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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.

The impact of human leukocyte antigen (HLA) mismatch after reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (RIT) using unrelated donors (UD) is unclear, and may be modulated by T-cell depletion. We therefore examined outcomes of 157 consecutive patients undergoing RIT after uniform conditioning with fludarabine, melphalan, and alemtuzumab (FMC). Donors were 10/10 HLA-matched (MUDs, n = 107) and 6 to 9/10 HLA-matched (MMUDs, n = 50), with no significant differences in baseline characteristics other than increased cytomegalovirus seropositivity in MMUDs. Rates of durable engraftment were high. Graft failure rates (persistent cytopenias with donor chimerism) were similar (8% vs 3%, P = .21), though rejection (recipient chimerism) was more frequent in MMUDs (8% vs 0%, P < .01). There were no significant differences between donors in the incidences of acute graft-versus-host disease (GVHD; 20% vs 22% grade 2-4, respectively, P = .83), chronic extensive GVHD (3-year cumulative incidence [CI] 23% vs 24%, P = .56), or treatment-related mortality (1-year CI 27% vs 27%, P = .96). Furthermore, there was no difference in 3-year overall survival (OS; 53% vs 49%, P = .44). Mismatch occurred at the antigenic level in 40 cases. The outcome in these cases did not differ significantly from the rest of the cohort. We conclude that RIT using HLA-mismatched grafts is a viable option using FMC conditioning.

Involved field (IF) radiation was compared with extended field (EF) radiation in Hodgkin lymphoma (HL) to ascertain whether reduced radiation fields would reduce the late sequelae of radiation without compromising disease control and survival. A total of 603 patients with stage I or II HL were entered into this trial; laparotomy was carried out in 380 (63%) patients. Stage I or IIA disease patients were randomised to receive IF or EF comprising a mantle or inverted Y fields alone. Stage I and IIB patients were randomised between mantle or inverted Y fields and total nodal irradiation (TNI). The dose was 35Gy to uninvolved sites and 40Gy to involved sites. The median follow-up of surviving patients was 25.2 years with only 3.3% lost to follow-up. The treatment failure rate at 25 years in stage IA and IIA was 44% after EF and 54% after IF ( P =0.01); in stage I and IIB this was 80% (EF) and 82% (TNI) at 25 years. No difference in overall survival between the randomised groups was seen. The incidence of second malignancies was 21% after IF and 20% after EF with a slight excess of lung cancer in the EF group. No significant differences in the causes of death between the randomised arms have emerged. In conclusion, IF radiotherapy for stage I and IIA HL results in a 11% greater risk of relapse compared with EF but has no effect on overall survival, risk of second malignancy or cause of death at 25 years.

Six patients with severe combined immunodeficiency were transplanted with bone marrow from their HLA haploidentical parents. T-lymphocytes were removed by complement mediated lysis with a monoclonal antibody (CAMPATH-I) followed by rosetting with sheep erythrocytes. The patients were pre-conditioned with marrow ablative chemotherapy, using busulphan and cyclophosphamide. There was graft take in five patients, with evidence of transient acute graft-versus-host disease only. Three patients are alive and well greater than 5 months after transplant, and one who is 12 months post graft has full reconstitution of cell mediated and humoral immunity, including specific antibody response to immunization. Transplantation across major histocompatibility barriers can be successfully achieved using this method of treatment of donor marrow.

Expression of receptors for sheep red blood cells and the ability to proliferate in response to phytohaemagglutinin (PHA) are the traditional properties of human T cells, but the function of the sheep red cell receptor (the T11 antigen) is controversial and the mechanism of PHA-induced mitogenesis unclear. Mitogenesis involves a complex series of cell-mediated and factor-dependent interactions, but a rise in intracellular free calcium concentration, [Ca2+]i, seems to be an important primary event in T-cell activation. We have now investigated the effects of three monoclonal antibodies, previously shown to inhibit mitogen-induced proliferation, on T-cell [Ca2+]i. We find that anti-LFA-2 and OKT11, which react with the sheep red cell receptor, have no effect on [Ca2+]i, nor do they inhibit the rise in [Ca2+]i induced by concanavalin A (Con A) or the mitogenic anti-T3 monoclonal antibody UCHT1 (ref. 11). They do, however, block PHA-induced Ca2+ mobilization. Anti-LFA-1, which reacts with the lymphocyte function-associated antigen, has no effect on intracellular Ca2+. These studies suggest that the sheep red blood cell receptor is an activation pathway for T cells and that the effects of PHA are mediated through this pathway.

There is a long-standing interest in the possible role of mitochondria in malignancy. We sought to discover whether amplification of mitochondrial DNA (mtDNA) occurred in leukaemia, and found it was often remarkably amplified in the blast cells of acute myeloid leukaemia (AML). We used gene dosage experiments to quantify the amount of mtDNA relative to nuclear DNA. DNA extracted from peripheral blood leucocytes or bone marrow of healthy individuals or patients was simultaneously hybridized with a probe for the mitochondrial genome and a control probe for the renin gene on human chromosome 1. Comparative densitometric ratios of approximately 1 were obtained between the two signals in 20 normal control peripheral blood samples. In contrast, comparative ratios in the range of 2-50 were observed in 25 AML samples and 13 of these showed 8-fold or greater amplification of mtDNA relative to normal peripheral blood controls. An additional four cases of AML were investigated at both presentation and remission and showed 3-10-fold amplification of mtDNA at presentation, but no amplification when in clinical remission. 18 cases of chronic granulocytic leukaemia (CGL) were also studied in chronic phase and showed mtDNA dosage levels equivalent to normal peripheral blood controls. However, 8/9 CGL patients showed mtDNA amplification during transformation from chronic phase. We conclude that amplification of mtDNA is an invariable feature of acute myeloid leukaemia and that it may be a useful marker for detecting transformation of CGL.

