Dual targeting of CD19 and CD22 with bicistronic CAR-T cells in patients with relapsed/refractory large B-cell lymphoma.

Relapse after CD19-directed chimeric antigen receptor T-cell (CAR-T) therapy for large B-cell lymphoma (LBCL) is commonly ascribed to antigen loss or CAR-T exhaustion. Multiantigen targeting and programmed cell death protein-1 blockade are rational approaches to prevent relapse. Here, we test CD19/22 dual-targeting CAR-T (AUTO3) plus pembrolizumab in relapsed/refractory LBCL (NCT03289455). End points include toxicity (primary) and response rates (secondary). Fifty-two patients received AUTO3 and 48/52 received pembrolizumab. Median age was 59 years (range, 27-83), 46/52 had stage III/ IV disease and median follow-up was 21.6 months. AUTO3 was safe; grade 1-2 and grade 3 cytokine release syndrome affected 18/52 (34.6%) and 1/52 (1.9%) patients, neurotoxicity arose in 4 patients (2/4, grade 3-4), and hemophagocytic lymphohistiocytosis affected 2 patients. Outpatient administration was tested in 20 patients, saving a median of 14 hospital days per patient. Overall response rates were 66% (48.9%, complete response [CR]; 17%, partial response). Median duration of remission (DOR) for CR patients was not reached and for all responding patients was 8.3 months (95% confidence interval [CI]: 3.0-not evaluable). 54.4% (CI: 32.8-71.7) of CR patients and 42.6% of all responding patients were projected to remain progression-free at ≥12 months. AUTO3 ± pembrolizumab for relapsed/refractory LBCL was safe and delivered durable remissions in 54.4% of complete responders, associated with robust CAR-T expansion. Neither dual-targeting CAR-T nor pembrolizumab prevented relapse in a significant proportion of patients, and future developments include next-generation-AUTO3, engineered for superior expansion in vivo, and selection of CAR binders active at low antigen densities.

Therapy for isocitrate dehydrogenase 2 (IDH2)R172 -mutant acute myeloid leukaemia.

Although we earlier reported a very poor outcome for younger adult patients with isocitrate dehydrogenase 2 (IDH2)R172 -mutated acute myeloid leukaemia (AML) entered into UK trials compared to IDH2WT and IDH2R140 -mutated patients, this was not corroborated by a study from the German-Austrian AML Study Group. We have therefore investigated a later cohort of IDH2-mutated patients to identify any changes in outcome and whether this could inform the optimal treatment for IDH2R172 AML. We found an improved outcome for IDH2R172 -mutated AML in the later trials and the data suggests that this may be due to the increased use of allogeneic transplantation to consolidate first remission.

Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS: Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS: Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease-negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4%-84.4%) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION: AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL.

Additional impact of mutational genotype on prognostic determination in resistant and relapsed acute myeloid leukaemia.

Outcome after failure of initial therapy in younger adult patients with acute myeloid leukaemia (AML) is highly variable. Cytogenetics, length of first remission (CR1) before relapse, and allogeneic transplantation are known prognostic factors, but the contribution of leukaemic genotype is less clear, particularly in resistant disease. Of 5,651 younger adult patients entered into UK MRC/NCRI AML trials between 1988 and 2014 with available FLT3ITD and NPM1 genotype, 326 (6%) had resistant disease and 2338 (41 %) relapsed after achieving CR1. Overall survival (OS) was significantly higher in relapsed compared to resistant disease (p = 0·03). Independent favourable prognostic factors for OS in resistant disease included lower blast cell percentage after two courses of induction therapy (p = 0.0006) and NPM1 mutant (NPM1MUT) (p = 0.04). In relapsed disease, longer CR1 was a favourable independent factor for attainment of CR2 (p < 0.0001) and OS from time of relapse (p < 0.0001), but CR2 rate and OS from relapse were significantly worse in those who had received an allograft in CR1 (respectively p < 0.05, p < 0·002). NPM1MUT was marginally beneficial for OS (p = 0.04). FLT3ITD and DNMT3AMUT were adverse factors for OS (respectively p < 0.0001, p = 0.02). Mutational analysis adds additional independent prognostic information to demographic features and previous therapy in patients with resistant and relapsed disease.

EZH2-Deficient T-cell Acute Lymphoblastic Leukemia Is Sensitized to CHK1 Inhibition through Enhanced Replication Stress.

Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2-deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small-molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2-deficient, but not EZH2 wild-type, T-ALL cells in vivo, as well as in a primary cell model of PRC2-mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene-expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Finally, we demonstrate this phenotype is mediated through derepression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2-mutated T-ALL. SIGNIFICANCE: Loss-of-function mutations of PRC2 genes are associated with chemotherapy resistance in T-ALL, yet no specific therapy for this aggressive subtype is currently clinically available. Our work demonstrates that loss of EZH2 activity leads to MYCN-driven replication stress, resulting in increased sensitivity to CHK1 inhibition, a finding with immediate clinical relevance.This article is highlighted in the In This Issue feature, p. 890.

