMT3A in CN-AML. More data regarding the prognostic impact of DNMT3A in prospective studies with better characterized clinical parameters are needed. How to Classify AML With the NPM1 Mutation That Also Meets the Criteria for AML-MRC? According to 2008 WHO classification, AML with morphologic evidence of multilineage dysplasia, MDSrelated cytogenetic changes, or preceded by MDS or MDS/ myeloproliferative neoplasm (MPN) meets the criteria for AML with myelodysplasia-related changes (AML-MRCs). AML with the NPM1 mutation is usually de novo, but a small subset of cases may be preceded by MDS or MDS/ MPN. 20 One study identified the NPM1 mutation in 37 (13.1%) of 283 patients at diagnosis of secondary AML following MDS or CMML. 21 In de novo AML with the NPM1 mutation, karyotypic abnormalities can be found in about 5% to 15% of cases, and multilineage dysplasia can be observed in about 25% of cases. 12 By definition, these cases also meet the WHO criteria for AML-MRC, a worse prognostic group. How should this discrepancy be reconciled? Case 398 was a 46-year-old woman who had AML associated with morphologic evidence of dysplasia and an abnormal karyotype: 47,XX,+4[9],46,XX[11]; FISH confirmed trisomy 4 in 64% of nuclei, and molecular studies detected the NPM1 mutation with 40% of the mutant allele. FLT3-ITD was wild type. The leukemic blasts expressed CD34 and other myeloid markers at diagnosis. The patient achieved complete remission after induction without evidence of disease on day 14 of induction but had persistent cytopenia after consolidation. Repeat bone marrow (BM) evaluation at 6 months showed 13.2% blasts and monocytosis with immunophenotypic evidence of monocytic differentiation and CG, and FISH found no trisomy 4. However, immunostain detected cytoplasmic NPM1-positive cells in a background of dysplasia Image 2. The patient was diagnosed with relapsed disease, underwent allogeneic SCT, and went into sustained remission. AML with isolated trisomy 4 is rare and carries a frequency of the NPM1 mutation similar to that of cases of CN-AML. 22 Trisomy of chromosome 4, 8, or 21 is reported to be one of the most commonly observed cytogenetic aberrations in AML with the NPM1 mutation and is considered a secondary event that does not confer additional prognostic value. 23 Of note, case 207 was a patient with AML who had trisomy 8 with NPM1 and FLT3-ITD mutations, and case 299 was a patient with AML who had extramedullary sarcoma involving the mandible associated with NPM1, FLT3- ITD, and CEBPA mutations and trisomy 21. Am J Clin Pathol 2015;144:

4 Lin and Falini / AML With Recurrent Genetic Abnormalities A B Wild-type mutated ,000 2,000 1,000 ITD Image 1 Case 91, acute myeloid leukemia with NPM1, FLT3-ITD, and DNMT3A mutations. A, The bone marrow aspirate smear shows numerous blasts. B, Molecular studies identified mutations in NPM1 (top), FLT3-ITD (center), and DNMT3A (bottom). The size of the FLT3-ITD mutation is 90 base pairs, and the allele burden (mutant/wild-type ratio) of FLT3-ITD is low at (Courtesy of X. Calvo.) R882H Based on the evidence (immunophenotype and prognosis) that points to AML with mutated NPM1 as a distinct disease, regardless of morphologic evidence of dysplasia or certain types of karyotypic aberrations as described above, the consensus diagnosis for case 398 was AML with the NPM1 mutation and trisomy 4, rather than AML with MRC. This diagnosis is supported by gene expression profiling data reported in the literature that have identified a unique signature for AML with the NPM1 mutation, regardless of the presence or absence of dysplasia. 24 In comparison, case 355 was a 68-year-old woman with a sudden onset of aggressive AML carrying mutations of NPM1, FLT3-ITD, DNMT3A, and TP53 but a normal diploid karyotype (46,XX) in 20 metaphases, following a history of stable MDS over 4 years that was managed only by observation. TP53 is described to be more common in secondary AML and signifies a high-risk disease. 