ALBERT

Description: AN and MM are co-senior authors. Background. Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two widely used reduced-intensity conditioning (RIC) regimens for allogeneic hematopoietic stem cell transplantation (allo-SCT). Patients and Methods. In the current survey, we compared transplantation outcomes in a cohort of 394 acute myeloid leukemia (AML) patients given grafts from HLA-identical siblings after FB (n=218; with a total busulfan dose ranging between 7.1 and 8.9 mg/kg p.o., or between 6.0 and 6.9 mg/kg i.v.) or FM (n=176; with a total melphalan dose ranging between 130 and 150 mg/m2). Patients given manipulated grafts and those given T cell depleting agents (ATG or alemtuzumab) were not included. At time of transplantation, 266 patients (68%) were in first complete remission (CR1), 69 (18%) in later CR, while 59 patients (15%) had advanced diseases. Three-hundreds and fifty-two patients (89%) received peripheral blood stem cells while the remaining 42 patients received bone marrows as stem cell source. Results. Three FB patients but no FM patients failed to engraft. Median time for reaching 500 neutrophils was 17 (1-50) days in FB patients versus 14 (9-43) days in FM patients (P

Description: The use of in vivo Alemtuzumab in reduced intensity conditioning (RIC) stem cell transplantation for AML has been reported to be associated with low non-relapse mortality (NRM) and favourable survival outcomes. However, Alemtuzumab depletes the alloreactive donor T cells and recipient antigen presenting cells that mediate graft versus leukaemia (GvL) and graft versus host disease (GvHD). We report the analysis of 90 patients from the British Society for Blood and Marrow Transplantation (BSBMT) registry comparing T-cell replete and Alemtuzumab-containing protocols in HLA-identical sibling RIC transplants for AML. Patient characteristics were: median age at diagnosis-50 years; 46%-male, 54%-female; diagnostic karyotypes according to MRC AML criteria-13% good risk, 76% standard risk, 11% poor risk; 67%-CR1, 24%-CR2, 9-refractory/relapsed disease/PR. Conditioning protocols were: fludarabine/melphalan (66%), fludarabine/busulphan (17%), fludarabine/cyclophosphamide (11%), others (6%). 51 patients (57%) received in vivo Alemtuzumab and 37 patients (41%) did not receive any T-depleting antibodies. 2 patients (2%) received ALG/ATG and were excluded from subsequent analyses comparing the effect of Alemtuzumab with T-replete transplants. The median CD34 cell dose was 4.41x106/kg (0.77–15.8). The actuarial overall survival (OS) and progression-free survival (PFS) at 5 years for all patients were 53% and 47% respectively. The NRM and relapse risk (RR) at 5 years were 16% and 49% respectively. The majority of the relapses were within 2 years of transplant. Acute GvHD was either absent or Grade I in 75 patients (84%). Grade II-IV acute GvHD developed in 15 patients (16%). Extensive chronic GvHD occurred in 18/65 surviving ≥100 days (28%). A complete hematological remission(CR) at transplant predicted for OS at 3 years with 54% survival for CR1, 57% survival for CR≥2 and 0% survival for PR/relapse/refractory disease (p

Description: Background Historically diagnosis and prognosis of myeloid disorders including acute myeloid leukemia (AML) have been determined using a combination of morphology, immunophenotype, cytogenetic and more recently single gene, if not single mutation, analysis. The introduction of NGS technology has resulted in an explosion in the quantity of mutation data available. However, the feasibility and utility of NGS technology with regards to decision-making in routine clinical practice of myeloid disorders is currently unknown. We therefore developed an advanced NGS tool for simultaneous assessment of multiple myeloid candidate genes from low amounts of input DNA and present clinical utility analysis below. Methods We designed a targeted resequencing assay using a TruSeq Custom Amplicon panel with the MiSeq platform (both Illumina) consisting of 341 amplicons (~56 kb) designed around exons of genes frequently mutated in myeloid malignancies (ASXL1, ATRX, CBL, CBLB, CBLC, CEBPA, CSF3R, DNMT3a, ETV6, EZH2, FLT3, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PDGFRA, PHF6, PTEN, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, WT1 & ZRSR2). Filtering, variant calling and