Case Study Interpretation Carrie Chenault Propath, Dallas TX and “Buddy” Frank Fuda University of Texas Southwestern Medical Center, Dallas TX

Clinical history CBC Data 67 yo F with a history of hepatocellular carcinoma s/p chemotherapy and cirrhosis presents with fever and pancytopenia. CBC Data Normal Ranges WBC: 3.37 K/mm3 4.0 – 11.0 K/mm3 RBC: 2.57 M/mm3 3.70 – 5.10 M/mm3 Hgb: 7.3 g/dL 12.0 – 15.0 g/dL Hct: 22.2% 34.0 – 44.0% MCV: 86.4 fl 80.0 – 98.0 fl MCHC: 32.9 g/dL 33.0 – 35.0 g/dL RDW: 16.1% 11.3 – 15.1% Plts: 20 K/mm3 150 – 450 K/mm3

A bone marrow biopsy was performed for morphology, cytogenetics, and flow cytometry.

Triage Flow cytometry morphologic preparations Smear Cytospin Numerous immature cells; suspect acute leukemia Thus, an Acute Leukemia Panel was chosen

Selected 4-color tubes from the Propath Acute Leukemia Panel run on the BD FACSCanto II™ Tube 1: CD14 FITC/CD64 PE/CD45 PerCP/CD34 APC Tube 2: HLA-DR FITC/CD13 PE/CD33 PerCP/CD45 APC Tube 3: CD15 FITC/CD117 PE/CD36 PerCP/CD45 APC Tube 4: CD16 FITC/CD71 PE/CD45 PerCP/CD11b APC Tube 5: IC MPO FITC/CD3 PE/CD34 PerCP/CD79a APC

A few extra 4-color tubes added from a second bone marrow sample performed two days later at a neighboring institution (UTSW). BD FACSCalibur ™ Tube 6: CD34 FITC/CD14 PE/CD45 PerCP/CD38 APC Tube 7: CD16 FITC/CD56 PE/CD45 PerCP/CD11b APC Tube 8: CD15 FITC/CD33 PE/CD34 PerCP/CD117 APC Tube 9: CD36 FITC/CD64 PE/CD34 PerCP/CD117 APC

Flow cytometry set up 4-Color Flow Cytometry BD FACSCanto II™ flow cytometer (first set of data/Propath) BD FACSDiva software was used to acquire data Files are in FCS2.0 format. BD FACSCalibur™ flow cytometer (second set of data/UTSW) BD CellQuest Pro software was used to acquire data Leukobyte Cytopaint ClassicTM software was used for data analysis

Leukobyte Cytopaint ClassicTM software The following examples from both laboratories were analyzed using CytopaintTM software. A freeform analysis, known as “cluster analysis,” was performed on ungated data. This analysis does not utilize predetermined gating strategies but rather allows the analyst to “paint and clean” clusters of cells in any manner they see fit.

A cell cluster of interest can be identified on almost any plot. Cluster analysis Cluster analysis assumes that cellular subsets form distinct clusters in multi-dimensional flow cytometry space. A cell cluster of interest can be identified on almost any plot. In this example, we will use side scatter verses forward scatter to begin, although any combination of markers and/or physical parameters can be used.

We can choose to investigate this cluster of large cells with moderate to high side scatter. We use cytopaint to color the cluster red with our “paint brush” cursor.

Some of the events that we colored on the SCC vs FSC plot clearly do not belong to the cluster on other plots. See the scattered events painted red on these plots.

We can color all the areas that fall outside our cluster of interest black on several plots.

Such a subset is not present in this example. The black can then be removed to erase scattered cells that do not belong to the cluster. Caveat We need to be careful not to erase a small subset of cells that actually belongs to the larger cluster but shows a different expression pattern for a particular marker. Such a subset is not present in this example.

This would presumably leave us with a cluster of like cells.

