ICCS e-Newsletter CSIWinter 2013

Jacqueline Emmons, MD

Department of Pathology University of Texas Southwestern Medical Center

Dallas, Texas

History

• 11 month old female with a history of fever and failure to thrive

• On physical exam, the child was pale and lethargic.

CBC DataNormal Ranges

WBC: 67 K/mm3 6 -- 17.5 K/mm3

RBC: 3.23 M/mm3 3.70 – 5.30 M/mm3

Hgb: 9.3 g/dL 11.1 – 14.2 g/dLHct: 26% 30 – 42%

MCV: 80.5 fl 70.0 – 84.0 fl

MCHC: 35.8 g/dL 32.0 – 36.0 g/dL

RDW: 14.1% 11.5 – 15.0%

Plts: 62 K/mm3 150 – 600 K/mm3

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

• 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.

• 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 PE PerCP APC

1 CD10 CD22 CD20 CD34

2 CD14 CD11b CD34 CD19

3 CD45 CD11c CD34 CD14

4 CD15 CD33 CD45 CD34

5 CD36 CD64 CD45 CD34

6 CD2 CD117 CD45 CD34

7(intracellular)

MPO CD79a CD45 CD34

TUBE 1Upon 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 plotcan 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.

doublets

granulocytes

debris

TUBE 1

The CD34(+) blasts (red) are negative for CD20 and CD10 and are partial dim positive for CD22.

TUBE 2

As anti-CD34 is included in tube 2 as well,the blasts can be isolated and analyzed ina 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 doublets

Exclusion of CD34(+)events

TUBE 2Granulocytes painted green on forward versus side scatter

Clean up with CD34(+)

eosinophils

Neutrophilic elements

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.

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

Color Key:Red: BlastsLight blue: LymphocytesDark blue: MonocytesGreen: Granulocytes

TUBE 2

The blasts are CD19(partial +) and CD11b(partial +). A few of the blasts appear to express CD14.

• 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(-)

• 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 CD79a

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

Evidence of MLL gene rearrangement by FISH

Intact MLL gene

Split MLL gene

Diagnosis:

Mixed phenotype acute leukemia, B/myeloid with MLL gene rearranged

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 lineages– Rare cases showing trilineage differentiation have been described

• Specific cytogenetic abnormalities may be associated with MPAL– t(9;22)(q34;q11.2);BCR-ABL1– t(v;11q23);MLL rearranged

• Flow cytometry is integral in the diagnosis of MPAL

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, CD10

T lineage: cCD3, preferably by flow cytometry (IHC may detect CD3 zeta chain which is not T cell specific) OR sCD3 (rare)

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 count

• The leukemia usually demonstrates evidence of both monocytic and B-lymphoid differentiation– Commonly, the leukemic blasts show two different populations

morphologically – a population of monoblasts and a population of lymphoblasts

– Some cases may present with one population of blasts with no clear distinguishing features by morphology

– CD10 is usually negative– CD22 and CD79a are often weak

• This type of acute leukemia is associated with a poor prognosis.

• MLL is not only implicated in MPAL in infants

• Rearrangements 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 characterized

Mixed Lineage Leukemia gene (MLL) on chromosome 11q23

• 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

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