Myelodysplastic syndromes (MDSs) are a group of hematopoietic disorders affecting the myeloid lineage, characterized by cytopenias and clonal evolution to acute myeloid leukemia (AML). We hypothesized that natural killer (NK) cells and their activating killer immunoglobulin-like receptors (aKIRs) influence the immune surveillance and clinical outcome of patients with MDSs. Here, we first examined the distribution of aKIR genes and haplotype in 2 independent cohorts of MDS and AML patients. The median number of aKIR genes was lower in MDS patients than healthy controls (2 vs 3 genes; P = .001), and lower in patients with secondary AML (progressed from MDSs) compared with de novo AML patients (2 vs 3; P = .008) and healthy controls (2 vs 3; P = .006). In a multivariate analysis, the presence of KIR haplotype A (characterized by low aKIR content 0-1) independently predicted a higher risk of conversion to AML (relative risk [RR] with 95% confidence interval [CI], 2.67 [1.13-6.71]; P = .02) and worse adjusted progression-free survival (RR with 95% CI, 2.96 [1.59-5.52]; P = .001) and overall survival (2.25 [1.17-4.31]; P = .02), compared with KIR haplotype B (multiple aKIR genes). These novel findings may help to identify MDS patients with a high risk of disease progression who would likely benefit from adoptive NK-cell therapy.

Acute myeloid leukaemia (AML) is a disorder characterized by a clonal proliferation derived from primitive haematopoietic stem cells or progenitor cells. Abnormal differentiation of myeloid cells results in a high level of immature malignant cells and fewer differentiated red blood cells, platelets and white blood cells. The disease occurs at all ages, but predominantly occurs in older people (>60 years of age). AML typically presents with a rapid onset of symptoms that are attributable to bone marrow failure and may be fatal within weeks or months when left untreated. The genomic landscape of AML has been determined and genetic instability is infrequent with a relatively small number of driver mutations. Mutations in genes involved in epigenetic regulation are common and are early events in leukaemogenesis. The subclassification of AML has been dependent on the morphology and cytogenetics of blood and bone marrow cells, but specific mutational analysis is now being incorporated. Improvements in treatment in younger patients over the past 35 years has largely been due to dose escalation and better supportive care. Allogeneic haematopoietic stem cell transplantation may be used to consolidate remission in those patients who are deemed to be at high risk of relapse. A plethora of new agents - including those targeted at specific biochemical pathways and immunotherapeutic approaches - are now in trial based on improved understanding of disease pathophysiology. These advances provide good grounds for optimism, although mortality remains high especially in older patients.

Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission.We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction.Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status.The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.).

Purpose To investigate breast cancer risk after supradiaphragmatic radiotherapy administered to young women with Hodgkin's lymphoma (HL) in a much larger cohort than previously to provide data for patient follow-up and screening individualized according to treatment type, age, and time point during follow-up. Patients and Methods Breast cancer risk was assessed in 5,002 women in England and Wales treated for HL with supradiaphragmatic radiotherapy at age < 36 years from 1956 to 2003, who underwent follow-up with 97% completeness until December 31, 2008. Results Breast cancer or ductal carcinoma in situ developed in 373 patients, with a standardized incidence ratio (SIR) of 5.0 (95% CI, 4.5 to 5.5). SIRs were greatest for those treated at age 14 years (47.2; 95% CI, 28.0 to 79.8) and continued to remain high for at least 40 years. The maximum absolute excess risk was at attained ages 50 to 59 years. Alkylating chemotherapy or pelvic radiotherapy diminished the risk, but only for women treated at age ≥ 20 years, not for those treated when younger. Cumulative risks were tabulated in detail; for 40-year follow-up, the risk for patients receiving ≥ 40 Gy mantle radiotherapy at young ages was 48%. Conclusion This article provides individualized risk estimates based on large numbers for patients with HL undergoing follow-up after radiotherapy at young ages. Follow-up of such women needs to continue for 40 years or longer and may require more-intensive screening regimens than those in national general population programs. Special consideration is needed of potential measures to reduce breast cancer risk for girls treated with supradiaphragmatic radiotherapy at pubertal ages.

In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short-term homing, leading to loss of cells in non-haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24 h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N-cadherin, osteopontin and vascular cell-adhesion molecule-1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo.

Activating mutations in the NOTCH1 pathway are frequent in pediatric T-cell acute lymphoblastic leukemia (T-ALL) but their role in refining risk stratification is unclear. We screened 162 pediatric T-ALL patients treated on the MRC UKALL2003 trial for NOTCH1/FBXW7 gene mutations and related genotype to response to therapy and long-term outcome. Overall, 35% were wild-type (WT) for both genes (NOTCH1(WT)FBXW7(WT)), 38% single NOTCH1 mutant (NOTCH1(Single)FBXW7(WT)), 3% just FBXW7 mutant (NOTCH1(WT)FBXW7(MUT)) and 24% either double NOTCH1 mutant (NOTCH1(Double)FBXW7(WT)) or mutant in both genes (NOTCH1(MUT)FBXW7(MUT)), hereafter called as NOTCH1±FBXW7(Double). There was no difference between groups in early response to therapy, but NOTCH1±FBXW7(Double) patients were more likely to be associated with negative minimal residual disease (MRD) post-induction than NOTCH1(WT)FBXW7(WT) patients (71% versus 40%, P=0.004). Outcome improved according to the number of mutations, overall survival at 5 years 82%, 88% and 100% for NOTCH1(WT)FBXW7(WT), NOTCH1(Single)FBXW7(WT) and NOTCH1±FBXW7(Double) patients, respectively (log-rank P for trend=0.005). Although 14 NOTCH1±FBXW7(Double) patients were classified as high risk (slow response and/or MRD positive), only two had disease progression and all remain alive. Patients with double NOTCH1 and/or FBXW7 mutations have a very good outcome and should not be considered for more intensive therapy in first remission, even if slow early responders or MRD positive after induction therapy.

The diagnosis of Burkitt Lymphoma (BL) and B-cell lymphomas unclassifiable with features intermediate between Diffuse Large B-cell Lymphoma and BL (BLU) in adults remains problematic even with immunophenotyping and MYC gene analysis. Gene expression profiling may improve categorization but is not routinely available. BL and its variants should be treated with specific regimens incorporating intensive courses of chemotherapy with fractionated alkylating agents and cell cycle phase-specific agents that readily cross the blood brain barrier. Subsequent courses should be given as soon as haematological recovery occurs, with the whole course completed within a few months. A number of regimens have been developed that encompass these principles but there have been no comparative randomized trials. The results from several studies suggest that the addition of rituximab is highly efficacious and this may be particularly valuable in older patients. It is usual to employ 'risk-adapted' strategies in the treatment of BL but these must be continually re-evaluated, and 'response-adapted' approaches should be explored. The role of transplantation is limited and largely confined to autologous transplants in patients who only achieve a partial response on front-line therapy or who have a chemosensitive relapse. Further advances will be greatly facilitated by randomized trials, which will require international collaboration.