Favourable outcomes for high-risk Burkitt lymphoma patients (IPI 3-5) treated with rituximab plus CODOX-M/IVAC: Results of a phase 2 UK NCRI trial.

Introduction: Outcomes after frontline treatment of Burkitt lymphoma (BL) have improved with the introduction of dose-intense chemotherapy regimens, such as CODOX-M/IVAC. While rituximab has increased survival rates for most forms of high-grade B-cell lymphoma, there has previously been hesitancy about incorporating it into BL treatment, partly due to concerns about increased toxicity. Prospective data using the standard dose CODOX-M/IVAC regimen in combination with rituximab are lacking. We conducted a single-arm phase 2 trial to assess the efficacy and toxicity of R-CODOX-M/R-IVAC. Methods: Eligible patients were aged 18-65 years, with newly diagnosed BL with MYC rearrangement as the sole cytogenetic abnormality, and high-risk disease, defined by an International Prognostic Index (IPI) score of 3-5. Patients received two cycles of R-CODOX-M chemotherapy alternating with two cycles of R-IVAC, followed by two further cycles of rituximab alone. The primary endpoint was 2-year progression-free survival. Results: Thirty-eight patients were registered but after central pathology review, 27 patients had confirmed BL and commenced study treatment. Median age was 35 years, 14.8% patients had central nervous system involvement and 18.5% were HIV positive. Twenty-two (81.4%) patients completed four cycles of chemotherapy. There were two treatment-related deaths (7.4%). Two-year progression-free and overall survival rates were 77.2% (90% confidence interval [CI]: 56.0-89.0) and 80.7% (90% CI: 59.6-91.5), respectively. Conclusions: This prospective trial demonstrates excellent survival rates with R-CODOX-M/R-IVAC in a high-risk BL cohort. It provides reassuring evidence regarding the feasibility of this regimen and also provides a benchmark for future studies.

Analysis of the clinical impact of NPM1 mutant allele burden in a large cohort of younger adult patients with acute myeloid leukaemia.

Although an NPM1 mutation is generally considered to be a good prognostic marker in acute myeloid leukaemia, it has recently been suggested that a higher level of NPM1 mutant (NPM1MUT ) alleles relative to wild-type alleles is associated with poor clinical outcome. We therefore sought to confirm this finding in a larger study of 876 NPM1MUT cases entered into UK national trials. In univariate analysis, the higher NPM1MUT allele burden was associated with a lower complete remission (CR) rate, higher relapse rate and reduced overall survival, but this was largely attributable to the association of the higher NPM1MUT allele burden with other known poor risk factors, particularly the presence of a concomitant FLT3 internal tandem duplication. In multivariate analysis, there was no significant impact of the NPM1MUT allele burden on CR rates, and the impact on relapse and overall survival, whilst still significant, was greatly reduced. This impact was similar in patients who did or did not receive an allogeneic transplant in first CR. We conclude that the binary presence or absence of an NPM1 mutation, combined with minimal residual disease levels following induction therapy, should continue to be used in therapeutic management rather than stratification according to the NPM1MUT level.

Allo-HSCT in transplant-naïve patients with Hodgkin lymphoma: a single-arm, multicenter study.

We evaluated the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in transplant-naïve patients with relapsed/refractory Hodgkin lymphoma (HL) who failed to attain metabolic complete response (mCR) to 1 to 2 lines of salvage chemotherapyThose with residual but nonprogressive disease assessed by positron emission tomography/computed tomography scanning were eligible. An additional 1 to 2 cycles of salvage therapy were permissible in those with progressive disease or when required to bridge to allo-HSCT, with additional imaging at baseline before transplantation. Conditioning consisted of carmustine, etoposide, cytarabine, melphalan, and alemtuzumab. Donor lymphocyte infusions (DLI) were administered for mixed chimerism or residual or relapsed disease. Eleven patients had sibling donors, 13 had HLA-matched unrelated donors, and 7 had HLA-mismatched unrelated donors. There were no graft failures, and no episodes of grade 4 acute graft-versus-host disease (GVHD); only 19.4% of patients had grade 2 to 3 GVHD, and 22.2% had extensive chronic GVHD. The non-relapse mortality rate was 16.1% (95% confidence interval [CI], 7.1%-34.5%). Relapse incidence was 18.7% (95% CI, 8.2%-39.2%). The study met its primary objective, with a 3-year progression-free survival of 67.7% (95% CI, 48.4%-81.2%). Survival outcomes were equivalent in those with residual metabolically active disease immediately before transplantation (n = 24 [70.8%; 95% CI, 17.2%-83.7%]). Two of the 5 patients who relapsed received DLI and remained in mCR at latest follow-up, with a 3-year overall survival of 80.7% (95% CI, 61.9%-90.8%). We demonstrate encouraging results that establish a potential role for allo-HSCT in selected high-risk patients with HL. This trial was registered at www.clinicaltrials.gov as #NCT00908180.

