Acute Leukemias of Ambiguous Origin

Home , CD1, CD16, CD19, CD20, CD33, CD3 (immunology)

AJCP / Review Article

Anna Porwit, MD, PhD,1 and Marie C. Béné, PharmSciD, PhD2

From the 1Department of Laboratory Medicine and Pathobiology, University Health Network, University of Toronto, Toronto, Canada, and 2Immunology Laboratory, University Hospital of Nancy-Brabois, Nancy, France.

Key Words: Acute leukemia; Immunophenotype; Lineage; Myeloid; Lymphoid; Mixed phenotype acute leukemia; Undifferentiated acute leukemia Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 Am J Clin Pathol September 2015;144:361-376

DOI: 10.1309/AJCPSTU55DRQEGTE

ABSTRACT What Is the Definition of Acute Leukemia of Ambiguous Lineage? Objectives: This session of the Society for Hematopathology/ European Association for Haematopathology Workshop In the 2008 World Health Organization (WHO) classifi- focused on acute leukemias of ambiguous origin. cation,1 acute leukemias of ambiguous lineage were defined as leukemias that show no clear evidence of differentiation Methods: We provide an overview of mixed-phenotype along a single lineage. The recognition of acute leukemia of acute leukemia (MPAL) as recognized in the current World ambiguous lineage requires extensive multiparametric flow Health Organization classification and summarize diagnostic cytometry (FCM) immunophenotyping disclosing the spe- criteria for major categories of MPAL: B/myeloid, T/myeloid, cific features of these diseases ❚Figure 1❚. In extremely rare B/T, and B/T/myeloid. cases of acute undifferentiated leukemia (AUL), although Results: Most MPAL cases submitted were B/myeloid markers of all lineages have been investigated, no signifi- and T/myeloid MPAL, the most frequent types, but three cant expression is detected on blast cells except for CD34 cases of B/T MPAL were also submitted, and examples and/or HLA-DR (present in most of these cases). A sec- of all categories are illustrated. We emphasize that a ond, slightly more frequent occurrence is that of blast cells comprehensive approach to immunophenotyping is labeling with several monoclonal antibodies, recognizing required to accurately establish the diagnosis of MPAL. antigens normally expressed on different lineages. Formerly Flow cytometry immunophenotyping using a large called biphenotypic acute leukemias (BALs), these neo- panel of antibodies is needed as well as confirmatory plasms have been renamed mixed-phenotype acute leukemia immunohistochemical analysis and cytochemistry studies (MPAL). On the basis of associated cytogenetic anomalies, for myeloperoxidase and nonspecific esterase. We discuss MPAL can be subdivided according to the presence of the technical issues in determining blast lineage and possible Philadelphia chromosome into subgroups: t(9;22)/BCR- pitfalls in MPAL diagnosis. In particular, rare cases of ABL1, t(v;11q23)/MLL, or not otherwise specified. All other B-acute lymphoblastic leukemia (B-ALL) can express unusual immunophenotypes, including early natural killer myeloperoxidase but are otherwise consistent with B-ALL (NK) leukemias, constitute the remainder of leukemias of and should be treated as such. Last, we review the differential ambiguous origin. diagnosis between acute undifferentiated leukemia and acute myeloid leukemia with minimal differentiation.

Conclusions: There was an agreement that diagnosis Introduction to MPAL of MPAL can be challenging, especially if applied flow cytometry panels are not comprehensive enough. The morphologic features of the blasts in most cases of MPAL are mostly uninformative, and therefore the

Extensive one other B-lineage–associated marker (eg, cCD79a, surface immunophenotyping or cCD22, or CD10) will be required. If CD19 expression is weak, however, at least two other B-lineage markers need to be expressed by the blasts. Assessment of T-lineage engage- Acute Mixed Others, undifferentiated phenotype including ment relies on the demonstration of cCD3 expression as leukemia acute leukemia natural killer mentioned above. Of note, the fluorochrome used for this determination should yield strong fluorescence, for instance, phycoerythrin or allophycocyanin. The immunophenotypic With With Not otherwise t(9;22) t(n;11q23) specied strategy to explore these cells involves the use of two monoclonal antibodies directed to CD3 (usually anti-e chain) con- ❚Figure 1❚ Schematic for the classification of acute leukemias jugated to different fluorochromes. Cells are first incubated of ambiguous origin. without manipulation, ideally using whole bone marrow Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 (BM). After incubation, permeabilizing reagents will allow diagnosis of MPAL relies exclusively on immunopheno- both lysis of erythrocytes and intracytoplasmic labeling with typic features. MPAL cells appear most often as morpho- the second anti-CD3 antibody. Analysis by FCM will then logically undifferentiated, but sometimes the neoplastic cells identify normal residual T lymphocytes as costained with may display more lymphoblastic or myeloblastic cytology. both antibodies, while T-lineage blast cells lacking surface In some cases, two types of blasts with a distinctive size and CD3 will be stained with the second anti-CD3 only. morphology point to the different, extremely rare occurrence of a bilineal or biclonal leukemia. These neoplasms can also be recognized by FCM analysis as two cell subsets with dif- What Possible Combinations of MPAL ferent immunophenotypes. Occur and What Are Their Frequencies? MPAL can be identified using the recommended panel of the European LeukemiaNet2 or other comprehensive All possible combinations of MPAL can be observed, combinations.3 Importantly, several markers specific for including B/myeloid, T/myeloid, B/T, or even rare B/T/ the myelo/monocytic lineage as well as for the B- and myeloid. In a series of 100 cases of MPAL published by the T-lymphoid lineages must be tested, excluding a restric- EGIL,7 B/myeloid cases were most frequent, representing tive strategy of quick orientation followed by selected 59% of all MPAL cases. The frequencies of T/myeloid, B/T, lineage-specific markers. Of note, as detailed below, at or B/T/myeloid were 35%, 4%, and 2%, respectively. The least two key cytoplasmic (c) markers must be investigated: blast cells in MPAL show a specific gene expression pat- myeloperoxidase (MPO) for myeloid lineage and cCD3 for tern, as illustrated by a microarray study of acute leukemias T lineage. Recommendations were first published by the performed at St Jude Children’s Research Hospital, where European Group for the Immunological Characterization of 35 childhood MPAL cases segregated in a specific cluster Leukemias (EGIL),4 with a scoring system identifying BAL between B-acute lymphoblastic leukemia (B-ALL), T-acute as cases with more than two immunophenotypic points in lymphoblastic leukemia (T-ALL), and acute myeloid leuke- more than one lineage. This proposal relied on 26 antibod- mia (AML).8 ies to perform a proper score, yet published reports seldom applied such extensive panels. In the 2008 WHO classification,1 the name of these leu- How Are Patients With MPAL kemias and their immunophenotypic definition (as above) Managed Clinically? were changed. MPO stands as the most robust marker that will identify myeloid engagement. Recent publications have The clinical management of MPAL cases is problem- redefined the threshold for MPO positivity,5,6 and yet most atic. Often, an initial course of corticosteroids is attempted, MPAL cases with a myeloid component will express MPO followed by an acute lymphoblastic leukemia (ALL)–like broadly in most blasts. An alternative, for MPAL with a approach. In the absence of response to corticosteroids, most strong monocytic differentiation, is represented by (very clinicians will switch to an AML-like approach with alkylat- rare) cases in which blasts will be positive for nonspecific ing agents. However, patients who fared well in the literature esterase and/or express at least two of the following anti- were clearly those who could benefit from allogeneic stem gens: CD14, CD11c, CD36, CD64, or cytoplasmic lyso- cell transplantation. zyme. Determination of the engagement of blast cells in the It is important to identify MPAL cases and not misdiag- B-lymphoid lineage will rely on the expression of surface nose these tumors as ALL or AML by using immunopheno- CD19. If the latter is bright, demonstration of expression of typic panels that are not sufficiently comprehensive. Indeed,