We have investigated the prognostic significance of isocitrate dehydrogenase 2 (IDH2) mutations in 1473 younger adult acute myeloid leukemia patients treated in 2 United Kingdom Medical Research Council trials. An IDH2 mutation was present in 148 cases (10%), 80% at R140 and 20% at R172. Patient characteristics and outcome differed markedly between the 2 mutations. IDH2R140 significantly correlated with nucleophosmin mutations (NPM1MUT), whereas IDH2R172 cases generally lacked other molecular mutations. An IDH2R140 mutation was an independent favorable prognostic factor for relapse (P = .004) and overall survival (P = .008), and there was no significant heterogeneity with regard to NPM1 or FLT3 internal tandem duplication (FLT3/ITD) genotype. Relapse in FLT3/ITDWTNPM1MUTIDH2R140 patients was lower than in favorable-risk cytogenetics patients in the same cohort (20% and 38% at 5 years, respectively). The presence of an IDH2R172 mutation was associated with a significantly worse outcome than IDH2R140, and relapse in FLT3/ITDWTNPM1WTIDH2R172 patients was comparable with adverse-risk cytogenetics patients (76% and 72%, respectively).

PURPOSE: This multicentre, prospective, randomised-controlled trial compared efficacy and toxicity of differing radiotherapy doses in non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: Patients with any histological subtype of NHL, requiring radiotherapy for local disease control, whether radical, consolidative or palliative, were included. Three hundred and sixty one sites of indolent NHL (predominantly follicular NHL and marginal zone lymphoma) were randomised to receive 40-45Gy in 20-23 fractions or 24Gy in 12 fractions. Six hundred and forty sites of aggressive NHL (predominantly diffuse large B cell lymphoma as part of combined-modality therapy) were randomised to receive 40-45Gy in 20-23 fractions or 30Gy in 15 fractions. Patients with all stages of disease, having first-line and subsequent therapies were included; first presentations of early-stage disease predominated. RESULTS: There was no difference in overall response rate (ORR) between standard and lower-dose arms. In the indolent group, ORR was 93% and 92%, respectively, (p=0.72); in the aggressive group, ORR was 91% in both arms (p=0.87). With a median follow-up of 5.6years, there was no significant difference detected in the rate of within-radiation field progression (HR=1.09, 95%CI=0.76-1.56, p=0.64 in the indolent group; HR=0.98, 95%CI=0.68-1.4, p=0.89 in the aggressive group). There was also no significant difference detected in the progression free or overall survival. There was a trend for reduced toxicities in the low-dose arms; only the reduction in reported erythema reached significance. CONCLUSION: In a large, randomised trial, there was no loss of efficacy associated with radiotherapy doses of 24Gy in indolent NHL and 30Gy in aggressive NHL, compared with previous standard doses of 40-45Gy.

Purpose Reduced-intensity conditioning has minimized nonrelapse-related mortality rates after allogeneic transplantation in patients with Hodgkin's lymphoma, and relapse has now become the major cause for treatment failure. We aimed to assess the impact of donor lymphocyte infusions (DLIs) on relapse incidence when administered for mixed chimerism and their utility as salvage therapy when given for relapse. Patients and Methods This study reports the outcomes of 76 consecutive patients with multiply relapsed or refractory Hodgkin's lymphoma who underwent allogeneic transplantation that incorporated in vivo T-cell depletion. Forty-two patients had related donors and 34 had unrelated donors. DLIs were administered in a dose-escalating fashion to 22 patients for mixed chimerism (median time of first dose, 9 months post-transplantation) and to 24 patients for relapse. Results Three-year donor lymphocyte–related mortality was 7%, relating mainly to the induction of graft-versus-host disease. Nineteen (86%) of 22 patients receiving donor lymphocytes for mixed chimerism converted to full donor status. Four-year relapse incidence was 5% in these 22 patients compared with 43% in patients who remained relapse free but full donor chimeras at 9 months post-transplantation (P = .0071). Nineteen (79%) of 24 patients receiving donor lymphocytes for relapse responded (14 complete responses, five partial responses). Four-year overall survival from relapse was 59% in recipients of donor lymphocytes, contributing to a 4-year overall survival from transplantation of 64% and a 4-year current progression-free survival of 59% in all 76 patients. Conclusion These data demonstrate the potential for allogeneic immunotherapy with donor lymphocytes both to reduce relapse risk and to induce durable antitumor responses in patients with Hodgkin's lymphoma after hematopoietic stem-cell transplantation that incorporates in vivo T-cell depletion.

