We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA-electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti-CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML.

BackgroundDiffuse large B-cell lymphoma (DLBCL) typically leads to effacement of the nodal architecture by an infiltrate of malignant cells. Rarely (<1%), DLBCL can present with an interfollicular pattern (DLBCL-IF) preserving the lymphoid follicles. It has been postulated that DLBCL-IF is derived from marginal zone B cells and may represent a large-cell transformation of marginal zone lymphoma (MZL), however no direct evidence has been provided to date. Here we describe a rare case of a diagnostically challenging DLBCL-IF involving a lymph node in a patient with a prior history of lymphadenopathy for several years and MZL involving skin.Case presentationA 53-year old man presented to our Dermatology Clinic due to a 1-year history of generalized itching, fatigue of 2–3 month’s duration, nausea and mid back rash that was biopsied. PET (positron emission tomography)/CT (computed tomography) was performed and revealed inguinal, pelvic, retroperitoneal, axillary, and cervical lymphadenopathy. The patient was referred to surgery for excisional biopsy of a right inguinal lymph node.Diagnostic H&E stained slides and ancillary studies were reviewed for the lymph node and skin specimens. B-cell clonality by PCR and sequencing studies were performed on both specimens.We demonstrate that this patient’s MZL and DLBCL-IF are clonally related, strongly suggesting that transformation of MZL to DLBCL had occurred. Furthermore, we identified a novel deletion of the long arm of chromosome 20 (del(20q12)) and a missense mutation in BIRC3 (Baculoviral IAP repeat-containing protein 3) in this patient’s DLBCL that are absent from his MZL, suggesting that these genetic alterations contributed to the large cell transformation.ConclusionsTo our knowledge, this is the first report providing molecular evidence for a previously suspected link between MZL and DLBCL-IF. In addition, we describe for the first time del(20q12) and a missense mutation in BIRC3 in DLBCL. Our findings also raise awareness of DLBCL-IF and discuss the diagnostic pitfalls of this rare entity.

