Genetic Testing in the Diagnosis and Biology of Acute Leukemia

2017 Society for Hematopathology/European Association for Haematopathology Workshop Report

Marian H. Harris, MD, PhD; David R. Czuchlewski, MD; Daniel A. Arber, MD; Magdalena Czader, MD, PhD


Am J Clin Pathol. 2019;152(3):322-346. 

In This Article

Genetic Abnormalities Indicating an Undiagnosed Underlying Neoplasm, Disease Evolution, Clonal Relationships, and Residual Disease

Genetic heterogeneity and clonal evolution are well known to occur in acute leukemias. Stepwise acquisition of driver and passenger mutations, genetic diversification, and selection of individual "most fit" clones occur during the natural disease course and during therapy. These phenomena have diagnostic, prognostic, and therapeutic implications and emphasize the importance of repeated testing at critical time points. In the following paragraphs, we discuss cases that illustrate advantages and challenges associated with sequential testing Table 4.

Evaluation or re-evaluation of original diagnostic samples is always recommended to establish a baseline. When patients present late in a disease or the original specimen is not available, targeted testing of individual cell populations and successive testing of follow-up samples may help to diagnose an underlying chronic myeloid neoplasm and determine clonal relationships. Several cases illustrating the value of such testing were presented in the workshop. An unusual scenario of severe thrombocytosis developing after induction therapy for AML with mutated NPM1 was described in case 240. Morphologic features were consistent with MPN, unclassifiable and RT-PCR demonstrated a rising JAK2 mutation. In this case, an absence of mutated JAK2 and lack of MPN-like megakaryocytes in the original AML sample supported the diagnosis of de novo AML with mutated NPM1 rather than blast crisis of MPN. Case 232 described T-ALL with BCR-ABL1 rearrangement presenting in a lymph node. BCR-ABL1–positive T-ALL accounts for 2% to 4% of T-ALL and can occur de novo or as a blast crisis of chronic myelogenous leukemia (CML).[63] The majority of BCR-ABL1–positive T-ALL cases reported previously showed a major breakpoint region raising a differential diagnosis of blast crisis of preexisting CML. In case 232, the bone marrow was unremarkable with a minimal BCR-ABL1 fusion by PCR (<1%), which may represent a minute population of leukemic blasts. Therefore a diagnosis of de novo BCR-ABL1 T-ALL was favored. In contrast, case 294 was originally diagnosed as AML with BCR-ABL1 and inv(16)(p13.1q22). However, when interphase FISH confirmed BCR-ABL1 fusion in segmented neutrophils, the diagnosis was updated to CML, BCR-ABL1–positive, in blast phase [with inv(16)(p13.1q22)].

Arguably, the most important clinical implication of clonal evolution is an emergence of therapy-resistant clones and appearance of genetic abnormalities amenable to targeted therapy. Thus, an important goal of repeated testing at disease progression is to detect such clones, identify therapeutic targets, and determine eligibility for clinical trials.

The classic tyrosine kinase fusion, BCR-ABL1, can develop late in a disease, either as a dominant clone or a subclone. BCR-ABL1–positive clones can appear in the course of AML as well as MDS, and are typically a poor prognostic indicator.[64] This phenomenon needs to be distinguished from BCR-ABL1–positive AML, a provisional entity in the current WHO classification, which is diagnosed in patients without prior history of CML, with no prior chemotherapy/radiation, and no recurrent abnormalities defining other types of AML. At times, when a detailed history is not available, this entity may be challenging to distinguish from blast phase of previously undiagnosed CML. In case 177, only postinduction bone marrow was available for review and FISH analysis showed BCR-ABL1 fusion and KMT2A gene rearrangement. No karyotype was reported. KMT2A gene rearrangement is exceedingly rare in CML; however, it can occur in chronic, accelerated, and blast phase.[65] Although case 177 may represent a de novo AML with BCR-ABL1 and KMT2A rearrangement, a review of the diagnostic sample, complete cytogenetic evaluation, and sequencing may be helpful to exclude a previously undiagnosed CML. In postinduction samples, preexisting undiagnosed CML can be excluded using FISH or RT-PCR of segmented neutrophils.[66] The BCR-ABL1–positive blast crisis in a patient with previously diagnosed BCR-ABL1–negative MPN (case 81, long-standing history of essential thrombocythemia) is a rare event, but nevertheless critical to recognize due to therapeutic implications.[67] At times, BCR-ABL1 rearrangement is only seen in a subset of blasts, suggestive of late acquisition of the BCR-ABL1 fusion. Case 187 described AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 and subclonal BCR-ABL1 (minor breakpoint). Subclonal populations were also seen in case 317, which described a B-ALL with the majority of cells harboring t(14;18)(q32;q21), a minor subclone with BCR-ABL1 p190 fusion, and a FLT3-ITD. The BCL2 rearrangement has been previously reported in rare cases of B-ALL and is frequently associated with MYC rearrangement. B-ALL with BCL2 rearrangements is reported to have dismal prognosis, which in the workshop case was further amplified by the presence of FLT3-ITD and a BCR-ABL1 subclone.[68] Case 94 provides an additional example of repeat testing detecting clonal evolution and leading to targeted therapy. A 64-year-old man initially presented with AML, NOS with FLT3-ITD. Following FLT3-directed therapy with gilteritinib, the patient experienced progressive disease with loss of FLT3-ITD and an acquisition of a new leukemic subclone with NUP214-ABL1 fusion. This rearrangement is well-described in ALL (particularly T-ALL) but has not been previously reported in AML. Sorafenib was added to the salvage chemotherapy regimen and the patient achieved sustained remission prior to and following HSCT. In case 184, stepwise acquisition of resistant clones with numerical abnormalities detected by conventional karyotyping and gene mutations in DNMT3A, IDH1, FLT3, and TP53 were seen in sequential samples of AML relapse. Blasts at the second relapse showed cytoplasmic vacuoles typically seen in association with TP53 mutations. Additional abnormalities acquired in the course of AML progression, in particular a complex karyotype, TP53 and FLT3 mutations, are associated with dismal prognosis. Similar progression to a complex karyotype was seen in case 279. The patient initially presented with AML with t(16;16)(p13.1;q22); CBFB-MYH11, FLT3-ITD, and FLT3-TKD. Even though AML with CBFB-MYH11 is in the favorable risk category, a presence of FLT3-ITD increases the risk of recurrence. Indeed, the patient relapsed after a 2-year long remission, at which time the bone marrow showed additional numerical chromosome abnormalities, JAK2 V617F, JAK2 exon 12, and WT1 mutations. FLT3-ITD and FLT3-TKD were no longer present. JAK2 V617F mutations have been reported in de novo AML; in contrast, however, JAK2 exon 12 mutations are exceedingly rare in AML.[69] In case 285 (AML, NOS [acute monoblastic leukemia]), there were three mutations within the same signaling pathway, FLT3-ITD, KRAS, and NRAS, and independent WT1 mutations. Follow-up samples demonstrated that only three mutations persisted throughout the disease course and therefore these likely represented driver events. The presence of different mutations affecting the same pathway, as seen in cases 279 and 285, seems redundant; it has nevertheless been previously reported.

