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T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)-

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) offers safe and effective therapy for certain viral infections and could prove useful in the eradication of tumor cells. Whether or not the infused T cells persist for extended periods, retaining their ability to expand in response to antigenic stimulation, is not known. We now report long-term detection of gene-marked Epstein-Barr virus (EBV)-specific CTLs in immunocompromised patients at risk for the development of EBV lymphoproliferative disease. Infusions of CTLs not only restored cellular immune responses against EBV, but also established populations of CTL precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months. Our findings support wider use of antigen-specific CTLs in adoptive immunotherapy.

Epstein-Barr virus (EBV) causes potentially lethal immunoblastic lymphoma in up to 25% of children receiving bone marrow transplants from unrelated or HLA-mismatched donors. Because this complication appears to stem from a deficiency of EBV-specific cytotoxic T cells, we assessed the safety and efficacy of donor-derived polyclonal (CD4+ and CD8+) T-cell lines as immunoprophylaxis and treatment for EBV-related lymphoma. Thirty-nine patients considered to be at high risk for EBV-induced lymphoma each received 2 to 4 intravenous infusions of donor-derived EBV-specific T lymphocytes, after they had received T-cell–depleted bone marrow from HLA-matched unrelated donors (n = 33) or mismatched family members (n = 6). The immunologic effects of this therapy were monitored during and after the infusions. Infused cells were identified by detection of the neo marker gene. EBV-specific T cells bearing theneo marker were identified in all but 1 of the patients. Serial analysis of DNA detected the marker gene for as long as 18 weeks in unmanipulated peripheral blood mononuclear cells and for as long as 38 months in regenerated lines of EBV-specific cytotoxic T cells. Six patients (15.5%) had greatly increased amounts of EBV-DNA on study entry (>2,000 genome copies/106 mononuclear cells), indicating uncontrolled EBV replication, a complication that has had a high correlation with subsequent development of overt lymphoma. All of these patients showed 2 to 4 log decreases in viral DNA levels within 2 to 3 weeks after infusion and none developed lymphoma, confirming the antiviral activity of the donor-derived cells. There were no toxic effects that could be attributed to prophylactic T-cell therapy. Two additional patients who did not receive prophylaxis and developed overt immunoblastic lymphoma responded fully to T-cell infusion. Polyclonal donor-derived T-cell lines specific for EBV proteins can thus be used safely to prevent EBV-related immunoblastic lymphoma after allogeneic marrow transplantation and may also be effective in the treatment of established disease. © 1998 by The American Society of Hematology.

Recent genomic profiling of childhood acute lymphoblastic leukemia (ALL) identified a novel high-risk subtype with a gene expression signature resembling Philadelphia chromosome-positive ALL and a poor prognosis (Ph-like ALL). However, the role of inherited genetic variation in Ph-like ALL pathogenesis remains unknown. In a genome-wide association study (GWAS) of 511 ALL cases and 6,661 non-ALL controls, we identified a single susceptibility locus for Ph-like ALL (GATA3, rs3824662, P=2.17×10−14, odds ratio [OR]=3.85, for Ph-like ALL vs. non-ALL; P=1.05×10−8, OR=3.25, for Ph-like ALL vs. non-Ph-like ALL) that was independently validated. The rs3824662 risk allele was associated with somatic lesions underlying Ph-like ALL (i.e., CRLF2 rearrangement, JAK mutation, and IKZF1 deletion) and directly influenced GATA3 transcription. Finally, GATA3 SNP genotype was also associated with early treatment response and the risk of ALL relapse. Our results provide insights into interactions between host and tumor genomes and their importance in ALL pathogenesis and prognosis.