362 Am J Clin Pathol 2015;144:361-376 © American Society for Clinical Pathology DOI: 10.1309/AJCPSTU55DRQEGTE AJCP / Review Article some refractory cases of acute leukemia, with poor response common cytogenetic variant of B/myeloid MPAL, associ- to therapy, may represent undetected cases of MPAL that ated with t(9;22)(q34;q11.2)/BCR-ABL1. Three cases had were incorrectly assigned to a single lineage. Using an FCM highly hyperdiploid and/or a near-tetraploid karyotype, and intracytoplasmic orientation panel combining MPO, cCD3, one case showed hypodiploidy. Highly hyperdiploid and/or and CD19 (possibly with cCD79a, cCD22, or CD10) could near-tetraploid karyotypes are rare in AML and ALL9,10 and be a good approach to detect such cases early in the process have been reported previously as common in MPAL and of immunophenotyping a neoplasm. It is important to point BAL classified according to the EGIL system.4,7,11 In the out that immunohistochemistry (IHC) on BM biopsy sec- case series collected for this workshop, the highly hyper- tions also can be useful to confirm B, T, or myeloid differ- diploid and/or near-tetraploid karyotype was also common entiation by using antibodies specific for PAX5, CD3, and (five [16%] of 32 cases: three B/myeloid, one T/myeloid, MPO and/or lysozyme, respectively. Immunohistochemical and one B/T). analysis may be of great help in cases with two or more dif- One of the near-tetraploid cases (case 285) carried also Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 ferent populations with specific distributions in BM or other the t(12;17)(p13;q11.2) involving the ZNF384/CIZ (chromo- tissue biopsy specimens since it allows direct visualization some 12p13) and TAF15 (chromosome 17q11.2) genes. The of cell location. t(12;17)(p13;q11) has been described as a rare but recurrent abnormality, predominantly observed in B-ALL cases that usually have an early pre–B immunophenotype and showing aberrant expression of the myeloid antigens CD13 and/ Discussion of Workshop Cases or CD33. The t(12;17) is less common in AML, and reported There were 32 cases included in this workshop ses- cases often showed aberrant expression of CD19.12-16 In a sion. Several cases submitted initially to this session did recent case report, a pro–B/B-I ALL, which switched to an not completely meet the criteria for MPAL and were moved AML at relapse, was described.17 Case 285 is the first case to other sessions, although the moving in some cases was with this translocation in which the WHO criteria for MPAL debated by members of the review panel, revealing the need are fulfilled. Another chromosomal abnormality hitherto for more robust criteria and greater consensus on definitions. not described in MPAL, inv(3)(q21q26.2), was detected in The workshop cases are discussed in the subtypes to which case 283. Inv(3)(q21q26.2) or t(3;3)(q21;q26.2) displays they were allocated. The summary of these cases (below) an inversion or homologous reciprocal translocation that illustrates the heterogeneity of MPAL presentation. The leads to juxtaposition of the ecotropic viral integration site 1 following discussion also describes the characteristics of (EVI1) gene with the ribophorin 1 (RPN1) gene. AML cases MPAL cases, technical issues involved in MPAL diagnosis, carrying this abnormality are classified as one of the catego- and groups of cases that can be linked by some common ries of AML with recurrent cytogenetic abnormalities in the characteristics or diagnostic issues. WHO classification and most commonly have a myeloid or myelomonocytic immunophenotype and poor prognosis.18,19 An MPAL phenotype has not been reported previously in a leukemia with inv(3) or t(3;3). Typical Examples of MPAL MPAL T/Myeloid MPAL B/Myeloid The characteristics of MPAL cases with T and myeloid Typical examples of MPAL with populations of blasts lineage markers are illustrated in ❚Image 3❚, ❚Image 4❚, and showing B lineage and myeloid differentiation are illus- ❚Table 2❚. Most T/myeloid MPALs were characterized by trated in ❚Image 1❚ and ❚Image 2❚. Immunophenotypes of all two populations of blasts: one with a T-precursor immuno- cases classified as B/myeloid MPAL are given in ❚Table 1❚. phenotype (usually coexpressing some myeloid markers but Expression of the B-cell marker CD19 was moderate to negative for MPO) and the other fulfilling MPAL criteria. In strong in cases 10, 208, 285, 323, and 328 and weak/par- two cases (case 139 and 200), the myeloid lineage showed tial in all other cases. B-cell lineage was most often cor- a monocytic differentiation pattern. In two patients (cases roborated by expression of cCD79a (demonstrated by FCM, 200 and 402), different immunophenotypes were observed IHC, or both), cCD22 (by FCM), and/or PAX5 (by IHC), in a lymph node biopsy specimen and the BM. In case 200, whereas CD10 was positive in three cases (cases 10, 208, BM showed an immunophenotype suggestive of AML with and 214). MPO expression was variable: most blast cells monocytic differentiation, but in the lymph node two aber- were MPO positive in four cases (cases 10, 76, 285, and rant populations were detected: one with cCD3 positivity 323), but other cases showed only subpopulations of MPO- and one with a myelomonocytic immunophenotype. Cytoge- positive cells. Cases 208, 328, and 214 represented the most netic analysis also showed two different clonal populations,