We use this approach to paint all the clusters of interest in all tubes. Red: Presumed atypical population Yellow: Myeloblasts Green: Granulocytes Blue: Lymphocytes Gray: All non-colored events

The Yellow cells are CD34(+) myeloblasts. TUBE 1 Forward scatter versus side scatter shows an expanded population of large, somewhat granular cells (red). On CD45 versus side scatter, this population sits in a similar region as the granulocytes (green), showing moderate expression of CD45. Therefore, our population of interest is hematolymphoid. The Yellow cells are CD34(+) myeloblasts. The Blue cells are lymphocytes.

TUBE 1 cont. The population shows evidence suggestive of monocytic differentiation with predominantly bright expression of CD14 and CD64. The population lacks expression of CD34, which is a marker of immaturity. Red: Presumed Atypical Cells; Yellow: Myeloblasts; Green: Granulocytes; Blue: Lymphocytes; Gray: All non-colored events

TUBE 2 The population expresses the myeloid marker CD13 and shows more evidence suggestive of monocytic differentiation with bright expression of CD33 and expression of HLA-DR. Red: Presumed Atypical Cells; Yellow: Myeloblasts; Green: Granulocytes; Blue: Lymphocytes; Gray: All non-colored events

TUBE 3 Here, the population shows variable expression of the myeloid marker CD15, which is characteristic of monocytes. It also shows expression of CD36, which is a marker expressed by monocytes (CD36 is also expressed by erythroid and megakaryocytic progenitors, platelets, and subsets of dendritic cells). A small subset of the population is CD117(+). CD117 is a stem cell marker present on the first two stages of myeloid differentiation (i.e., the blast stage and the promyelocyte stage). As such, in the appropriate context, CD117 can be considered a marker of immaturity. Red: Presumed Atypical Cells; Yellow: Myeloblasts; Green: Granulocytes; Blue: Lymphocytes; Gray: All non-colored events

TUBE 4 Up to this point, our population has shown evidence suggesting an expanded population of monocytes, with a very small subset showing evidence of immaturity. In this tube, we see that the population expresses CD11b but not CD16. Being a monocyte population, a significant level of CD16 expression is not expected, as only a small proportion of monocytes normally express CD16. CD11b, however, is typically expressed by monocytes at a high intensity (or brightly). Here, the population shows partial dim to moderate expression. Although subsets of normal monocytes can show decreased expression of CD11b, the intensity of CD11b on our population is unusual and raises concern for a neoplasm.

TUBE 5 The cells in this tube have been permeabilized to allow investigation for cytoplasmic antigen expression. The population expresses the myeloid marker myeloperoxidase and lacks expression of the B-lineage marker CD79a and the T-lineage marker CD3.

TUBE 6 The remaining tubes were additional tubes performed at a neighboring institution on a second bone marrow sample performed within a couple days. While the features are the essentially the same between the two institutions, notice that the plots look slightly different. This is due to differences in the flow cytometry machines, the processing methods, available fluorochromes, and the level of peripheral blood contamination. Notably, the UTSW sample was hemodilute, as evidenced by an increased proportion of T-cells, NK-cells and CD16(+) neutrophils. Our case Normal Bone Marrow Physical parameters show similar features on both machines. The monocyte population is large with relatively high side scatter. This plot is from a normal bone marrow for comparison. Notice, the increased side scatter of our monocyte population verses normal. Red: Presumed Atypical Cells; Yellow: Myeloblasts; Green: Granulocytes; Blue: Lymphocytes; Gray: All non-colored events

TUBE 6 Compare the bright intensity of CD38 on our population (left) to the moderately bright intensity usually seen on monocytes in benign bone marrow (red population below). In this tube, our population shows unusually bright expression of CD38. Our case Normal Bone Marrow Red: Presumed Atypical Cells; Yellow: Myeloblasts; Green: Granulocytes; Blue: Lymphocytes; Gray: All non-colored events

Tube 7 In this tube, CD56 is expressed by nearly the entire monocyte population. While normal regenerating or reactive monocyte populations often express CD56, in our experience, CD56 is usually on a portion of these monocytes (i.e., partial CD56) rather than the entire population. Here, almost the entire monocyte population expresses CD56. Although not definitively diagnostic, this expression pattern raises a higher level of suspicion for a neoplastic process.

from normal bone marrow Typical pattern from normal bone marrow Tube 7 BM monocyte populations are usually predominantly negative for CD56; however, reactive/regenerative monocytes can show significant expression of CD56. In this normal BM case, nearly all monocytes are CD56(-). BM monocyte populations are mostly bright positive for CD11b. Our case Our case Here, we see decreased expression of CD 11b. Once again, this is concerning for a neoplasm but by itself is not specific. Here, we see expression of CD56 on nearly all the monocytes. While this is not specific, it is concerning for a neoplasm.

