Acute Myeloid Leukemia - AML | Choose the Right Test

Medical Experts

Contributor

Li

Associate Professor of Pathology (Clinical), University of Utah

Medical Director, Hematopathology, ARUP Laboratories

Acute myeloid leukemias (AMLs) are heterogeneous blood cancers characterized by myeloid blasts (eg, undifferentiated myeloid precursors) in the blood, bone marrow, and/or other tissues.^, These conditions include AML with myelodysplasia-related gene mutations (MDS/AML) and acute promyelocytic leukemia (APL).^,^, In addition to morphologic evaluation of the bone marrow, immunophenotypic, cytogenetic, and molecular genetic studies are standard of care in AML and are necessary for accurate classification and risk stratification and to guide therapy.^,^,

Quick Answers for Clinicians

How is laboratory testing used in the evaluation of minimal residual disease in acute myeloid leukemia?

The evaluation of minimal residual disease (MRD) in acute myeloid leukemia (AML) usually involves flow cytometry for specific immunophenotypes or real-time quantitative polymerase chain reaction (PCR) for specific genetic abnormalities.^,^, MRD should be assessed following initial induction, before allogeneic hematopoietic stem cell transplantation, and at other times as appropriate for the treatment used. Refer to the Minimal Residual Disease section for more information.

How does laboratory testing for pediatric acute myeloid leukemia differ from testing for adult acute myeloid leukemia?

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDSs) are rare in children. The majority of the laboratory testing strategy for pediatric AML and/or MDSs is the same as that for adult AML and/or MDSs and is discussed in the same clinical guidelines, although the prognostic implications of certain somatic genetic findings may not be established in pediatric populations or may vary between adults and children.^, Many cases of pediatric AML and/or MDSs are associated with hereditary syndromes; therefore, germline genetic testing for such syndromes should be considered in individuals diagnosed with AML before 40-50 years of age.^, There may also be differences between adult and pediatric testing for specific hereditary syndromes associated with bone marrow failure; for example, serum trypsinogen is tested in the evaluation of suspected Shwachman-Diamond syndrome in children, but not in adults. Individuals with Down syndrome are also at increased risk of pediatric AML.

How does laboratory testing inform clinical trial enrollment in acute myeloid leukemia?

The National Comprehensive Cancer Network (NCCN), European LeukemiaNet (ELN), and European Society for Molecular Oncology (ESMO) encourage clinical trial participation,^,^, particularly in clinical trials that offer minimal residual disease (MRD) monitoring. The identification of specific genetic findings in patients with acute myeloid leukemia (AML) may allow for targeted clinical trial enrollment (and treatment selection).^, The NCCN therefore suggests molecular testing for clinically actionable mutations at each AML relapse or progression, and the ELN recommends banking samples to facilitate research.

What is the role of human leukocyte antigen (HLA) testing in acute myeloid leukemia?

Human leukocyte antigen (HLA) typing is recommended for individuals with acute myeloid leukemia (AML) for whom hematopoietic stem cell transplantation is being considered.^, For more information, refer to the ARUP Consult HLA Testing topic.

Indications for Testing

Laboratory evaluation for hematologic malignancies, including AML, should be considered in individuals who present with signs of bone marrow infiltration on a CBC (eg, anemia, thrombocytopenia), constitutional symptoms (eg, fever, fatigue, anorexia, weight loss), leukocytosis with circulating peripheral blasts, and/or AML-related complications (eg, tumor lysis syndrome, disseminated intravascular coagulation [DIC], leukostasis).

Criteria for Diagnosis and Classification

The diagnosis and classification of AML are based on the results of bone marrow examination, bone marrow immunophenotyping (flow cytometry and immunohistochemistry), cytogenetic testing (karyotyping or fluorescence in situ hybridization [FISH]), and molecular genetic tests (eg, massively parallel sequencing, also referred to as next generation sequencing, or NGS).^,^, These tests are used to determine the percentage of blasts and identify recurrent genetic abnormalities, findings which define and distinguish between AML, MDS/AML, APL, myelodysplastic syndromes (MDSs), and B-cell or T-cell lymphoblastic leukemias and lymphomas.^,^,^, The World Health Organization (WHO) and International Consensus Classification (ICC) vary in their classifications of AML and related entities.^, The National Comprehensive Cancer Network (NCCN) does not advocate for one system over the other.

