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

Acute Myeloid Leukemia

The CAP/ASH guidelines on the diagnostic workup of acute leukemia, the European LeukemiaNet (ELN) guidelines on the diagnosis and management of AML in adults, and the National Comprehensive Cancer Network (NCCN) clinical practice guidelines on AML all agree that karyotype and FISH (as appropriate) should be performed for new diagnoses of AML.[2–4] In addition, the CAP/ASH guidelines recommend that the workup of AML should include testing for FLT3-internal tandem duplication (ITD) mutations, and may also include testing for mutations in IDH1, IDH2, TET2, WT1, DNMT3A, and/or TP53, which may be prognostically or therapeutically significant.[2] Leukemias with core binding factor rearrangements (core binding factor [CBF]-AML, RUNX1-RUNX1T1 or CBFB-MYH11) should also be tested for KIT mutations, while leukemias other than CBF-AML, acute promyelocytic leukemia (APL), or AML with myelodysplasia-related changes (AML-MRC) should be tested for mutations in NPM1, CEBPA, and RUNX1, which, if positive, would place the leukemia in a specific subtype. The ELN recommendations include testing for mutations in ASXL1, CEBPA, FLT3 (ITD and TKD, including the allelic ratio), IDH1, IDH2, KIT, NPM1, RUNX1, and TP53.[3] The NCCN guidelines recommend screening for mutations in ASXL1, CEBPA, FLT3 (ITD and tyrosine kinase domain [TKD]), IDH1, IDH2, KIT, NPM, RUNX1, and TP53.[4] The field is changing rapidly,[5–7] and all three organizations acknowledge that our understanding of the diagnostic, prognostic, and/or therapeutic significance of genetic alterations will continue to evolve.

AML With Recurrent Genetic Abnormalities

Session 3 included 14 cases of AML with recurrent genetic abnormalities (AML-RGA) as defined by the WHO classification Table 1. Among these cases, there were three cases of APL, one RUNX1-RUNX1T1 AML (co-occurring with chronic lymphocytic leukemia [CLL]), one inv(3)(q21q26.2) AML, three cases of AML with BCR-ABL1, one AML with KMT2A rearrangement, and five cases of AML with mutated RUNX1. The workup of all 14 cases included karyotype and either FISH or polymerase chain reaction (PCR)-based screening for recurrent chromosomal rearrangements. Ten cases used DNA sequencing (with substantial variation in panel size) and two cases used RNA sequencing for fusion detection.

Both cases that used RNA sequencing were cases of APL in which testing for the classic PML-RARA fusion had been negative. All three cases of APL in this session had difficult to detect RARA rearrangements, with normal karyotypes and negative FISH studies. The RARA rearrangements were ultimately detected by advanced molecular techniques, including RT-PCR, RNA sequencing, and single-nucleotide polymorphism (SNP) array, illustrating the power of combining clinical, morphologic, and immunophenotypic information with molecular genetics. For example, case 180 described a case in which APL was suspected, but karyotype, FISH, and RT-PCR were negative for a RARA translocation. RNA sequencing revealed a novel IRF2BP2-RARA translocation, confirming the diagnosis of APL Image 1. The leukemia was sensitive to all-trans retinoic acid (ATRA), both at diagnosis and at a subsequent relapse, underscoring the clinical relevance of this molecular genetic finding.

Image 1.

Case 180. A, Hypercellular marrow with sheets of atypical cells with irregular nuclei and abundant eosinophilic cytoplasm (H&E). B, Bone marrow aspirate smear shows these atypical cells have distinct cytoplasmic granules. No Auer rods were noted (Wright-Giemsa). C, Primer design covering IRF2BP2 exon 2 and RARA exon 3 or intron 2 for detection of IRF2BP2-RARA fusion in cDNA and genomic DNA (gDNA), respectively. D, Results from reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR for detection of IRF2BP2-RARA fusion. RT-PCR using forward primer from IRF2BP2 exon 2 and reverse primer from RARA exon 3 gave rise to an amplicon of 310 bp. Genomic PCR using forward primer from IRF2BP2 exon 2 and reverse primer from RARA intron 2 gave rise to an amplicon of 323 bp. E, Direct sequencing of genomic PCR products revealed a IRF2BP2-RARA fusion with a distinct breakpoint and part of sequences from IRF2BP2 exon 2 and RARA intron 2. FP, forward primer; RP reverse primer. D, Courtesy of Cameron Yin, Keyur P. Patel, Zhuang Zuo, Sa A. Wang, Shaoying Li, L. Jeffrey Medeiros, and Carlos E. Bueso-Ramos.