Introduction: Patients with Acute Myeloid Leukaemia (AML) who harbour a FLT3-ITD mutation have a worse prognosis characterised by increased early relapse. Outcomes following relapse in such patients are typically poor, not only because of earlier relapse, but also because of worse performance post-relapse than those who have the same remission duration but are FLT3-ITD WT. In a single centre retrospective study among relapsed patients, Ravandi et al [1] found a remission rate of 24% and median survival of only 13 weeks. There are therefore twin challenges in this population: first to reduce the early relapse rate, and also to develop more effective treatments post relapse. In the recent QUANTUM-R trial [2] for patients with relapsed or refractory disease, single agent quizartinib (AC220) was found to significantly improve median survival from 20.4 weeks to 27 weeks when compared to "doctor's choice" treatment (low-dose ara-C, MEC or FLAG-Ida). There is however, no directly comparable data for the population in the QUANTUM-R trial. To contextualise these results, especially given the potentially different outcomes by control treatment, we looked at outcomes in the UK NCRI AML15,16,17 trials in patients satisfying the eligibility criteria of QUANTUM-R. Methods: Patients aged 18+ in the UK NCRI AML15,16,17 trials were identified who harboured a FLT3 ITD mutation, were treated with intensive chemotherapy, and were either refractory to two courses of induction therapy, or relapsed within six months of transplant, or did not receive a prior transplant and had a remission duration of 6 months or less. Patients were grouped hierarchically as refractory, relapsed post transplant, or relapsed without prior transplant. Eligibility was established at the point a patient first became eligible for analysis. The primary outcome was overall survival (OS), measured from point of eligibility, with subsequent remission with or without count recovery as secondary outcome. A sensitivity analysis was performed excluding those who died within 21 days of eligibility to eliminate patients who might be thought of as too unwell to enter a post-relapse trial. Cox regression was used to identify prognostic factors for survival. Results: A total of 264 patients were identified (refractory n=58, relapsed post SCT n=49, relapsed without SCT n=157). The median age was 51 (range 18-84); 25% of patients were aged 60 or older; 44% were male, 95% had intermediate cytogenetics; 11% had secondary disease. Split by age, among those under 60 45 were refractory, 44 relapsed post SCT and 110 relapsed without SCT; for ages 60+ the figures were 13 vs 5 vs 47. Overall 17% of patients experienced a subsequent remission; with median survival of 86 days and 1 year OS of 13%. If deaths within 21 days were excluded, the remission rate improved to 21%; with a median survival of 133 days and 1 year OS of 16%. In multivariable Cox regression, age group HR for age>60 1.81 (1.33-2.47) p=0.0002) and route to eligibility (HR refractory vs relapsed no SCT 0.77 (0.55-1.07); relapsed post SCT vs no SCT 1.58 (1.11-2.25) p=0.003) were the only factors affecting survival - in particular sex, secondary disease, and ITD allelic burden were not significant. In the sensitivity analyses, only age was significant (HR 1.77 (1.24-2.53) p=0.001); with route to eligibility not significant (p=0.14). Among patients with post-relapse treatment information, 65% were treated intensively, 8% non-intensively, and 20% with palliation - other patients received experimental therapies. When restricting attention to those treated intensively, median survival was 130 days with 17% 1 year OS. Figures were not materially changed if early death was excluded. Of 215 patients who had not relapsed post transplant, 53 (25%) received a transplant post-eligibility. In these 56 patients, median survival was 301 days with 42% alive at one year. Conclusions: In relapsed/refractory AML, outcomes for FLT3-ITD mutated patients are generally poor and worse for older patients. Applying the eligibility criteria of QUANTUM-R and excluding early deaths gives outcomes comparable to the control group of the QUANTUM-R study. In the 25% of patients who proceeded to transplant survival was extended indicating that a treatment which can deliver patients to transplant has the potential to improve patient outcomes. References: [1] Ravandi F, et al. Leuk Res. 2010;34(6):752-756; [2] Cortes et al. EHA 2018, Abstr LB2600. Table. Table. Disclosures Hills: Daiichi Sankyo: Consultancy, Honoraria. Russell:Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Speakers Bureau.

Introduction: The increasing delineation of acute myeloid leukemia (AML) has identified a number of genetic mutations which may be amenable to targeted therapies. However, such mutations typically only occur in a minority of patients, and this relative paucity presents challenges in drug development. Even for more common mutations such as FLT3 ITD, randomised trials can take many years to complete, and there is the issue of how to deal with patients who are tested but not eligible. Earlier phase trials therefore tend to be single arm studies, and often recruit in the relapsed/refractory population, where eligibility is known up front, and it is possible to obtain an early read out for efficacy. Such is the case for the recent evaluations of enasidenib and ivosidenib in IDH1/2 mutated patients. However, with single-arm studies there a need to contextualise results. We therefore looked at outcomes from the United Kingdom NCRI trials of AML for patients with IDH1/IDH2 mutations who were relapsed or refractory to therapy. Methods: A database search identified patients within the UK NCRI AML trials with an IDH1/IDH2 mutation, who had received intensive induction and who were either: in second relapse, relapsed post-transplant, refractory to two courses of induction, or who relapsed within 1 year of remission. Outcomes were measured from the point of eligibility: patients who were multiply eligible were included only once, at their first point of eligibility. The primary outcome was overall survival, with achievement of complete remission, with or without peripheral count recovery, as secondary outcome. Cox regression analysis was used to identify prognostic factors within the cohort of patients. Cytogenetics are evaluated using the MRC classification. Results: A total of 757 patients were identified with IDH1/2 mutation (IDH1 alone n=247; IDH2 alone n=504, both n=6). Of these 211 patients satisfied the relapsed/refractory criteria (IDH1 alone n=81; IDH2 alone n=128; both IDH1/2 n=2; refractory n=28; relapsed post SCT n=34; relapsed within 1 year with no SCT n=138; second relapse n=11 - Table). Median age was 54 years (range 22-77); 51% were male; and 95% of patients had intermediate risk cytogenetics. Remissions were achieved in 43/211 patients (20%; refractory 50%; relapsed post SCT 15%; relapsed within 1 year 17%; second relapse 9% - Table). Patients with IDH1 mutations had a remission rate of 23%; for IDH2 mutated patients, the rate was 18%. Median survival was 4.4 months for IDH1 mutated patients, and 6.6 months for IDH2 mutations; 2 year survival was 17%, 21% respectively. Split by age, median survival was 4.0 and 9.4 months respectively (2-year survival 19%; 27%) for patients aged Conclusions: These results give context to the recent findings in single arm studies of ivosidenib for relapsed/refractory IDH1 mutated patients, and enasidenib for patients harbouring an IDH2 mutation. In the two studies reported, median survival was respectively 8.8 and 9.3 months, compared to 4.4 and 6.6 months in a younger group of patients identified from the UK NCRI AML trials treated with a variety of therapies. In both monotherapy trials the median survival was extended: however, reported one-year survival was not greatly improved (enasidenib 1 year survival 39% vs 34% for the NCRI cohort; ivosidenib, approximately 35% vs 32%). The difference in survival for IDH2 mutated patients in the NCRI cohort, by age and route to eligibility indicates that the interpretation of the results of single arm studies, in a heterogeneous condition such as AML, is fraught with difficulties. Ideally the magnitude of benefit should be assessed using randomised data from large scale collaborations and platform trials. Table: Outcomes for IDH1/IDH2 mutated relapsed/refractory patients in the UK NCRI AML trials. Disclosures Hills: Daiichi Sankyo: Consultancy, Honoraria. Russell:Daiichi Sankyo: Consultancy; Jazz Pharma: Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau.