Response Criteria

The therapeutic response criteria for AML are based on the results of bone marrow examination (including blast enumeration) and peripheral blood counts and may include minimal residual disease (MRD) assessment (by polymerase chain reaction [PCR] or flow cytometry).^, A suitable bone marrow specimen must be obtained to evaluate for response.

Laboratory Testing

Initial Workup

In addition to a clinical assessment that includes history and physical examination, recommended laboratory testing in the initial workup of suspected AML includes a CBC with differential and platelet count, peripheral smear, metabolic panel, and uric acid, lactate dehydrogenase, vitamin B12 and B9, fibrinogen, prothrombin time, and partial thromboplastin time tests.^,

Imaging and other nonlaboratory tests may be useful, depending on the patient’s history and presentation.^,

Bone Marrow Biopsy and Aspirate

Use of bone marrow core biopsy and aspirate samples for morphologic assessment, blast enumeration, immunophenotyping, cytogenetics, and molecular genetic testing is recommended.^,^,^, The same pathologist should interpret the results of tests of all specimens, if possible. These procedures are performed in the evaluation and diagnosis of AML, to determine treatment response, to aid in prognosis and therapeutic decision-making, and in ongoing monitoring.^,^,^,

If adequate aspirate cannot be obtained or bone marrow examination cannot be performed for clinical reasons, peripheral blood may be used for diagnosis and other studies if there are enough blasts.^, If a core biopsy specimen is available but aspirate is not, touch imprints and an unfixed, disaggregated core biopsy specimen may be used.

If recommended testing cannot be performed at diagnosis, preservation of an initial sample is recommended for later testing to inform ongoing monitoring. Collection and storage of both bone marrow and blood at diagnosis, remission, and relapse may be useful for future testing and to facilitate research.

Immunophenotyping

Immunophenotyping of bone marrow is recommended in the evaluation of suspected AML and is used in diagnosis, risk stratification, and therapeutic decision-making.^,^, Multiparameter flow cytometry using a panel that includes markers that can distinguish between AML and other acute leukemias is the preferred test.^, Peripheral blood may be used if adequate bone marrow aspirate cannot be obtained and there are enough peripheral blasts. If adequate bone marrow aspirate or peripheral blood cannot be obtained for flow cytometry, immunohistochemistry on an unfixed, disaggregated bone marrow core biopsy specimen may be used.^,

Testing for Cytogenetic Abnormalities

Cytogenetic testing is recommended as part of the evaluation of suspected AML and is used to classify AML and identify abnormalities relevant to prognosis and therapeutic decision-making.^,^, Karyotyping should be performed, and if not successful, FISH testing for markers associated with AML may be performed.

WHO and ICC criteria differ in terms of the somatic genetic rearrangements/abnormalities that define AML; these rearrangements/abnormalities may also be important in prognosis and therapeutic decision-making^,^,^, and are detailed in the following table.

