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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

Supplementary Table 2: Cell viability data from the small-molecule drug screening in EZH2-WT and EZH2-KO1 cells (related to Fig. 1)

Supplementary Table 3: Mutations identified by exome sequencing in Liu et al., 2017 T-ALL cohort (related to Fig. 5G)

Supplementary Figures and Legends

Supplementary Table 1: List of the main targets and number of probes included in the small-molecule inhibitors library (related to Fig. 1)

Supplementary information provided in this work.

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.

We compared the International Prognostic Index (IPI), Revised (R)-IPI and age-adjusted (aa)-IPI as prognostic indices for patients with diffuse large B-cell lymphoma (DLBCL) in the UK National Cancer Research Institute (NCRI) R-CHOP 14 versus 21 trial (N = 1080). The R-IPI and aa-IPI showed no marked improvement compared to the IPI for overall and progression-free survival, in terms of model fit or discrimination. Similar results were observed in exploratory analyses incorporating the Grupo Español de Linfomas/Transplante de Médula Ósea (GELTAMO)-IPI, where baseline β2-microglobulin data were available (N = 655). Although our findings support current use of the IPI, a novel prognostic tool to better delineate a high-risk DLBCL group in the rituximab era is needed.

Although we earlier reported a very poor outcome for younger adult patients with isocitrate dehydrogenase 2 (IDH2)

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.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we performed a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identified a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10-8; KMT5B). We also identified a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N=1287) at 6p21.32 (rs3916765; P = 1.51 × 10-10; HLA). Our results inform on AML etiology by identifying putative functional genes operating in histone methylation (KMT5B) and immune function (HLA).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

IntroductionOutcomes 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.MethodsEligible 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.ResultsThirty‐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.ConclusionsThis 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.

Risk-stratification and treatment intensification according to minimal residual disease (MRD) analysis has improved outcomes of patients with acute lymphoblastic leukemia (ALL).[1][1],[2][2] However, a significant proportion of patients with T-cell ALL (T-ALL) still experience early relapse or

INTRODUCTION We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR-T immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL. Cumulatively, this data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Recently, CAR-T therapy has been explored in indolent lymphomas such as follicular (FL) and mantle cell lymphoma (MCL), but a high incidence of toxicity including Grade 3-4 ICANS has been reported. We have initiated testing of AUTO1 in the setting of indolent and high-grade B-NHL and CLL (NCT02935257). METHODS Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised leukapheresate. Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m 2 x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x10^6 CD19 CAR T-cells at day 0 and day 9 is employed in the CLL cohort. A single dose of 200 x10^6 CD19 CAR T-cells is delivered to patients with B-NHL. Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months. RESULTS As of 17th May 2021, we recruited 13 patients: 7 with FL, 4 with MCL, 1 DLBCL and 1 CLL. Apheresis and product manufacture was successful in all 13 patients and 9 patients were infused: 7 with FL and 2 with MCL. Three patients (1 DLBCL, 1 CLL and 1 MCL) were pending infusion at time of data cut-off and 1 patient (MCL) died due to Covid-19 prior to infusion. Patients treated with AUTO1 had a median age of 56 years (range 39-68y), had received a median of 3 prior lines of treatment (range 2-5) and all patients had stage IV disease at screening. Grade 1 CRS was reported in 4/9 and Grade 2 CRS in 1/9. 1/9 developed MAS which resolved with anakinra/dexamethasone. No ICANS was observed on study. Excellent CAR engraftment was observed and 9/9 patients were in CMR by 18FDG PET-CT post-treatment. At a median of 6.1 months (range 4.0-8.1m), 8/9 patients were disease free at last follow-up. One patient died in CMR at month 5.6 of COVID-19. CONCLUSION AUTO1 has a tolerable safety profile in adult patients with r/r B-NHL despite high disease burden. Early data shows 100% complete remission rates and excellent CAR engraftment/expansion. Additional MCL, CLL and DLBCL patients, updated data and longer follow up will be presented. Disclosures Roddie: Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau. Hartley: Astra Zeneca: Ended employment in the past 24 months; ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Farzaneh: Autolus: Consultancy, Current equity holder in publicly-traded company. Lowdell: Autolus: Consultancy, Current equity holder in publicly-traded company. Linch: Autolus: Consultancy, Current equity holder in publicly-traded company. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company. Peggs: Autolus: Consultancy, Current equity holder in publicly-traded company.

