ALBERT

Description: Introduction: Leukemia cells are able to escape from immunosurveillance using immune tolerance mechanisms as the majority of leukemia antigens are either shared or aberrantly expressed self-proteins. T cells reactive to these antigens are purged during thymic selection. CD2, a pan-T-cell antigen, is expressed early during T cell developments in thymus and is found on all subsets of mature T cells. Recent studies show that there are low levels of extrathymic CD2 negative (CD2-) T cells, which show immature T cell features and can be induced to differentiate into mature helper and cytotoxic T cells in vitro. Since circulating CD2- T cells could represent pre-selection immature T cells, they may play an important role in tumor immunity. Methods: 81 pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients, 22 pediatric acute myeloid leukemia (AML) patients and 22 normal controls were included in this study. B-ALL group included 45 NCI-standard risk (SR) patients and 36 NCI-high risk patients. All the leukemia patients were diagnosed at Children's Mercy Hospital in the past ten years with a diagnostic peripheral blood (PB) specimen. The PB specimens were studied by four-color multiparameter flow cytometry with antibodies for T cell markers (CD2, CD3, CD4, CD5, CD7 and CD8) and CD45, and analyzed by BD FACSDiva 8.0.1. CD2- and CD3+ T cells were recorded as % of total T cells. Student's t-test was used to compare results. Results: The percentages of CD2- T cells in AML (mean ± STD: 1.31% ± 1.41%) and B-ALL (0.84% ± 0.67%) were significantly higher than that seen in control group (0.51% ± 0.52%, p

Description: Tisagenlecleucel is a CD19 directed immunotherapy approved for the treatment of young patients with relapsed or refractory precursor B-cell acute lymphoblastic leukemia (ALL). The most important toxicity related to tisagenlecleucel therapy is cytokine release syndrome (CRS). CRS is an exaggerated systemic inflammatory response that occurs frequently along with T-cell expansion following the administration of tisagenlecleucel. Tisagenlecleucel guidelines recommend delaying treatment when an active infection is present due to the concern that the pre-existing inflammatory response associated with infection may predispose patients to severe CRS. We describe two cases where tisagenlecleucel was successfully administered to patients in the setting of life-threatening infection. Patient 1 is a 23-year-old Caucasian male with refractory Philadelphia chromosome negative B-cell ALL who had received prior treatment with chemotherapy, blinatumomab, inotuzumab ozogamicin and a haploidentical stem cell transplant (SCT) followed by multiple Zalmoxis infusions. Five months following SCT he relapsed. At relapse, he underwent leukapheresis followed by bridging chemotherapy with ifosfamide and etoposide. He developed severe neutropenia and respiratory failure associated with a right lower lung consolidation. A biopsy demonstrated a mucormycosis infection and he required surgical debridement including resection of portions of the lung, diaphragm and liver. At this time, he had 92% blasts in the bone marrow. Eleven days after his surgery he received tisagenlecleucel despite being persistently febrile. Prior to the infusion, he received a modified lymphodepleting chemotherapy regimen including two days of fludarabine and cytarabine. Due to severe neutropenia, he was receiving granulocyte transfusions. These were discontinued prior to the infusion and resumed after 12 days. CRP and ferritin the day prior to infusion were 26.2 mg/dL and 18,419 ng/mL. He remained persistently febrile for 13 days post-infusion. He received a single dose of tociluzimab 7 days following his infusion due to high fevers. He did not require any treatment with corticosteroids for CRS. The absolute neutrophil count recovered to 〉500x103/µL at 31 days post infusion. A bone marrow aspirate done 26 days post-infusion did not show any evidence of leukemia by multicolor flow cytometry (MFC). He remains alive without evidence of disease 11 months after treatment with tisagenlecleucel. Patient 2 is a 4-year-old Hispanic female with refractory B-cell ALL found to have a TP53 deletion and t(1;19). She had received prior treatment with chemotherapy, blinatumomab and local radiation therapy to the site of extramedullary disease found in the left maxillary sinus at relapse. She underwent leukapheresis and received bridging chemotherapy with mercaptopurine and methotrexate. After 3 days of lymphodepleting chemotherapy she developed septic shock and E. Coli bacteremia. She became severely ill requiring continuous renal replacement therapy for 5 days and extracorporeal membrane oxygenation (ECMO) for 6 days. Shortly after ECMO decannulation she developed fever and was found to have multiple pulmonary opacities concerning for fungal infection. Blasts were noted in the peripheral blood. Sixteen days after presenting with septic shock and 11 days from ECMO she received tisagenlecleucel. CRP at the time of infusion was 22.9 mg/dL. She developed persistent fevers post-infusion for 17 days. She received two doses of tociluzimab 20- and 21-days post-infusion due to recurrence of high fever and reactive lymphadenopathy with third spacing and concern for renovascular compromise. She did not require any treatment with corticosteroids for CRS. Bone marrow aspirate done 32 days post-infusion did not show any evidence of leukemia by MFC. The absolute neutrophil count recovered to 〉500x103/µL at 59 days post infusion. The patient remained without evidence of disease for 7 months following treatment but died due to infectious complications with persistent pancytopenia. Tisagenlecleucel is a potentially life-saving treatment for relapsed and refractory B-cell acute lymphoblastic leukemia in children and young adults 24 years of age or younger. Tisagenlecleucel is an option for the treatment of patients with active infection and/or inflammation with progressive leukemia when no other therapeutic alternative exists. Disclosures August: Novartis Pharmaceuticals: Speakers Bureau. Myers:Novartis Pharmaceuticals: Consultancy, Speakers Bureau.