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C D

❚Image 1❚ Example of B/myeloid mixed-phenotype acute leukemia, BCR-ABL1 positive, with a heterogeneous population of blasts with myeloperoxidase (MPO)–positive larger myeloid blasts (arrows) and MPO-negative smaller lymphoid blasts (arrowheads) (A, May-Grünwald-Giemsa; B, MPO). Bone marrow biopsy specimen shows a mixed population of CD79+ smaller blasts (C) and MPO+ larger blasts (D). See Table 1 for details and Image 2 for flow cytometry findings. (Case 208, courtesy of E. Moore, MD, and colleagues.) one with 46,XY,der(12)t(11;12)(q21;p11.2) and the other in the other cases. CD7 was expressed in one of the cases, with 45,XY,dic(12;18)(p11.2;p11.2). In case 402, a lymph and no other T-cell–associated markers were seen in the node biopsy specimen showed a T-precursor immunopheno- other. One of these cases had a normal karyotype, and one type, whereas in the BM, a population of blasts fulfilling the was hyperdiploid. The third MPAL B/T case had trisomy criteria of MPAL was detected. 11 and two populations of blasts: one with an immunophenotype of B-precursor ALL (EGIL B-II) and one of T-ALL MPAL B/T with early T-cell precursor immunophenotype (EGIL T-I).20 MPAL cases that express both B- and T-lineage markers are rare (<5% of MPALs).7 Three examples (cases 312, MPAL B/T/Myeloid 359, and 390) were submitted to the workshop ❚Table 3❚ and Case 268 was the only case of acute leukemia that ❚Image 5❚. In two cases, a population of blasts was detected expressed markers of all three lineages fulfilling criteria that coexpressed CD19 (strong) and cCD3. B-cell lineage for B/T/myeloid MPAL. The patient was a 58-year-old was supported by CD22 expression in one case and CD79a man with two separate leukemic cell populations. One

364 Am J Clin Pathol 2015;144:361-376 © American Society for Clinical Pathology DOI: 10.1309/AJCPSTU55DRQEGTE AJCP / Review Article

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100 101 102 103 –2 0 2 101 102 103 01100 01 102 103 100 101 102 103 CD19 CD10 CD3 CD3 ❚Image 2❚ Flow cytometry immunophenotypic findings in case 208 (see Table 1 for details): Complicated case where it was difficult to differentiate CD45 low blasts from other leukocytes (upper row left plots). Granulocytes are intermingled with blasts, and there is also a continuum between lymphocytes and blasts. CD34 allows discriminating the myeloid blast population (upper row, right plots, CD34: dark blue dots, back-gated). CD34+ cells are CD19+ and express an unusual heterogeneous CD10 pattern (lower row, left plots). Myeloperoxidase expression is dim but definitively positive, and cytoplasmic CD79 is positive at a similar level as for normal B cells (lower row, right plots). SSC, side scatter. Red, neutrophils; green, monocytes; pink, lymphocytes; light blue, blasts. population corresponded to an AML and was positive for myeloid. Case 256 illustrated a rare occurrence of a T/ CD7, CD4 (dim), CD11b, CD13, CD33, CD34, CD117, myeloid MPAL (Table 4). The other three cases demon- MPO, and HLA-DR, and the other population corresponded strated an immunophenotype switch, previously reported in to MPAL with a B/T immunophenotype positive for TdT, MLL rearranged leukemias.22-27 CD19, CD20, CD10, cCD79a, CD22, PAX5, CD33 (dim), MLL rearrangement juxtaposes the amino-terminus of cCD3, and CD5 (dim) and negative for MPO ❚Image 6❚ the histone methyltransferase MLL with a variety of differ- and ❚Image 7❚. Conventional cytogenetics showed a normal ent fusion partners. To date, more than 70 fusion partners karyotype, and BCR-ABL1 was negative. Another case ful- of the MLL gene have been characterized.28 Deregulation filling criteria for T/myeloid MPAL and also showing weak of HOX seems to be one of the most important factors in CD19 expression was case 356 (Table 2). The patient was a MLL-induced leukemogenesis. Since HOX expression 14-year-old girl in whom both a lymph node biopsy speci- is high in stem cells and early precursors and needs to men and a BM showed blasts positive for cCD3 and MPO be downregulated for maturation, a continuous ectopic with weak expression of CD19 and cCD79a. However, other HOX expression will create a basis for the development B-cell markers were not found and the criteria for B-lineage of abnormal preleukemic precursors that may progress differentiation were not fulfilled. to various forms of acute leukemia.28,29 In cases 180 and 370 submitted to this session, the primary diagnosis was MLL Rearrangement and Immunophenotype Switch AML with t(9;11)(p22;q23), but the relapse presented Cases 180, 256, 370, and 392 were examples of MPAL as ALL/lymphoma with an EGIL B-I immunophenotype associated with MLL rearrangement ❚Table 4❚. MPAL with (CD10 negative). In the third patient (case 392), a therapy- MLL rearrangement is considered a separate entity in the related myeloid neoplasm presented as a t(4;11)(q21;q23) WHO classification,21 with a frequency of approximately B-ALL/lymphoma with an EGIL B-I immunophenotype, 10% of adult and 12% to 18% of pediatric MPAL cases.7 whereas AML with monocytic differentiation was seen at Most cases of MPAL with MLL rearrangement are B/ first relapse and B/myeloid MPAL at second relapse. The

❚Table 1❚ Summary of Cases of MPAL, B/Myeloid Patient Consensus Case Age, y/ WHO Diagnosis/ No. Sex Immunophenotype Cytogenetics/Molecular Genetics *Comments 208 36, F Two populations of blasts (75% of BM cells in total): B t(9;22)(q34;q11.2); BCR-ABL1 (by FISH) MPAL (B/ lymphoblastic (39%) CD19+, CD10+, CD13+, CD34+, myeloid), cCD79a+, HLA-DR+, TdT+ with variable expression of with t(9;22) cCD22 IgM, negative for CD4, CD11b, CD14, CD15, (q34;q11.2) CD64, MPO Myelo(mono)blastic (36%) with the following e1a2 BCR-ABL1 by RT-PCR immunophenotype: CD4+, CD13+, CD15+, CD19+, CD33+, CD34+, CD64+, HLA-DR+, MPO+ with variable expression of CD10, CD11b, cCD79a, and TdT and