25 The overall features in this case suggest AML arising from transformation of underlying MDS, and the consensus diagnosis for this tumor was AML-MRC despite a normal diploid karyotype. Key points: In de novo AML, morphologic evidence of myelodysplasia or trisomy 4, 8, or 21 may not confer further prognostic impact in patients with otherwise low-risk AML with the NPM1 mutation. However, in patients with a history of MDS or related diseases, the diagnosis of AML- MRC is more appropriate, particularly when supported by molecular genetic findings. Younger and older patients likely have different molecular mechanisms involved in AML pathogenesis. Does CMML With the NPM1 Mutation Represent an Early Phase of De Novo AML? The NPM1 mutation is uncommon in CMML. 26,27 Cases of acute myelomonocytic leukemia (AMML) with the NPM1 mutation may be preceded by CMML. Is the antecedent CMML actually AMML detected at an early phase? If so, should these neoplasms be classified as de novo AML regardless of a blast percentage rather than AML with MRC? Two cases submitted to the workshop brought this issue to the forefront. Case 255 was a 79-year-old man who initially had CMML that rapidly progressed to AML. An initial CBC showed mild leukocytosis with monocytosis (WBC count, /L; 25% monocytes) and mild anemia without thrombocytopenia. Examination of BM revealed 13% blasts/ promonocytes and erythroid dysplasia in the background. The patient was diagnosed with CMML type 2 (CMML-2) and observed with supportive care. Three months later, the patient developed a WBC count of /L with 44% monocytes, 8% promonocytes, and 18% blasts (hemoglobin level, 7.6 g/dl; platelet count, /L). BM aspirate smears found 71% blasts/promonocytes. Further workup showed a normal karyotype and NPM1 and FLT3-ITD 22 Am J Clin Pathol 2015;144:19-28

5 AJCP / Review Article A B Image 2 Case 398, acute myeloid leukemia with NPM1 mutation and trisomy 4. A, The bone marrow aspirate shows numerous blasts and a dysplastic erythroid precursor (Wright-Giemsa, 1,000). B, After induction and consolidation, the disease remained in a smoldering status with persistent cytopenia and NPM1+ cells in the bone marrow by immunohistochemical stain for NPM1 ( 1,000). (Courtesy of R. E. Hutchison.) mutations. Immunostain demonstrated cytoplasmic NPM1- positive cells in both the CMML-2 and AMML samples. The patient relapsed after induction therapy and died 12 months after diagnosis. Similarly, case 428 was a 26-year-old woman who initially had chronic bronchitis, an elevated WBC count, and 11% blasts/promonocytes in BM (CMML-2). Initial cytogenetic studies identified 47,XX,+21[11],46,XX[9]. Treatment with lenalidomide and azacitidine was recommended. Within 4 weeks, during which the patient received supportive care only, the disease evolved to AMML with 82% blasts in the bone marrow and 41% blasts in the peripheral blood. This time, CG showed 48,XX,+2,+6 [1];XX[19], with only 4% of cells positive for trisomy 21 by FISH analysis, suggesting a preferential expansion of the diploid subclone. Mutations of NPM1, DNMT3A, and N-RAS were also identified. The patient achieved CR after induction therapy but died due to complications of an allogeneic transplant 9 months after the initial diagnosis. These cases suggest that in at least a subset of patients, the initial CMML may, in fact, represent an early phase of de novo AML with the NPM1 mutation rather than transformation from underlying MDS/MPN. Evidence supporting this notion includes clusters of NPM1c+ (cytoplasmic positive) blasts in BM biopsy specimens at the time of putative CMML and a short latency interval between the diagnosis of CMML and a subsequent diagnosis of AML. AML with the NPM1 mutation frequently displays AML M5b morphology with extreme peripheral blood monocytosis. However, there are also reports in the literature suggesting that the NPM1 mutation may be a transforming event that heralds progression from CMML to AMML. Some cases of CMML with the NPM1 mutation remain stable without progression for more than 2 years. 21 Thus, some cases may represent true AML-MRC, whereas others are an early phase of de novo AML. Key points: Screening of newly diagnosed cases of CMML for the NPM1 mutation may be warranted and, when positive, an early phase of AML should be suspected, particularly in young patients. Patients with NPM1-positive CMML tend to progress to AMML within a short time interval, but the clinical course can be variable. AML With the CEBPA Mutation Around 10% of CN-AML cases carry the CEBPA mutation. Most CEBPA-mutated cases are double mutated (CEBPAdm), involving a combination of N-terminal and C-terminal mutations; only a few cases of CN-AML carry a singly mutated CEBPA (CEBPAsm). 28 Studies in the literature suggest that the prognosis for patients with CEBP- Adm AML, usually biallelic, is favorable. 29 Patients with CEBPAsm have a significantly increased frequency of the FLT3-ITD mutation compared with both wild-type and CEBPAdm AML. Unlike the NPM1 mutation, the FLT3-ITD mutation is associated with a significantly worse outcome, irrespective of CEBPA mutation status. Outcome in NPM1- positive patients with CEBPAsm is reported to be comparable to CEBPAdm. 30 Am J Clin Pathol 2015;144:

