1. eCSI case Weijie Li, MD Department of Pathology & Laboratory Medicine
Children’s Mercy Hospital

2. Clinical History
A 16 year old girl was transferred to the PICU from a local community hospital over the weekend. She was critically ill with septic shock and multi organ failure including respiratory failure, renal failure on dialysis, hepatic insufficiency, hemodynamic instability requiring vasopressors, and coagulopathy with a DIC picture. In addition, she also had lymphadenopathy, organomegaly, anemia, thrombocytopenia, low fibrinogen, fever and elevated ferritin.
Hemophagocytic lymphohistiocytosis (HLH) was diagnosed. A STAT bone marrow examination was performed to rule out malignancy before starting therapy for HLH.

3. Peripheral blood (PB) smear:
PB smear showed frequent medium to large sized atypical or “immature” “monocytic cells”

4. Bone marrow (BM) smear:
BM smear revealed many similar atypical monocytoid cells.
Based on morphology, acute monocytic leukemia was suspected.

5. Flow cytometry analysis:
A 4-color acute leukemia panel was performed on the BM specimen using BD FACSCanto A flow cytometer and the data was acquired using BD FACSDiva software.
Results of the selected tubes are provided for review (FITC/PE/PerCP/APC).
Tube 1: CD14/CD13/CD45/CD117
Tube 2: CD56/CD34/CD45/CD33
Tube 3: CD10/CD22/CD45/HLA-DR
Tube 4: CD61/CD11b/CD45/CD19
Tube 5: CD5/CD8/CD45/CD4
Tube 6: CD2/CD7/CD45/CD3
Tube 7: TCRab/TCRgd/CD45/CD3
Tube 8: CD2/CD1a/CD45/CD3
Tube 9: TdT/cCD22/CD45/cCD3
Tube 10: CD3/CD25/CD45/CD5

6. CD45 vs side scatter (SSC)
There are essentially no cells in the blast gate (dim CD45 intensity with low SSC).
Besides granulocytes (in pink) and erythroids (in green), there is a big cell population (in blue) in the monocyte gate (bright CD45 with low to intermediate SSC), which is not separated from mature lymphocytes.
This population was gated as “blasts” and analyzed.

7. Tube 1:
All the gated cells are negative for CD117. Most of the gated cells are negative for CD14 and CD13.
The CD13 and CD14 double positive cells are mostly consistent with mature monocytes.
There is a small subset of cells positive for CD13 and negative for CD14.

8. Tube 2:
All the gated cells are negative for CD34. The majority of the gated cells are negative for CD33 and CD56.
There are small subsets of cells positive for CD33 or CD56 only.

9. Tube 3:
Almost all the gated cells are strongly positive for HLA-DR, and negative for CD10 or CD22.
There are rare CD22 positive B cells.

10. Tube 4:
Almost all the gated cells are positive for CD11b, negative for CD19 and CD61.
There are rare CD19 positive B cells.
Some cells are falsely positive for CD61 due to the attachment of platelets.

11. Tube 5:
The majority of the gated cells are negative for CD5, and most of the gated cells are negative for CD4 and CD8.
There are small numbers of normal CD5+ CD8+ T cells (in red) and CD5+ CD4+ T cells (in green).
There are subsets of the abnormal cells positive for CD8 or CD4 only.

12. Summary of the immunophenotype so far:
Positive: CD11b, HLA-DR, CD13 (small subset), CD56 (small subset), CD4 (small subset), CD8 (partial), CD45
Negative: CD34, CD117, CD5, CD22, CD19, CD14, CD33, CD10, CD61
The results of other tubes not shown include CD38+, CD15-, CD20-, myeloperoxidase-, lysosome -.
Is the phenotype consistent with acute monocytic leukemia? Or should acute monocytic leukemia be ruled out now?