negative for cCD22 and IgM Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 328 35, M FCM: 13% blasts CD34+, CD117s+, CD33+, CD13s+, 46,XY,t(9;22)(q34;q11.2) [15] and 46,idem, MPAL (B/ CD15s+, CD19+, CD22s+, cCD79a dim+, CD25s+, del(20)(q11.2q13.3)[5] myeloid), TdT+, MPOs+, CD10–, CD66c–, CD2–, CD5–, CD7–, with t(9;22) CD56– (q34;q11.2) IHC: PAX5+ (80%-90%), CD19+ (50%), TdT+ (90%), e1a2 BCR-ABL1 by RT-PCR MPO+ (30%), CD117–, CD20– 214 63, F Two populations of blasts (60% of BM cells in total): 45,XX,del(7)(p15), t(9:22)(q34:q11.2), –16 MPAL (B/ B-lymphoblastic: CD19+, CD10+, CD34+, CD33+, [19]/46,XX[1] myeloid), HLA-DR+, and CD38+, TdT+, PAX5+, CD45 dim with t(9;22) (q34;q11.2) Myelo(mono)blastic: HLA-DR, CD33, CD4, partial CD5, dim CD11B, CD14, CD64, partial CD15, CD38, CD45 bright, increased SSC, MPO, and lysozyme positive by IHC 285 41, F One population of blasts: CD19+, cCD79a+, MPO+, 82-84,XXXX,–1,–2,–3,–4,–7,+8,–9,–10, MPAL, NOS (B/ CD33+, CD34, HLA-DR+, CD38+, and with partial t(12;17)(p13;q11.2)x2, der(12)t(12;17) myeloid) expression of CD13 and CD15 (p13;q11.2),+13,–15,–16,–17,–20,– 21,+22[cp7]/46,XX[13] Negative for BCR-ABL1 by FISH *Associated with t(12;17) (p13;q11.2) 283 40, M One blast population 40% CD2–, surface CD3–, cCD3–, 46,XY,inv(3)(q21q26.2), inv(9) [20] MPAL, NOS, B/ CD4–, CD5–, CD7+, CD8–, CD10–, CD11bs+, CD11cs+, myeloid CD13+, CD14–, CD15s+, CD16–, CD19s+, CD20–, CD22s+, cCD22s+, CD33+, CD34+, CD36–, CD38+, CD45+, CD56–, CD64–, CD79as+, CD117+, HLA-DR+, MPOs+ (6%-8%), TdTs+ *Associated with inv(3) (q21q26.2) 323 90, F One population of blasts: CD19 dim, CD14 dim, CD15+, 35,XX,–3,–4,–5,–7,–8,–9,–13,–15,–16,–17,– MPAL, NOS, B/ CD45 dim, sCD22 dim, CD56 dim, CD71 dim, CD79a+ 20[9] myeloid and CD34+, lCD13–, CD20– or CD33–, cCD79a+, cCD3– By IHC: MPO+, PAX5+, TdT+, CD79a+/–, and negative for 46,XX,?t(5;6)(q23;q24)[1] *With lysozyme, CD10, and CD20 hypodiploidy 46,XX[10] FISH for t(9;22) and MLL: neg 10 5, F One population of blasts: CD45 dim/–, low SSC, CD10+, 63-64<3n>,XXX,add(1)(p13),–2,–3,–7,+10,– MPAL, NOS, B/ CD19+, CD22+, partial CD13+, partial CD33+, CD34+, 13,–14,–15,–16,–19,–20,+21,+21[cp6]/63– myeloid cCD79a+, nTdT+, HLA-DR, MPO+, cCD3–, CD15–, and 65<3n>,idem,?add(2)(p23),– CD117– 10,+mar[cp2]/46,XX,[26] FISH for t(9;22) and MLL: neg *Associated with hyperdiploidy 76 26, M One population of blasts: BM FCM: CD34+, CD33+, 80-85<4n>,XXY,–Y,add(1)(p36),–2,add(2) MPAL, NOS, B/ HLA-DR+, CD7+, MPO+, cCD79a+, cCD22 dim+, cCD3 (q31)x1-2,–3,add(3)(q12-21)x2,–6,–7,der(7) myeloid dim/–, sCD5+ and sCD19+, TdT–, CD2–, CD4–, CD8–, t(7;8)(p15;q13),–8,–8,–9,–9,add(12)(p11.2) and mCD3– x2,–13,–13,–15,–15,–16,–16,–17,–17,– 18,+3-9mar[cp9]/46,XY[16] Lymph node IHC: CD34+, CD33+, CD99+, CD5+, CD56+ *Associated weak, CD7+, MPO+, CD79+a, PAX5+; negative for with near cCD3, CD4, CD8, CD2, CD10, CD123, CD117, and TdT tetraploidy 206 51, F Two populations of blasts (90% of BM cells in total): 46,XX; negative for BCR-ABL1 gene fusion MPAL, NOS, B/ main population: CD4s+, CD7 dim+, CD13s+, CD14+, and MLL rearrangement by FISH myeloid CD15+, CD36+, CD38+, CD45 dim+, CD58+, CD117–/ dim, HLA-DRs+, MPOs+, cCD3–, CD5–, CD8–, CD19–, CD20–, CD22–, CD34– , CD56–, CD61–, nTdT–, cCD79a–

366 Am J Clin Pathol 2015;144:361-376 © American Society for Clinical Pathology DOI: 10.1309/AJCPSTU55DRQEGTE AJCP / Review Article

❚Table 1❚ (cont) Summary of Cases of MPAL, B/Myeloid Patient Consensus Case Age, y/ WHO Diagnosis/ No. Sex Immunophenotype Cytogenetics/Molecular Genetics *Comments 206 51, F Minor population (5%-7%): CD19+, CD22 s–/bright, CD34+, CD38+, CD45 dim+, CD58+, nTdT+, cCD79a+, CD10–, CD14–, CD33–, cCD3–, MPO– 365 41, M Blasts 42% of BM cells: CD34+, CD33 dim+, CD38s+, 45,XY,–7 [20] MPAL, NOS, B/ MPO dim+ (18%), CD7+, HLA-DR dim+, CD117+, CD64 myeloid dim+, CD11bs+, CD19s+ (30%), CD79a dim+, nTdT–, cCD3–, CD15–, CD13–, CD20–, CD14– IHC: MPO+, lysozyme+, CD68+, PAX5s+ FISH tri8 2%

BM, bone marrow; c, cytoplasmic; FCM, flow cytometry; FISH, fluorescence in situ hybridization; IgM, immunoglobulin M; IHC, immunohistochemistry; MPAL, mixed- Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 phenotype acute leukemia; MPO, myeloperoxidase; neg, negative; NOS, not otherwise specified; RT-PCR, reverse transcriptase polymerase chain reaction; SSC, side scatter; WHO, World Health Organization.

A B

C D

❚Image 3❚ Example of T/myeloid mixed-phenotype acute leukemia showing in both bone marrow smears (A, May-Grünwald- Giemsa) and biopsy sections (B, H&E) presence of small myeloperoxidase (MPO)–negative blasts and approximately 25% larger MPO+ blasts. Positivity for both cytoplasmic CD3 (C) and MPO (D) is illustrated in the lymph node sections from the same patient. See Table 4 for details and Image 4 for flow cytometry findings. (Case 279, courtesy of R. E. Alexander, MD, and M. Czader, MD, PhD.)

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CD2-FITC 0 CD8-FITC 0 0 0 CD45-ECD 10 10 CD71-FITC 10 10

100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 CD7-PE CD4-PE CD33-PE CYTOTDT-FITC ❚Image 4❚ Flow cytometry immunophenotypic findings in case 279: bilineal case with clearly different immunophenotypes on lymphoblasts (cyan dots, cCD3+, CD7+, CD2+, TdT+) and myeloblasts (dark blue dots, CD33+, CD15+, CD117+, MPO+). FITC, fluorescein isothiocyanate; MPO, myeloperoxidase. Red, neutrophils; green, monocytes; pink, lymphocytes; light blue, lymphoblasts; dark blue, myeloid blasts.