6 Lin and Falini / AML With Recurrent Genetic Abnormalities A B Image 3 Case 409, acute myeloid leukemia with CEBPA mutation. A, Numerous blasts in the peripheral blood (WrightGiemsa, 1,000). B, Bone marrow trephine biopsy specimen shows sheets of immature cells (H&E, 400). (Courtesy of V. E. Klepeis.) Three cases of CN-AML submitted to the workshop demonstrated a heterozygous CEBPA mutation (cases 299, 399, and 409). Case 299 was diagnostically challenging, as the patient had a myeloid sarcoma involving the mandible and also had mutations of NPM1 and FLT3-ITD. Case 409 was a 68-year-old man who had a WBC count of /L and 79% circulating blasts Image 3. Screening of 23 common cancer genes, including FLT3, NPM1, IDH1, IDH2, KRAS, NRAS, and TP53, found no evidence of mutations. After standard induction with idarubicin and cytarabine, the patient achieved complete remission without evidence of disease at day 34. Consolidation consisted of decitabine, but the clinical course was implicated by delayed recovery of the neutrophil count. The patient underwent nonmyeloablative allogeneic SCT and remained in complete remission documented by bone marrow examination at day 100 posttransplant. Recent studies have shown the prognostic significance of cooperating mutations (GATA2, TET2) in CEBPAdm AML, suggesting that testing for these genes may be helpful in predicting the clinical outcome of patients with CEBPA-mutated AML.7 Should AML Risk Stratification Be Based on a More Dynamic Model Incorporating Clonal Evolution at Relapse? Comparison of primary and relapsed samples has shown that AML is an oligoclonal expansion of leukemic cells that evolve over time; relapse is related to dominance of competing subclones or emerging new clones.4,31 NPM1 and DNMT3A are likely primary events, whereas cells carrying secondary mutations such as FLT3-ITD may 24 Am J Clin Pathol 2015;144:19-28 expand over the course of disease, causing relapse and drug resistance. Case 186 was a 65-year-old man whose AML was positive for the NPM1 mutation without detectable FLT3-ITD at diagnosis, but the FLT3-ITD mutation was identified 9 months later and coincided with rapidly progressive and refractory disease and death 2 months later. Similarly, case 360 was a 68-year-old man who initially had CN-AML positive for NPM1 and FLT3-ITD mutations. He achieved morphologic CR after induction but had a persistent low level of FLT3-ITD, for which he received allogeneic SCT. This patient had a short interval of molecular CR with no detectable FLT3-ITD, before the disease relapsed 1 month later with newly detected translocation t(12;13) involving ETV6 and FLT3, along with return of the FLT3-ITD mutation. The patient s overall survival was 8 months. These cases support the notion that clonal evolution is responsible for a more aggressive disease and underscores a need for a dynamic model of risk assessment and targeted therapy.32 Is There a Need for Additional Risk Assessment Beyond Gene Mutation Analysis? Advances in molecular techniques have discovered not only gene mutations but also other underlying genetic abnormalities that contribute to pathogenesis or prognosis. Emerging data from RNA sequencing (RNA-Seq), microrna expression profiling, DNA methylation profiling, and genome-wide single-nucleotide polymorphism based arrays have implicated abnormal RNA splicing, epigenetic modification, copy number changes, and other molecular mechanisms in AML pathogenesis.33,34 Although published