Background: The International Prognostic Index (IPI) was first described in 1993 and is the most widely used prognostication tool in diffuse large B-cell lymphoma (DLBCL). Five independent risk factors for OS: age (≤60 yrs versus >60 yrs), stage (I/II versus III/IV), number of extranodal (EN) sites (≤1 versus >1), performance status (PS) (0-1 versus ≥2) and serum lactate dehydrogenase (LDH) (normal versus elevated) were used to design a prognostic model which stratified patients into 4 prognostic groups according to the number of risk factors present: low (0-1), low-intermediate (2), high-intermediate (3), and high-risk (4-5); predicting 5-yr OS rates of 73%, 51%, 43% and 26% and 5-yr relapse-free survival (RFS) rates of 70%, 50%, 49%, 40% respectively. For patients aged ≤60 yrs 3 risk factors (stage, PS and LDH level) remained independently significant for OS, and an age-adjusted (aa) model (the aa-IPI) was proposed which predicted 5-yr OS for the low (0), low-intermediate (1), high-intermediate (2) or high-risk (3) groups of 83%, 69%, 46% and 32% respectively (Shipp et al, 1993). The British Columbia Cancer Agency re-evaluated the role of IPI in the rituximab era in their registry-based cohort of 365 patients in 2007. Although the IPI remained predictive, only 2 (rather than 4) prognostic groups were identified and the authors recommended use of the Revised-IPI (R-IPI) where redistribution of the absolute number of IPI risk factors better stratified patients into 3 prognostic groups: very good (0) good (1,2) and poor (3-5) (Savage et al, 2007). However, a subsequent analysis of pooled data from 3 prospective trials (Ziepert et al, 2010) confirmed that the IPI remained highly predictive of outcomes in the rituximab era. Here we report an evaluation of prognostic indices within the UK NCRI R-CHOP 14 vs 21 prospective trial cohort (Cunningham et al, 2013). Methods: The IPI and R-IPI were applied to the R-CHOP 14 versus 21 trial cohort (n=1,080) and for the subgroup of patients aged ≤60 yrs (n=515) and correlated with OS and progression-free survival (PFS). For patients aged ≤60 yrs the aa-IPI was applied in addition. It was not possible to evaluate the NCCN-IPI in our cohort as absolute LDH values were not collected at enrolment. The association between baseline clinical factors (age, gender, PS, stage, presence of >1 EN site of disease, presence of an elevated LDH, disease bulk and B symptoms) and patient outcomes were investigated in univariable and multivariable analysis (MVA). Performance of the prognostic indices was compared using the Concordance Probability Estimate (CPE) and Akaike's Information Criterion (AIC): CPE evaluates discriminatory power to assess the strength of statistical models (higher values indicate better discrimination); AIC estimates the quality of statistical models relative to each other in terms of fitting the data (lower values indicate a better model fit). Results: After a median follow-up of 6.5 years, both the IPI and R-IPI were significantly associated with OS (Figure 1) and PFS; the IPI performed better than the R-IPI for OS and PFS in terms of discrimination and model fit (Table 1). All IPI factors were significantly associated with OS, and remained in MVA with the exception of disease stage. For patients aged ≤60 yrs, the IPI, R-IPI and aa-IPI were all strongly associated with OS and PFS; the IPI performed best overall of the 3 prognostic indices in terms of discrimination and model fit (Table 2). All individual IPI risk factors, excepting stage, were again found to be significantly associated with OS in MVA for patients aged ≤60 yrs. Conclusion: For the entire DLBCL cohort both the IPI and R-IPI identified meaningful prognostic groups for OS and PFS. Although the IPI outperformed the R-IPI, neither index identified a patient subgroup with an OS of Disclosures Cunningham: Roche pharmaceuticals: Research Funding. Hawkes:Bristol Myers Squibb: Other: Speaker fee, Research Funding; Merck KGA: Research Funding; Astra Zeneca: Research Funding; Merck Sharpe Dohme: Research Funding; Celgene: Other: Advisory board, Research Funding; Takeda: Other: Speaker fee; Merck: Other: Advisory board; Roche: Other: Speaker fee; advisory board. Pocock:Kent & Canterbury Hospital: Employment. Ardeshna:Celgene: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Conference expenses, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Radford:Celgene: Research Funding; Takeda: Consultancy, Research Funding, Speakers Bureau; ADC Therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Speakers Bureau; GlaxoSmithKline: Equity Ownership; Novartis: Consultancy, Speakers Bureau; AstraZeneca: Equity Ownership; BMS: Consultancy, Speakers Bureau; Pfizer: Research Funding. McMillan:Celgene: Honoraria, Other: travel support; BMS: Honoraria; Amgen: Honoraria; Takeda: Other: travel support; Roche: Consultancy, Honoraria, Other: travel support; Pfizer: Research Funding; MSD: Honoraria. Johnson:Janssen: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria, Research Funding; Boeringher Ingelheim: Consultancy; Kite: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria, Travel, accommodations, expenses; Novartis: Honoraria; Celgene: Honoraria; Eisai: Research Funding; Zenyaku Kogyo: Other: Travel, accommodations, expenses; Genmab: Consultancy.

Mutations in the isocitrate dehydrogenase gene (IDH1) were recently described in patients with acute myeloid leukemia (AML). To investigate their prognostic significance we determined IDH1 status in 1333 young adult patients, excluding acute promyelocytic leukemia, treated in the United Kingdom MRC AML10 and 12 trials. A mutation was detected in 107 patients (8%). Most IDH1+ patients (91%) had intermediate-risk cytogenetics. Mutations correlated significantly with an NPM1 mutation (P < .0001) but not a FLT3/ITD (P = .9). No difference in outcome between IDH1+ and IDH1− patients was found in univariate or multivariate analysis, or if the results were stratified by NPM1 mutation status. However, when stratified by FLT3/ITD status, an IDH1 mutation was an independent adverse factor for relapse in FLT3/ITD− patients (P = .008) and a favorable factor in FLT3/ITD+ patients (P = .02). These results suggest that metabolic changes induced by an IDH1 mutation may influence chemoresistance in a manner that is context-dependent.