TO THE EDITOR: Addition of the CD33-targeted immunoconjugate gemtuzumab ozogamicin (GO; Pfizer) has been shown to improve the response to standard-induction chemotherapy and results in better long-term survival in adult patients with acute myeloid leukemia (AML).[1][1] The greatest impact was

Introduction: Prognosis for adult B-cell Acute Lymphoblastic Leukaemia (B-ALL) is poor and there is currently no licensed CD19 Chimeric Antigen Receptor (CAR) therapeutic. We developed a novel CD19 CAR (CAT-41BBz CAR) with a fast off-rate, designed to result in more physiological T-cell activation, reduce toxicity and improve engraftment. We describe updated data from the Phase I ALLCAR19 (NCT02935257) study of AUTO1 in relapsed/refractory adult B-ALL. Methods: Manufacturing: AUTO1 utilises non-mobilised autologous leukapheresate. The first 6 products were generated using a standard dynabead/WAVE bioreactor process and subsequent products using a semi-automated closed process. Study design: Patients aged >16y underwent lymphodepletion with fludarabine (30mg/m2 x3) and cyclophosphamide (60mg/kg x1) followed by split dose CAR T-cell infusion (Day 0: if ≥20% Bone Marrow (BM) blasts, infuse 10 x 106 CAR T-cells; if Results: As of 13 May 2020, 24 patients have been leukapheresed, 23 products manufactured and 19 patients received at least 1 dose of AUTO1. The median age was 43y (range 18-62), 26% had prior blinatumomab, 47% had prior inotuzumab ozogamicin and 63% had prior hematopoietic stem cell transplantation (HSCT). At the time of pre-conditioning, 42% had ≥50% BM blasts. No patients experienced ≥Grade 3 CRS (Lee criteria), 3/19 (16%) experienced Grade 3 ICANS that swiftly resolved with steroids. Of 19 infused patients, 16/19 (84%) achieved Minimal Residual Disease (MRD) negative complete response (CR). Currently 6 patients have died, none related to AUTO1. 11/19 (58%) patients remain on study and continue in MRD negative remission at a median follow up of 12.2 months (range 0.6-24.4m). To date, only 2 patients underwent HSCT whilst in remission. For all treated patients, the event-free survival (EFS) at 6 months was 62% and 76% for those whose products were manufactured using the closed process. Patients exhibited robust CAR expansion (mean peak CAR T levels 716,769 copies/µg DNA). Conclusions: AUTO1 has a tolerable safety profile in adult patients with r/r B-ALL despite high disease burden. Early data shows high remission rates with 84% achieving MRD negative CR. This preliminary data supports the further development of AUTO1 as a standalone treatment in patients with r/r B-ALL. Data from additional patients and longer follow up will be presented. Furthermore, data from extension cohorts of patients with low- and high- grade B-cell Non-Hodgkin's lymphoma and chronic lymphocytic leukaemia will be presented. Figure Disclosures Roddie: Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria. O'Reilly:Gilead: Honoraria; Novartis: Honoraria, Other: Travel support. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Linch:Autolus: Consultancy. Pule:UCLB: Patents & Royalties; Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL; Autolus: Current Employment, Other: owns stock in and receives royalties, Patents & Royalties. Peggs:Autolus: Consultancy.

Summary This phase II trial was designed to determine the safety and efficacy of a modified paediatric risk‐stratified protocol in young adults (18–30 years) with classical Hodgkin Lymphoma. The primary end‐point was neurotoxicity rate. The incidence of grade 3 neurotoxicity was 11% (80% CI, 5–19%); a true rate of neuropathy of >15% cannot be excluded. Neuropathy and associated deterioration in quality of life was largely reversible. The overall response rate was 100% with 40% complete remission (CR) rate. Twelve months disease‐free survival (DFS) was 91%. We demonstrate that a risk‐stratified paediatric combined modality treatment approach can be delivered to young adults without significant irreversible neuropathy.

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.

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.

We performed a subgroup analysis of the phase III UK National Cancer Research Institute R-CHOP-14 versus R-CHOP-21 (two- versus three-weekly rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) trial to evaluate the outcomes for 50 patients with World Health Organization 2008 classified primary mediastinal B-cell lymphoma identified from the trial database. At a median follow-up of 7·2 years the 5-year progression-free survival and overall survival was 79·8% and 83·8%, respectively. An exploratory analysis raised the possibility of a better outcome in those who received R-CHOP-14 and time intensification may still, in the rituximab era, merit testing in a randomised trial in this subgroup of 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. KIThigh 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 KIThigh 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.