7 AJCP / Review Article A B C Image 4 Case 183. A, Acute myeloid leukemia with MYC amplification: a blast contains pseudo- Chediak-Higashi like granules. Inset: A blast contains numerous Auer rods (arrow). B, Karyotype showing numerous double minutes (arrow). C, Fluorescence in situ hybridization using a MYC breakapart probe shows MYC gene amplification. (Courtesy of N. Aggarwal.) data are largely based on investigational rather than routine clinical studies, the data highlight the complex interplay of different molecular mechanisms involved in leukogenesis. Case 218 was a 62-year-old woman who had AML with the NPM1 mutation, and FLT3-ITD was wild type. Therefore, this disease belongs to a favorable subgroup according to ELN criteria. However, hypermethylation of the GSTM1 gene was identified, which has been reported to be an adverse biomarker. 35 The patient achieved complete remission with standard therapy but relapsed three times within 41 months of initial diagnosis, with the first relapse occurring early at 18 months. This case suggests that risk assessment beyond the incorporation of gene mutation data is needed. Although this topic was not extensively discussed during the workshop, the future will likely see comprehensive molecular characterization of AML as a part of the initial diagnostic workup and possibly also at relapse. Are There Distinct Morphologic and FLT3-ITD Response Patterns Induced by FLT3 Inhibitor Treatment? Case 254 described one of two divergent patterns of response to an FLT3 inhibitor, quizartinib (AC220), designated as differentiation or cytotoxic by the contributors. The differentiation pattern is marked by progressive maturation of hematopoietic elements in cellular BM, accompanied by stable or, paradoxically, a rising FLT3-ITD burden, suggesting differentiation rather than elimination of the leukemic clone. By contrast, the cytotoxic pattern is marked by reduced BM cellularity and minimal hematopoiesis with a falling FLT3-ITD burden. Both response patterns are associated with symptomatic alleviation, but only the former was associated with improved peripheral blood counts. These response patterns appear to be related to or depend on the underlying genetic makeup of the disease: a differentiation response is seen in CN-AML, whereas a cytotoxic response largely occurs in AML with an abnormal karyotype. Am J Clin Pathol 2015;144:

8 Lin and Falini / AML With Recurrent Genetic Abnormalities What Is the Minimal Screening Panel for Gene Mutations in CN-AML? Although an expert panel has already recommended testing for NPM1, CEBPA, and FLT3-ITD mutations in patients with CN-AML who are treated with intention to cure, discussion at the workshop suggests that the minimal screening panel for routine practice needs to be expanded to include at least NPM1, FLT3-ITD, CEBPA, and DNMT3A. As deep sequencing becomes more affordable, a more extensive panel that will include other newly identified and recurrently mutated genes will likely become available. This practice will hopefully lead to recognition of more molecularly refined subtypes of AML. What Is the Best Approach for MRD Monitoring in CN-AML in Routine Clinical Practice? Since the NPM1 mutation is a primary event and usually stable over time, quantitative polymerase chain reaction (PCR) assays for mutant NPM1 are considered an excellent approach for monitoring MRD in patients with NPM1-mutated AML and has prognostic value. Detection of aberrant cytoplasmic nucleophosmin in leukemic cells by IHC correlates with the NPM1 mutation in tissue biopsy specimens may be used as a screening tool, 36 but tissue fixation is critical for this approach (using B5/EDTA fixative is optimal). Concomitant FLT3-ITD and DNMT3A mutations may also affect the depth of MRD in NPM1- mutated AML, but FLT3-ITD is reported to be unstable for MRD monitoring. One study described persistence of the DNMT3A mutation in five patients who lost the NPM1 mutation at relapse, indicating DNMT3A should also be included in MRD monitoring. 31,37 The CEBPA mutation is also considered a primary event that is usually stable over the course of disease. Many methods can be used for detecting the CEBPA mutation, including PCR, denaturing length performance liquid chromatography, and direct Sanger sequencing; the latter is considered the gold standard. 