13. Tube 6: The result of this tube clearly points to a T cell malignancy.
The majority of the gated cells are positive for CD7 (bright) and CD2. Surface CD3 expression is partial and variable.
A small number of normal T cells are painted red.
The result of this tube clearly points to a T cell malignancy.

14. Tube 7:
The abnormal cells show partial surface CD3 expression and are negative for both alpha beta and gamma delta T cell receptors.

15. Tube 8:
Abnormal cells are negative for CD1a.

16. What kind of T cell malignancy?
T lymphoblastic leukemia? Near-early T-cell precursor (ETP) leukemia?
CD7+ (bright), CD2+, CD5-, CD3+ (partial), mostly CD4/8-, CD11b+, HLA-DR+, CD1a-
Since CD34 and CD1a are negative, other T cell precursor markers such as TdT and CD99 should be tested.

17. Tube 9:
Abnormal cells are positive for intracellular CD3 and negative for TdT and CD22.
The cCD3 negative cells (in green) are possible intermixed normal monocytes, B cells and/or granulocytes in the gate.

18. Should T lymphoblastic leukemia be diagnosed?
Immunohistochemistry stains of the BM confirmed the absence of TdT, but showed strong CD99 expression (see the microscopic picture below).
Should T lymphoblastic leukemia be diagnosed?
Correlation with morphology.
The morphology of the abnormal cells is not typical for lymphoblasts.
A mature T cell leukemia/lymphoma should be ruled out.
CD99 can be expressed in mature T cell lymphomas such as anaplastic large cell lymphoma.

19. Tube 10:
Abnormal cells are separated into three populations: CD3+CD5-CD25-, CD3-CD5-CD25-, and CD3-CD5-CD25+.
Normal T cells (in red circles) are CD3+CD5+CD25-.

20. STAT Immunostains for CD30 and ALK1 on BM smears showed frequent CD30 positive and ALK1 positive cells.

21. Diagnosis: Leukemic phase ALK+ anaplastic large cell lymphoma.
A selected flow cytometry panel was performed on the PB sample and showed 35% of the abnormal cells with a similar immunophenotype.
Diagnosis: Leukemic phase ALK+ anaplastic large cell lymphoma.
STAT FISH analysis confirmed ALK gene rearrangement and NPM-ALK fusion.

22. Acute Monocytic Leukemia (FAB: AML-M5b)
A myeloid leukemia with monocytic lineage in more than 80% of the leukemic cells.
The majority of the monocytic cells are promonocytes instead of monoblasts (acute monoblastic leukemia, AML-M5a).
More common in adults.
May have extramedullary lesions.
Associated with 11q23 rearrangement affecting KMT2A(MLL) gene.
Hemophagocytosis associated with t(8;16)(p11.2;p13.3).
Seen in some therapy-related acute myeloid leukemia.

23. Acute Monocytic Leukemia (FAB: AML-M5b)
Morphology:
Increased monoblasts (round nuclei with open chromatin and nucleoli, abundant basophilic cytoplasm), promonocytes (folded nuclei) and atypical monocytes (increased N:C ratio, immature chromatin pattern).
Flow Cytometry study:
Variably express myeloid antigen CD13, CD33 (bright), CD15 and CD65.
At least two monocytic markers: CD14, CD4, CD11b, CD11c, CD64, CD68, CD36 and lysozyme.
Positive for HLA-DR.
Variable expression of CD34 and CD117.
Aberrant expression of CD7 or CD56 in some cases.

24. T Lymphoblastic leukemia/lymphoma (T-ALL/LBL)
15% of ALL in Children; 25% of ALL in adults.
85-90% of all lymphoblastic lymphoma (LBL).
Mediastinal mass common; peripheral lymph nodes and extranodal involvement.
T-ALL: >25% blasts in PB or BM.
More immature immunophenotype in T-ALL than in T-LBL.
Morphology: similar to B lymphoblasts, medium size, high N:C ratio, condensed or dispersed chromatin and inconspicuous nucleoli.

