PMC6904788_01
Major events of the case: (A) timeline with key stages highlighted in red color; (B) inductions and bone marrow disease status at different time points with relevant cytogenetic features. MSK=Memorial Sloan Kettering Cancer Center. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Bone marrow (BM) aspirate ( 1, Fig. 1) differential showed > 80% blasts and core biopsy revealed hypercellular marrow (100%) with blasts diffusely infiltrating the marrow space (Table S1). CD45 expression, uniformly positive for CD34, CD10, CD19, HLA-DR, and CD38, and negative for CD20, T-cell, and myeloid markers. Cytogenetic studies showed normal karyotype; FISH revealed trisomy 17 at low level and was negative for t(9;22), the Philadelphia (Ph) chromosome (Fig. 1B). He also experienced multiple episodes of neutropenic fever (course timeline in Fig. 1A). He was transferred to a second institution, where he was found to have persistent B-ALL; BM FISH was also notable for trisomy 8 and del(20q) ( 2, Fig. 1). In preparation for possible blinatumomab, he then began cyclophosphamide and vincristine (Table S2), with day 15 BM studies revealing persistent B-ALL (100% cellular marrow, diffusely infiltrated by B-lymphoblasts,  4, Fig. 1), with course complicated by Klebsiella pneumoniae bacteremia. Restaging BM biopsy performed day 27 post-IO demonstrated variable cellularity (<5%-50%) with foci of regenerative changes ( 6, Figs. 1B and 2G-K); FACS detected no abnormal immature B-cell population (Fig. 2M). BM studies confirmed continued MRD-negative CRi of B-ALL ( 8, Fig. 1B), but morphologic/immunophenotypic findings of myelodysplastic syndrome (MDS) became evident. On day 248 from the initial diagnosis of B-ALL, he successfully underwent alloHCT from a matched unrelated donor without significant complication (Fig. 1A).
Representative histologic sections, immunostains, flow cytometry plots, and genomic profiles prior to and following inotuzumab ozogamicin (IO). (A) Bone marrow core biopsy ( 5) on arrival to this institution demonstrated markedly increased blasts with fine chromatin and distinct nucleoli (H&E, 400x), (B) patchy fibrosis and large aggregates of atypical megakaryocytes (H&E, 400x), and (C) increased background fibrosis as noted by reticulin stain. The blasts expressed (D) CD34 and (E) TDT by immunohistochemistry (400x); (F) aspirate smears showed blasts of variable size. Following one cycle of inotuzumab ozogamicin, core biopsy ( 6) demonstrated (G) osteosclerotic changes in the bony trabeculae and essentially acellular marrow (H&E, 400x), with (H&I) patchy regeneration of hematopoiesis and mildly increased megakaryocytes (H&E, 400x) without an increased blast population expressing (J) CD34 or (K) TDT. Multiparameter flow cytometry from (L) prior to treatment ( 5) revelated the blasts expressed CD45 (dim), CD19, CD22, CD20 (partial), CD10 (partial), and CD34, consistent with B-lymphoblastic leukemia, and (M) following treatment disclosed only rare events ( 6) that represent plasma cells and rare B cells, without any immunophenotypic evidence of B-lymphoblastic leukemia. (N) Results of matched tumor/normal comprehensive genomic profiling for somatic mutations, of bone marrow biopsies A) prior to IO treatment ( 5, Fig. 1A) and B) after blinatumomab consolidation ( 8, Fig. 1A); persistence of BRCA1 and U2AF1 mutations is highlighted in yellow. *The NRAS exon 2 mutations were noted to occur on different alleles.  Mutations in CDKN2A reflect the same mutation affecting p14 and p16 through alternative splicing.  The IKZF1 copy number losses fell slightly below our criteria for deletion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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BM studies ( 5, Fig. 2A-F) noted 90% blasts on aspirate with variably cellular marrow (0%-90%) with patchy sheets of blasts. BM FACS again identified B-lymphoblasts expressing CD19, CD22 and CD34, CD45 (dim), and partial expression of CD10 and CD20 (Fig. 2L). Next-generation sequencing studies using the IMPACT-Heme platform (Supplementary Methods), with matched normal control (nail clippings), revealed 3 distinct, somatic NRAS mutations on different alleles, CDKN2A mutation, and IKZF1 loss (Fig. 2N). Restaging BM biopsy performed day 27 post-IO demonstrated variable cellularity (<5%-50%) with foci of regenerative changes ( 6, Figs. 1B and 2G-K); FACS detected no abnormal immature B-cell population (Fig. 2M).