7 As deep sequencing becomes more affordable and clinically feasible, its broad application in clinical practice is anticipated. AML With MYC Amplification MYC amplification was described in two cases of AML with unique cytogenetic features, including double minutes (cases 129 and 183). Case 183 was particularly distinctive; the blasts contained multiple Auer rods and pseudo-chediak-higashi type granules and were negative for CD34 and HLA-DR expression mimicking M3 blasts. FISH analysis confirmed MYC gene amplification in both cases Image 4. Summary Since the introduction of the 2008 WHO classification and ELN system of AML risk stratification, new recurrent genetic aberrations in AML have been discovered. This session of the workshop focused on cases of CN-AML and illustrated the morphologic spectrum and molecular genetic features of AML cases with NPM1, FLT3-ITD, DNMT3A, CEBPA, and other common AML-associated mutations. De novo CN-AML with the NPM1 mutation is best classified according to the mutation rather than as AML-MRC, despite morphologic evidence of dysplasia or additional chromosomal aberrations such as trisomy 4, 8, or 21. An early phase of de novo AML should be suspected, especially in younger patients, when a short latency period is noted and blast percentage is in the range of CMML. In the setting of AML relapse, dominance of expanded subclones or new clones may alter the risk group, usually manifested as lowrisk subgroup (NPM1 mutation only), moving up to a higher risk subgroup (NPM1 and FLT3-ITD). Currently, screening for NPM1, CEPBA, FLT3-ITD, and DNMT3 mutations is recommended in routine clinical practice. Quantitative PCR for mutant NPM1 is an excellent marker for monitoring MRD. As deep sequencing becomes more affordable and available, more genes of clinical significance can be screened. The future of AML classification is likely to be based on a more comprehensive molecular characterization of neoplastic clones and subclones for better risk stratification and targeted therapy. Corresponding author: Pei Lin, MD, Unit 72, Dept of Hematopathology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX Acknowledgments: The following were contributors or co-contributors of cases to this session: N. Aggarwal, S. Ahamd Alghamdi, X. Andrade, D. A. Arber, H. Aviv, M. Aymerich, A. Bagg, B. Bai, A. Bakhirev, S. Bhagavathi, J. D. Brazelton, C. E. Bueso-Ramos, X. Calvo, M. Carroll, J. Cerny, D. Colomer, L. C. Contis, S. Cordelia, J. E. Cortes, E. L. Courville, D. Costa, J. Esteve, S. H. Faderl, T. C. Gentile, R. Hasserjian, J. Hussong, R. E. Hutchison, T. Kelley, J. King, V. E. Klepeis, S. Konoplev, M. Konopleva, G. Lu, L. J. Medeiros, J. Morrissette, T. Muzzafar, G. E. Nybakken, R. Ohgami, K. Patel, D. Peker, S. L. Perkins, A. Perl, S. Pullarkat, V. G. Robu, M. Routbort, D. Roy, M. Rozman, M. Salama, R. A. Schulz, Q. Shen, J. S. Sidhu, V. Sriganeshan, C. K. Stein, N. Villamor, D. Wakefield, X. I. Wang, B. A. Woda, W. Xing, M. Yabe, S. Yea, C. Yin, H. Yu, D. Zhang, and Z. Zuo. References 1. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114: Am J Clin Pathol 2015;144:19-28

9 AJCP / Review Article 2. Dohner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115: The Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368: Ding L, Ley TJ, Larson DE, et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature. 2012;481: Welch JS, Ley TJ, Link DC, et al. The origin and evolution of mutations in acute myeloid leukemia. Cell. 2012;150: Cagnetta A, Adamia S, Acharya C, et al. Role of genotypebased approach in the clinical management of adult acute myeloid leukemia with normal cytogenetics. Leuk Res. 2014;38: Grossmann V, Haferlach C, Nadarajah N, et al. CEBPA double-mutated acute myeloid leukaemia harbours concomitant molecular mutations in 76.8% of cases with TET2 and GATA2 alterations impacting prognosis. Br J Haematol. 2013;161: Mendler JH, Maharry K, Radmacher MD, et al. RUNX1 mutations are associated with poor outcome in younger and older patients with cytogenetically normal acute myeloid leukemia and with distinct gene and microrna expression signatures. J Clin Oncol. 2012;30: Metzeler KH, Becker H, Maharry K, et al. ASXL1 mutations identify a high-risk subgroup of older patients with primary cytogenetically normal AML within the ELN Favorable genetic category. Blood. 2011;118: Metzeler KH, Maharry K, Radmacher MD, et al. TET2 mutations improve the new European LeukemiaNet risk classification of acute myeloid leukemia: a Cancer and Leukemia Group B study. J Clin Oncol. 