annotation were performed using Basespace and Variant Studio (Illumina) with additional indel detection achieved using Pindel. A cohort of samples previously characterised with conventional techniques was used for validation and the lower limit of detection established using qPCR. Post-validation, DNA from 152 diagnostic blood or bone marrow samples from patients with confirmed or suspected myeloid disorders; both AML (n=46) and disorders with the potential to transform to AML i.e. myelodysplasia (confirmed n=54, suspected n=10) and myeloproliferative neoplasms (n=42), were analysed using this assay. To gather clinical utility data we developed a reporting algorithm to feed back information to clinicians; only those variants with a variant allele frequency (VAF) of 〉10% and described as acquired in publically available databases were reported with the exception of novel mutations predicted to result in a truncated protein. Further utility data was obtained using published mutation algorithms to determine the proportion of patients in whom mutation data altered prognosis. Results In the validation cohort, initial concordance for detection of clinically significant mutations was 88% rising to 100% once Pindel was used to identify FLT3 ITDs. The lower limit of detection was 3% VAF, and mean amplicon coverage was 390 reads. Using our reporting algorithm 66% of patients in the post-validation cohort had a suspected pathogenic mutation relevant to a myeloid disorder, rising to 74% in patients with confirmed diagnoses. The median number of reported variants per sample for all diagnoses was one (range 0-6). When mutation data for patients with AML with intermediate risk cytogenetics was analysed using the algorithm of Patel et al (N Engl J Med. 2012;366:1079-1089), 4/22 (18%) moved into another risk category. A further two patients had double CEBPA mutations, improving their prognosis. Identification of complex mutations in KIT exon 8 in 2/6 patients with core binding factor AML resulted in more intensive MRD monitoring due to the increased risk of relapse. Interpretation of mutation data for patients with confirmed myelodysplasia using the work of Bejar et al (N Engl J Med. 2011;364:2496-2506) revealed 13/54 (24%) had a high risk mutation independently associated with poor overall survival. 2/8 (25%) patients with chronic myelomonocytic leukemia and 1/12 (8.3%) patients with primary myelofibrosis had high risk ASXL1 exon 12 mutations, independently associated with a poor prognosis. Among suspected diagnoses confirmatory mutations were found in 2/19 (11%), while the absence of mutations reduced the probability of myeloid disease in 11/19 (58%), in some cases sparing elderly patients invasive bone marrow sampling. A further 20 patients had clinically relevant mutations. Conclusions The NGS Myeloid Gene Panel provided extra information to clinicians in 57/152 patients (38%) helping inform diagnosis, individualize disease monitoring schedules and support treatment decisions. The targeted panel approach requires rigorous validation and standardisation in particular of bio-informatics pipelines, but can be adapted to incorporate new genes as their relevance is described and will become central to treatment decisions. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION The prognosis for children with refractory acute myeloid leukemia (AML) treated with chemotherapy is dismal and data on the outcome after allogeneic haematopoietic stem cell transplant (SCT) are scanty with reported leukemia free survival (LFS) rates of 10-20%. Thus there is significant controversy about whether SCT is appropriate in such patients (pts). We performed a retrospective, national study to analyse outcomes and prognostic factors for children undergoing SCT for refractory AML in the UK. METHODS A retrospective analysis of all pts 5% blasts in the bone marrow (BM) or proven extramedullary disease (EM) was performed. Source data verification (SDV) was performed to ensure pts were indeed refractory. The primary end-point was 5 year LFS. Secondary end-points were Relapse Rate (RR), Treatment Related Mortality (TRM), Graft Versus Host Disease (GVHD) and Overall Survival (OS). The Kaplan Meyer method was used to estimate survival data and Fisher's exact and Mantel-Cox Log Rank tests were used to compare disease- transplant- and survival-related variables. RESULTS Following SDV, a total of 44 pts from 13 centres were included in the study. The median age at SCT was 11.5 yrs and the median number of prior lines of chemotherapy was 3. The median time from diagnosis to SCT was 197 days. 23 pts had primary refractory AML and 21 had relapsed refractory AML. 12 pts showed adverse risk cytogenetics, 26 standard risk and 6 favourable. EM disease was documented in 5 pts. 42 children had 〉5% myeloid blasts in the BM immediately prior to conditioning and refractory disease was confirmed by cytogenetics/molecular genetics in 23. 