ICCS - PowerPoint Presentation

Slide1

ICCS e-Newsletter CSIWinter 2013

Jacqueline Emmons, MD

Department of Pathology

University of Texas Southwestern Medical Center

Dallas, TexasSlide2

History 11 month old female with a history of fever and failure to thriveOn physical exam, the child was pale and lethargic.Slide3

CBC Data

Normal Ranges

WBC: 67 K/mm

3

6

-- 17.5 K/mm3RBC: 3.23 M/mm33.70 – 5.30 M/mm3Hgb: 9.3 g/dL11.1 – 14.2 g/dLHct: 26%30 – 42%MCV: 80.5 fl70.0 – 84.0 flMCHC: 35.8 g/dL32.0 – 36.0 g/dLRDW: 14.1%11.5 – 15.0%Plts: 62 K/mm3150 – 600 K/mm3

A manual differential of the peripheral blood revealed 69% blasts.Slide4

A peripheral blood sample was received in the flow cytometry lab with the indication to “rule out acute leukemia.”Selected tubes

from the diagnostic analysis are included in this case study for review.Slide5

A four color analysis was performed using a BD FACScalibur™ flow cytometer. An ungated

, cluster analysis was performed with BD Paint-a-Gate™ software. Files are in FCS2.0 format.

Tubes with the following antibody combinations are included for review.

Tube

FITC

PEPerCPAPC1CD10CD22CD20CD342CD14CD11bCD34CD193CD45CD11cCD34CD144CD15CD33CD45CD345

CD36

CD64

CD45

CD34

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CD2

CD117

CD45

CD34

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(intracellular)

MPO

CD79a

CD45

CD34Slide6

TUBE 1

Upon opening the file, it is apparent

that there are a large number of CD34(+) events.

After using CD34 to isolate the cells of interest

(painted in red), the forward versus side scatter plot

can be used to further define or “clean up” thepopulation. Here, all events outside of the “blast gate” are highlighted in black and will be excluded.doubletsgranulocytes

debrisSlide7

TUBE 1

The CD34(+) blasts (red) are

n

egative for CD20 and CD10 and

are partial dim positive for CD22.Slide8

TUBE 2

As anti-CD34 is included in tube 2 as well,

the blasts can be isolated and analyzed in

a similar fashion to tube 1.

In addition, other cell types can also be

studied in this tube.To look for any CD34(-) monocytic cells, CD14 can be used to gate. The population can then be better defined using theforward versus side scatter and CD34.Exclusion of debris, lymphocytes, granulocytes,and doubletsExclusion of CD34(+)eventsSlide9

TUBE 2Granulocytes painted green on forward versus side scatter

Clean up with CD34(+)

eosinophils

Neutrophilic

elementsSlide10

TUBE 2

Small lymphocytes can be

isolated by first painting on the forward versus side scatter. Some of the blasts are small and overlap with the lymphocyte gate in this plot. The CD34 can be used to exclude blasts. Then, CD14 can be used to exclude monocytes. Slide11

TUBE 2

A complete analysis of tube 2 with all cell populations analyzed would look like this:

Color Key:

Red: Blasts

Light blue: Lymphocytes

Dark blue: MonocytesGreen: GranulocytesSlide12

TUBE 2

The blasts are CD19(partial +) and CD11b(partial +). A few of

the blasts appear to express CD14.Slide13

Tubes 3 – 7 can all be analyzed in a similar manner to tubes 1 and 2.The immunophenotype of the blasts is as follows:

CD34(+)

CD2(-)

CD10(-)

CD11b(partial +)

CD11c(partial +)CD14(few cells +) CD15(partial +)CD19(partial +)CD20(-)CD22(partial dim +)CD33(variably +)CD34(+)CD45(moderately +)CD64(partial +)Cytoplasmic CD79a(partial +)CD117(few cells +)Cytoplasmic MPO(-)Slide14