25. T-ALL/LBL Flow Cytometry study: Early T-cell precursor (ETP) leukemia
Usually positive: TdT (immature marker), CD7, cytoplasmic CD3.
Variably positive (immature markers): CD1a, CD34, CD99.
Variably positive (mature T-cell markers): CD2, surface CD3, CD4, CD5, CD8.
Variably positive: CD10, TAL-1
Aberrant expression: CD79a, CD13, CD33
Early T-cell precursor (ETP) leukemia
Absent CD1a and CD8, absent or weak CD5 (<75% positive cells), and expression of at least one of the following myeloid or stem-cell markers: CD117, CD34, HLA-DR, CD13, CD33, CD11b, or CD65.
Associated with worse prognosis (some studies).

26. Anaplastic Large Cell Lymphoma, ALK+
3% of adult non-Hodgkin lymphomas, 10-20% of childhood lymphomas, slight male predominance.
Lymph node as well as extranodal involvement.
10-30% bone marrow involvement (usually subtle).
Favorable prognosis compared with ALCL, ALK-.
Morphology:
Broad spectrum.
Hallmark cells: eccentric, horseshoe- or kidney-shaped nuclei and abundant cytoplasm.
Five morphologic patterns: common (60%), lymphohistiocytic (10%), small cell (5- 10%), Hodgkin-like (3%), composite (15%).
Leukemic presentation more common in small cell variant.

27. Anaplastic Large Cell Lymphoma, ALK+
Flow cytometry immunophenotype:
CD45+, CD30+, HLA-DR+
Variable T cell antigen expression
CD2+ (67-72%), CD4+ (33-80%), CD7+ (32-60%), CD3+(32-45%), CD5+ (14- 32%), CD8+ (14-32%)
CD25+ (80%).
Aberrant expression: CD13+ (40-80%); some cases show expression of CD56, CD15 or other myeloid markers.
Though the definite diagnosis of ALCL cannot be established by flow cytometry study without an ALK1 antibody, flow cytometry can quickly identify the abnormal cells and provide a preliminary diagnosis together with morphology.

28. Anaplastic Large Cell Lymphoma, ALK+
Immunohistochemistry:
ALK1+, staining pattern depending on ALK gene translocation, nuclear and cytoplasmic staining pattern in t(2;5)(p23;q35).
EMA+ in most of the cases.
Cytotoxic associated antigen expression: TIA1, granzyme B, perforin.
BCL2- LMP1-.
Cytogenetics:
90% with TCR gene rearrangement.
Translocations and fusion proteins involving ALK at 2p23:
t(2;5)(p23;q35), most common (84%), partner gene: NPM.
t(1;2)(q25;p23), 13%, partner gene: TPM3.
Inv(2)(p23q35), 1%, partner gene: ATIC.
Other rare translocations: t(2;3)(p23;q21), t(2;17)(p23;q23), t(2;X)(p23;q11-12), t(2;19)(p23;p13.1), etc.
No difference in prognosis.

29. References:
Swerdlow SH, et al. WHO classification of Tumors of Hematopoietic and Lymphoid Tissue. 4th ed. 2008
Juco J, et al. Immunophenotypic Analysis of Anaplastic Large Cell Lymphoma by Flow Cytometry. Am J Clin Pathol 2003;119:
Kesler MV, et al. Anaplastic Large Cell Lymphoma, A Flow Cytometric Analysis of 29 Cases. Am J Clin Pathol 2007;128:
Muzzafar T, et al. Flow Cytometric Immunophenotyping of Anaplastic Large Cell Lymphoma. Arch Pathol Lab Med. 2009;133:49–56

30. Acknowledgements:
I would like to thank Ruth E. Morgan, our flow cytometry lab coordinator, and Refat J. Ali and Roxanne Nieder, our flow cytometry technologists, for performing the tests and preparing the dot plot data.