ICCS e-Newsletter CSI Hamid Zia, MD Andrew Chu, MD Franklin Fuda, DO Department of Pathology University of Texas Southwestern Medical Center Dallas, Texas

Clinical History A 29 year-old pregnant female (G3P1A1) was found to have leukocytosis, anemia and thrombocytopenia on routine CBC at ~6 week-gestation She had night sweats and nausea for a week She experienced an episode of gum bleeding while brushing her teeth approximately one week ago A peripheral blood smear review showed blasts

CBC Results CBC ResultNormal Range WBC: 55 K/mm K/mm 3 RBC: 3.29 M/mm – 5.10 M/mm 3 Hgb: 11 g/dL12.0 – 15.0 g/dL HCT: 31.8% % MCV: 96.7 fl80.0 – 98.0 fl MCH: 33.4 pg27.0 – 33.0 pg MCHC: 34.6 g/dL33.0 – 35.0 g/dL RDW: 13.7%11.3 – 15.1 % Platelets: 33 K/mm – 450 K/mm 3

A peripheral blood specimen was received in the flow cytometry lab with history of leukocytosis, anemia & thrombocytopenia Four color flow cytometry was performed using a BD FACScalibur™ flow cytometer. Ungated, cluster analysis was performed with BD Paint-a-Gate™ software. Files are in FCS2.0 format.

Selected tubes* with the following antibody combinations are included for review. TubeFITCPEPerCPAPC 1CD10CD22CD20CD34 2 CD14CD45CD38 3CD36CD64CD45CD34 4CD16CD56CD45CD11b 5CD15CD33CD45CD34 6CD2CD117CD45CD34 7CD7CD13HLA-DRCD34 *Seven selected representative tubes from a 10 tube analysis

Looking at the first tube file, a large population of CD34(+) events becomes apparent The CD34(+) events are isolated in red as the “population of interest” The CD34(+) events are then “cleaned” to isolate a “cluster” of like cells. In this example, the events are cleaned on forward vs side scatter using black to exclude all cells that do not appear to belong to the cluster. Any combination of markers and/or physical parameters can be used in a similar fashion to clean the cluster. Cluster of interest granulocytes and monocytes debris/mature red blood cells doublets Tube 1

The CD34(+) blasts (in red) lack expression of CD10, CD20 and CD22 Mature B-cells Neutrophils Basophils Mature B-cells Other cell populations can be isolated in a similar step-wise fashion of gating and cleaning.

Tube 2 Red = Blasts Green = Granulocytes Blue = Monocytes Maturing Eosinophils Maturing Neutrophils 15% population of variably-sized blasts: CD34(+), CD38(variably +), CD45(moderately +) 54% monocytes with variable expression of CD14 14% maturing granulocytic elements with decreased orthogonal light scatter properties in the maturing neutrophils (suggesting hypogranularity)

Tube 3 Expanded population of monocytes that are show increased variability for CD36 & CD64 In our lab, mature monocytes are typically double bright for CD36 and CD64 Few blasts are CD64(+) Few blasts are CD36(+) Typical location of mature monocytes Red = Blasts Green = Granulocytes Blue = Monocytes

Tube 4 Blasts are CD16(-), CD56(-) & 11b(-) Monocytes show increased variability for CD11b Granulocytic maturation curve for CD11b/CD16 looks normal There is no expression of CD56 on monocytes & granulocytes Red = Blasts Green = Granulocytes Blue = Monocytes

Tube 5 Red = Blasts Green = Granulocytes Blue = Monocytes Cyan = Lymphocytes Blasts stain positive for the myeloid markers CD15 & CD33 but have a slightly atypical maturation curve for CD15, CD33 AND CD34 Monocytes are expanded but look relatively normal CD15(+) with moderate CD33 rather than bright Double bright expression of CD15 and CD34

Tube 6 Tube 7 Blasts express the myeloid associated marker CD117 A minute proportion of monocytes express CD34 and CD117, which is evidence of immaturity for this proportion Blasts express the myeloid marker CD13 A large proportion of the blasts show unusual dim expression of HLA-DR The monocytes also show increased variability for HLA-DR and CD13

Overall immunophenotypic Findings: 15% population of variably-sized blasts with the following immunophenotype: CD34(+), CD2(few +), CD13(+), CD15(partial +), CD25(few +), CD36(few +), CD38(variably +), CD45(moderately +), CD64(few +), CD117(+), CD123(variably +), HLA-DR(variable +), MPO(dim +), TdT(-), other myeloid and lymphoid antigens (predominantly -) 54% monocytes with variable expression of CD11b, CD13, CD14, CD36, and HLA-DR and with a minute proportion showing expression of CD34 and CD117