Purpose To determine the clinical relevance of mutations in the CCAAT/enhancer binding protein α (CEBPA) gene in acute myeloid leukemia (AML) and to examine factors that might modify prognostic impact. Patients and Methods The entire CEBPA coding sequence was screened in 1,427 young adult patients with AML, excluding acute promyelocytic leukemia, using denaturing high-performance liquid chromatography and direct sequencing. Results Of 107 patients (7%) with CEBPA mutations, 48 patients (45%) had one mutation (CEBPA-single), and 59 patients (55%) had two mutations (CEBPA-double). The incidence of CEBPA-double patients was similar in intermediate cytogenetic risk patients with and without a normal karyotype (6% and 5%, respectively). CEBPA-double patients had evidence of a lower coincidence with FLT3/ITDs (P = .04) and were highly unlikely to have an NPM1 mutation (P < .0001). CEBPA-double but not CEBPA-single patients had a significantly better overall survival (OS) at 8 years (34%, 31%, and 54% for CEBPA–wild-type [WT], CEBPA-single, and CEBPA-double, respectively, P = .004). This benefit was lost in the presence of a FLT3/ITD (OS for CEBPA-WT, CEBPA-single, and CEBPA-double FLT3/ITD-negative patients: 36%, 35%, 59%, respectively, P = .002; OS for CEBPA-WT, CEBPA-single, and CEBPA-double FLT3/ITD-positive patients: 26%, 21%, 14%, respectively, P = .05). There was no evidence of any additional favorable benefit for a CEBPA-single mutation in the presence of an NPM1 mutation (OS, 45%, 44%, and 56%, P = .2, for NPM1-positive/CEBPA-WT, NPM1-positive/CEBPA-single, and NPM1-negative/CEBPA-double patients, respectively). Conclusion Screening for CEBPA mutations can be restricted to patients with intermediate-risk cytogenetics lacking an FLT3/ITD or NPM1 mutation. Only the presence of a CEBPA-double mutation should be used for therapy risk stratification.

We investigated the benefit of adding all-trans retinoic acid (ATRA) to chemotherapy for younger patients with nonacute promyelocytic acute myeloid leukemia and high-risk myelodysplastic syndrome, and considered interactions between treatment and molecular markers. Overall, 1075 patients less than 60 years of age were randomized to receive or not receive ATRA in addition to daunorubicin/Ara-C/thioguanine chemotherapy with Ara-C at standard or double standard dose. There were data on FLT3 internal tandem duplications and NPM1 mutations (n = 592), CEBPA mutations (n = 423), and MN1 expression (n = 195). The complete remission rate was 68% with complete remission with incomplete count recovery in an additional 16%; 8-year overall survival was 32%. There was no significant treatment effect for any outcome, with no significant interactions between treatment and demographics, or cytarabine randomization. Importantly, there were no interactions by FLT3/internal tandem duplications, NPM1, or CEBPA mutation. There was a suggestion that ATRA reduced relapse in patients with lower MN1 levels, but no significant effect on overall survival. Results were consistent when restricted to patients with normal karyotype. ATRA has no overall effect on treatment outcomes in this group of patients. The study did not identify any subgroup of patients likely to derive a significant survival benefit from the addition of ATRA to chemotherapy. This study is registered at http://www.controlled-trials.com under ISRCTN17833622.

PurposeThis multicenter, prospective, randomized controlled trial compared the efficacy and toxicity of two chemotherapy regimens in advanced Hodgkin's lymphoma (HL): the weekly alternating Stanford V and the standard, twice-weekly regimen of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD).Patients and MethodsPatients had stage IIB, III, or IV disease or had stages I to IIA disease with bulky disease or other adverse features. Radiotherapy was administered in both arms to sites of previous bulk (> 5 cm) and to splenic deposits, although this was omitted in the latter part of the trial for patients achieving complete remission (CR) in the ABVD arm. A total of 520 patients were randomly assigned and were assessed for the primary outcome measure of progression-free survival (PFS). Five hundred patients received protocol treatment, and radiotherapy was administered to 73% in the Stanford V arm and to 53% in the ABVD arm.ResultsThe overall response rates after completion of all treatment were 91% for Stanford V and 92% for ABVD. During a median follow-up of 4.3 years, there was no evidence of a difference in projected 5-year PFS and overall survival (OS) rates (76% and 90%, respectively, for ABVD; 74% and 92%, respectively, for Stanford V). More pulmonary toxicity was reported for ABVD, whereas other toxicities were more frequent with Stanford V.ConclusionIn a large, randomized trial, the efficacies of Stanford V and ABVD were comparable when given in combination with appropriate radiotherapy.

4 patients are described with rheumatoid arthritis, splenomegaly, neutropenia and an unusual proliferation of T cells in the blood and marrow. These patients are clinically similar to patients with classical Felty's syndrome but can be distinguished from them by staining blood and marrow mononuclear cells with a panel of monoclonal anti-T cell antibodies. The T cells from patients with T cell proliferations stain with UCHT1 (OKT3 equivalent) and UCHT4 (OKT8 equivalent but do not stain with a panel of OKT1-like antibodies. In 7 patients with classical Felty's syndrome there was no increase of UCHT4 cells in the blood and the large majority of T cells stained with OKT1-like antibodies. The marrows from the patients with T cell proliferations contain plentiful haemopoietic progenitor cells and it is probable that the T lymphocytes suppress their normal maturation. There was a poor response to splenectomy in 2 patients with T cell proliferations, and single cytotoxic drug therapy may be more appropriate when therapy is required. The literature is reviewed and it is suggested that the T cell proliferations may be secondary to the rheumatoid process.

A direct comparison of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) effects on neutrophil adhesiveness has been carried out. In vitro, GM-CSF and G-CSF upregulate neutrophil CD11b to a similar degree (to 227 +/- 69%, and 232 +/- 70% of control cells, respectively, p < 0.0005), but GM-CSF is more effective in downregulating neutrophil leucocyte adhesion molecule-1 (LAM-1), reducing levels to 33 +/- 4% (p < 0.0005), while G-CSF causes a fall to only 65 +/- 17% (p < 0.005) of control. The concentration of GM-CSF needed to achieve maximal activity is at least one log less than that of G-CSF. In vivo, both GM-CSF and G-CSF upregulate neutrophil CD11b (to 296 +/- 45% and 370 +/- 150%, respectively of baseline), but surface levels of LAM-1 on circulating cells are unchanged. GM-CSF increased neutrophil adhesion to cultured human endothelium in vitro (from 9.3 +/- 0.7% to 15.4 +/- 1.3%, p < 0.0005, n = 10), while G-CSF was without effect. In vivo, both GM-CSF and G-CSF produce a transient leucopenia, but recovery of peripheral counts occurs much earlier (by 60 minutes) with G-CSF, than with GM-CSF (only 50% of cells have demarginated at 120 min). GM-CSF appears to be greater proadhesive agonist for neutrophils than G-CSF.