Sequencing can help to confirm clonal relationship across various disease stages (chronic vs blast phase), in relapse specimens, in different anatomic sites, or even between samples with distinct diagnoses. An excellent illustration of the value of repeat testing in demonstrating clonal relationships was shown in case 73, in which an underlying CMML was first diagnosed after induction therapy for AML. The postinduction bone marrow showed morphologic features of CMML and ASXL1, NRAS, SRSF2, and KRAS mutations. These mutations were also seen at the time of AML diagnosis. During the course of the disease, the patient developed papules and plaques, and was diagnosed with leukemia cutis with features of CMML with Langerhans cell differentiation. The latter specimen showed several mutations identical to those seen in prior samples. An interesting evolution of t-MDS with progression to overt acute leukemia was reported in case 297. At the time of progression to t-AML, a prominent proliferation of small plasmacytoid dendritic cells negative for CD56, CD2, and TdT was seen in the bone marrow. FISH showed 7/7q deletion in 87% of nuclei, and TET2 and ZRSR2 with VAF of 51% and 83%, respectively. Eighteen months later, bone marrow showed 15% blasts and plasmacytoid dendritic cells with blastic morphology and positivity for CD56 and partial TdT. The frequency of 7/7q deletion, and TET2 and ZRSR2 mutations remained unchanged. Another example of clonal relationship between therapy-related CMML and previously diagnosed B-ALL was suggested in case 56 due to a common deletion at 3′ of the KMT2A gene present in both neoplasms.

Quantitative evaluation of residual disease is well established in a number of hematopoietic neoplasms. Determination of residual disease is critical especially during therapy leading to HSCT. In AML, the significance of minimal residual disease (MRD) determined by flow cytometry or molecular methods has been recognized and is slowly making its way into clinical practice. Molecular evaluation of residual disease in AML has been confounded by a complex dynamic, including evolution of leukemic clones, background clonal hematopoiesis of indeterminate potential (CHIP), and germline abnormalities. Two cases discussed in session 7 highlighted challenges of genetic testing in posttreatment samples. Case 69 described AML with mutated NPM1, and KIT, DNMT3A, and TET2 mutations. Despite a complete morphologic remission, two of the mutations, DNMT3A and TET2, persisted on day 31 after treatment. The patient subsequently relapsed with AML showing the same karyotype and mutations as seen at the time of original diagnosis. The persistence of DNMT3A and TET2 mutations most likely represented background clonal hematopoiesis, which in previous studies was not associated with a risk of relapse.[70] The phenomenon of clonal hematopoiesis adds complexity to the genetic evaluation of patients with acute leukemias and can be particularly challenging when evaluating MRD. In this context, mutations known to occur most commonly in CHIP should be regarded with caution and interpretation of next-generation sequencing results should focus on leukemia- and patient-specific alterations. A combination of genetic testing and multiparameter flow cytometry can also be helpful and has been shown to enhance the predictive value of MRD in AML.[70]

Evaluation of blood and bone marrow samples obtained post-HSCT can be complicated by preexisting mutations of donor origin, including germline mutations and CHIP. Therefore, care must be taken not to over-interpret such abnormalities as clonal evolution. Case 155 illustrated this issue in a patient with a previous diagnosis of t-AML with a complex karyotype and FLT3 mutations. After engraftment, the patient was diagnosed with a recurrent leukemia showing several previously seen chromosomal abnormalities, a new CSF3R mutation of unknown significance at VAF of 11%, and a pathogenic mutation in STAG2 at 39%. The latter corresponded to the proportion of blasts in the sample and likely represented clonal evolution. The CSF3R mutation was confirmed to be a germline mutation of donor origin and nonpathogenic.