Flow Cytometry Interpretation An expanded population of immunophenotypically aberrant myeloblasts in a background of expanded monocytic cells with some aberrant features and some features of immaturity Consistent with a high grade myeloid neoplasm with differential diagnoses including (but not limited to) evolving acute myeloid leukemia with monocytic differentiation or chronic myelomonocytic leukemia-2

Peripheral Blood Blasts- 22% Monocytic cells- 44% Blasts and immature cells Promonocyte

Dysplastic eosinophil Bone Marrow Aspirate Courtesy of Weina Chen, MD University of Texas Southwestern Medical Center Dallas, Tx Myeloblast Immature Monocytes

Dysplastic eosinophils Bone Marrow Aspirate Courtesy of Weina Chen, MD University of Texas Southwestern Medical Center Dallas, Tx

Bone Marrow Core Biopsy Marrow packed with immature mononuclear cells and scattered eosinophilic precursors

46,XX,inv(16)(p13q22)[20] Karyogram

CBFB 16q22 (3' tel) CBFB 16q22 (5' cent) FISH panel with evidence of a CBFB [inv(16)] gene rearrangement along with a deletion of the 3' (telomeric) end of the CBFB gene on interphase nuclei

Final Diagnosis ACUTE MYELOID LEUKEMIA (AML) WITH INV(16)(p13q22).

AML WITH INV(16)(p13q22) A Core Binding Factor Leukemia: Defined by a recurrent cytogenetic abnormality irrespective of blast count at diagnosis- inv(16)(p13q22) or t(16;16)(p13;q22) WHO 2008 Classification Definition: Acute myelomonocytic leukemia with abnormal eosinophils (AML-M4EO by FAB) in the bone marrow

Epidemiology: 5-10% of both adult & pediatric AML Clinical Features Peripheral blood & bone marrow always involved May present with myeloid sarcoma in up to 50% of cases (highest incidence for any AML) Lymphadenopathy and hepatomegaly are particularly common AML WITH INV(16)(p13q22)

AML WITH INV(16)(p13q22) Prognosis Overall good prognosis Good response to Cytarabine based Rx KIT mutations are present in approximately 30% of cases; worse overall survival and higher rates of relapse. Trisomy 22 - Specific and seen in 10-15% cases  favorable outcome

AML WITH INV(16)(p13q22) Morphology Peripheral Blood & Bone Marrow: Heterogeneous neoplastic population of myeloblasts, monoblasts & promonocytes Maturing granulocytes are sparse and do not show dysplasia Hypercellular marrow often with more than 20% blasts (may be lower than 20% in some cases) Auer rods may be seen in blasts

AML WITH INV(16)(p13q22) Morphology Peripheral Blood & Bone Marrow: Most striking abnormality: Eosinophils and eosinophilic promyelocytes and myelocytes have large immature basophilic granules

AML WITH INV(16)(p13q22) Molecular /Cytogenetic The inv(16)(p13q22) or t(16;16)(p13;q22) Classical cytogenetics could be negative: Use FISH & RT-PCR to identified submicroscopic case if index of suspicion is high Locus 16q22 - beta subunit of the core binding factor (CBFβ) Locus 16p13 - smooth muscle myosin heavy chain (MYH11) Both cytogenetic alterations generate the fusion transcript CBFβ–MYH11

AML WITH INV(16)(p13q22) Molecular /Cytogenetic Multiple fusion transcripts exist CBFβ–MYH11 fusion transcript prevents the normal differentiation process by: Sequestering CBFα2 in the cytoplasm Acting as a transcriptional repressor by recruiting co-repressors Acquiring chromatin-modifying histone deacetylase activities upregulate NF-kappa B signaling pathway

AML WITH INV(16)(p13q22) Molecular /Cytogenetic The deletion of the 3′ end of the CBFB gene in inv(16) confirmed by FISH in the present case is a rare finding, and has been described only in AML M4; its prognostic significance is as yet undetermined. In contrast, deletion of the 5′ region of the MYH11 (16p13) is relatively common, detected in ∼ 20% of AML with inv(16).

Double Population of Pathological cells: Myeloid blast cells: CD34, CD117, CD13, CD33, CD15, myeloperoxidase. Monocytic cells: CD4, CD11b, CD11c, CD14, CD64, CD36, lysozyme Aberrant coexpression of CD2 in the blast population and monocytic cells occurs in a subset of cases but it is not specific for this type of AML with inv(16)(p13q22) or t(16;16)(p13;q22) AML WITH INV(16)(p13q22) Immunophenotype

References Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW (Eds.): WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues. IARC: Lyon Ortolani C. Flow Cytometry of Hematological Malignancies. Wiley-Blackwell, Tirado, Carlos A., et al. "Acute myeloid leukemia with inv (16) with CBFB–MYH11, 3′ CBFB deletion, variant t (9; 22) with BCR–ABL1, and del (7)(q22q32) in a pediatric patient: case report and literature review." Cancer genetics and cytogenetics (2010):