Key Points• A genome-wide study of the association of over 5 million SNPs with methotrexate clearance in 1279 patients treated with HDMTX in multicenter COG trials 9904 and 9905.• We replicated the finding that inherited variations in SLCO1B1 are the most important genetic variations influencing methotrexate clearance.Methotrexate clearance can influence the cure of and toxicity in children with acute lymphoblastic leukemia (ALL). We estimated methotrexate plasma clearance for 1279 patients with ALL treated with methotrexate (24-hour infusion of a 1 g/m 2 dose or 4-hour infusion of a 2 g/m 2 dose) on the Children's Oncology Group P9904 and P9905 protocols. Methotrexate clearance was lower in older children (P ‫؍‬ 7 ؋ 10 ؊7 ), girls (P ‫؍‬ 2.7 ؋ 10 ؊4 ), and those who received a delayed-intensification phase (P ‫؍‬ .0022). A genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in the organic anion transporter gene SLCO1B1 (P ‫؍‬ 2.1 ؋ 10 ؊11 ). This replicates findings using different schedules of high-dose methotrexate in St Jude ALL treatment protocols; a combined meta-analysis yields a P value of 5.7 ؋ 10 ؊19 for the association of methotrexate clearance with SLCO1B1 SNP rs4149056. IntroductionHigh-dose methotrexate (HDMTX) is an important element of chemotherapy in the treatment of acute lymphoblastic leukemia (ALL) and other malignancies. [1][2][3][4] Mikkelsen et al 5 showed that a 1 g/m 2 dose given over 24 hours resulted in significantly greater active methotrexate polyglutamates in leukemic cells than the same dose given over 4 hours. In a randomized clinical trial, investigators from the Children's Oncology Group (COG) tested the efficacy and toxicity of a 2 g/m 2 dose over 4 hours versus a 1 g/m 2 dose over 24 hours. 6,7 The systemic exposure to methotrexate (ie, plasma concentration over time) is related to cure and toxicity in children with ALL. 8,9 In a group of children with ALL who were treated and monitored at a single institution (St Jude Children's Research Hospital), with extensive prospective therapeutic drug monitoring, even including pharmacokinetically guided dosage adjustments, we identified variants in the SLCO1B1 gene that were associated with methotrexate clearance in a genome-wide association study (GWAS). 10,11 Herein, we sought to test whether we could replicate these GWAS findings in a large cohort of patients treated with alternative HDMTX schedules on the COG multi-institutional trials P9904 and P9905. MethodsThis study was approved by the institutional review boards of all participating institutions, and informed consent was obtained in accordance with the Declaration of Helsinki. Patients in P9904 included National Cancer Institute (NCI) standard-risk (age 1.00-9.99 years and initial white blood cell count [WBC]Ͻ 50 000/L) patients with an ETV6-RUNX1 translocation or simultaneous trisomies of chromosomes 4 and 10, whereas patients in P9905 included a mixture of NCI standard-risk patients without favorable genetic lesions, NCI high-risk (age...

There is a growing interest in using antigen-specific T cells for the treatment of human malignancy. For example, adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been effective prophylaxis and treatment of EBV-associated lymphoproliferative disease in immunocompromised patients. For all immunotherapies, however, there has been a hypothetical concern that mutations in tumor-specific antigens may lead to tumor escape. We now demonstrate that such events may indeed occur, with lethal outcome. A patient who developed lymphoma after marrow transplantation received donor-derived, EBV-specific CTLs but died with progressive disease. The tumor cells proved substantially less sensitive to cytolysis than the EBV-transformed B-cell line used for CTL generation. The major cytolytic activity of the donor CTL was directed against 2 HLA-A11-restricted epitopes in the viral EBNA-3B antigen. Sequence analysis of this gene in the tumor virus revealed a 245-base pair deletion, which removed these 2 CTL epitopes. Hence, the viral antigen in the tumor had mutated in a way that allowed escape from CTLs. Analysis of EBV polymorphisms demonstrated that before CTL infusion, more than one virus was present, including a virus with wild-type EBNA-3B. After CTL infusion, only the virus with the EBNA-3B deletion could be detected, suggesting that the infused CTLs had selected a resistant strain in vivo. Such an occurrence, even when polyclonal CTL lines are used against genetically stable virus antigens, suggests that escape mutants may be a serious problem when CTL therapy is directed against more unstable tumor cell-derived targets.

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and leukemia cell resistant to glucocorticoids confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the sensitivity to prednisolone of primary leukemia cells from 444 newly diagnosed ALL patients, revealing significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases.