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 Leukemias of Ambiguous Lineage

The WHO classification defines acute leukemias of ambiguous lineage (ALAL) as those that do not show clear immunophenotypic evidence of differentiation in any single lineage.[1] This category includes AUL, which shows no evidence of lineage-specific differentiation, and MPAL, which shows differentiation along multiple different lineages. Both t(9;22)(q34.1;q11.2); BCR-ABL1 and t(v;11q23.3); KMT2A-rearranged are recurrent and define specific subtypes of MPAL. The CAP/ASH guidelines recommend karyotype and assessment for both these alterations in the genetic workup of MPAL.[2] The WHO defines other subtypes of ALAL descriptively, including AUL, MPAL B/myeloid NOS (MPAL with evidence of both B-cell and myeloid lineage), and MPAL T/myeloid NOS (MPAL with evidence of both T-cell and myeloid lineage). Recent work has shown frequent ZNF384 rearrangements in pediatric B/myeloid MPAL,[38] and recurrent NOTCH1, DNMT3A, IDH2, and PHF6 (T/myeloid) and RUNX1 (B/myeloid) alterations in adult MPAL.[39,40] Although there are no molecular genetic changes known to be diagnostic of MPAL or AUL at this point, as genomic analysis is used more widely, no doubt more will be learned about their genetic landscapes.

Sessions 3 and 7 included nine cases of ALAL: one case of AUL and seven cases of MPAL (one KMT2A-rearranged, two B/myeloid, three T/myeloid, one B/T) Table 3, and one additional case of ALAL vs blastic plasmacytoid dendritic cell neoplasm (BPDCN). A karyotype was performed in all cases, and at least seven cases employed FISH. Five cases used DNA sequencing, while none included RNA sequencing. Case 243 described MPAL with t(v;11q23.3); KMT2A-rearranged with a B/myeloid immunophenotype and a complex karyotype. The KMT2A rearrangement was seen only by FISH. Another cryptic fusion, NUP98-NSD1, t(5;11)(q35;p15), was seen in two cases: B/myeloid MPAL in a 13-year-old patient using SNP microarray analysis and confirmed by FISH (case 113), and in an 11-year-old patient with ALAL using RNA sequencing (case 203). Interestingly, this fusion was also reported in two recent series of MPAL in a single case each.[39,40] B/T MPAL is a rare subtype of leukemia, even within ALAL. Two small series have noted recurrent PHF6 truncating mutations in B/T MPAL, as seen in workshop case 163.[40,41] Although not strictly diagnostic, DNA sequencing in these cases often helped to support a diagnosis of MPAL when aberrations were identified that are typically associated with different lineages. In case 348, B/myeloid MPAL Image 4 with an unusual rearrangement involving EWSR1, FISH demonstrated that both the myeloid and B lymphoblastic population harbored the EWSR1 rearrangement. EWSR1 rearrangements have been reported in rare cases of B- and T-ALL, and in AML.[42–44] The significance of this rearrangement in leukemogenesis is unknown; however, it has been suggested that in select cases it plays a role in pathogenesis through a p53/p21-dependent pathway.[44]

Image 4.

Case 348. Flow cytometry illustrating a heterogeneity of scatter and antigen expression in a typical case of mixed phenotype acute leukemia, B/myeloid, with expression of B-cell and myeloid lineage markers. A, Side scatter/forward scatter (SSC/FSC) showing two populations of blasts. B, Variable density of CD19 and to lesser extent CD38 on myeloid and lymphoid blasts. C, Expression of myeloperoxidase (MPO) in myeloid blasts. D, Myeloid blasts showing expression of another myeloid antigen, CD117. Blue, lymphoid blasts; red, myeloid blasts. APC, allophycocyanin; BV421, Brilliant Violet; FITC, fluorescein isothiocyanate; PECy7, phycoerythrin cyanine 7; PerCPCy5.5, peridinin chlorophyll protein cyanine 5.5. Courtesy of Weina Chen, Jo Ellen Krueger, Franklin Fuda, and Prasad Koduru.

T/myeloid MPAL is a challenging diagnostic category, as there is immunophenotypic overlap with early T precursor (ETP) ALL: both show coexpression of myeloid antigens and cytoplasmic CD3. As defined in the WHO classification, ETP-ALL does not express myeloperoxidase (MPO). Recent data suggest that ETP-ALL and T/myeloid MPAL may exist on a biological spectrum between T-ALL and AML,[38] with alterations in ETV6, EZH2, WT1, and FLT3 being common in both, while NOTCH1 alterations are more common in ETP-ALL and CEBPA and CUX1 alterations are more common in T/myeloid MPAL. These genetic trends are not diagnostic, however; indeed, both cases of T/myeloid MPAL presented in session 3 showed NOTCH1 mutations (cases 319 and 188).

Finally, case 30 was considered by the panel as ALAL vs BPDCN. The neoplasm had exclusively leukemic presentation and weak CD123 expression, unusual features for BPDCN.[45] Additional studies such as CD303 or other plasmacytoid dendritic cell markers could not be performed due to exhausted tissue. According to the WHO classification, neoplasms that do not show all typical immunophenotypic features of BPDCN may be better diagnosed as ALAL.[1]