Submitting Author: Xian, Rena Ruiyu, MD
Institution: Hospital of the University of Pennsylvania
Additional authors:David L. Porter, M.D., Bruce L. Levine, Ph.D., Michael Kalos, Ph.D., Carl H. June, M.D., and Adam Bagg, M.D.
Session: B Lymphoblastic Leukemia/Lymphoma

A 66-year-old man was originally diagnosed with chronic lymphocytic leukemia (CLL) in 1996. Following a six-year period of asymptomatic surveillance, he experienced progressive leukocytosis and lymphadenopathy requiring therapy. Between 2002 and 2010 various agents, including rituximab, fludarabine, bendamustine, and alemtuzumab, were used with approximately 2-year progression-free intervals between treatments. However, his disease continued to evolve with persistent widespread lymphadenopathy. In July 2010, the patient was enrolled in a phase I clinical trial evaluating chimeric antigen receptor-modified T cells directed against CD19 (CART-19). In September 2010, he received pentostatin/cytoxan induction and a total of 1.42×10^7 autologous CART-19 cells (infused over a three day period). The patient initially experienced fevers, chills, anorexia, nausea and diarrhea, which was followed by delayed tumor lysis syndrome on day 22. Bone marrow studies performed on day 23 showed no evidence of CLL by histology, immunophenotype, and cytogenetics/FISH. By day 28 the patient no longer had palpable adenopathy. Subsequent serial CT scans confirm sustained clinical remission, and bone marrow studies demonstrate no residual disease. The patient is now 2 years and 4 months post autologous CART-19 therapy without any evidence of residual disease, using sensitive (10^-6) molecular techniques. He does have sustained hypogammaglobulinemia, which is treated with periodic IVIG therapy.

Bone marrow biopsies were fixed in B5 and decalcified prior to processing and H&E staining. The first marrow submitted to the workshop (labeled HS-10-10547) was obtained five months prior to CART-19 therapy, which showed a hypercellular marrow (60%) extensively involved (40%) by CLL. Post-therapy bone marrow studies up to two years following infusion, including the second bone marrow submitted for review (labeled HS-11-6512) obtained at six months post treatment, have all remained histologically negative*.

(*Note: Since this patient is part of an ongoing clinical trial, with a need to perform multiple additional studies on the limited bone marrow biopsies, we are unfortunately unable to submit unstained slides to the workshop.)

Day 0 immunohistochemical stains on the bone marrow core biopsy show numerous CD20+ lymphoid aggregates, while flow cytometry performed on the bone marrow aspirate shows an expansion (65%) of variably-sized bright surface kappa restricted CD5+ CD10- CD19+ CD20+ CD23+ CD38- IgM+ B cells. Bone marrow samplings obtained at day +23 and +180 reveal virtually no B cells by immunohistochemistry and flow cytometry. Subsequent bone marrow biopsies up to one-year post therapy continue to demonstrate a virtual absence of B cells and certainly no immunophenotypic evidence of CLL by flow cytometry.

One day prior to receiving CART-19 infusion, cytogenetics studies were performed on a bone marrow aspirate, and showed 46,XY,del(17)(p12)[5]/46,XY,der(17)t(17;21)(q10;q10)[5]/46,XY[14]. FISH studies confirmed the deletion of TP53. Follow-up cytogenetics studies of the bone marrow up to two years post therapy demonstrate a normal male karyotype, 46,XY, and FISH remains negative for deletion of TP53.

Real-time PCR was performed on peripheral blood to detect DNA encoding the anti-CD19 chimeric antigen receptor starting on day 1 of CART-19 infusion. Using this approach, by day 21 a 3-log expansion of the modified CART-19 T cells was seen in vivo. At this peak transgene time-point, CART-19 cells accounted for over 20% of circulating lymphocytes, which also coincided with clinical tumor lysis syndrome. Copies of the transgene in peripheral blood remained high six months post infusion. The circulating half-life of these T cells is estimated to be 35 days. Quantitative PCR also detected transgene DNA in the bone marrow up to two years post therapy. Next generation sequencing of the IGH@ gene (with a sensitivity of ~10^-6) shows no minimal residual disease in the bone marrow two years post CART-19 infusion.

This is the first ever case to demonstrate the utility of a novel and highly effective form of therapy, which can also be applied to treat other CD19+ neoplasms, including B lymphoblastic leukemia. Unlike first-generation CART cells, the modified receptor against CD19 used in this case is linked to the 4-1BB domain of CD137 and the cytoplasmic zeta domain of CD3, which are T cell-specific co-stimulatory and signaling domains. These CART cells are observed to have unique qualities such as a capacity for in vivo replication, acquisition of a memory T cell phenotype, and long-term in vivo persistence resulting in sustained tumor control.

This patient is now two years and four months post therapy with durable clinical, pathologic, and exquisitely sensitive molecular remission.

Refractory chronic lymphocytic leukemia apparently cured by autologous chimeric antigen receptor-modified T cell therapy against CD19 (CART-19).

Refractory chronic lymphocytic leukemia apparently cured by autologous chimeric antigen receptor-modified T cell therapy against CD19 (CART-19)

Case 264