MLL-AF4 fusion has been reported in therapy-related ALL, controls. In a study comparing MPO expression detected by after treatment with either topoisomerase II inhibitors or FCM and cytochemistry in cases of AML and ALL, a 13% alkylating agents, and seems to be associated with a worse threshold was found to be relevant using an isotype control clinical outcome.30,31 as a background reference (sensitivity, 95.1%; specificity, 91.7%).5 If residual normal lymphocytes were used as reference, a threshold of 28% had to be applied, yielding an improved 97.4% sensitivity and 96.1% specificity in dis- What Are Some of the Important tinguishing between ALL and AML. The WHO criteria for Technical and Interpretation Issues MPAL do not indicate any lower limit for MPO expression in the Diagnosis of MPAL? in leukemic blasts,1 but it seems reasonable to use published thresholds.3,5 Since MPAL cases often show more than MPO one population of blasts, MPO could be present in only a The issue of MPO expression in the diagnosis of MPAL minor population, which has to be identified as the myeloid has been discussed in the literature.32-34 Previous French- component. Another issue is that AML with minimal dif- American-British group guidelines, based on cytochemistry, ferentiation and no MPO expression could be involved in used 3% of MPO-positive blasts in BM smears as sufficient MPAL. Therefore, as stated in the WHO criteria, when there to call a leukemia MPO positive.35,36 A threshold of 10% are two or more distinct populations of leukemic cells, one expression has been used by the EGIL group.3 However, with coexpression of a number of myeloid markers, no MPO discordant cases are found when both methods are com- and no lymphoid markers also can be accepted to define the pared,6,37 leading to the conclusion that the 10% threshold myeloid component of MPAL.1 may be conservative but not very sensitive. It should be Rarely, cases of otherwise typical B-ALL/lymphoma emphasized that MPO expression can be a difficult test to can express MPO by FCM or IHC (case 259) or least often establish by FCM immunophenotypic analysis since differ- by cytochemistry.32-34,41 These cases are best classified ences in permeabilization reagents and various antibodies and treated as B-ALL/lymphoma. In contrast, most MPAL have been reported.38-40 Thus, MPO expression in leukemic cases show expression of other myeloid-associated markers blasts has to be compared with internal negative and positive and also are characterized by the presence of two or more

368 Am J Clin Pathol 2015;144:361-376 © American Society for Clinical Pathology DOI: 10.1309/AJCPSTU55DRQEGTE AJCP / Review Article

❚Table 2❚ Summary of Cases of MPAL, T/Myeloid Case Patient Consensus WHO No. Age, y/Sex Immunophenotype Cytogenetics/Molecular Genetics Diagnosis/ *Comments 279 13, M Two populations of blasts: T lymphoblasts: CD2+, 45-46,XY,add(1)(p22), der(3)dup(3) MPAL, NOS; T/ CD7+, CD5+, cCD3+, nTdT+, CD38+ (q21q29)r(3)(p26q29), add(5)(q13),add(7) myeloid (q34),del(8)(22), t(9;14)(p22;q11.2),–11, –15,+add(19)(q13.3), +mar2[cp20]/ Nonclonal[1]/46,XY[4] The myeloid/T-cell population: CD33+, CD15+, CD117+, CD64+, CD2+, CD7+, CD38+, MPO+, cCD3s+ 92 17, F Two populations of blasts: 90% cCD3+, nTdT+, 46,XX MPAL, NOS; T/ CD34+ , CD13+, CD117+. 10% also cMPO+ myeloid

(cytochemistry MPO+ 10%) Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 225 7, M Two populations: all CD34 and cCD3, subpopulation 46,XY MPAL, T/myeloid, also CD117+ and MPO+ by FCM and IHC NOS 303 67, M CD34+, partial CD117+, CD13+, CD33+, CD11b+, 45,XY,–7,del(20)q11.2q13.1) MPAL, T/myeloid, HLA-DR–, TdT– and MPO–, cCD3+/dim, CD5+ [4]/47,idem,+2mar[2]/46,XY[3] NOS and CD7+, cCD79a– *Relapse as AML 139 80, M Two populations: 17% blast gate CD4+/7+/11b+/ High hyperdiploid karyotype: trisomies MPAL, NOS T/myeloid 13+/33+/34+/56s+/64s+ and cCD3 (partial), 50% for chromosomes 8, 11, and 12 and an (bilineal) monocyte gate: CD4+/11b+/13+/14+/33+/64+ extra chromosome X. In addition, five *Monocytic IHC: lysozyme+ of these abnormal cells had trisomies differentiation and for chromosomes 10 and 21. hyperdiploidy 200 58, M BM: FCM CD33 dim, CD7 dim, CD4 dim, CD38+, 46,XY,der(12)t(11;12)(q21;p11.2) MPAL, NOS T/myeloid HLA-DR+, CD11cs+, CD11bs+; AML was [6]/45,XY,dic(12;18)(p11.2;p11.2) favored. IHC: CD68+, CD34-MPO–, CD117–, [5]/46,XY[9] CD3–, CD20–, lysozyme– Lymph node: FCM CD33dim, CD7+, CD4 dim, *Bilineal in the lymph CD5s, dim CD11b dim, CD38+, HLA-DR+. IHC: node, monocytic two distinct blast populations, nodules CD3+, differentiation CD4+, CD5+, CD7+, CD99+, at the periphery of the nodules CD7+, lysozyme+, CD33+, TdT+, CD99+, MPO+, CD11c+ 402 10, F Lymph node FCM: 74%, CD7+, CD5 dim, CD2+, 45,XX,der(5)t(5;12)(q22;q13),–7,– MPAL, NOS T/myeloid CD34+, CD117+, CD11b+, CD3+, CD71+, 12,der(13)t(7;13)(q11.2;q34),+der(?) nTDT+, HLA-DRs+, cCD3+, CD1a–, CD4–, CD8–, t(?;7)(?;p?15)[9]/46,XX[11] MPO– BM FCM: 7%-8%, CD7+, CD34+, cCD3+, MPOs+ *T-LBL in the lymph node, MPAL in BM 259 14, F Lymph node: two populations: 53% T-lymphoblasts, BM aspirate: complex karyotype. MPAL, NOS T/myeloid CD45 dim, cCD3+,CD2+, CD5 dim, CD7+, 46,X,t(X;9)(q13;p24),del(3)(q12),t(4;12) CD13+, CD19 dim+, other T-cell and myeloid (q12;p13),t(11;14)(p13;q11.2),del(13) markers including MPO (q13q31),del(16)(q22),del(18)(q21) [16]/46,XX[4] IHC showed also myeloid cell area: CD33+, CD15+, FISH: Homozygous deletion of *Bilineal; CD19 dim CD68 (KP1+), MPO+, CD34–, CD117– CDKN2A(9p21) in 92% of 200 cells; and CD79 dim do BM, two populations: 36% lymphoid blasts CD2+, loss of 3′ETV6 (12p13) in 87.5% not fully corroborate CD7+, cCD3, CD19 dim, cCD79a dim, CD13+, of cells; loss of 3′TCR (14q11.2) in B lineage CD33 dim, CD45 dim+ 90% of cells studies. No evidence Myeloid blasts with intermediate SSC (37%): of a BCR-ABL fusion gene or CD33 bright, CD4+, MPO+, CD64+, HLA-DR+, rearrangements involving the MLL, CD11bs+, CD11Cs+, CD2+, CD7+, CD13+, FGRR1, or IGH loci. CD19s– dim, CD45 dim, cCD3–