The high number of submitted cases of AML with mutated RUNX1 in this session (cases 53, 164, 281, 313, and 370), in addition to the high number of cases of myeloid neoplasms with germline RUNX1 mutation in session 1, highlights the need to consider family history and germline testing as part of the diagnostic workup, which is of particular relevance in this diagnosis as similar RUNX1 mutations may be seen in the somatic and germline settings.[8]

Session 7 cases discussed in this section include three cases of AML-RGA with unusual mutations that together highlight the importance of careful integration of morphologic and molecular genetic findings (Table 1). Case 116 described AML with t(8;21)(q22;q22.1);RUNX1-RUNX1T1 and FBXW7 mutation. FBXW7 mutations are exceedingly rare in AML, and have been primarily reported in T-ALL, CLL, and solid tumors. FBXW7 is a tumor suppressor gene responsible for tagging oncoproteins for degradation by the ubiquitin-proteasome system and is the most commonly affected gene of the ubiquitin-proteasome system.[9] Case 301 described AML with mutated NPM1 and additional mutations in PTPN11, IDH1, and RUNX1. Whereas NPM1 mutation is frequently accompanied by mutations in genes responsible for DNA methylation (DNMT3A, TET2, IDH1, IDH2) and signaling (FLT3, NRAS, PTPN11), NPM1 and classic RUNX1 mutations are typically considered mutually exclusive. Rare reported instances of coexisting RUNX1 and NPM1 mutations showed atypical RUNX1 variants, similar to the RUNX1 p.R19K mutation seen in case 301.[10] Case 144 demonstrated a response of a leukemic clone to FLT3 inhibitor in a patient with AML with mutated NPM1 and FLT3-ITD (as well as mutations in DNMT3A and TET2). The patient achieved morphologic remission Image 2, but molecular genetic analysis revealed that the variant allele fraction (VAF) of the FLT3-ITD, NPM1, DNMT3A, and TET2 mutations remained unchanged. The discordance between the morphology and next-generation sequencing results suggests that the blasts underwent differentiation under the influence of quizartinib, similar to the pattern seen in APL treated with ATRA therapy. Such differentiation response has been previously described in patients with AML with normal karyotype and FLT3-ITD after FLT3 inhibitor therapy.[11] In contrast, patients with a complex karyotype responded with clearance of blasts and showed a hypocellular marrow indicative of a cytotoxic response to the FLT3 inhibitor.

Image 2.

Case 144. A, Pretherapy bone marrow biopsy with 85% blasts. B, Bone marrow aspirate posttherapy with FLT3 inhibitor showing differentiation of the leukemic clone to segmented neutrophils. Courtesy of Siddharth Bhattacharyya, Grant E. Nybakken, Darshan Roy, Jennifer Morrissette, Christopher Watt, Alexander Perl, Martin Carroll, Jonathan Canaani, and Adam Bagg.

AML With Myelodysplasia Related Changes, Therapy-related MDS/AML, and Acute Myeloid Leukemia, Not Otherwise Specified

Ten additional cases of AML were presented in session 3 Table 2, including five cases of AML-MRC, three cases of therapy-related myeloid neoplasms (therapy-related myelodysplastic syndrome [t-MDS]/AML), one case of AML not otherwise specified (AML-NOS), and one case that could not be classified. The diagnostic workup of all 10 cases included karyotype, with additional FISH studies performed in eight cases. Eight cases included DNA sequencing, and three used RNA sequencing for fusion detection. The cases in this group showed a wide variety of genetic alterations. Some alterations were diagnostic of a particular subtype of leukemia, for example the i(17q) detected in case 54 is diagnostic of AML-MRC. Other alterations carried significant prognostic and/or therapeutic information, such as the FLT3-ITD mutation in case 54, which led to treatment with sorafenib, or the identification of a CBFA2T3-GLIS2 rearrangement in case 276, which is associated with a poor prognosis in pediatric acute megakaryoblastic leukemia, and led to a decision to transplant the patient in first remission.[12,13]