2 pts were in BM remission but had frank EM disease. 38 pts (86%) received myeloablative conditioning (14 TBI based) and 6 (14%) had reduced intensity conditioning (RIC). In vivo T cell depletion was used in 25 pts. 15 pts (35%) were transplanted from an HLA identical family donor, 15 from a matched unrelated donor and 14 (32%) a mismatched donor. BM was used as the stem cell source in 18 (41%), peripheral blood in 20 (46%) and cord blood in 6 cases (14%). Median follow up was 4 years 10 months. 5 pts never achieved engraftment and had disease progression. The remaining 39 pts engrafted at a median of 15 days post-SCT. 30 pts (68%) achieved a complete remission (CR) following SCT. TRM at 1 year was 18% (5 infections, 1 cardiac failure, 1 GVHD-related). Acute GVHD occurred in 23 pts and was severe (grade ≥III) in 8 (19%). The incidence of chronic GvHD was low (1 limited, 2 extensive). Relapse was the major cause of treatment failure and occurred in 17 pts (39%) at a median 2.3 months post SCT. At last follow-up, 18 pts remain alive and in continuous complete remission (CCR). In this cohort, the 5 year OS and LFS were both 43% (95%CI 31-61%) (Figure1). Outcomes in pts with primary refractory disease (9/23, 39% in CCR) and those with relapsed refractory AML (9/21, 43% in CCR) were equivalent. Outcome for pts with cytogenetic confirmation of refractory disease was not statistically different (7/23, 30% in CCR) from the overall group. Pts transplanted with ≤30% blasts in the BM had improved outcomes (5-year LFS 52% vs 27%, p= 0.05). Likewise, the development of aGVHD of any grade was associated with a significantly better LFS (5-year LFS 56% vs 30%, p= 0.05). Cytogenetics including monosomy 7 (n=7) and molecular risk classification did not translate into a significant prognostic factor for relapse. Since RIC was used in only 6 pts, the impact of the intensity of conditioning cannot be determined. CONCLUSIONS This is the largest series of outcomes for SCT for refractory paediatric AML reported to date. Our data indicate that for selected pts, particularly those with a lower disease burden, SCT offers a realistic chance of salvage in both primary refractory and relapsed refractory AML (5 year LFS 43%) with acceptable toxicity. The association of aGVHD with improved LFS suggest a possible role in engineering a graft-versus-leukemia effect in this patient group. Figure 1. Leukemia-free survival for pediatric patients with refractory acute myeloid leukemia undergoing allogeneic stem cell transplantation. Figure 1. Leukemia-free survival for pediatric patients with refractory acute myeloid leukemia undergoing allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Telomere shortening is associated with disease evolution in chronic myelogenous leukemia (CML). We have examined the relationship between diagnostic telomere length and outcome in 59 patients with CML who entered into the MRC CMLIII Trial by Southern blot hybridization using the (TTAGGG)4 probe. Age-adjusted telomere repeat array (TRA) reduction was found to significantly correlate with time from diagnosis to acceleration, such that patients with a larger TRA reduction entered the accelerated phase more rapidly (r = −0.50; P = .008). Cox-regression analysis for this group was suggestive of a relationship between a greater TRA-reduction and a shorter time to acceleration (P = .054). Age-adjusted TRA reduction did not significantly affect either the time to blast crisis or overall survival. Our results show that telomere shortening observed at the time of diagnosis in CML significantly influences the time to progress to the accelerated phase. The measurement of diagnostic TRA may prove to be clinically important in the selection of patients at high risk of disease transformation in CML.

