Download eCSI Case Discussion

ICCS e-Newsletter CSI
Winter 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 Data
Normal 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/dL
Hct:
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 1
Upon opening the file, it is apparent
that there are a large number of CD34(+) events.
granulocytes
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.
debris
doublets
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 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
TUBE 2
Granulocytes painted green on forward versus side scatter
eosinophils
Neutrophilic elements
Clean up with CD34(+)
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 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
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
Split MLL gene
Intact 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 Blymphoid 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.
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
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(-).
References
Swerdlow 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