Description: CMV, Adenovirus and EBV are major viral pathogens causing morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. Previous studies have shown that prophylactic adoptive immunotherapy with donor-derived Cytotoxic T Lymphocytes (CTL) directed against EBV and CMV can effectively prevent the clinical manifestations of these viruses. We have extended these studies by generating CTL from normal donor PBMC that can restore cellular immunity to CMV, EBV and adenovirus simultaneously. We have developed a protocol utilizing an initial round of stimulation with autologous mononuclear cells transduced with a recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber carrying a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65). This is followed by 2 rounds of weekly stimulation with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector using MOIs of 10 and 100 respectively. After 3 rounds of stimulation, 9 CTL cultures contained a mean of 83% (range 8.4–98.99%) CD8+ve and a mean of 19.6% (range 2.2–91.6%) CD4+ve cells. In chromium release and/or IFNg ELISPOT assays, all CTL lines showed specific activity against CMV and EBV targets. 8/9 lines also showed specific lysis against adenovirus targets. Further, using MHC-peptide multimers we have been able to demonstrate the simultaneous presence of CD8+ve cells recognizing peptide epitopes from CMV pp65 (range 2.32–21%) and Adenovirus hexon (1.07–8.08%) in the CTL cultures. So far we have treated 6 patients in this phase I CMV prophylaxis study, 3 on dose level 1 (1x10e7/m2) and 3 on dose level 2 (5x10e7/m2). Patients received 1 infusion of virus-specific CTL from 54–120 days post transplant. We observed up to a 28-fold increase in CMV pentamer positive CD8+ve cells post CTL. At last follow-up (7–35 weeks post CTL infusion) all patients are CMV and EBV negative. 2 patients were transiently positive for CMV by PCR 4–9 weeks post CTL but both were negative 7 days later without anti-viral therapy, with a corresponding rise in CMV-specific CTL detected in the peripheral blood. 2 patients were culture positive for adenovirus in their stool pre CTL therapy. One of these patients was infected with adenovirus species from subgroups A, C and D. In both patients, we observed a 2-log reduction of adenovirus copies/g stool within 2–3 weeks post CTL infusion at which time their symptoms (fever, loose stools) resolved. In summary, we have developed a protocol for the efficient generation of multi-virus specific CTL: infusion of small numbers of these cells increased virus-specific CD8+ve T cells in the peripheral blood post CTL infusion. Further, reduction in adenovirus load in stool suggests efficacy of adenovirus specific CTL in vivo. However, expansion of virus-specific CTL in vivo may require presence of antigen. We will therefore complete this prophylaxis study and then proceed to using virus-specific CTL for the treatment of CMV and adenovirus disease post transplant.