AML, acute myeloid leukemia; BM, bone marrow; c, cytoplasmic; FCM, flow cytometry; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; MPAL, mixed- phenotype acute leukemia; MPO, myeloperoxidase; NOS, not otherwise specified; SSC, side scatter; T-LBL, T-lymphoblastic; WHO, World Health Organization. subpopulations of blasts with slightly different immunophe- diagnose T-cell malignancies since the 1980s.42,43 FCM notypes. In some cases of MPAL, MPO may be expressed immunophenotyping methods to detect cCD3 were estab- only in a small subset of blasts, with or without lymphoid lished in the 1990s when permeabilization reagents became markers (biphenotypic or bilineal presentation). available.39,40 As in the case of MPO, expression of cCD3 in leukemic blasts has to be determined in comparison with Cytoplasmic CD3 internal negative and positive controls (B cells, monocytes, Cytoplasmic CD3 (cCD3) is considered the most spe- granulocytes, and normal T lymphocytes, respectively). At cific marker for the T-cell lineage and has been used to least a fraction of blasts should express cCD3 at the level of

❚Table 3❚ Summary of Cases of MPAL, B/T Patient Age, Consensus WHO Case No. y/Sex Immunophenotype Cytogenetics/Molecular Genetics Diagnosis/ *Comments 390 82, M One population of blasts: cCD3+, CD7+, CD19+, 46,XY, FISH negative for:5p15.2 MPAL, NOS, B/T CD22h, CD13+, CD117+, CD15s+, CD5–, (D5S23), 5q31 (EGR1), 7cen (D7Z1), CD11b–, CD14–, CD33–, CD56–, CD61–, CD79a–, 7q31 (D7S486), 8cen (D8Z2), CD235a–, MPO– 11q23(MLL), 13q14 (D13S319), 13q34 (LAMP1), and 20q12 (D20S108) 359 44, M One population of blasts: CD34+, CD19+, CD33+, 50,XY,dup(1)(p22p36.1),+4,+10,– MPAL, NOS, B/ CD56+, HLA-DRs+, CD49d+, cCD79a+, 15,+21,+22,+mar[12]50,idem, CD123s+, CD45 dim, CD38 bright, cCD3s+, del(11)(q12),add(19)(q13.1)[3]; CD1–, CD2–, mCD3–, CD4–, CD5–, CD7–, CD8–, diploid male karyotype 46,XY[5]

CD10–, CD13–, CD14–, CD15–, CD20–, CD22–, Negative for all recurrent *With hyperdiploidy Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 CD25–, CD36–, CD41–, CD64–, CD66c–, CD81–, translocations, negative for CD117–, TdT–, sIg–, cIgM– , MPO– mutations in IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, FLT3 (including ITD), and CEBPA 312 11, F Two blast populations: 1: CD45 dim+, CD19+, 47,XX,+11[19]/46,XX[1]. FISH negative MPAL, NOS, B/T CD20–/dim+, CD10+, TdT+, CD34 dim+, CD117–, for monosomy 7, BCR-ABL1, ETV6- MPO–, CD13–, CD33–, CD38+ , CD79a, PAX5 RUNX1, MLL rearrangement 2: cCD3+, mCD3–, CD7+, CD34+, CD117+, *Bilineal HLA-DR+, CD38+, CD19–, CD20–, CD10–, TdT–, MPO–, CD13+, CD33– c, cytoplasmic; FISH, fluorescence in situ hybridization; MPAL, mixed-phenotype acute leukemia; MPO, myeloperoxidase; NOS, not otherwise specified; WHO, World Health Organization.

A B

C 103 D 103 E1 E2 E1 E2 ❚Image 5❚ Example of rare B/T mixed-phenotype acute 102 102 leukemia with medium- to large-sized blasts: (A, May-Grünwald-Giemsa) bone marrow smear and (B, H&E) 101 101 bone marrow biopsy specimen. By flow cytometry, blasts CD10 cy79a were positive for cCD3 (T lineage, C) and CD19, CD10 and 100 100 E3 E4 E3 E4 CD79a (B lineage, C and D). See Table 3 for details. (Case 390, courtesy of H. Ayyad, MD, and colleagues.) 100 101 102 103 100 101 102 103 cyCD3 CD19

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A B Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021

C D

❚Image 6❚ Immunohistochemistry on bone marrow biopsy sections of case 268 shows CD117+ PAX5– area (A, ×100) corresponding to acute myeloid leukemia and CD117– PAX5+ area (B, ×100) corresponding to mixed-phenotype acute leukemia B/T (see Image 7 for flow cytometry findings corroborating B- and T-lineage characteristics). C and D, Higher magnification (×400) of outlined area as shown in A and B, respectively. (Case 268, courtesy of S. A. Ely, MD, and colleagues.)

normal T cells, and weak expression in a minor fraction of case 349 (Table 5), expression of cCD3 was very weak and blasts is insufficient to diagnose MPAL. Many AML cases could not be confirmed by IHC. Also, in case 303 (Table 2), show aberrant expression of other T-cell–associated mark- heterogeneous expression of cCD3 was not confirmed by ers, such as CD2, CD4, CD5, or CD7, or NK-cell–associated IHC, although a fraction of blasts that showed adequately markers, such as CD56.44,45 Thus, the correct interpreta- high expression to qualify as MPAL was observed by FCM, tion of cCD3 staining is important for final diagnosis. If a together with CD5 and CD7. No cCD3 expression was seen BM biopsy specimen is available, the expression of cCD3 at relapse in this patient. may be confirmed by IHC. In case 261, positive for CD2, CD4, and CD56, review of the FCM files revealed no sufficient cCD3 expression, and no confirmatory IHC could CD19 and Other B-Cell Markers be performed. Thus, considering the presence of a complex karyotype, a consensus diagnosis of AML with myelodys- The WHO classification provides no definite thresh- plasia-related changes was rendered ❚Table 5❚. Similarly, in old for the size of the population expressing CD19 that is

A 1,000 1,000 1,000

800 800 800

600 600 600

400 400 400 SSC-Height SSC-Height SSC-Height 200 200 200

0 0 0 100 101 102 103 100 101 102 103 100 101 102 103 CD45 PerCP CD45 PerCP CD45 PerCP Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021

B 3 3 10 10 1,000

102 102 800

600 101 101 400

CD19 PE 0 0 cyTdT FITC 10 10 SSC-Height 200

0 100 101 102 103 100 101 102 103 100 101 102 103 cyCD3 APC CD19 PE CD45 PerCP

C 3 3 10 10 1,000

102 102 800

600 101 101 400

CD13 PE 0 0 SSC-Height

10 cyMPO FITC 10 200

0 100 101 102 103 100 101 102 103 100 101 102 103 CD34 APC CD34 APC CD45 PerCP

D 3 3 10 10 1,000

102 102 800 600 101 101 400 0 0 CD34 APC CD20 APC 10 10 SSC-Height 200

0 100 101 102 103 100 101 102 103 100 101 102 103 CD38 FITC CD38 FITC CD45 PerCP

❚Image 7❚ Flow cytometry immunophenotypic findings in case 268: CD45 dim (blast) area contains complex populations of normal B-cell precursors, myeloid blasts, and an abnormal B/T population coexpressing cytoplasmic CD3 and surface CD19. A, Apparently homogeneous blast (Bermudes) cell population. B, B/T compartment. C, Myeloid M0 blasts. D, Hematogones. FITC, fluorescein isothiocyanate. Red, neutrophils; green, monocytes; pink, lymphocytes; light blue, blasts; dark blue, CD34+ cells; yellow, hematogones. (Case 268, courtesy of S. A. Ely, MD, and colleagues.)