Tubes 8 and 9 Tubes 8 and 9 were added to verify the identity of the CD117(+) subset of cells The monocyte population shows a relatively typical pattern of expression for monocytes on the CD15 vs CD33 plot (tube 8) and the CD36 vs CD64 plot (tube 9). Tube 8 Tube 9 CD117(+) subset CD117(+) subset

Tubes 8 and 9 Closer look at the expression pattern for both the CD15 vs CD33 plot (tube 8) and the CD36 vs CD64 plot (tube 9) actually reveals that our population slightly deviates from a normal monocyte expression pattern. Tube 8 Typical pattern from normal BM Our population Here, nearly the entire population shows bright expression of CD15. While not specific, we often see this feature associated with neoplastic monocytes. Usually, monocytes show variable expression of CD15 from negative to moderate to bright positive.

Typical pattern from normal BM Tubes 8 and 9 Tube 8 Typical pattern from normal BM Our population Also notice that the minute myeloblast population shows unusually dim expression of CD33.

Typical pattern from normal BM Tube 9 Typical pattern from normal BM Our population Normally, there is only a small proportion of CD36 negative to moderately positive monocytes. More mature forms show nice bright expression of CD36. In our population, the CD36 negative to moderately positive monocytes are expanded.

Compare and Contrast The following slide shows further examples of the current case versus a non-neoplastic normal bone marrow. Color code for all the plots Red: Presumed atypical population Yellow: Myeloblasts Green: Granulocytes Blue: Lymphocytes Gray: All non-colored events

from normal bone marrow Typical pattern from normal bone marrow As monocytes mature from monoblasts to mature monocytes, they gain expression of CD14. Most monocytes in bone marrow are at a mature stage with bright expression of CD14. Monocyte populations in bone marrow usually show predominantly bright of expression of CD45. Our case Our case Here, we see slightly dim expression of CD45. This is a common finding in immature monocyte populations but is not specific. In our case, there is a relatively normal expression pattern for CD14 but with an increased proportion of CD14(dim +) cells.

Overall flow findings Note: If there is no deviation from the way normal monocytes express a marker, we label the marker as (+) without further description. Immunophenotype of the aberrant monocytes in our case: CD4(+) CD11b(partial +) CD13(+) CD14(predominantly +) CD15(bright +) CD16(-) CD33(+) CD34(-) CD36(variably +) CD38(bright +) CD45(slightly dim +) CD56(predominantly +) CD64(+) CD117(predominantly - /small subset +) HLA-DR(+) MPO(+) Aberrancies (unusual findings) relative to normal monocytes: Partial and dim expression of CD11b Bright CD38 expression Slightly dim expression of CD45 CD56 expression on most cells CD117 expression on a small subset of cells Overrepresentation of CD15(bright +) cells Overrepresentation of CD36( to moderately +) cells Increased proportion of CD14(dim +) cells

Monocytes Determining the significance of aberrancies on monocytes can be difficult. Many of the aberrancies on monocytes are not absolute and can be seen in reactive or regenerative populations of monocytes. Aberrancies on monocytes must be considered in conjunction with the overall immunophenotype. In this case, there are several aberrancies that alone might not raise a high level of suspicion but taken together raise concern for a monocytic neoplasm. Immature neoplastic monocyte populations often do not express markers of immaturity such as CD34 and CD117. Mature neoplastic monocytes and immature neoplastic monocytes may show the same immunophenotype. This can create difficulty in differentiating between subcategories of monocytic leukemia such as chronic myelomonocytic leukemia and acute myeloid leukemia with monocytic differentiation. In this case, only a small subset of the aberrant monocytes (i.e., only 2% of the monocyte population) expressed a marker of immaturity (i.e., CD117). Immature neoplastic monocytes may or may not show other features associated with immaturity. Monoblasts often lack expression of CD14 and CD13 and show bright expression of CD15. The aberrant monocytes in this case, which are monoblasts morphologically, retain CD13 expression and mostly CD14 expression. They show bright expression of CD15.

Overall flow findings (Cont.) In addition to the aberrant monocytes, there was a minute population (0.09% of total events) of myeloblasts with mild variation CD33 dim(+) Variable CD38 expression Few cells that lacked expression of HLA-DR By flow cytometry, the granulocytes were approximately 10-20% (depending on the level of peripheral blood contamination) A careful bone marrow differential needs to be performed on morphologic material to determine the proportion of monocytic elements versus granulocytic elements for final disease classification Additional information from morphologic, genetic, and clinical features is particularly important in subclassifying monocytic neoplasms.

Flow Cytometry Interpretation The results show an expanded population of immunophenotypically aberrant monocytes. In addition, there is a minute population of myeloblasts showing immunophenotypic variation. In association with the numerous immature cells/blasts identified on the morphologic preparations, these findings suggest a high grade myeloid neoplasm and would support a diagnosis of acute myeloid leukemia with monocytic differentiation. Final interpretation requires correlation with a complete morphologic work up as well as genetic and clinical features.

Morphology Work Up Bone marrow aspirate Blasts with morphologic features suggestive of monocytic lineage

Bone marrow aspirate Blast with erythrophagocytosis

Bone marrow core biopsy Sheets of immature cells

Cytogenetic findings

Reminder of clinical history Hepatocellular carcinoma s/p chemotherapy

Therapy related acute myeloid leukemia with t(8;16)(p11;p13) Final Diagnosis Therapy related acute myeloid leukemia with t(8;16)(p11;p13)

Leukemia 2009:23:934–943. Leukemia Research 2013 Jan;37(1):32-6. AML with t(8;16)(p11;p13) The (8;16) translocation is observed in acute myeloid leukemia (AML) including AML-M4 and AML-M5. This is a rare subtype, 0.4% of all acute leukemias, but it is more common in therapy related AML with an incidence of 1.6%. It can occur after a history of solid tumors (breast cancer) or hematological diseases (chronic myelomonocytic leukemia and lymphomas) and is associated with previous therapy with alkylating drugs in combination with topoisomerase-II-inhibitors Interval from chemotherapy to acute leukemia is short, often without a preleukemic phase. Patients may present with leukemia cutis and have a high incidence of DIC. It has a poor prognosis, median overall survival 4.7 months. Leukemia 2009:23:934–943. Leukemia Research 2013 Jan;37(1):32-6.

AML with t(8;16)(p11;p13) Blasts have the characteristic morphologic finding of erythrophagocytosis. Blasts have characteristic flow findings including increased side scatter; expression of MPO, CD4, CD11b, CD13, CD14, CD15, CD33, CD56, CD64; and lack of markers of immaturity (CD34 and CD117). Cytochemical stains often show coexpression of MPO and NSE. Leukemia 2009;23:934–943.

Summary AML with t(8;16)(p11;p13) is rare entity with unusual and unique clinical, morphologic, flow cytometric, and cytochemical features. It has been reportedly mistaken for acute promyelocytic leukemia, but the immunophenotype is more likely to be mistaken for mature aberrant monocytes. Prognosis is poor with a median survival of 4.7 months1 1. Haferlach T, Kohlmann A, Klein HU, Ruckert C, Dugas M, Williams PM, Kern W, Schnittger S, Bacher U, Löffler H, Haferlach C. AML with translocation t(8;16)(p11;p13) demonstrates unique cytomorphological, cytogenetic, molecular and prognostic features. Leukemia. 2009 May;23(5):934-43.

References Haferlach T, et al. Leukemia 2009;23:934–943. Diab A, et al. Leukemia Research 2013 Jan;37(1):32-6.