BACKGROUND: The cell of origin (COO) of diffuse large B cell lymphoma (DLBCL), germinal center (GC) or non-germinal center (NGC), may have prognostic significance for treatment outcome in first-line and relapsed settings (Lenz et al NEJM 2008; Thieblemont et al JCO 2011). "Double hit" DLBCL (DHL), defined by chromosomal breakpoints affecting the MYC/8q24 locus and BCL2/18q21 and/or BCL6/3q27 loci and arising either from transformation of follicular lymphoma (tFL) or de novo, has no standard effective therapy in the relapsed setting. Since new therapies are needed for poor prognostic groups of relapsed DLBCL patients (pts), we examined the efficacy of treatment with autologous T cells genetically modified to express a chimeric antigen receptor consisting of an external anti-CD19 single chain murine antibody domain with CD3ζ and 4-1BB signaling domains (CTL019 cells) in pts with relapsed/refractory GC and NGC DLBCL, DHL, and tFL as part of an ongoing phase IIa clinical trial (NCT02030834). METHODS: Eligible pts had CD19+ DLBCL with measurable residual disease after primary and salvage therapies, were not eligible for autologous stem cell transplant (ASCT) or had relapsed/residual disease after ASCT, and had responsive or stable disease with most recent therapy. COO of DLBCL was defined immunohistochemically using the Hans algorithm (Hans et al Blood 2004) and included the DLBCL subtypes DHL and tFL that met this definition. Fluorescence in-situ hybridization was performed on diagnostic tissue using Vysis MYC (8q24), BCL2 (18q21) and BCL6 (3q27) break apart probes to determine DHL. DHL was defined by a MYC locus chromosomal translocation with a second translocation involving either BCL2 or BCL6. After steady state apheresis to collect peripheral blood leukocytes, pts received lymphodepleting chemotherapy based on clinical features and past therapies. 1 to 4 days after chemotherapy, pts received a single dose of CTL019 cells. Following CTL019, pts received no further therapy for DLBCL. Initial tumor response assessment was performed 3 months (mo) after CTL019 using IWG criteria. Enrollment started in Feb 2014; data are reported through 24 Jul 2016. RESULTS: 13 pts with DLBCL are enrolled and evaluable for response (7 pts GC, 5 pts NGC, 1 undetermined). The median age is 59 years (range: 25-77), male:female ratio 10:3, median number of prior therapies 5 (range: 2-8), and number of pts with prior transplant 7 (54%). At enrollment, Ann Arbor stages were: Stage IV 8 pts (61%), Stage III 1 pt (8%), and Stage II 3 pts (23%) Stage IE 1 pt (8%); 3 pts (23%) had bone marrow involvement. LDH was increased in 8 pts (62%). ECOG PS was 0 in 4 pts (31%) and 1 in 9 pts (69%).Lymphodepleting chemotherapy regimens were bendamustine (90 mg/m2 x 2; 1 pt), cyclophosphamide (1 gm/m2; 2 pts), radiation-cyclophosphamide (4,000cGy-750 mg/m2; 1 pt), modified-EPOCH (3 pts), and hyper-fractionated cyclophosphamide (300 mg/m2 q12 hours x 6; 6 pts). 12 pts received 5.00E+08 (5.10 - 6.75E+06 cells/kg) CTL019 cells; 1 pt received 1.93E+08 (3.10E+06 cells/kg).Median peak CTL019 cell expansion in blood occurred 7 days after infusion (range: 2-28 days); there is no difference in peak expansion between responders and non-responders or pts with GC or NGC DLBCL.Cytokine release syndrome occurred in 9 pts (8 grade 2; 1 grade 3) and did not predict response. Transient neurotoxicity included delirium in 2/13 pts (1 grade 2; 1 grade 3) and cognitive disturbance in 1/13 pts (1 grade 1). At 3 mo post CTL019, overall response rate (ORR) is 52% (7/13 pts); ORR at 3 mo for GC 71% (5/7 pts) and NGC 40% (2/5 pts). Complete response rate (CR) at 3 mo is 38% (5/13 pts); CR at 3 mo for GC 43% (3/7 pts) and NGC 40% (2/5 pts). Best response for all pts is CR in 6 of 13 pts (46%); CR for GC 57% (4/7 pts) and NGC 40% (2/5 pts). 3 of 7 pts with GC DLBCL had tFL and all 3 achieved CR; 2 of 7 pts with GC DLBCL had DHL and both achieved CR. To date, no pt achieving CR has relapsed. Median progression-free survival is 5.8 mo for all pts, 3.0 mo for NGC pts, and not reached for GC pts (57.1% [95%CI: 17.2%-83.7%] progression-free at median follow-up 21.9 mo). At median follow-up 23.3 mo for responding pts, 85.7% [95%CI: 33.7-97.9%] maintain response. CONCLUSIONS: A single treatment with CTL019 cells can achieve durable remissions in pts with relapsed/refractory GC and NGC DLBCL, DHL and transformed FL. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Schuster: Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Research Funding; Genentech: Consultancy, Honoraria; Merck: Research Funding; Janssen Research & Development: Research Funding; Hoffman-LaRoche: Research Funding; Gilead: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Svoboda:Pharmacyclics: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding. Nasta:Millennium Pharmaceuticals: Research Funding. Porter:Genentech/Roche: Employment; Genentech/Roche: Equity Ownership; Incyte: Honoraria; Novartis: Consultancy; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties; Immunovative Therapies: Other: Travel, Accommodations, Expenses. Mato:ProNAi: Research Funding; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Theradex: Research Funding; Gilead Sciences: Research Funding; Abbvie: Research Funding; TG Therapeutics: Consultancy; Pharmacyclics: Consultancy; Gilead Sciences: Consultancy; Abbvie: Consultancy. Wasik:Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding. Chew:Novartis: Research Funding. Hasskarl:Novartis: Employment. Marcucci:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. June:Pfizer: Honoraria; Johnson & Johnson: Research Funding; University of Pennsylvania: Patents & Royalties; Tmunity: Equity Ownership, Other: Founder, stockholder ; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership.

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The genetic and molecular abnormalities underlying histological transformation (HT) of nodal marginal zone lymphoma (NMZL) to diffuse large B-cell lymphoma (DLBCL) are not well known. While del(20q12) is commonly deleted in myelodysplastic syndrome it has not previously been associated with DLBCL. We recently described a case of DLBCL harboring del(20q12) in a patient with a history of MZL involving lymph nodes and skin. Here we report eight matched cases of transformed MZL(tMZL): six from nodal MZL (tNMZL) and two from splenic MZL (tSMZL). We found >20% del(20q12) in 4/6 tNMZL, but not in tSMZL, nor in unmatched DLBCL, MZL with increased large cells (MZL-ILC), or MZL cases. To examine whether transformation is associated with a specific gene signature, the matched cases were analyzed for multiplexed gene expression using the Nanostring PanCancer Pathways panel. The differential gene expression signature revealed enrichment of inflammatory markers, as previously observed in MZL. Also, tMZL and de novo DLBCL were enriched for extracellular matrix proteins such as collagen and fibronectin, vascular development protein PDGFRβ, DNA repair protein RAD51, and oncogenic secrete protein Wnt11. A subset of genes is expressed differentially in del(20q12) tMZL cases vs non-del(20q12) tMZL cases. These results suggest a specific pathway is involved in the histological transformation of NMZL, which could serve as an indicator of aggressive clinical course in this otherwise indolent neoplasm.