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Chapter 58 Pathobiology of Acute Myeloid Leukemia 921

Clonal Hierarchy of Acute Myeloid Leukemia A broader understanding of the mutational landscape of AML has
provided a basis for development and testing of novel targeted thera-
The recognition that some mutations may be lost or acquired at relapse pies and monitoring of disease. The role of residual disease monitor-
(e.g., FLT3-ITD), while others are present at diagnosis and remain ing may occur through cytogenetic, flow cytometric, and now also
stable (e.g., NPM1, PML-RARA, and CBF rearrangements) suggests molecular assessment. The incorporation of these residual disease
that distinct clonal populations can coexist in an AML sample. markers into clinical practice remains under intense investigation;
Sequencing of serially obtained samples has revealed a clonal hierarchy similar to patients with chronic myeloid leukemia, it may eventually
in AML, beginning with a founding clone that represents the initial inform treatment duration and maintenance therapies.
population that becomes dominant in the bone marrow. The founding
clone spawns subclones that retain founding clone mutations, but gain Molecular Diagnostics in Acute Myeloid Leukemia
additional mutations that confer a growth advantage. The clonal
architecture evolves over time as a feature of the natural history of the The various mutations and dysregulated pathways integral to the
disease, or in response to selective pressure imposed by therapy. Analysis pathobiology of acute myeloid leukemia (AML) also yield specific
of AML cases that entered morphologic remission after cytotoxic therapeutic targets and offer clear prognostic implications for patients.
chemotherapy, and later relapse, revealed that the dominant clone at As such, molecular testing at the time of AML diagnosis has become
relapse retains founding clone mutations. These observations raise the the standard of care, to assist with subclassification of disease, risk
hypothesis that therapies directed at eradicating drivers of the founding stratification, selection of an induction regimen, and consolidation
clone may be more effective than therapies targeting subclones. preferences. Diagnosis of AML requires analysis of a specimen demon-
strating excess myeloblasts. For patients with high levels of circulating
Somatic mutations accumulate in an age-dependent manner in disease, some studies may be performed with the use of peripheral
normal individuals without perturbing hematopoiesis in most cases. blood samples; however, assessment of a bone marrow biopsy and
However, several large cohort sequencing studies have demonstrated aspirate is essential.
that up to 10% of apparently healthy individuals over 65 years of age
have detectable clonal populations that are marked by somatic muta- Standard Evaluation
tions, often in genes that are recurrently mutated in MDS/AML The cornerstone of the diagnosis of AML remains morphologic assess-
(particularly in epigenetic regulators, including DNMT3A, TET2, and ment. In the current WHO guidelines, at least 20% of the bone marrow
cellularity must be comprised of myeloblasts, except in the presence
ASXL1). Although clonally skewed hematopoiesis has been associated of the t(8;21), t(16;16)/inv(16), or t(15;17) rearrangements, which
with an elevated risk of later developing a hematologic malignancy, the are sufficient for an AML diagnosis regardless of blast count. In addi-
vast majority of these individuals do not progress to MDS or AML. tion, promonocytes in acute monocytic leukemia, megakaryoblasts in
Additional epidemiologic study will be required to determine how acute megakaryocytic leukemia, and abnormal promyelocytes in acute
often clonal hematopoiesis is a precursor to MDS/AML and whether promyelocytic leukemia are added to the blast percentage. Only in pure
surveillance and/or early intervention are warranted. erythroleukemia are erythroblasts included in the blast count.
Flow cytometry utilizes multiparametric analysis of single cells to
assess cellular granularity and size, cell surface, intracellular antigen
Leukemia Stem Cells expression, and other features. The coexpression of certain cell surface
markers may help to confirm myeloid cell origin, identify immature
+
blasts, typically CD34 and CD117 , and also to distinguish an aberrant
+
Normal HSCs are characterized by their capacity for self-renewal and phenotype of a leukemic blast population. Flow cytometry can enumer-
multilineage differentiation, properties that can only be assessed in ate small populations of leukemic cells, below the limit of detection
vivo using functional assays such as xenotransplantation into immune by morphology. For this reason, flow cytometric analysis has been
deficient mouse models. The pioneering studies of John Dick and developed as a platform to monitor minimal residual disease (MRD).
colleagues demonstrated that a similar cellular hierarchy exists in The role of MRD assessment by flow cytometry in routine AML care
AML, in which rare cells with an immunophenotype similar to remains under investigation.
normal stem/progenitor cells are enriched for the capacity to initiate A critical element in the initial laboratory assessment of AML is
leukemia in xenotransplantation models. Further work has led to the cytogenetic analysis of a bone marrow aspirate specimen. This provides
important prognostic data for risk stratification and informs therapeutic
identification of several cell surface antigens that are preferentially strategies. Cells from the aspirate are cultured, mitosis is interrupted,
expressed on leukemia-initiating cells compared with normal HSCs, and the paired chromosomes are arranged to identify missing, trans-
including CD123, CD99, and TIM3. located, or duplicated segments. Fluorescence in situ hybridization
Interestingly, cells with an HSC immunophenotype that are utilizes fluorescently labeled DNA probes and can identify gains and
capable of engraftment in mice and multilineage differentiation in losses of chromosomal material, as well as rearrangements that may
vivo can be recovered from the bone marrow of patients with AML. be cryptic using conventional banding techniques.
These “preleukemic stem cells” may harbor the same class of muta- Detection of somatic mutations that are known drivers of AML
tions detected in individuals with clonally skewed hematopoiesis biology can aid in the initial risk classification of patients with AML,
(e.g., DNMT3A), but lack the full complement of mutations found particularly those with intermediate-risk cytogenetics. Current National
Comprehensive Cancer Network guidelines recommend testing of four
in the bulk AML sample, suggesting that they are ancestral. The genes (KIT, FLT3, NPM1, and CEBPA) at diagnosis, because their
contribution of these preleukemic stem cells to chemotherapy resis- prognostic significance has been validated in large cohorts (level 2A
tance and relapse remains to be determined. evidence). Testing for mutations in RUNX1 is recommended for WHO
classification. In addition, use of targeted therapies is increasingly
dependent on detection of a specific tumor genotype.
FUTURE DIRECTIONS Investigational Testing
Given the rapidly expanding number of genes that are recognized
AML comprises a heterogeneous spectrum of disease, which shares a targets of recurrent somatic mutation in AML, more comprehensive
common myeloid phenotype and is characterized by accumulation of mutational profiling is beginning to enter routine clinical practice. With
abnormal leukemic myeloblasts. The development of AML occurs increasing numbers of genes to query, next-generation sequencing
through serial acquisition of somatic mutations, resulting in clonal approaches offer advantages in sensitivity, cost, and efficiency over
expansion and genetic evolution of this disease. As our understanding traditional testing methods (e.g., polymerase chain reaction, Sanger
of the genetic underpinnings has grown, it is clear that there are sequencing). Large panels of genes can be tested simultaneously
various pathways to the development of AML (Fig. 58.3). Moreover, by pre-enriching for the targets of interest (by automated amplicon
some mutations appear to be earlier, founding events, while other generation, or hybridization capture). With further improvements in
mutations are typically acquired during disease progression. The analytical workflow and cost reduction, whole-genome and transcrip-
tome sequencing could displace some existing diagnostic tools, as
further identification of the sequence of mutation acquisition, as well these platforms can provide simultaneous detection of mutations, gene
as cooccurring and mutually exclusive genetic pathways, will help to expression, copy number alteration, and structural variation.
better distinguish different subsets of this disease.