Description: Lack of progress in curing AML is likely, in part, to be due to the genetic and functional heterogeneity of AML. ~13 Tier 1 mutations occur per patient sample arrayed in 2-5 clones (CGARN, N Engl J Med, 2013). Not all AML cell populations may be equally chemosensitive; for example, leukemia-propagating leukemic stem cells (LSC) are more chemoresistant. (Ishikawa et al., Nat Biotechnol, 2007). Additionally, the impact of genetic heterogeneity on LSC function is unclear. In ~ 70% of primary human AML with 〉2% CD34+ cells, LSCs exist within both CD34+CD38- and CD34+CD38+ compartments (Taussig et al., Blood, 2008), and have progenitor-like transcriptional programmes (Goardon et al., Cancer Cell, 2011). ~30% of AML with

Description: Telomere shortening is associated with disease evolution in chronic myelogenous leukemia (CML). We have examined the relationship between diagnostic telomere length and outcome in 59 patients with CML who entered into the MRC CMLIII Trial by Southern blot hybridization using the (TTAGGG)4 probe. Age-adjusted telomere repeat array (TRA) reduction was found to significantly correlate with time from diagnosis to acceleration, such that patients with a larger TRA reduction entered the accelerated phase more rapidly (r = −0.50; P = .008). Cox-regression analysis for this group was suggestive of a relationship between a greater TRA-reduction and a shorter time to acceleration (P = .054). Age-adjusted TRA reduction did not significantly affect either the time to blast crisis or overall survival. Our results show that telomere shortening observed at the time of diagnosis in CML significantly influences the time to progress to the accelerated phase. The measurement of diagnostic TRA may prove to be clinically important in the selection of patients at high risk of disease transformation in CML.

Description: The allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment in patients with MDS. Indeed, many retrospective studies have shown that the higher risk patients according to IPSS (intermediate-2 and high) have a better overall survival with HSCT if performed at time of diagnosis while HSCT in lower risk patients (low and intermediate-1) should be postponed. Actually, some lower risk patients have poor prognostic features leading to propose the transplantation before they evolve to a higher risk. In this registry study from the CMWP, we analyzed outcome of patients who were diagnosed low risk MDS and who finally were transplanted without evolving to a higher risk. As more and more of these patients are proposed to the transplantation, their survey may give information about their prognostic risks. All adult patients transplanted between 2000 and 2011 and registered in the EBMT registry could be included if they have a low risk MDS at time of diagnosis and at time of transplantation. Overall and progression-free survival (OS and PFS) were estimated using Kaplan-Meier product limit estimator. For competing risk, cumulative incidence functions were estimated using usual methodology. Cox proportional hazard model were used to test the potential risk factors (age, gender, CMV recipient/donor, disease, IPSS, transfusion, donor type, conditioning). Missing data were handled through multiple imputations by chained equations methods. 291 patients met the inclusion criteria: 126 women and 165 men. Median age at diagnosis was 53 years and inter-quartile range (IQR) from 44 to 59. The majority of patients have refractory anemia or refractory cytopenia with multilineage dysplasia (66%), while one third of patients have refractory anemia with excess blasts. Most patients were intermediate-1 (80%) and required transfusions before the transplantation (84%). Median age at time of transplantation was 55 years (IQR: 46-60). Median time from diagnosis to transplantation was 11 months (IQR: 7-22). Donor was an HLA matched sibling in 122 (42%) patients and an unrelated donor in the remaining patients. The preferred source of stem cells was peripheral blood stem cells (PB) (78%) followed by bone marrow (19%) and cord blood (3%). Conditioning regimen consisted in a reduced intensity conditioning regimen in the majority of patients (59%) and 58% received an in vivo T depletion. PFS and OS of the whole patients are shown in Figure 1 A and 1 B. Cumulative incidence of grade II-IV acute GVHD, chronic GVHD, non-relapse mortality and relapse are shown in Figure 2 A-D. Multivariate analysis with original data set (excluding missing data) found the following factors as poor prognostic for PFS: age 〈 35 years (Hazard ratio (HR): 4.22, p=0.009) or 〉 45 years (p 5% (HR: 1.67, p=0.05), bone marrow (HR: 2.18, p=0.003) or cord blood (HR: 5.28, p=0.014) as sources of stem cells rather than PB and CMV serostatus positive for the recipient and negative for the donor (HR: 2.50, p=0.002) while transfusion before the transplantation (HR): 0.51, p=0.013) and T-depletion (HR: 0.60, p=0.43) were protective. The second multivariate analysis included all patients with imputed datasets showing similar results. To conclude, outcome after HSCT in patients with lower risk IPSS are better than those observed in higher risk. In favorable transplant condition (PB, T-depletion), expected OS reaches 70% with a low mortality rate after the second year which could be a valid option in some lower risk patients presenting some high risk factors as poor molecular biology or resistance to agents stimulating erythropoiesis. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Blaise: Sanofi: Honoraria, Research Funding.