2011;29: Patel JP, Gonen M, Figueroa ME, et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med. 2012;366: Falini B, Martelli MP, Bolli N, et al. Acute myeloid leukemia with mutated nucleophosmin (NPM1): is it a distinct entity? Blood. 2011;117: Dang H, Chen Y, Kamel-Reid S, et al. CD34 expression predicts an adverse outcome in patients with NPM1-positive acute myeloid leukemia. Hum Pathol. 2013;44: Pratcorona M, Brunet S, Nomdedeu J, et al. Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy. Blood. 2013;121: El Ghannam D, Taalab MM, Ghazy HF, et al. DNMT3A R882 mutations in patients with cytogenetically normal acute myeloid leukemia and myelodysplastic syndrome. Blood Cells Mol Dis. 2014;53: Renneville A, Boissel N, Nibourel O, et al. Prognostic significance of DNA methyltransferase 3A mutations in cytogenetically normal acute myeloid leukemia: a study by the Acute Leukemia French Association. Leukemia. 2012;26: Thol F, Damm F, Ludeking A, et al. Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia. J Clin Oncol. 2011;29: Gaidzik VI, Schlenk RF, Paschka P, et al. Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML Study Group (AMLSG). Blood. 2013;121: Ribeiro AF, Pratcorona M, Erpelinck-Verschueren C, et al. Mutant DNMT3A: a marker of poor prognosis in acute myeloid leukemia. Blood. 2012;119: Falini B, Nicoletti I, Martelli MF, et al. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features. Blood. 2007;109: Schnittger S, Bacher U, Haferlach C, et al. Characterization of NPM1-mutated AML with a history of myelodysplastic syndromes or myeloproliferative neoplasms. Leukemia. 2011;25: Bains A, Lu G, Yao H, et al Molecular and clinicopathologic characterization of AML with isolated trisomy 4. Am J Clin Pathol. 2012;137: Haferlach C, Mecucci C, Schnittger S, et al. AML with mutated NPM1 carrying a normal or aberrant karyotype show overlapping biologic, pathologic, immunophenotypic, and prognostic features. Blood. 2009;114: Verhaak RG, Goudswaard CS, van Putten W, et al Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood. 2005;106: Milosevic JD, Puda A, Malcovati L, et al. Clinical significance of genetic aberrations in secondary acute myeloid leukemia. Am J Hematol. 2012;87: Itzykson R, Kosmider O, Renneville A, et al. Prognostic score including gene mutations in chronic myelomonocytic leukemia. J Clin Oncol. 2013;31: Courville EL, Wu Y, Kourda J, et al. Clinicopathologic analysis of acute myeloid leukemia arising from chronic myelomonocytic leukemia. Mod Pathol. 2013;26: Green CL, Koo KK, Hills RK, et al. Prognostic significance of CEBPA mutations in a large cohort of younger adult patients with acute myeloid leukemia: impact of double CEBPA mutations and the interaction with FLT3 and NPM1 mutations. J Clin Oncol. 2010;28: Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, et al. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood. 2009;113: Dufour A, Schneider F, Hoster E, et al. Monoallelic CEBPA mutations in normal karyotype acute myeloid leukemia: independent favorable prognostic factor within NPM1 mutated patients. Ann Hematol. 2012;91: Kronke J, Bullinger L, Teleanu V, et al. Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia. Blood. 2013;122: Warren M, Luthra R, Yin CC, et al. Clinical impact of change of FLT3 mutation status in acute myeloid leukemia patients. Mod Pathol. 2012;25: Adamia S, Haibe-Kains B, Pilarski PM, et al. A genomewide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets. Clin Cancer Res. 2014;20: Adamia S, Bar-Natan M, Haibe-Kains B, et al. NOTCH2 and FLT3 gene mis-splicings are common events in patients with acute myeloid leukemia (AML): new potential targets in AML. Blood. 2014;123: Ohgami RS, Ma L, Ren L, et al. DNA methylation analysis of ALOX12 and GSTM1 in acute myeloid leukaemia identifies prognostically significant groups. Br J Haematol. 2012;159: Am J Clin Pathol 2015;144:

10 Lin and Falini / AML With Recurrent Genetic Abnormalities 36. Konoplev S, Huang X, Drabkin HA, et al. Cytoplasmic localization of nucleophosmin in bone marrow blasts of acute myeloid leukemia patients is not completely concordant with NPM1 mutation and is not predictive of prognosis. Cancer. 2009;115: Kronke J, Schlenk RF, Jensen KO, et al. Monitoring of minimal residual disease in NPM1-mutated acute myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group. J Clin Oncol. 2011;29: Am J Clin Pathol 2015;144:19-28