1. eCSI case 2 – November 2016 Jyotinder Nain Punia, MD
Assistant Professor of Pathology and Immunology
Baylor College of Medicine/Texas Children’s Hospital
Houston, Texas

2. Clinical History
5 year old boy with pancytopenia and circulating blasts.
Bone marrow was performed and submitted for morphologic examination, flow cytometry and cytogenetics
Flow cytometry was performed using 6 color leukemia panel on a BD FACSCanto with two lasers and acquired using BC FACSDiva software.
Flow plots were analyzed using FCS Express V4

3. Bone marrow aspirate showing multiple blasts (X400)

4. Bone marrow biopsy showing sheets of blasts (200X)

5. 6 Color Flow Panel For Leukemia
FITC PE
PerCP-Cy5.5
PE-Cy7
APC
APC-H7 1
CD7
CD2
CD3
CD8
CD4
CD45 2
Lambda
Kappa
CD19
CD10
CD5 3
CD15
CD42+61
CD34
CD38
CD11b 4
CD64
CD16+56
CD14
CD13
CD117 5
CD99
CD58
CD33
CD25
CD52 6
CD71
GlyA
HLA-DR
CD20
CD22

6. Flow Cytometry
Follow red population (blasts)

7. Flow cytometry (contd)
Partial CD10 expression
Heterogenous
CD34
expression
Heterogenous expression of HLA-DR

8. Flow Cytometry (contd)
Dim CD99 and CD58 expression
Negative for expression of CD33
Subset with CD13 expression
Note: .fcs files with CD33, CD99, CD58 are not provided for preview

9. Immunophenotypic Findings
Markedly increased blasts detected (90%).
Blasts are positive for
CD45 (moderate), CD19, CD22, CD10 (subset), CD34 (heterogenous), HLA-DR (heterogenous), CD99, CD58, CD38 (dim)
Coexpression of myeloid marker CD13 (subset)

10. Cytogenetics (FISH)
ETV6-RUNX1 fusion in a total of 92% and loss of the uninvolved ETV6 gene in 3.5% of cells.

11. Final Diagnosis
B-Acute Lymphoblastic Leukemia (B-ALL)with t(12;21)(p13;q22);TEL-AML1 (ETV6-RUNX1)

12. Clinical History (contd)
Patient enrolled in protocol AALL0932
Day 29 bone marrow was negative for leukemic clone
Two and a half years later, presents with circulating blasts
Bone marrow for morphology, flow cytometry (6 color panel plus intracytoplasmic tube with CD79a, CD3, TdT and myeloperoxidase) and cytogenetics was performed
Morphologically blasts were similar to the patient’s original clone

13. Flow cytometry Partial and dim CD7 expression
Follow red population (blasts)
Partial and dim CD2 expression
Partial and dim CD4 expression

14. Flow cytometry (contd)
No expression of CD19 or CD10
Heterogenous expression of HLA-DR
CD34 expression

15. Flow Cytometry (contd)
Dim expression of CD99
Expression of CD33
Expression of CD117 and CD13
Note: .fcs files with CD33, CD99, CD58 are not provided for preview

16. Flow Cytometry (contd)
Partial expression of CD16+56
Partial expression of CD11b
Partial expression of CD15

17. Flow Cytometry (contd)
Blasts show no expression of cytoplasmic CD79a, CD3 or myeloperoxidase (MPO)
Normal B-cells with expression of cytoplasmic CD79a
Normal Granulocytes with expression of cytoplasmic MPO
Normal T-cells with expression of cytoplasmic CD3
Note: .fcs files intracytoplasmic markers are not provided for preview

18. Immunophenotypic Findings
Increased blasts detected (27%).
Blasts are positive for
CD45 (moderate), CD13 (dim), CD33, CD15 (partial), CD11b (partial), CD16+56 (partial), CD34, CD117, HLA-DR, CD99 (dim), CD38, CD2 (partial),CD7 (partial), CD4 (partial)

19. Cytogenetics
Deletion 7q31
No ETV6-RUNX1 fusion.
Monosomy 7
46,XY,r(7)
45,XY,-7
7q31 gene deletion pattern in 60% and a monosomy 7 pattern in 27.5% of cells.
No evidence of ETV6-RUNX1 fusion

20. Final Diagnosis
Acute myeloid leukemia (Therapy related myeloid neoplasm)

21. Lineage Switch?
This is unusual as B-ALL with t(12;21) is considered good prognosis, but this case showed
recurrence of leukemia
lineage switch from B-lymphoblastic leukemia to myeloid leukemia (development of a therapy-related myeloid neoplasm)
cytogenetics showed a completely different clone on relapse

22. Possible Mechanisms of Lineage Switch in Acute Leukemias
Bipotential progenitors
Rather than a strict divergence of lymphoid and myeloid lineages, a potential of lineage switch exists
Such bipotential progenitors are target for leukemic translocations
Cell reprogramming and dedifferentiation
Lineage commitment is controlled by transcription factors, such as:
PU.1, CEBPA for myeloid cell commitment
Notch1, GATA3 and PAX5 for T-and B- cell development
Deletion or aberrant expression of such regulators can result in reprogramming and dedifferentiation

23. Possible Mechanisms of Lineage Switch in Acute Leukemia (cont’d)
Clonal selection
Heterogeneous population is present, but initially the dominant leukemic clone is apparent
Chemotherapy selectively suppresses or eradicates the apparent leukemic clone, hence the minor subclonal expansion results in a lineage switch
Microenvironment
Competition of tumor cells for the niche
Environmental manipulation
Disruption of the stem cell-niche communication

24. References
Dorantes-Acosta, E., Pelayo R. Lineage Switching in Acute Leukemias: A Consequence of Stem Cell Plasticity? Bone Marrow Research. 2012;2012: doi: /2012/
Rossi, J. G., Bernasconi, A. R., Alonso, C. N., Rubio, P. L., Gallego, M. S., Carrara, C. A., Guitter, M. R., Eberle, S. E., Cocce, M., Zubizarreta, P. A., Felice, M. S.. Lineage switch in childhood acute leukemia: An unusual event with poor outcome. Am. J. Hematol., 87: 890–897. doi: /ajh
So, C.W., Cleary, M.L. Dimerization: a versatile switch for oncogenesis. Blood, 15 August 2004;104, (4). Doi: /blood
Imataki, O., Ohnishi, H., Yamaoka, G. et al. Lineage switch from precursors B cell acute lymphoblastic leukemia to acute monocytic leukemia at relapse. Int J Clin Oncol (2010) 15: 112. doi: /s