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❚Table 4❚ Summary of Cases of MPAL With MLL Rearrangement Case Patient Consensus WHO Diagnosis/ No. Age, y/Sex Immunophenotype Cytogenetics/Molecular Genetics *Comments 256 15, F Lymph node: FCM: 37% blasts, CD3 dim, CD5 FISH on the lymph node, MPAL, T/myeloid, with t(v;11q23); dim, CD7+, CD33+, CD34+, CD79a+, TdT+, break-apart probe MLL MLL rearranged CD11s+, MPOs+. IHC: CD3 (weak), CD34, rearrangement+, BCR-ABL1 TdT, MPO (weak), CD20–, lysozyme – , negative on blood NOTCH1 negative, suggesting the absence of activating alterations in this pathway. BM 30% blasts not available for review. 180 63, M Initial: CD34–, cMPO–, CD117–, CD45+ , HLA– t(9;11)(p22;q23) Acute leukemia with recurrent DR+, CD4+, CD11b+, CD15+, CD36+, cytogenetic abnormality, t(9;11) CD64+, CD3–, CD10–, CD19– (p22;q23)

Relapse: CD34–, cMPO–, CD117–, CD45+, *Presented with acute monocytic Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 HLA-DR+, CD4–, CD11b–, CD15–, CD36–, leukemia with lymph node CD64–, CD3–, CD10–, CD19+, cCD79a+, involvement, relapse as B-acute nTdT+, cCD22+/– lymphoblastic leukemia, CD10 negative 370 9, M 2005 BM: FCM, 74% blasts CD33+, CD15+, BM 2005: 46,XY,add(12)(p11.2) Acute leukemia with MLL CD4+, CD16+, CD56+, HLA-DR+, CD11b. [9]ish ins(9;11)(p22;q23q23) rearrangement with cryptic variant The blasts were negative for CD13 and CD14, (5′MLL+,9cen+,WCP11–; t(9;11)(p22;q23), MLTT3-MLL B and T markers. MLL+, WCP11+) /46,XX[15] fusion with evolving new clone nuc ish 11q23(MLLx2)(5′MLL of IGH rearrangement presenting spx1)[124]/11q23(MLLx2)[76] as (1) acute myeloid leukemia 2006 BM: FCM, 91% blasts CD19+, CD22+, BM 2006: 46,XY,t(14;19) in 2005, (2) first relapse as HLA-DR+. Myeloid, other B and T markers (q32;p13)ish t(14;19) B-acute lymphoblastic leukemia negative (q32;p13)(3′IGH+,5′IGH in 2006, and (3) second relapse con 3′IGH+)[7]/46,XY[23] as extramedullary (vertebral nuc ish (5′MLLx3,3′MLLx2) mass) involvement by B-acute (5′MLL con 3′MLL x2)[45]/ lymphoblastic leukemia (MLLx2)[155]/12p13(TELx2), 21q22(AML1x2)[200] 2012: Extradural vertebral mass, FCM: 65% 2012: Extradural vertebral mass blasts CD45 dim, CD19s+, CD11bs+, 46,XY,inv(3)(p21q27),inv(12) CD79as+, CD38+, HLA-DR, negative: m and (q13q24.3),t(14;19)(q32;p13) cCD3, CD2, CD5, CD7, CD4, CD8, CD10, [8]/46,XY[1]ish ins(9;11) CD20, m and cCD22, sIg, CD13, CD14, (p22;q23q23)(5′MLL+;MLL+) CD33, CD23, CD15, CD16, CD56, CD117, nuc ish(5′MLLx3,3′MLLx2) CD36, CD64, TdT and MPO. IHC: MPO+, (5′MLL con 3′MLLx2) CD68–, lysozyme–, CD19: weakly (70%), [134/200]/ (ETV6x2)[200]/ CD79a+(90%), PAX5+ (90%) (5′IGHx2,3′IGHx3)(5′IGH con 3′IGHx2) [136/200] 392 68, M Previous therapy for B-lymphoma. Diagnosis: Diagnosis: 46,XY,t(4;11) Therapy-related myeloid neoplasm, FCM: CD45 dim, HLA-DR+, CD19 bright, (q21;q23)[16]/46,XY,t(4;11) with t(4;11), MLL rearranged, CD22h+, CD38+, CD34s+, CD10–. IHC: TdT+, (q21;q23),del(7)(q22q34)[4] presenting as B-lymphoblastic PAX5+, and CD79as+, negative: CD10, CD34, leukemia with relapse as acute CD117, CD22, CD68, lysozyme, and BCL6 myeloid leukemia (monocytic differentiation) and second relapse as MPAL B/myeloid with t(v;11q23); MLL rearranged (bilineal) Relapse 1: FCM CD45+, HLA-DR+, CD14+, Relapse 1: 46,XY,t(4;11)(q21;q23) CD11c+, CD11b+, CD13+ , CD33+, CD34–, [13]/46,XY[7] CD117– expression B markers negative. IHC: CD68+, lysozyme+, CD163 weak, negative TdT, CD34, CD117, CD10, PAX5, CD45RA, CD123, MPO Relapse 2: FCM, two abnormal populations: B-LBL Relapse 2: MLL confirmed by 50% CD45 dim, HLA-DR+, CD19+, CD22+, FISH CD38+, CD34s+, CD5–, CD10–, CD11c–, CD11b–, CD13–, CD33–, CD20–, CD23–, SIg–. Monocytic: 20%-30% CD45h+, CD14+, HLA-DR+, CD4h, CD11c+, CD11b+, CD13h+, CD33+, CD38+. IHC: TdT+, PAX5 50%+, CD68 40%+, lysozyme 80%+, negative CD79a, CD20, CD22, CD10, CD34, CD117, MPO

B-LBL, B-lymphoblastic; BM, bone marrow; c, cytoplasmic; FCM, flow cytometry; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; MLL, mixed-lineage leukemia; MPAL, mixed-phenotype acute leukemia; MPO, myeloperoxidase; WHO, World Health Organization.