AML-Defining Cytogenetic Abnormalities
Cytogenetic Abnormality	WHO Classification	ICC
RUNX1::RUNX1T1	AML with RUNX1::RUNX1T1 fusion	AML with t(8;21)(q22;q22.1)/RUNX1::RUNX1T1^a
CBFB::MYH11	AML with CBFB::MYH11 fusion	AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/CBFB::MYH11^a
PML::RARA	APL with PML::RARA fusion APL with a variant RARA rearrangement	APL with t(15;17)(q24.1;q21.2)/PML::RARA^a
PML::RARA		APL with other RARA rearrangements^a,b
KMT2A rearrangement	AML with KMT2A rearrangement	AML with t(9;11)(p21.3;q23.3)/MLLT3::KMT2A^a
KMT2A rearrangement	AML with KMT2A rearrangement	AML with other KMT2A rearrangements^a,c
DEK::NUP214	AML with DEK::NUP214 fusion	AML with t(6;9)(p22.3;q34.1)/DEK::NUP214^a
MECOM rearrangement	AML with MECOM rearrangement	AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)/GATA2; MECOM(EVI1)^a
MECOM rearrangement	AML with MECOM rearrangement	AML with other MECOM rearrangements^a,d
BCR::ABL1	AML with BCR::ABL1 fusion^e	AML with t(9;22)(q34.1;q11.2)/BCR::ABL1^e,f
NUP98 rearrangement	AML with NUP98 rearrangement	AML with other rare recurring translocations^a,g
RBM15::MRTFA	AML with RBM15::MRTFA fusion
Other cytogenetic abnormalities	AML with other defined genetic alterations^h
^aAt least 10% blasts required for diagnosis, per ICC. ^bOther RARA rearrangements include t(1;17)(q42.3;q21.2)/IRF2BP2::RARA; t(5;17)(q35.1;q21.2)/NPM1::RARA; t(11;17)(q23.2;q21.2)/ZBTB16::RARA; cryptic inv(17q) or del(17)(q21.2q21.2)/STAT5B::RARA, STAT3::RARA. Other genes are rarely rearranged with RARA:TBL1XR1 (3q26.3), FIP1L1 (4q12), BCOR (Xp11.4). ^cOther KMT2A rearrangements include t(4;11)(q21.3;q23.3)/AFF1::KMT2A; t(11;19)(q23.3;p13.3)/KMT2A::MLLT1; t(6;11)(q27;q23.3)/AFDN::KMT2A; t(10;11)(p12.3;q23.3)/MLLT10::KMT2A; t(10;11)(q21.3;q23.3)/TET1::KMT2A; t(11;19)(q23.3;p13.1)/KMT2A::ELL. ^dOther MECOM rearrangements include t(2;3)(p11~23;q26.2)/MECOM::?; t(3;8)(q26.2;q24.2)/MYC, MECOM; t(3;12)(q26.2;p13.2)/ETV6::MECOM; t(3;21)(q26.2;q22.1)/MECOM::RUNX1. ^eAt least 20% blasts required for diagnosis, per WHO. ^fPer the ICC, the entity with BCR::ABL1 and ≥10-19% blasts is not classified as MDS/AML because it overlaps with chronic myeloid leukemia. ^gOther rare recurring translocations include t(1;3)(p36.3;q21.3)/PRDM16::RPN1; t(3;5)(q25.3;q35.1)/NPM1::MLF1; t(8;16)(p11.2;p13.3)/KAT6A::CREBBP; t(10;11)(p12.3;q14.2)/PICALM::MLLT10; t(16;21)(p11.2;q22.2)/FUS::ERG; t(16;21)(q24.3;q22.1)/RUNX1::CBFA2T3; and the following found mainly in children and infants: t(1;22)(p13.3;q13.1)/RBM15::MRTF1; t(5;11)(q35.2;p15.4/NUP98::NSD1; t(11;12)(p15.4;p13.3)/NUP98::KMD5A; NUP98 and other partners; t(7;12)(q36.3;p13.2)/ETV6::MNX1; inv(16)(p13.3q24.3)/CBFA2T3::GLIS2. ^hOther defined genetic alterations include RUNX1T3(CBFA2T3)::GLIS2; KAT6A::CREBBP; FUS::ERG; MNX1::ETV6; NPM1::MLF1. Sources: Arber, 2022; Khoury, 2022

Other cytogenetic abnormalities important in AML include myelodysplasia-related cytogenetic abnormalities, specifically, complex karyotype and/or del(5q)/t(5q)/add(5q), -7/del(7q), +8, del(12p)/t(12p)/add(12p), i(17q), -17/add(17p)/del(17p), del(20q), or idic(X)(q13).^,

Refer to the European LeukemiaNet (ELN) recommendations for specific variant-based risk stratification information.^,

Molecular Genetic Testing for Other Somatic Variants

An evaluation of somatic molecular markers by PCR (for individual mutations) or NGS (for multigene panel testing) is recommended in all individuals with AML.^, This testing is important for classification, risk assessment, and prognosis in AML and may guide treatment decisions.^,^, A platform that tests for molecular genetic mutations and chromosomal rearrangements at the same time may be considered. Caution is recommended when interpreting the results of panel tests performed on tumor tissue because somatic reversion may yield false-negative results. Copy number variants (CNVs) are important mutations in AML but may not be detected by all panels.

WHO and ICC criteria differ in terms of the somatic gene mutations that define AML; these mutations may also be important in prognosis and therapeutic decision-making^,^,^, and are detailed in the following table.