8 patients with acute myeloid leukaemia (AML), either at presentation or in remission, had normal T-cell subsets as defined by Fc receptors and monoclonal antisera. In contrast, a persistant excess of T lymphocytes staining with Leu 2a (defining suppressor cells) was noted in a case of AML in which persistant amegakaryocytic thrombocytopenia occurred after other haemopoietic lines had recovered from ablative chemotherapy. It is suggested that the 2a+ cells were involved in the suppression of megakaryocytopoiesis, and the phenotype of the lymphoid cells is compared and contrasted with previously described cases of other cytopenias associated with an excess of suppressor cells.

The aggressive non-Hodgkin's lymphomas are a group of malignancies with rapid symptom onset and a short natural history. Presenting features vary depending on the site(s) affected. Investigations are aimed at making an accurate diagnosis and classification, assessing stage and prognostic features, and assessing the patient's ability to tolerate aggressive therapy. The WHO classification has been widely adopted in routine practice, and has been recently updated. Diffuse large B-cell non-Hodgkin's lymphoma, Burkitt's lymphoma and T-cell lymphomas will be discussed in this contribution, with brief mention of other entities. The mainstay of treatment is multi-agent chemotherapy. The advent of monoclonal antibody therapy has improved outcomes in many B-cell malignancies. However, some aggressive lymphomas, including many T-cell disorders, still carry a poor prognosis, and new treatment strategies are needed.

Purpose The role of allogeneic transplantation with reduced-intensity conditioning in diffuse large B-cell lymphoma (DLBCL) is currently unclear, with relatively little published data. We report the outcome of reduced-intensity transplantation (RIT) in a cohort of 48 consecutive patients with relapsed/refractory DLBCL (30 patients with de novo disease and 18 patients with transformed follicular lymphoma) who underwent transplantation with an alemtuzumab-containing regimen, with a median follow-up of 52 months. Patients and Methods Patients had experienced treatment failure with a median of five lines of prior therapy, including autologous transplantation in 69%, and 17% of patients were chemotherapy refractory at transplantation. Median age was 46 years, and 38% of patients had matched/mismatched unrelated donors. Conditioning was with alemtuzumab, fludarabine, and melphalan, and additional graft-versus-host disease (GVHD) prophylaxis was with cyclosporine. Results All patients were successfully engrafted. Only 17% of patients developed grade 2 to 4 acute GVHD, with 13% experiencing extensive chronic GVHD. Four-year estimated nonrelapse mortality was 32%, and relapse risk was 33%. Twelve patients received donor lymphocyte infusions ± chemoimmunotherapy for relapse, and five patients obtained durable remissions, giving current progression-free survival (PFS) and overall survival (OS) rates at 4 years of 48% and 47%, respectively. Patients who had chemotherapy-sensitive disease before RIT had current PFS and OS rates at 4 years of 55% and 54%, respectively. Chemotherapy-refractory patients had a poor outcome. Conclusion The encouraging survival rates with extended follow-up suggest a role for RIT in chemotherapy-sensitive relapsed DLBCL, even in patients who have previously experienced treatment failure with autologous transplantation. Future studies will be required to determine whether any subset of patients with relapsed DLBCL should be considered for RIT versus autologous transplantation.

The National Cancer Research Network (NCRN) is currently coordinating a Phase III randomised study (LY05) comparing fludarabine and cyclophosphamide (FC) with or without rituximab (R) for previously untreated mantle cell lymphoma (MCL). The combination of FC is well-recognised as significantly immunosuppressive and there are concerns that adding rituximab may increase infection risk further. The impact of rituximab on other markers of toxicity is also unclear. We analysed the toxicity data on 139 patients treated within the NCRN LY05 trial. Non-hematological toxicity was similar between the two treatment arms. The only difference in hematological toxicity was a higher rate of lymphocytopenia with fludarabine cyclophosphamide and rituximab (FCR), which did not translate into increased febrile episodes or infections. In conclusion, the addition of rituximab to FC for previously untreated MCL has no significant impact on toxicity.

Purpose To determine the clinical relevance of Wilms’ tumor 1 (WT1) gene mutations in acute myeloid leukemia (AML) with normal karyotype (NK). Patients and Methods Exons 7 and 9 of WT1 were screened in samples from 470 young adult NK AMLs using a combination of direct sequencing and high-resolution capillary electrophoresis. Results Overall, 51 mutations were detected in 47 cases (10%): 46 frameshift mutations with insertion/deletion of one to 28 base pairs in exon 7 (n = 45) or exon 9 (n = 1), with a median mutant level of 45% (range, 8% to 86%), and five substitutions in exon 9: D396N (n = 3), H397Y (n = 1) and H397Q (n = 1). Patients with WT1 mutations had an inferior response to induction chemotherapy compared with wild-type cases (complete remission rate, 79% v 90%, odds ratio [OR] = 3.02; 95% CI, 1.17 to 7.82; P = .02), a higher rate of resistant disease (15% v 4%; OR = 9.33; 95% CI, 2.38 to 36.6; P = .001), an increased cumulative incidence of relapse (67% v 43%, hazard ratio [HR] = 3.02; 95% CI, 1.69 to 5.38; P = .0008), with a reduction in both relapse-free survival (22% v 44%; HR = 2.16; 95% CI, 1.32 to 3.55; P = .005) and overall survival (26% v 47%; HR = 1.91; 95% CI, 1.23 to 2.95; P = .007) at 5 years. In multivariate analysis, which included FLT3 internal tandem duplication and NPM1 mutation status, the presence of a WT1 mutation remained an independent adverse prognostic factor. Conclusion WT1 mutations are a negative prognostic indicator in NK AML and may be suitable for the development of targeted therapy.

FLT3 internal tandem duplication mutant levels >50%, indicative of bi-allelic disease in some cells, are associated with a particularly poor prognosis in acute myeloid leukaemia; lower levels have an intermediate prognosis relative to wild-type FLT3. To examine whether a small population of homozygous mutant cells is responsible for the worse relapse risk rather than heterozygous disease per se, we determined the genetic composition of 34 intermediate mutant level (25-50%) samples. Only two had evidence of mutant homozygosity; only one had more homozygous than heterozygous mutant cells. Bi-allelic disease in intermediate mutant level cases is uncommon and heterozygous disease is sufficient for adverse outcome.