The blasts express markers of both myeloid and B-lymphoid differentiation.In addition to nonspecific myeloid markers such as CD15 and CD33, the blasts express markers suggesting monocytic differentiation (CD11c, dual expression of CD64 and CD36, a few cells positive for CD14)

B-lymphoid markers include partial strong expression of CD19, partial expression of CD22, and partial expression of CD79aSlide15

Blasts in the peripheral blood smear.An NSE cytochemical stain supports monocytic differentiation (inset)Slide16

Evidence of MLL gene rearrangement by FISH

Intact MLL gene

Split MLL geneSlide17

Diagnosis: Mixed phenotype acute leukemia, B/myeloid with MLL gene rearrangedSlide18

Mixed phenotype acute leukemia (MPAL)MPALs express markers of one or more lineages to a significant degree

MPALs may be:

Bilineal

: two separate blast populations each of a different lineage

Biphenotypic

: one blast population expressing markers of two different lineagesRare cases showing trilineage differentiation have been describedSpecific cytogenetic abnormalities may be associated with MPALt(9;22)(q34;q11.2);BCR-ABL1t(v;11q23);MLL rearrangedFlow cytometry is integral in the diagnosis of MPALSlide19

Requirements for assigning more than one lineage to a blast population (2008 WHO Classification)

Myeloid lineage:

Myeloperoxidase

(MPO) by flow

cytometry, IHC, or cytochemistry OR Evidence of monocytic differentiation (2 or more of the following: NSE, CD11c, CD14, CD64, lysozyme) B lineage: Strong CD19 with strong expression of at least one of the following: CD79a, cCD22, CD10 OR Weak CD19 with strong expression of at least 2 of the following: CD79a, cCD22, CD10T lineage: cCD3, preferably by flow cytometry (IHC may detect CD3 zeta chain which is not T cell specific) OR sCD3 (rare)Slide20

MPAL with MLL gene rearranged

The case presented is characteristic for an MPAL with MLL gene rearrangement

MPAL with MLL rearranged is more common in the pediatric population, particularly in

infants

Patients typically present with a

high white blood cell countThe leukemia usually demonstrates evidence of both monocytic and B-lymphoid differentiationCommonly, the leukemic blasts show two different populations morphologically – a population of monoblasts and a population of lymphoblastsSome cases may present with one population of blasts with no clear distinguishing features by morphologyCD10 is usually negativeCD22 and CD79a are often weakThis type of acute leukemia is associated with a poor prognosis.Slide21

MLL is not only implicated in MPAL in infantsRearrangements of the MLL gene occur in acute leukemias in patients of all ages and often in therapy-related hematopoietic neoplasms after treatment with topoisomerase II inhibitors

MLL rearrangements can be seen in ALL, AML, and MPAL

Account for >70% of infant

leukemias

Large and diverse group of translocation partners

Over 50 translocation partners have been characterizedMixed Lineage Leukemia gene (MLL) on chromosome 11q23 Slide22

AML with MLL rearrangement usually shows evidence of monocytic differentiation BLL with MLL rearrangement tends to occur in infants presenting with a high white blood cell count and CNS involvement. The leukemic cells are characteristically CD10(-).

Mixed Lineage Leukemia gene (MLL) on

chromosome 11q23 Slide23

ReferencesSwerdlow SH, Campo E, Harris NL, et al., editors. WHO Classification of

Tumours

of

Haematopoietic

and Lymphoid Tissues. Lyon

, France: IARC; 2008.Muntean AG, Hess JL. The pathogenesis of mixed-lineage leukemia. Annu Rev Pathol. 2012;7:283-301. Matutes E., Pickl WF, et al. Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood. 2011 Mar 17;117(11):3163-71.Sam TN, Kersey JH, Linabery AM, et al. MLL gene rearrangements in infant leukemia vary with age at diagnosis and selected demographic factors: a Children's Oncology Group (COG) study. Pediatr Blood Cancer. 2012 Jun;58(6):836-9