AML-Defining Somatic Mutations
WHO	ICC
AML with NPM1 mutation	AML with mutated NPM1^a
AML with CEBPA mutation^b,c	AML with in-frame bZIP CEBPA mutations^a
Not included	AML^b,dand MDS/AML14 with mutated TP53
^a≥10% blasts required for diagnosis. ^b≥20% blasts required for diagnosis. ^cCEBPA mutations include biallelic mutations (biCEBPA) and single mutations in the bZIP region (smbZIP-CEBPA). ^dAt least 1 pathogenic TP53 mutation with VAF ≥10%. VAF, variant allele frequency Sources: Arber, 2022; Khoury, 2022

Other somatic gene mutations that are important in AML include^,:

c-KIT (for risk stratification)
FLT3-ITD and FLT3-TKD mutations (for targeted therapy)
IDH1/IDH2 mutations (for targeted therapy)
Myelodysplasia-related gene mutations: ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2

Refer to the ELN recommendations for specific variant-based risk stratification information.^,

Monitoring and Surveillance

Minimal Residual Disease

MRD, also referred to as measurable residual disease, refers to small populations of leukemic cells that remain after treatment. The detection of MRD may be useful in determining response, remission, or relapse; in relapse risk assessment; in prognosis; in treatment decision-making; and as a surrogate endpoint in clinical trials.^,^, In AML, MRD testing is usually performed via real-time quantitative PCR, droplet digital PCR (ddPCR), or multiparameter flow cytometry for specific abnormalities detected at diagnosis.^,^, In most clinical laboratories, NGS is generally not used for MRD assessment because it is not as sensitive as flow cytometry and PCR tests, although the data from error-corrected NGS is promising in ongoing research.^,^, Thresholds for detecting MRD are different from thresholds used for diagnosis; therefore, MRD-specific assays are recommended.^,

MRD testing is recommended after initial induction and before stem cell transplantation and may be performed at other times, depending on the specific treatment administered. The use of a high-quality bone marrow sample (an early pull of bone marrow aspirate) is especially important for MRD assessment in AML and is recommended in most cases, although peripheral blood may be used for some assays.

Flow cytometry for specific immunophenotypes or PCR for specific genetic abnormalities on bone marrow aspirate specimens is used in ongoing MRD monitoring. The specific strategy for and cadence of monitoring depends on the diagnosis and type of treatment. Multigene panel testing may be useful to aid in treatment decision-making and clinical trial enrollment and should be considered at each progression or relapse.

Other Monitoring Tests

Bone marrow aspiration and biopsy are performed during monitoring in AML.^, Other tests used in monitoring include, but are not limited to, blood urea nitrogen, CBCs with differentials, coagulation tests, liver and kidney function tests, and creatinine, electrolyte, phosphorous, platelet, and uric acid tests. The frequency of testing depends on the specifics of therapy and other clinical indications. Refer to the NCCN guidelines for recommended testing cadences.

Surveillance

CBCs with differentials and peripheral smears are used to surveil for relapse in AML. Bone marrow aspiration and biopsy may be performed depending on the results of other tests. Refer to the NCCN guidelines for the recommended testing cadence.

The role of molecular testing in surveillance is a matter of ongoing research.

Hereditary Cancer Assessment

Germline molecular genetic testing for hereditary hematologic malignancies may be informative, particularly in individuals who:

Have been diagnosed with AML at any age
Have a somatic variant associated with a potential germline variant
Have a personal history of two or more cancers
Have a personal history of a hematologic cancer with a family history of hematologic abnormalities, any cancer, or early cancer diagnosis

Germline molecular testing in AML may take the form of a targeted NGS panel or whole exome or genome sequencing (WES/WGS) and assessment for CNVs (eg, as part of WES/WGS or via I microarray).^,

Several genes have been associated with inherited AML, MDS/AML overlap conditions, and related conditions (eg, some anemias).^,^,^, Such genes, which may be included in a panel test, include ACD, ANKRD26, ATG2B, BLM, BRCA1, BRCA2, CBL, CEBPA, CHEK2, CTC1, DDX41, DKC1, DNAJC21, EFL1, ELANE, ERCC6L2, ETV6, FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BRIP1/BACH1, FANCL, FANCM, FANCN/PALB2, FANCO/RAD51C, FANCP/SLX4, FANQ/ERCC4, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCU/XRCC2, FANCV/REV7/MAD2L2, G6PC3, GATA1, GATA2, GFI1, GSKIP, HAX1, JAGN, KRAS, MDM4, MLH1, MPL, MSH2, MSH6, NAF1, NBN NF1, NHP2, NOP10, NPM1, NRAS, PARN, PMS2, POT1, PTPN11, RECQL4, RPA1, RPL5, RPL11, RPL15, RPL23, RPL26, RPL27, RPL31, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS28, RPS29, RTEL1, RUNX1, SAMD9/SAMD9L, SBDS, SRP54, TCRG1, TERC, TERT, TINF2, TSR2, VPS45A, WAS, WRAP53, and ZCCHC8.^,^,^,^, Additional germline variants associated with AML and related disorders are also being researched.