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients.

Acute myeloid leukemia with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40% of patients. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least 1 mutation, in addition to t(8;21), was identified in 95%, with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes: GIGYF2, DHX15, and G2E3 Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTKmutated patients, we identified multiple mutations in the same pathway. Deep sequencing (∼42 000×) of 126 mutations in 62 complete remission samples from 56 patients identified 16 persisting mutations in 12 patients, of whom 5 lacked RUNX1-RUNX1T1 in quantitative polymerase chain reaction analysis. KIT high mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (hazard ratio, 1.96; 95% confidence interval, 1.22-3.15; P = .005). Together with age and white blood cell counts, JAK2, FLT3-internal tandem duplicationhigh, and KIT high mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n = 13) and unstable (n = 6) subgroups could be identified.

Immunophenotypic analysis of cell cycle status in acute myeloid leukaemia: relationship to cytogenetics, genotype and clinical outcome.

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P < 0·0001), of cycling cells beyond G1 (P < 0·0001) and the speed of cycling (P < 0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P = 0·002) and faster speed of cycling (P = 0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P = 0·0002), as was a higher relapse rate (P = 0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Variable outcome and methylation status according to CEBPA mutant type in double-mutated acute myeloid leukemia patients and the possible implications for treatment.

Although CEBPA double-mutated (CEBPADM) acute myeloid leukemia is considered to be a favorable-risk disease, relapse remains a major cause of treatment failure. Most CEBPADM patients have a classic biallelic mutant combination with an N-terminal mutation leading to production of p30 protein plus a C-terminal loss-of-function in-frame indel mutation (CEBPAClassic-DM), but approximately one-third of cases have one or more non-classic mutations, with diverse combinations reported, and there is little information on the consequences of such mutants. We evaluated outcome in a cohort of 104 CEBPADM patients, 79 CEBPAClassic-DM and 25 with non-classic mutants, and found that the latter may have poorer survival (5-year overall survival 64% vs. 46%; P=0.05), particularly post relapse (41% vs. 0%; P=0.02). However, for this analysis, all non-classic cases were grouped together, irrespective of mutant combination. As CEBPADM cases have been reported to be hypermethylated, we used methylation profiling to assess whether this could segregate the different mutants. We developed a CEBPAClassic-DM methylation signature from a preliminary cohort of 10 CEBPADM (including 8 CEBPAClassic-DM) and 30 CEBPA wild-type (CEBPAWT) samples, and independently validated the signature in 17 CEBPAClassic-DM cases. Assessment of the signature in 16 CEBPADM cases with different non-classic mutant combinations showed that only 31% had a methylation profile equivalent to CEBPAClassic-DM whereas for 69% the profile was either intermediate between CEBPAClassic-DM and CEBPAWT or equivalent to CEBPAWT These results suggest that CEBPADM cases with non-classic mutants may be functionally different from those with CEBPAClassic-DM mutants, and should not automatically be included in the same prognostic group. (AML12 is registered under ISRCTN17833622 and AML15 under ISRCTN17161961).

Targeting the T cell receptor ß-chain constant region for immunotherapy of T cell malignancies.

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan-T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor ß-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity.

Activation of the LMO2 oncogene through a somatically acquired neomorphic promoter in T-cell acute lymphoblastic leukemia.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Optimisation and quality control of cell processing for autologous stem cell transplantation.

Clinical practice and the technology of cell processing for autologous stem cell transplantation has continued to evolve over the last two decades and merits review of current quality control expectations. The external regulatory era has improved quality and safety standards but there is still variable practice, with specific risks illuminated by a number of clinical incidents. Viable CD34+ cell assays may fail to indicate significant losses in progenitor function during storage, particularly after cryopreservation, and there is a need to develop an alternative, real time functional assay to replace colony assays. The ultimate guide to potency and successful cell processing for haematopoietic progenitor cell products is prompt and reproducible engraftment and close monitoring is essential for safety and quality control.

Re-evaluation of progenitor thresholds and expectations for haematopoietic recovery based on an analysis of 810 autologous transplants: Implications for quality assurance.

Haematological engraftment was assessed in 804 autologous transplants. Neutrophil recovery occurred in over 99% within 14 d but platelet recovery was delayed beyond this time in 14·8%. Time to recovery was dependent on the progenitor cell dose infused. The minimum CD34+ cell threshold adopted in this study (2 × 106 /kg) was safe although recovery was faster with a dose >5 × 106 /kg. CD34+ cell doses of between 1 and 2 × 106 /kg were also acceptable if either the granulocyte-macrophage colony-forming cell dose exceeded 2 × 105 /kg or this dose was due to splitting a higher yield harvest. Prompt neutrophil recovery affords important quality assurance for laboratory processing.