1. ICCS e-Newsletter CSI Winter 2013
Jacqueline Emmons, MD
Department of Pathology
University of Texas Southwestern Medical Center
Dallas, Texas

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

3. CBC Data Normal Ranges WBC: 67 K/mm3 6 -- 17.5 K/mm3 RBC: 3.23 M/mm3
Hgb: g/dL
11.1 – 14.2 g/dL
Hct: %
30 – 42%
MCV: fl
70.0 – 84.0 fl
MCHC: g/dL
32.0 – 36.0 g/dL
RDW: %
11.5 – 15.0%
Plts: K/mm3
150 – 600 K/mm3
A manual differential of the peripheral blood revealed 69% blasts.

4. 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.

5. 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
CD19 3
CD45
CD11c 4
CD15
CD33 5
CD36
CD64 6
CD2
CD117 7
(intracellular)
MPO
CD79a

6. 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” the
population. Here, all events outside of the
“blast gate” are highlighted in black and will
be excluded.
granulocytes
doublets
debris

7. TUBE 1 The CD34(+) blasts (red) are negative for CD20 and CD10 and
are partial dim positive for CD22.

8. 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 the
forward versus side scatter and CD34.
Exclusion of debris, lymphocytes, granulocytes,
and doublets
Exclusion of CD34(+)events

9. TUBE 2 Granulocytes painted green on forward versus side scatter
eosinophils
Neutrophilic elements
Clean up with CD34(+)

10. 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.

11. 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: Monocytes
Green: Granulocytes

12. TUBE 2 The blasts are CD19(partial +) and CD11b(partial +). A few of
the blasts appear to express CD14.

13. 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 +)
CD45(moderately +)
CD64(partial +)
Cytoplasmic CD79a(partial +)
CD117(few cells +)
Cytoplasmic MPO(-)

14. 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

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

16. Evidence of MLL gene rearrangement by FISH
Split MLL gene
Intact MLL gene

17. Diagnosis: Mixed phenotype acute leukemia, B/myeloid with MLL gene rearranged

18. 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

19. 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)

20. 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.

21. Mixed Lineage Leukemia gene (MLL) on chromosome 11q23
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

22. 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(-).

23. References
Swerdlow SH, Campo E, Harris NL, et al., editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC; Muntean AG, Hess JL. The pathogenesis of mixed-lineage leukemia. Annu Rev Pathol. 2012;7: 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 Mar 17;117(11): 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 Jun;58(6):836-9