Diffuse large B-cell lymphoma with secondary involvement of the central nervous system (SCNS-DLBCL) is a rare condition carrying a poor prognosis. No optimal therapeutic regimen has been identified. We retrospectively analysed 23 patients with SCNS-DLBCL treated with R-IDARAM (rituximab 375 mg/m(2) IV day 1; methotrexate 12·5 mg by intrathecal injection day 1; idarubicin 10 mg/m(2) /day IV days 1 and 2; dexamethasone 100 mg/day IV infusion over 12 h days 1-3; cytosine arabinoside 1000 mg/m(2) /day IV over 1 h days 1 and 2; and methotrexate 2000 mg/m(2) IV over 2 h day 3. Ten out of 23 (44%) patients had CNS involvement at initial presentation ('new disease'), 10/23 (44%) had relapsed disease and 3/23 (13%) had primary refractory disease. 14/23 (61%) of patients responded - 6 (26%) complete response, 8 (35%) partial response. Grade 3-4 haematological toxicity was seen in all cycles, with no grade 3-4 or long-term neurological toxicity. Median follow-up for surviving patients was 49 months. At 2 years, estimated progression-free survival (PFS) was 39% and overall survival (OS) was 52%. Encouraging outcomes were reported in patients with new disease, with 5-year estimated PFS of 50% and OS 75%. R-IDARAM is a well-tolerated regimen with encouraging efficacy in patients with SCNS-DLBCL, although patients with relapsed or refractory disease continue to fare poorly.

Mantle cell lymphoma is an incurable and generally aggressive lymphoma that is more common in elderly patients. Whilst a number of different chemotherapeutic regimens are active in this disease, there is no established gold standard therapy. Rituximab has been used widely to good effect in B-cell malignancies but there is no evidence that it improves outcomes when added to chemotherapy in this disease. We performed a randomized, open-label, multicenter study looking at the addition of rituximab to the standard chemotherapy regimen of fludarabine and cyclophosphamide in patients with newly diagnosed mantle cell lymphoma. A total of 370 patients were randomized. With a median follow up of six years, rituximab improved the median progression-free survival from 14.9 to 29.8 months (P

Leukemia and lymphoma cell lines have been pivotal in the cytogenetic and molecular analysis of recurring chromosomal translocations, elucidating the pathogenesis of several hematological malignanc...

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.

Background Central nervous system (CNS) relapse of diffuse large B-cell lymphoma (DLBCL) is associated with a dismal prognosis. Here, we report an analysis of CNS relapse for patients treated within the UK NCRI phase III R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) 14 versus 21 randomised trial. Patients and methods The R-CHOP 14 versus 21 trial compared R-CHOP administered two- versus three weekly in previously untreated patients aged ≥18 years with bulky stage I–IV DLBCL (n = 1080). Details of CNS prophylaxis were retrospectively collected from participating sites. The incidence and risk factors for CNS relapse including application of the CNS-IPI were evaluated. Results 177/984 patients (18.0%) received prophylaxis (intrathecal (IT) methotrexate (MTX) n = 163, intravenous (IV) MTX n = 2, prophylaxis type unknown n = 11 and IT MTX and cytarabine n = 1). At a median follow-up of 6.5 years, 21 cases of CNS relapse (isolated n = 11, with systemic relapse n = 10) were observed, with a cumulative incidence of 1.9%. For patients selected to receive prophylaxis, the incidence was 2.8%. Relapses predominantly involved the brain parenchyma (81.0%) and isolated leptomeningeal involvement was rare (14.3%). Univariable analysis demonstrated the following risk factors for CNS relapse: performance status 2, elevated lactate dehydrogenase, IPI, >1 extranodal site of disease and presence of a ‘high-risk’ extranodal site. Due to the low number of events no factor remained significant in multivariate analysis. Application of the CNS-IPI revealed a high-risk group (4-6 risk factors) with a 2- and 5-year incidence of CNS relapse of 5.2% and 6.8%, respectively. Conclusion Despite very limited use of IV MTX as prophylaxis, the incidence of CNS relapse following R-CHOP was very low (1.9%) confirming the reduced incidence in the rituximab era. The CNS-IPI identified patients at highest risk for CNS recurrence. ClinicalTrials.gov ISCRTN number 16017947 (R-CHOP14v21); EudraCT number 2004-002197-34.

Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

A compact marker/suicide gene that utilizes established clinical-grade reagents and pharmaceuticals would be of considerable practical utility to T-cell cancer gene therapy. Marker genes enable measurement of transduction and allow selection of transduced cells, whereas suicide genes allow selective deletion of administered T cells in the face of toxicity. We have created a highly compact marker/suicide gene for T cells combining target epitopes from both CD34 and CD20 antigens (RQR8). This construct allows selection with the clinically approved CliniMACS CD34 system (Miltenyi). Further, the construct binds the widely used pharmaceutical antibody rituximab, resulting in selective deletion of transgene-expressing cells. We have tested the functionality of RQR8 in vitro and in vivo as well as in combination with T-cell engineering components. We predict that RQR8 will make T-cell gene therapy both safer and cheaper.

Some studies have suggested that cases of acute myeloid leukemia (AML) with low levels of FLT3 internal tandem duplications (FLT3(ITD)) do not have a worse prognosis if there is a concomitant NPM1 mutation, although this is controversial. To clarify this therapeutically important issue, we have analyzed FLT3(ITD) and NPM1(MUT) levels in 1609 younger adult cases of cytogenetically intermediate-risk AML. The cumulative incidence of relapse was increased in NPM1(MUT) cases by the presence of a FLT3(ITD), but did not differ markedly according to FLT3(ITD) level. This remained true when allowance was made for poor leukemic cell purity by adjustment of the FLT3(ITD) level to the measured NPM1(MUT) level. If consolidation therapies are to be determined by relapse risk, then NPM1(MUT) cases with low-level FLT3(ITD) should not be considered as good risk without further studies. AML 12 and AML 15 are registered at http://www.controlled-trials.com under ISRCTN17833622 and ISRCTN17161961, respectively.

Purpose The treatment of patients with diffuse large B-cell lymphoma (DLBCL) with cardiac comorbidity is problematic, because this group may not be able to receive anthracycline-containing chemoimmunotherapy. We designed a single-arm phase II multicenter trial of rituximab, gemcitabine, cyclophosphamide, vincristine, and prednisolone (R-GCVP) in patients considered unfit for anthracycline-containing chemoimmunotherapy because of cardiac comorbidity. Patients and Methods Sixty-one of 62 patients received R-GCVP, administered on day 1 with gemcitabine repeated on day 8 of a 21-day cycle. Median age was 76.5 years. All patients had advanced disease; 27 (43.5%) had left ventricular ejection fraction of ≤ 50%, and 35 (56.5%) had borderline ejection fraction of > 50% and comorbid cardiac risk factors such as ischemic heart disease, diabetes mellitus, or hypertension. Primary end point was overall response rate at the end of treatment. Results Thirty-eight patients (61.3%; 95% CI, 49.2 to 73.4) achieved disease response (complete response [CR], n = 18; undocumented/unconfirmed CR, n = 6; partial response, n = 14). Two-year progression-free survival for all patients was 49.8% (95% CI, 37.3 to 62.3), and 2-year overall survival was 55.8% (95% CI, 43.3 to 68.4). Thirty-four patients experienced grade ≥ 3 hematologic toxicity. There were 15 cardiac events, of which seven were grade 1 to 2, five were grade 3 to 4, and three were fatal, reflecting the poor cardiac status of the study population. Conclusion Our phase II multicenter trial showed that the R-GCVP regimen is an active, reasonably well-tolerated treatment for patients with DLBCL for whom anthracycline-containing immunochemotherapy was considered unsuitable because of coexisting cardiac disease.

