1. eCSI case Fall 2014 Andrea M. Sheehan, MD Associate Professor of Pathology & Immunology Baylor College of Medicine

2. Clinical History 17 year old boy with a history of B-acute lymphoblastic leukemia (B-ALL) – Expressed CD45(dim), CD19, CD22, CD38, CD58, CD52, CD34, CD99, and HLA-DR – 46,XY,t(4;11)(q21;q23) Treated according to AALL1131, and did well One year later, relapses with B-ALL with same immunophenotype – Underwent reinduction chemotherapy

3. Clinical History Now, three months later, the patient presents with neutropenic fever CBC shows ParameterResultUnitReference Range WBC0.05K/ul4.5-13.5 Hgb9.6g/dL13-16 Hct27.7%37-49 Platelet12K/ul150-450 Differential: Lymphocytes54.5%15-55 Monocytes9.1%0-4 Blasts36.4%0 ANC0.0K/ul1.8-80

4. Clinical History A bone marrow was performed and submitted for morphology, flow cytometry, and cytogenetics Flow cytometry was performed using a 6 color leukemia panel on a BD FACSCanto with two lasers and acquired using BD FACSDiva software Data was analyzed using FCS Express V4

5. Bone Marrow Aspirate 40x

6. Bone Marrow Biopsy 20x

7. 6 Color Panel

8. Flow Cytometry Large CD45 dim blast population with increased side scatter Also note smaller CD45 dim population with lower side scatter

9. T-cells (Tube 1) Dim CD4 expression Dim CD4 expression is present on the larger myeloid blast population, but other T cell markers are negative (CD3, CD2, CD7, CD8). A small population of normal T cells is present in the background Normal background T cells

10. B-cells (Tube 2) The larger myeloid blast population is negative for B cell markers, but back gating on the CD19+ population shows that smaller CD45 dim/low side scatter population are immature B cells that are CD10 negative and negative for surface light chains. Normal T cells are in blue. CD19+ back gate B cells are CD10 negative

11. B-cells (Tube 7) CD19+ back gate shows that the B cell population is composed of aberrant immature B lymphoblasts with strong expression of CD34 and overexpression of CD58. CD22 is also positive. The larger myeloid blast population is also positive for CD58 but negative for B cell markers. Aberrant immature B cells Two distinct blast populations are present! Those are not just hematogones!

12. Myeloid Tube (Tube 3) The larger myeloid blast population is CD15+, CD38+ with a small subpopulation having CD11b. See that the smaller B lymphoblast population is CD15 positive (back gate on CD34) CD42+61 positivity likely represents platelets stuck to the larger blasts rather than true expression.

13. Monocytic Tube (Tube 4) Both blast populations are negative for CD13 and CD117. The larger myeloid blast population shows dim expression of CD64 with essentially no CD14 or CD16+56. Dim CD64 expression Normal T cells are CD64 negative

14. B/erythroid (Tube 6) The smaller B lymphoblast population expresses CD22 and HLA-DR but not CD20. (back gate based on CD22 vs CD45) There is no increase in CD71. The larger myeloid blast population expresses heterogenous HLA-DR and dim CD71 but no B cell markers CD22 back gate Dim CD71 expression In these plots, we lowered the resolution, making the dots larger. This helps make the smaller lymphoblast population easier to see

15. Tube 5 The larger myeloid blast population expresses CD58 and heterogenous CD33. The smaller B lymphoblast population overexpresses CD58 but is largely CD33 negative, which is easier to see on the lymphoblast gate based on CD45 dim/low SSC (no B lineage specific marker is present in this tube). All events CD45 dim/low SSC gate

16. Immunophenotypic Findings Two distinct blast populations #1 – 97% - myeloid with monocytic differentiation – Positive for CD45 (moderate), increased side scatter, CD33 (heterogenous), CD15, CD11b (dim, subpopulation), CD64 (dim), CD14 (small subpopulation), HLA-DR (heterogenous), CD99, CD58, CD38, CD71, CD4 (dim) #2 – 3% - B lymphoblast – CD45 (moderate), low side scatter, CD19, CD22, CD34, HLA-DR, CD99 (dim), CD58, CD38, CD15