Session 7 cases discussed in this section include four cases of AML-MRC, four cases of therapy-related myeloid/lymphoid neoplasms, and five cases of AML, NOS (Table 2). These cases presented with rearrangements and mutations seen in unusual clinical contexts, some with prognostic significance or important for selection of targeted therapy. Case 148 reported a 24-year-old male who initially presented with a myeloid sarcoma and was subsequently diagnosed with AML-MRC. Blasts showed monocytic differentiation and prominent erythrophagocytosis Image 3, which prompted further analysis of the complex karyotype by FISH. A three-way translocation, t(1;16;8)(q21;p13;p11), resulting in a cryptic KAT6A-CREBBP fusion, was detected. This fusion is typically created by t(8;16)(p11;p13), which can occur in de novo or t-AML. AML with KAT6A-CREBBP has been described in children, frequently less than 2 years old, and in adults. Blasts typically show monocytic differentiation and erythrophagocytosis. There is frequent extramedullary involvement including leukemia cutis, myeloid sarcoma, and central nervous system disease. In adults, KAT6A-CREBBP fusion has been associated with worse outcome presumably due to its association with therapy-related disease; however, one case of spontaneous regression has been reported.[14,15] In the pediatric group, KAT6A-CREBBP fusion does not appear to adversely affect survival and has been more frequently reported in cases of spontaneous regression,[16] which may prompt expectant management. An interesting presentation of AML with t(8;16)(p11;p13) was reported in case 357, describing an 80-year-old male with a history of plasma cell myeloma and chronic myelomonocytic leukemia (CMML). This patient showed stepwise acquisition of genetic abnormalities including SETBP1 mutation upon a progression of CMML and KAT6A-CREBBP fusion at the time of a subsequent diagnosis of AML. These rare cases underscore the key role of morphology: the finding of erythrophagocytosis by blasts can drive the identification of the cryptic KAT6A-CREBBP rearrangement, which in a pediatric population may trigger a wait and watch approach.

Image 3.

Case 148. Prominent erythrophagocytosis by leukemic blasts seen in case with KAT6A/CREBBP rearrangement. Courtesy of Madhu M. Ouseph, Zakaria Grada, Karen A. Ferreira, Mark P. LeGolvan, Olga K. Weinberg, and Christopher P. Elco.

A number of AML cases included unusual mutations, which could be therapeutic targets. BRAF mutations are characteristic of hairy cell leukemia and may also be seen in plasma cell myeloma, histiocytic neoplasms, CLL, B-ALL, and unclassifiable splenic B-cell lymphoma. While they are rare in de novo AML, they have been described in AML with monocytic differentiation and in t-AML with KMT3A rearrangements.[17–20] Similar to case 37, the majority of BRAF mutations in AML occur in the kinase domain, causing constitutive activation of the MEK/ERK pathway. One reported case of BRAF-mutated, KMT2A-rearranged, therapy-related myeloid neoplasm (t-MN) showed short-term improvement after therapy with a BRAF inhibitor and recurred with new mutations in KRAS, suggesting bypassing of the BRAF blockade of the MEK/ERK pathway.[21]

Two cases, diagnosed as AML-MRC and AML, NOS, showed JAK2 V617F mutation, which occurs in approximately 1% of de novo AML. These leukemias show important differences when compared to JAK2 V617F mutated AML arising in a setting of a myeloproliferative neoplasm (MPN).[22–24] AML ex MPN more frequently presents with splenomegaly, megakaryocytic atypia, complex karyotype, and higher VAF of the JAK2 V617F mutation. De novo AML with JAK2 mutation shows additional abnormalities, including mutations in genes associated with DNA methylation.[25] Consistent with these findings, in workshop case 57, de novo AML, NOS with JAK2 V617F, there were two additional mutations, in ASXL1 and IDH2, which occurred at slightly higher VAF than JAK2 V617F. An unusual feature of this case was that, at relapse, the ASXL1 and IDH2 variants persisted, but the JAK2 V617F was not detected. This supports a hypothesis that JAK2 V617F occurring in de novo AML is not a driver mutation. Another case of de novo AML with JAK2 V617F mutation, case 96, was classified as AML-MRC with a complex karyotype including loss of chromosome 17, JAK2 and DNMT3A mutations, and a TET2 frameshift variant.