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

Introduction: In adults, prognosis for B-ALL is poor, patients are more vulnerable to CD19 CAR immunotoxicity and there is currently no CD19 CAR therapeutic with acceptable toxicity and durable efficacy. We have developed a novel second generation CD19CAR (CAT-41BBz CAR), with a faster off-rate but equivalent on rate than the FMC63-41BBz CAR (Kd 116 nM vs 0.9 nM, T1/2 9s vs 4.2 hours) designed to result in more physiological T-cell activation, reduce toxicity and improve engraftment. Preliminary paediatric clinical data of this novel CD19 CAR (AUTO1) supports this assertion. We here describe preliminary data from ALLCAR19 (NCT02935257), a multi-centre, Phase I clinical study of AUTO1 as therapy for r/r adult B-ALL. Methods: Manufacturing: AUTO1 utilises non-mobilised autologous leucapheresate. The first 6 trial products were generated using a standard dynal bead/WAVE Bioreactor process and subsequent products using a semi-automated closed process. Study design: ALLCAR19 is a phase I/II study recruiting subjects 16-65y with r/r B ALL. Lymphodepletion with fludarabine (30mg/m2 x3) and cyclophosphamide (60mg/kg x1) is followed by split dose CAR T cell infusion (Day 0: if ≥20% BM blasts, infuse 10 x 106 CAR T cells ; if Results: As of 24 July 2019, 16 patients have been leukaphresed, 14 products manufactured (one failed leukaphresis and one currently in manufacture) and 13 patients have received at least 1 dose of AUTO1. Of the 16 patients, median age was 35.5 (range 18-63), 10/16 (63%) had prior blinatumomab or inotuzumab ozogamicin and 12/16 (75%) had prior HSCT. At the time of pre-conditioning, 9/13 (69%) patients were in morphological relapse with >5% leukemic blasts of which 6/13 (46%) had ≥50% blast. 9/13 patients (69%) received the total target split dose of 410 x 106 CAR T cells while 1/13 patients (8%) received a reduced split total dose of 51.3 x 106 CAR T cells due to manufacturing constraints. 3/13 patients (23%) received only a first dose of 10 x 106 CAR T cells. The dose was administered safely to date: No patients experienced ≥Grade 3 CRS (using Lee criteria) and only 1/13 (8%) experienced Grade 3 neurotoxicity (dysphasia) that resolved swiftly with steroids. All patients had robust CAR expansion (median peak expansion 172 CAR/uL blood). Of the 13 patients dosed (1/13 pending 28 day follow up), 10/12 (83%) achieved MRD negative CR at 1 month and all patients had ongoing CAR T cell persistence at last follow up. Two patients experienced CD19 negative relapse (one at M3, one at M6), 1 patient died on D17 before first response evaluation, 1 died in molecular CR from sepsis, and 1 died from persistent disease. Currently, 7/12 remain on study and continue in flow/molecular MRD negative remission with a median follow up of 9.0 months (range 1.2-14.8). Conclusions: AUTO1 delivers excellent early remission rates with initial data showing 83% MRD negative CR and robust CAR expansion and persistence. Despite high tumour burden, the safety profile compares favourably to other CD19 CARs, with no cases of severe CRS and only one case of Gr3 neurotoxicity. This is consistent with experience in the paediatric cohort. Updated results will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

ASXL1 mutations are recurrent in acute myeloid leukemia (AML), but it is unclear whether ASXL1 genotype might influence patient management. We analyzed frequency and impact in younger (15-59 years) and older (≥ 60 years) patients with primary or secondary disease. Overall, 9% had truncating mutations. Incidence was significantly lower in younger patients with primary than with secondary disease (4%, 12%; p = 0.03). In older patients it did not differ significantly (11%, 15%; p = 0.5). In univariate analysis, ASXL1-mutated patients had a worse outcome (5-year relapse 83% vs. 56%, p = 0.01; overall survival [OS] 6% vs. 22%, p = 0.02). However in multivariate analysis, ASXL1 mutations had no prognostic significance (for OS, p = 0.3), because age was a major confounding factor. The low incidence of mutations in younger patients with primary disease and the lack of significance in multivariate analysis indicate that there is a limited role for screening at diagnosis for ASXL1 mutations for the purpose of prognostic stratification.

Background: Women treated with supradiaphragmatic radiotherapy (sRT) for Hodgkin lymphoma (HL) at young ages have a substantially increased breast cancer risk. Little is known about how menarcheal and reproductive factors modify this risk. Methods: We examined the effects of menarcheal age, pregnancy, and menopausal age on breast cancer risk following sRT in case–control data from questionnaires completed by 2497 women from a cohort of 5002 treated with sRT for HL at ages

Non-Hodgkin's lymphoma (NHL) is the fifth most common cancer in the UK, and represents a heterogeneous group of malignancies. This article will give an overview of lymphoid neoplasms, concentrating on the common subtypes of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Clinical outcomes in B-cell NHL have significantly improved in the past decade, largely due to the availability of the monoclonal antibody, rituximab. However, some aggressive lymphomas, including many T-cell disorders, still carry a poor prognosis.

