Case History
A two-and-a-half-year-old female child presented with fever and pallor of 1-week duration. Examination revealed hepatomegaly (span 11 cm) and splenomegaly (2 cm below the left subcostal margin). The hemoglobin was 46 g/L, platelet count 14.0 x109 /L and total leucocyte count 14.7 x 109/L. Peripheral blood film showed 62% circulating blasts (Fig. 1A-B ). Bone marrow aspiration revealed 82% blasts with scanty, agranular basophilic cytoplasm without Auer rods; 2% of the blasts showed cytochemical myeloperoxidase positivity. On flow cytometry, the blasts expressed bright CD19 and dim CD10 along with cytoplasmic CD79a and CD22. There was cross-lineage antigen expression of CD33 (bright; 90% cells) and CD13 (dim; 14% cells). The blasts were also positive for CD34, HLA-DR, CD38, CD81 and CD86; and were negative for CD20, CD117, T-lineage associated markers, monocytic & megakaryocytic-lineage associated markers. Cytoplasmic myeloperoxidase (anti-MPO) expression was also not noted. A working diagnosis of BCP-ALL with aberrant CD33 expression was rendered, following which the child was initiated on the Standard Risk (SR) BCP-ALL protocol of the Indian Childhood Collaborative Leukemia Group (ICiCLe), comprising of a pre-phase of prednisolone 60 mg/m2/d in three divided doses. 4
Fluorescent in-situ hybridization (FISH) testing with dual-colour dual-fusion probes for BCR-ABL1 and ETV6-RUNX1, dual break-apart probes for KMT2A and TCF3 (CytoTest Inc. Rockville, United States) revealed presence of TCF3 translocation in more than 80% of cells (Fig. 1E) . Subsequently, bothTCF3-PBX1 and TCF3-HLF translocations were excluded using a tri-colour probe (Cytocell Ltd, Cambridge).
After one week of pre-phase steroid therapy, although the toddler clinically appeared well, her leukocyte count increased to 63x109/L with 60% circulating blasts. On peripheral blood smear examination, in contrast to the morphology at diagnosis, these blasts were larger with indented nuclei, prominent nucleoli and moderate amount of granular basophilic cytoplasm without Auer rods. The blasts showed unequivocal cytochemical myeloperoxidase positivity in 20% of the cells (Fig. 1C-D ). The smaller lymphoblasts noted at the time of diagnosis were absent. Repeat flow cytometry performed on peripheral blood showed that 90% of the blasts expressed myeloid antigens (CD13, CD33, CD64, CD14, CD36, CD15 and cytoplasmic myeloperoxidase. The remaining 10% blasts showed the initial immunophenotype of BCP-ALL. The initial cytochemical myeloperoxidase and anti-MPO (flow cytometry) results were reviewed and the negative report (<3% positivity) was reverified.
Repeat FISH testing revealed the TCF3 rearrangement in blasts as well as granulocytes (Fig. 1F) . In view of absence of metaphase, further FISH testing was performed using a commercially available dual-colour break-apart probe (ZytoVision GmbH, Germany) that confirmed rearrangement of ZNF384 (Fig. 1G) . The diagnosis was revised to TCF3-ZNF384 [t(12;19)(p13;p13)] rearranged MPAL, B/myeloid. The patient’s risk stratification was upgraded to High-Risk (HR) and appropriate management initiated4.
Her remaining induction phase chemotherapy was interrupted by two episodes of febrile neutropenia, both managed with antimicrobials. End-of-induction bone marrow revealed 03% blasts and no measurable residual disease (MRD) on high-resolution multiparameter flow cytometry. She had a stormy course prior to consolidation too, developing a fungal brain abscess requiring surgical drainage, leading to a 2-week delay in start of consolidation therapy. Currently, the patient is on an augmented BFM (Berlin-Frankfurt-Munster) consolidation protocol and continues her antifungal prophylaxis.