Submitting Author: Xian, Rena Ruiyu, MD
Institution: (1)The Children’s Hospital of Pennsylvania, (2)Hospital of the University of Pennsylvania, and (3)Penn State Milton S. Hershey Medical Center
Additional authors:Stephan A. Grupp, M.D., Ph.D.(1), Michael G. Bayerl, M.D.(3), Michele E. Paessler, D.O. (1), Rebecca L. King, M.D. (1), Jaclyn Biegel, Ph.D.(1), Adam Bagg, M.D.(2), Gerald Wertheim, M.D., Ph.D.(1)
Session: B Lymphoblastic Leukemia/Lymphoma

The patient is a 7-year-old female who initially presented in June 2010 with bruising, epistaxis, bleeding gums, lymphadenopathy, and was diagnosed with standard-risk B lymphoblastic leukemia (B-ALL). She was treated with vincristine, dexamethasone, PEG-asparaginase in combination with intrathecal cytarabine and methotrexate (following the AALL0331 protocol), and experienced a rapid response. Minimal residual disease (MRD) flow cytometry performed by a COG-approved center demonstrated no MRD at the end of induction. Sixteen months post therapy, she relapsed while on maintenance, and was reinduced with Ara-C, doxorubicin, vincristine, PEG-asparaginase, intrathecal methotrexate, and prednisone (following the AALL01P2 protocol). Although she achieved complete remission at the end of induction, she relapsed again five months later. At this time she was treated with clofarabine, VP-16, and cyclophosphamide. Autologous T cells were also collected for possible future cell therapy. The patient did not achieve complete remission on this regimen as demonstrated by persistent bone marrow blasts at two-months-post-induction. Since residual disease precluded a stem cell transplant, she was enrolled in a phase I clinical trial to evaluate a new chimeric antigen receptor-modified T cell technology against CD19 (CART-19). In April 2012, she received a total of 1.1 x 10^8/kg modified T cells in divided infusions over a three-day period. Following infusion, the patient developed severe cytokine release syndrome requiring PICU admission and anti-IL-6 treatment. After recovery, a day 23 post-treatment bone marrow aspirate was negative for leukemia by morphology and by MRD flow cytometry. Subsequent bone marrow studies at three, six, and nine months post-therapy were also negative by several studies, including MRD flow cytometry. The patient is currently nine months post CART-19 infusion, and shows no evidence of acute leukemia.

Wright-Geimsa smears were prepared from a bone marrow aspirate obtained two months prior to CART-19 therapy (not submitted for review) at the patient’s second relapse. The aspirate was hypercellular with numerous lymphoblasts (87%). Bone marrow biopsies and aspirates were also obtained at three-, six-, and nine-months post-CART-19 treatment. Bone marrow biopsies were fixed in AZF and decalcified prior to processing and staining with H&E. Wright-Geimsa stains were prepared from the bone marrow aspirates at these time points. The three-month bone marrow biopsy (case # S-12-005152) was hypocellular (30-40%) with trilineage hematopoiesis, and showed no evidence of leukemia. The bone marrow study at six months post-therapy (case # S-12-007268) was normocellular with left-shifted granulopoiesis, and again showed no evidence of leukemia. The most recent bone marrow sampling obtained at nine months post CART-19 infusion continues to be histologically negative for leukemia.

Flow cytometry performed on the bone marrow aspirate obtained two months prior to CART-19 therapy revealed a distinct population of cells with CD45 vs. side scatter characteristics of blasts that represented 84% of total events with the following immunophenotype: HLA-DR+, CD9+, CD19+, CD10+, CD20-, CD34(subset), CD38+, cCD79a+, CD24+, and TdT+. Flow cytometry performed on a bone marrow aspirate obtained two days prior to CART-19 infusion demonstrated 62% blasts with a similar immunophenotype. Day 23, 90, 180, and 270 post-infusion bone marrow aspirates were obtained and sent to the University of Washington hematopathology laboratory for MRD testing. All four were negative for leukemic cells. Additionally, flow cytometry performed at our institution on the three-month bone marrow aspirate revealed immunophenotypically normal T cells, and virtually no B cells.

Cytogenetics performed on the diagnostic bone marrow aspirate (2010) showed 46,XX,t(9;14)(p13;q24)?c[20]. FISH performed on this specimen was positive for 9p deletion (CDNK2A probe). Clonal evolution at the first relapse in 2011 was evident as the cytogenetics showed 49,XX,+5,t(9;14)(p12;24)?c,+11,+17[17]/49, XX,+X, t(9;14)(p12;24)?c,+11,+16[13]. Cytogenetic and FISH studies performed at her second relapse, two months prior to CART-19 therapy, showed additional abnormalities: 48,XX,del(9)(p21.3),+11,del(14)(q2?q24),+16/46,XX[4]. A heterozygous deletion within the IKZF1 locus at 7p12.2 was identified by SNP analysis, as was a homozygous deletion of the region containing the CDKN2A locus at 9p21.3.

We present a highly effective modified autologous T cell therapy directed against CD19 in the treatment of refractory B lymphoblastic leukemia. The cell-based technology uses a lentiviral vector encoding a chimeric protein composed of a CD19-specific T cell receptor linked to both the 4-1BB domain of CD137 and the cytoplasmic zeta domain of CD3. Linkage of these T cell-specific co-stimulatory and signaling domains allows for T cell activation in the absence of major histocompatibility complexes, which may contribute to their observed capacity for bone marrow homing, conversion to a memory T cell phenotype, and in vivo replication and long-term persistence. CART-19 has been used to treat advanced cases of adult chronic lymphocytic leukemia with remarkable success. This is the first case in which CART-19 is used to treat refractory B lymphoblastic leukemia. In this patient, the cell-based therapy has shown a robust and durable response.

Treatment-refractor B lymphoblastic leukemia successfully controlled with a novel chimeric antigen receptor-modified T cell therapy against CD19.

B-acute lymphoblastic leukemia

Case 224