17. Differential Diagnosis Therapy related myeloid neoplasm with small clone of persistent B-ALL Lineage switch from a B-lymphoblast clone to a myeloid clone related to the patient’s MLL rearrangement

18. Cytogenetics 56-63,XY,+X,+Y,+2,+3,t(4;11)(q21;q23),der(4)(t(4;11), +der(4)t(4;11),+6,+7,+7,del(7)(q22q36)+8, der(9)t(9;11)(q34;q13)t(4;11)(der(11)t(9;11),+12,+13,+13,+13,+14, +17,del(17)(p11.1),+18,+19,+21,+22,+mar[cp20] FISH – MLL probe - pattern consistent with MLL gene rearrangement in 200 out of 200 (100%) cells studied, 162 showed additional copy of 3’MLL gene and 5 cells showed 2 additional copies of the 3’MLL gene Complex karyotype with presence of t(4;11) clone plus multiple new aberrations including a secondary translocation with 9q34 and 11q13

19. Diagnosis Myeloid component represents a lineage switch from original clone of B-acute lymphoblastic leukemia Bottom line: mixed phenotype acute leukemia with t(4;11), MLL rearranged, with two distinct clones present – B – lymphoblastic (3%) – Myeloid (monocytic) (97%)

20. Lineage Plasticity Cross lineage expression of antigens is common in acute leukemias – Lymphoid antigen expression in AML CD19 in t(8;21)+ AML CD4 in monoblastic leukemias CD2, CD7 in various subtypes of AML – Myeloid expression in ALL CD13, CD33 in B-ALL CD117 in T-ALL – At genetic level, clonal TCR or IgH rearrangements can be seen in B- ALL, T-ALL, and AML True mixed phenotype acute leukemias are rare – Most common genetic associations t(9;22) or MLL rearrangements – B/myeloid most common phenotype, but T/myeloid, B/T, or B/T/myeloid can be seen – Usually either one blast population with mixed markers (“biphenotypic”) or two distinct blast populations (“bilineal”)

21. Lineage Switch in Acute Leukemias True “lineage switch” is a rare event Mixed lineage leukemia gene (MLL) rearrangements most common genetic abnormality in these cases – Variety of translocation partners noted – t(4;11), t(9;11), t(11;19), ins(11;4), among others Also can be seen with t(9;22) BCR-ABL1, especially in setting of blast crisis of chronic myeloid leukemia Relatively more common in infantile acute lymphoblastic leukemia – MLL rearrangements are seen in 70-80% of these leukemias – May occur early in treatment course with emergence of aberrant monocytic clones Lineage switch most commonly B to myeloid – Monocytic differentiation is most common – Rare cases of T to myeloid or myeloid to B have been reported Very poor prognosis when this happens

22. Lineage Switch in Acute Leukemias Theories as to how lineage switch occurs – Chemotherapy induced selection of a minor subclone present at diagnosis but not detected – Leukemia arises from a precursor or stem cell with lymphoid and myeloid potential and changes phenotype under treatment pressure or with the accumulation of additional genetic abnormalities Gene expression profile studies of MLL rearranged acute leukemias show patterns in-between ALL and AML B-macrophage progenitors are present in normal bone marrow – Transdifferentiation from one lineage to another Has been reported in other settings (follicular lymphoma and follicular dendritic cell sarcomas)

23. Follow Up Patient treated on an individualized acute myeloid leukemia/high risk ALL chemotherapy protocol Patient’s disease was refractory to therapy Passed away a few weeks later due to infectious complications

24. References Rossi JG et al. “Lineage switch in childhood acute leukemia: an unusual event with a poor outcome.” Am J Hematol 2012 87:890-897. Stasik C et al. “Infant acute lymphoblastic leukemia with t(11;16)(q23;p13.3) and lineage switch into acute monoblastic leukemia” Cancer Genetics Cytogenetics 2006 168:146-149. Slamova L et al. “CD2 positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage” Leukemia 2014 28:609-620. Pui C, ed. Childhood Leukemias, 3 rd ed. 2012 Cambridge University Press.