Summary. Venous blood T cell phenotypes were analysed with monoclonal antibodies after 11 allogeneic and 17 autologous bone marrow transplants. In seven cases studied in the early regenerative period, cells with a thymocyte phenotype were present in the blood. In the large majority of patients treated with both allografts and autografts there was an imbalance of phenotypic ‘helper’ and ‘suppressor’ T cell subsets with initially a relative and later an absolute increase of ‘suppressor’ T cells. This imbalance was still present at over 250 d in eight out nine cases. Suppressor T cells bearing HLA-Dr antigens were abundant in one case of fatal GVHD but not in another, and were also increased following two autografts. It is concluded that T cell phenotyping is not of diagnostic value in sick patients following bone marrow transplantation when graft-versus-host disease is suspected.

The acquisition of uniparental disomy (aUPD) in acute myeloid leukemia (AML) results in homozygosity for known gene mutations. Uncovering novel regions of aUPD has the potential to identify previously unknown mutational targets. We therefore aimed to develop a map of the regions of aUPD in AML. Here, we have analyzed a large set of diagnostic AML samples (n = 454) from young adults (age: 15-55 years) using genotype arrays. Acquired UPD was found in 17% of the samples with a nonrandom distribution particularly affecting chromosome arms 13q, 11p, and 11q. Novel recurrent regions of aUPD were uncovered at 2p, 17p, 2q, 17q, 1p, and Xq. Overall, aUPDs were observed across all cytogenetic risk groups, although samples with aUPD13q (5.4% of samples) belonged exclusively to the intermediate-risk group as defined by cytogenetics. All cases with a high FLT3-ITD level, measured previously, had aUPD13q covering the FLT3 gene. Significantly, none of the samples with FLT3-ITD(-)/FLT3-TKD(+) mutation exhibited aUPD13q. Of the 119 aUPDs observed, the majority (87%) were due to mitotic recombination while only 13% were due to nondisjunction. This study demonstrates aUPD is a frequent and significant finding in AML and pinpoints regions that may contain novel mutational targets.

Lymphoplasmacytic lymphoma (LL) is incurable by standard therapy (median survival: 60 months). UK transplant registry data 1984-2003 identified 18 cases of histologically verified LL (median age: 50 years, range: 38-58 years). Nine patients received high dose chemotherapy [plus total body irradiation (TBI) in 1/9] and autologous peripheral blood stem cells (PBSC). Disease status at transplant was complete remission (2), partial remission (5), primary refractory (1) or relapse (1). Transplant related mortality (TRM) at 12 months was 0%. Median follow-up is 44 months with 4 year disease free survival 43% and overall survival 73%. Karnofsky performance status (KPS) is 80-100%. The nine allografted patients (median age: 49 years, range: 39-56 years) were conditioned with standard TBI (2), BEAM (2) or FLU-MEL (5) and received PBSC from HLA-matched sibling (8) or unrelated (1) donors. Disease status at transplant was partial remission (7) or primary refractory (2). TRM at 12 months was 44%. Complications included graft failure (2), grades I-II acute graft versus host disease (aGVHD) (2), grades III-IV aGVHD (3) and chronic GVHD (4). Median follow-up is 32 months with 4 year disease free survival 44% and overall survival 56%. KPS is 70-100%.

An internal tandem duplication in the fms-like tyrosine kinase 3 gene (FLT3/ITD) is associated with poor prognosis in acute myeloid leukemia (AML), but the impact of mutant level, size, and interaction with nucleophosmin 1 (NPM1) mutations remains controversial. We evaluated these characteristics in a large cohort of young adult AML patients. There was a highly significant trend for worsening in relapse risk (RR) and overall survival (OS) with increasing FLT3/ITD mutant level (P < .001 for both), and even in the low level mutant group (1%-24% of total FLT3 alleles), RR was significantly worse than in the FLT3 wild-type (WT) group (P < .001). In multivariate analysis, mutant level was the most powerful prognostic factor for RR. Mutant size and number had no significant impact on outcome. The beneficial impact of an NPM1 mutation on RR and OS was seen in FLT3/ITD(+) as well as FLT3/WT patients; both markers were highly significant independent predictors of outcome (P < .001). Stratification using both markers identified 3 prognostic groups: good (FLT3/ITD(-)NPM1(+)), intermediate (FLT3/ITD(-)NPM1(-) or FLT3/ITD(+)NPM1(+)), and poor (FLT3/ITD(+)NPM1(-)). Patients with high FLT3/ITD mutant level (greater than 50%) or FLT3/ITD(+) in the absence of an NPM1 mutation may be good candidates for more experimental therapeutic approaches.

Purpose: Activating Notch-1 mutations are frequent in T-cell acute lymphoblastic leukemia (T-ALL), occurring in >50% of patients. In murine models of T-ALL, Notch-1 activation can both directly initiate leukemia and cooperate secondarily to other primary events. Whether acquisition of Notch-1 mutations is an early initiating event or a secondary event in the pathogenesis of human T-ALL is unclear. Experimental Design: We used denaturing high-performance liquid chromatography, sequencing, and fragment analysis to analyze Notch-1 mutational status and mutant level in 62 patients at presentation as well as 16 matched presentation-relapse samples. Results: We detected Notch-1 mutations in 47 patients (76%). Seven of these were low-level mutations (quantified at ≤10%), despite high blast counts, suggesting that they were acquired as a secondary event in a subclone. Of 16 matched presentation-relapse samples studied, 7 were wild-type at both presentation and relapse. Five of nine mutant-positive patients at presentation relapsed with the same mutation(s) at the same high level. Four patients had evidence of a change in mutant at relapse. One lost a PEST mutation and became wild-type. Two others lost mutations at relapse but acquired different mutations, despite unchanged T-cell receptor rearrangements, suggesting that the latter event predated the acquisition of the Notch-1 mutation. One relapsed with a secondary T-cell leukemia and different Notch mutation. Conclusions: These results suggest that Notch-1 mutations can sometimes be acquired as secondary events in leukemogenesis and must be used cautiously as solitary minimal residual disease markers.