Case 217, diagnosed as AML, NOS, with an IDH2 mutation, showed a differentiation response upon treatment with an IDH2 inhibitor. Such responses are associated with retention of the original diagnostic karyotype and mutational profile, as described above for case 144 treated with a FLT3 inhibitor, and require a careful correlation of morphology and cytogenetic/molecular genetic results. ALK rearrangement is another potential therapeutic target that has been reported in rare cases of AML with monocytic differentiation. Anecdotal cases of AML with ALK rearrangement have been treated with crizotinib.[26] Case 224 described an acute monocytic leukemia with t(2;2)(p23;q12), which, in contrast to previously reported myeloid neoplasms with ALK rearrangement, was not associated with monosomy 7.[27] Case 329 showed an unusual CSF3R T640N mutation, which has been previously reported in rare cases of chronic neutrophilic leukemia and AML. This mutation was shown to activate JAK/STAT pathway and is sensitive to JAK2 inhibition.[28]

One case of AML, NOS and two cases of therapy-related lymphoblastic leukemia/lymphoma showed unusual KMT2A rearrangements. Case 136 described an 11-month-old infant who presented with a testicular mass and was diagnosed with AML, NOS (acute monocytic leukemia). Cytogenetic analysis demonstrated t(X;11)(q26;q23) and KMT2A rearrangement was confirmed by FISH. The authors suggested CT45A2 as the fusion partner due to its location on chromosome Xq26 and previous report of a similar translocation in an infant with MPAL.[29]CT45A2 is a member of gene family encoding immunogenic proteins expressed in normal testis. Its role in leukemogenesis is not clear. The cases of therapy-related ALL with variant KMT2A rearrangement included one B-ALL following Ewing sarcoma (case 302) and one T-ALL after therapy for BCR-ABL1 rearranged B-ALL (case 306). In both cases, MAML2 was identified as the fusion partner. The KMT2A-MAML2 fusion is rare and has been previously reported in t-MN, in two cases of therapy-related T-ALL and one case of therapy-related B-ALL.[30–32] The MAML2 protein is a transcriptional coactivator for NOTCH1, and is involved in self-renewal of hematopoietic stem cells and T-cell commitment. MAML2 is also expressed in B-cell lymphomas including classic Hodgkin lymphoma.[33]

The prognostic implications of a rare balanced chromosomal abnormality were highlighted by a case of a t-MN with features of MPAL, B/myeloid with t(16;21)(q24;q22)/RUNX1-CBFA2T3 and a complex karyotype in an elderly man (case 83). This rare fusion has been seen in less than 50 cases and its prognostic significance is dependent on patient age. In children, it is associated with higher event-free survival and lower risk of relapse as compared to a general AML group.[34] In adults, it frequently occurs in t-MDS/AML and predicts a poor outcome.[35]

Another case of t-AML had t(1;3)(p36,q21) and trisomy 21 (case 252). This uncommon translocation is associated more frequently with MDS and secondary AML, has rarely been reported in de novo AML, and is exceedingly rare in ALL. It is typically associated with multilineage dysplasia. In patients with de novo AML, the presence of t(1;3)(p36,q21) is sufficient for a diagnosis of AML-MRC, even in absence of morphologic dysplasia. Most patients with AML with t(1;3)(p36,q21) do not respond well to conventional chemotherapy, therefore more aggressive approaches such as hematopoietic stem cell transplantation (HSCT) have been suggested.[36] In contrast, trisomy 21 is relatively common in both pediatric and adult AML occurring at a frequency of 1% to 5%.[37] It is most frequently associated with other karyotypic abnormalities such as trisomy 8, complex karyotype, and deletion 7, and in this context has been associated with adverse prognosis. However, when present as a sole abnormality or in conjunction with favorable cytogenetics, it may indicate a favorable outcome.[37]