❚Table 5❚ Summary of AUL and AML Cases Discussed in the Session Patient Case Age, y/ Consensus WHO Diagnosis/ No. Sex Immunophenotype Cytogenetics/Molecular Genetics *Comments 199 89, F FCM, one blast population CD34+, CD117+/–, 46,XX AUL (consensus not reached) CD13, CD33 dim, HLA-DR, TdT, CD11b dim, CD4, CD7s dim, and cCD22 dim, negative for CD3, CD19, CD20, CD10, CD2, CD5, and MPO. IHC: PAX5s and CD79as *Infiltrates of plasmacytoid dendritic cells 250 47, F FCM, one blast population (86%): CD7+, 92-97<4n>,XXXX,+4,+4,+6,– AML with minimal differentiation, CD33+, CD34+, CD38+, HLA-DR+ 7,?t(10;11)(p12-13;q14-21) with t(10;11); PICALM/MLLT10

x2,–13,i(17) Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 (q10),+20[cp4]/46,XX[16] Negative: cCD3, sCD3, CD10, CD11c, CD14, FISH for BCR-ABL1 negative, four CD19, CD22, CD56, CD61, CD64, cCD79a, signals each for BCR and ABL1; MPO FISH for PML-RARA negative, four signals each for PML and RARA 396 38, M FCM, one blast population (96%): CD34+, Insufficient AML with minimal differentiation HLA-DR+, CD7+, CD33 dim, CD38s dim, Molecular: Negative for b3a2, b2a2, CD9s dim, CD52, CD58, CD81, CD49b, and e1a2 transcripts of bcr-abl/ CD184s dim t(9;22)(q34;q11), the short and Negative: CD19, CD20, CD22, cCD79a, long forms of PML-RARA/t(15;17) CD10, sIg, CD1, CD2, CD3, cCD3, CD4, (q22;q21), the A and D forms CD8, CD5, CD25, CD56, TdT, CD13, CD14, of CBFb-MYH11/inv(16), CD15, CD41, CD64, CD117, and MPO AML1-ETO/t(8;21)(q22;q22), E2A-PBX-1/t(1;19)(q23;p13), MLL-AF4/t(4;11)(q12;q23), and TEL-AML1/t(12;21)(p12;q22) 349 7 mo, M FCM: one population (skull mass and BM) 46,XY AML with minimal differentiation cCD3 dim, CD13 dim, CD33, CD34, CD56 bright, and CD117. Negative for CD1a, CD2, mCD3, CD5, CD4, CD7, CD8, CD11b, CD11c, CD14, CD16, CD41, CD61, CD64, glycophorin A, HLA-DR, MPO, and TdT 261 37, F FCM one population (82%) CD2+, CD4+, 46,XX,inv(21)(p13q22.1)[8]/45- AML with myelodysplasia related CD13+, CD33+, CD34+ dim, CD56+, CD6, 46,sl,add(6)(p22),i(8)(q10)[2],der(11) changes (complex karyotype) and MPO t(11;11)(p15;q13),del(12)(p11.2) [2],add(16)(p13.1),–17[3],add(17) (p13)[3],del(17)(p13)[4],del(18) (q21.1)[2][cp10]/46XX[2]

AML, acute myeloid leukemia; AUL, acute unclassifiable leukemia; BM, bone marrow; c, cytoplasmic; FCM, flow cytometry; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; MPO, myeloperoxidase; WHO, World Health Organization.

necessary to define B-lineage involvement. In two cases of What Criteria Are Helpful in the bilineal B/myeloid MPAL (cases 206 and 365), only minor Differential Diagnosis of AUL vs AML CD19+ populations were detected (5%-7% and 12.5% in With Minimal Differentiation? cases 206 and 365, respectively). Since B-cell lineage was Five cases submitted to this session (cases 199, 250, confirmed by other markers (Table 1), criteria were met for 261, 349, and 396) illustrated classification difficulties the diagnosis of MPAL. The intensity of CD19 expression between AUL and AML with minimal differentiation (Table by blasts should be compared with that expected by normal 5). True AUL is very rare, and these neoplasms should not B cells and, if comparable, considered bright. It has to be express markers of B, T, or myeloid lineage and should not stressed that in cases in which the whole blast cell popula- fulfill criteria for B- or T-ALL, AML with minimal differen- tion strongly expresses CD19, with the immunophenotype tiation, or MPAL. Nevertheless, AUL cases can be positive otherwise being consistent with B-ALL/lymphoma, the for CD34, CD38, HLA-DR, and/or TdT. Importantly, there presence of MPO expression as a sole aberrant marker may be cases that cannot be adequately classified due to should not dissuade therapy using a B-ALL regimen, and insufficient immunophenotyping data or discordant expres- patient prognosis does not seem to differ from other patients sion of various markers, rendering definitive classification with B-ALL.32 impossible. The latter cases should be designated as acute

374 Am J Clin Pathol 2015;144:361-376 © American Society for Clinical Pathology DOI: 10.1309/AJCPSTU55DRQEGTE AJCP / Review Article unclassifiable leukemia, which is different from AUL.46 8. Rubnitz, JE, Onciu M, Pounds S, et al. Acute mixed lineage The diagnosis of acute unclassifiable leukemia could be leukemia in children: the experience of St Jude Children’s Research Hospital. Blood. 2009;113:5083-5089. applied to case 199, for which the review panel could not 9. Bene MC, Castoldi G, Derolf A, et al. Near-tetraploid acute reach a consensus. In three other cases (Table 5), the panel myeloid leukemias: an EGIL retrospective study of 25 cases. consensus diagnosis was AML with minimal differentiation Leukemia. 2006;20:725-728. due to the expression of CD33 with no expression of B- or 10. Lemez P, Attarbaschi A, Bene MC, et al. Childhood near- T-lineage–associated markers. tetraploid acute lymphoblastic leukemia: an EGIL study on 36 cases. Eur J Haematol. 2010;85:300-308. 11. Manola KN. Cytogenetic abnormalities in acute leukaemia Corresponding author: Anna Porwit, MD, PhD, Dept of Pathobiology of ambiguous lineage: an overview. Br J Haematol. and Laboratory Medicine, University of Toronto, University Health 2013;163:24-39. Network, Toronto, ON, Canada; [email protected]. 12. La SR, Aventin A, Crescenzi B, et al. CIZ gene Acknowledgments: The following were contributors or rearrangements in acute leukemia: report of a diagnostic Downloaded from https://academic.oup.com/ajcp/article/144/3/361/1760621 by guest on 25 September 2021 co-contributors of cases to this session: S. Acree, R. E. Alexander, FISH assay and clinical features of nine patients. Leukemia. S. Al-Quran, S. A. Alghamdi, D. Arber, H. Ayyad, A. Bagg, F. 2005;19:1696-1699. H. Barakat, A. Basen, A. R. Brothman, C. E. Bueso-Ramos, L. 13. Martini A, La SR, Janssen H, et al. Recurrent rearrangement Cannizzaro, C. L. Cantu, C. Carter, W. Z. Chen, P. Cohen, A. 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