GATA2 mutations have recently been reported in acute myeloid leukaemia (AML) patients with CEBPA-double mutations. To explore their impact on this favourablerisk disease, we determined GATA2 status in 153 sporadic AML patients and three members of a germ-line CEBPA-mutant family at AML presentation. Overall, 27% (15/55) CEBPA-double, 16% (7/43) CEBPA-single and 0% (0/55) normal karyotype/ CEBPA-wild-type patients were GATA2-mutant. All familial AML patients acquired both a second CEBPA and a GATA2 mutation. CEBPA and GATA2 mutant levels indicated that both mutations were likely to be early events in leukaemogenesis. GATA2 status did not impact on the favourable outcome of CEBPAdouble/ FLT3-inernal tandem duplication-negative patients.

Objectives Elderly patients with diffuse large B cell lymphoma (DLBCL) without prohibitive co-morbidities may be cured with standard immuno-chemotherapeutic regimens, as used in younger patients. Less is known about the survival prospects in older people, if first-line therapy fails. This study aimed to provide additional information regarding prognosis in this group. Materials and Methods Databases were collated from three randomized trials of first-line therapy in those aged 60 and over, deemed fit enough for standard therapy. Overall survival from the point of treatment failure was calculated and comparisons were made between age groups and types of treatment failure. Results Overall survival (OS) at 2 years in 862 patients was 46%, 38%, 37% and 23%, respectively, for those aged 60–64, 65–69, 70–74 and > 74. Type of treatment failure impacted on 2 year OS as follows: initial partial remission (PR): 48%; complete response (CR) with late relapse: 37%; CR with early relapse: 17%; and less than PR to initial therapy: 12%. Conclusion Older patients failing first-line therapy for DLBCL should be counseled differently regarding prognosis depending upon age and type of treatment failure. The chance of survival was greater in those achieving PR or CR with relapse more than 12 months from diagnosis. This data may support the consideration of aggressive salvage therapy in fit patients in these categories, regardless of biological age per se. Palliative management may be more appropriate for those achieving less than PR to initial therapy or who enter CR but relapse within one year of diagnosis.

PURPOSE: The primary aim was to compare the diagnostic performance of PET/MRI (performed with basic anatomical MRI sequences) in detecting sites of disease in adult patients with lymphoma compared with the current standard of care, PET/CT. Secondary aims were to assess the additional value of diffusion-weighted imaging to PET/MRI in disease detection and to evaluate the relationship between the standardized uptake value on PET/MR and the apparent diffusion coefficient on diffusion-weighted imaging. METHODS: Sixty-eight studies in 66 consecutive patients with histologically proven Hodgkin or non-Hodgkin lymphoma were prospectively evaluated. Each patient had whole body PET/CT, followed by whole body PET/MR. Two experienced readers independently evaluated the PET/MRI studies, and two other experienced readers independently evaluated PET/CT. Site of lymphoma involvement and SUVmax at all nodal sites more avid than background liver were recorded. Readers provided stage (in baseline cases) and disease status (remission vs active disease). The apparent diffusion coefficient mean value corresponding to the most avid PET site of disease was recorded. RESULTS: Ninety-five nodal and 8 extranodal sites were identified on both PET/CT and PET/MRI. In addition, 3 nodal and 1 extranodal sites were identified on PET/MRI. For positive lesion detection, reader agreement in PET/MR was perfect between the 2 readers and almost perfect between PET/CT and PET/MR (k > 0.978). Intermodality agreement between PET/CT and PET/MRI was also near perfect to perfect for staging/disease status k = (0.979–1.000). SUVmax from PET/CT and PET/MRI correlated significantly (Spearman rho correlation coefficient, 0.842; P < 0.001). Diffusion-weighted imaging did not alter lesion detection or staging in any case. A negative correlation was demonstrated between ADC mean and SUVmax (Spearman rho correlation coefficient r, -0.642; P < 0.001). CONCLUSIONS: PET/MRI is a reliable alternative to PET/CT in the evaluation of patients with lymphoma. Diffusion-weighted imaging did not alter diagnostic accuracy. With comparable accuracy in detection of disease sites and added benefit of radiation dose reduction, PET/MRI has a potential to become part of routine lymphoma imaging.