Description: Allogeneic hematopoetic stem cell transplantation (allo-HSCT) offers long-term remission for patients with lymphoid malignancies. In particular, reduced intensity conditioned (RIC) protocols with Alemtuzumab T-cell depletion lead to durable engraftment and reduced graft versus host disease (GvHD). However, such regimens may be associated with reduced graft-versus-lymphoma (GvL) effect and higher incidence of disease progression, leading to increased use of donor lymphocyte infusion (DLI). This study examined transplant outcomes and use of DLI in a large UK RIC-Alemtuzumab conditioned HSCT cohort. 288 consecutive adult patients from three UK transplant centres (Birmingham, Glasgow, Oxford) undergoing first Alemtuzumab-based RIC HSCT for Hodgkin's lymphoma (57%), Non-Hodgkin's lymphoma (24%) and chronic lymphocytic leukemia (19%) between 2000 and 2012 were included. Overall survival (OS), current progression-free survival (CPFS), relapse incidence (RI), transplant-related mortality (TRM), incidence of GvHD and effects of DLI were analyzed. Median age was 49y (range 17-68) and 60% were male. Patients were heavily pre-treated with 58% having received ≥3 prior lines of treatment and 49% having failed a previous autograft. 55% received stem cells from an HLA-matched unrelated donor and 94% received a peripheral blood stem cell harvest. The most frequent conditioning regimen was Fludarabine, Melphalan and Alemtuzumab (FMC) (70%) and the median total Alemtuzumab dose was 50mg (range 30-100). Median duration of follow-up for survivors was 64 months. 3y OS and CPFS were 53% and 50%, respectively. Factors negatively associated with OS in univariate (UV) analysis were previous autograft (p=0.01), no requirement for DLI (p=0.05), disease status not CR (p=0.05), previous treatment number ≥3 (p=0.007) and failure to engraft plts (p=0.001). On multivariate (MV) analysis, only previous autograft (HR 1.58 p=0.01) and failure to engraft plts (HR 2.3 p=0.0001) retained significance. Factors negatively associated with CPFS on UV analysis were no requirement for DLI (p=0.006), failure to engraft plts (p=0.0003) and previous treatment lines ≥3 (p=0.04). On MV analysis, no DLI remained a significant predictor of adverse CPFS (HR 1.8 p=0.01), as did failure to engraft plts (HR 1.77 p=0.005) and ≥3 treatment lines (HR 1.46 p=0.04). At 3y, the cumulative incidence of relapse and TRM was both 27%. The incidence of grade 2-4 acute GvHD (aGvHD) and extensive chronic GvHD (cGvHD) was 22% and 18%, respectively. UV predictors of RI were unrelated donor (p=0.0009), HLA mismatch (p=0.04), age 〉 median (p=0.04) and lack of aGvHD (p=0.04). The presence of cGvHD may have been protective against relapse (p=0.08). No variables retained significance on MV modelling. UV predictors of TRM were failure to engraft plts (p=0.001), HLA mismatch (p=0.005), age 〉 median (p=0.004), previous autograft (p=0.02), FMC conditioning (p=0.03), ≥3 previous lines of treatment (p=0.001) and development of aGvHD (p=0.04). On MV modelling, failure to engraft plt (p=0.001), HLA mismatch (p=0.03), age 〉 median (p=0.003) and treatment number ≥3 (p=0.04) retained significance. 62/288 patients received DLI; 37 receiving prophylactic DLI (pDLI) for mixed chimerism and 25 receiving therapeutic DLI (tDLI) for disease relapse. The median dose of DLI was 1x 10^6 CD3+ cells/kg (range 0.1 - 10 x 10^6). Median time from HSCT to first DLI was 10.5 months (range 3-30), to 2nd DLI 15 months (6-51) and to 3rd DLI 19 months (9-30). At 3y, OS after pDLI was 75% and after tDLI was 29%. 29/37 (78%) of pDLI and 14/25 (56%) of tDLI recipients achieved full or stable mixed chimerism. 3y RI after pDLI was 29% and after tDLI was 59% (p=0.02). Overall, the incidence of GvHD post-DLI was 32% (48% tDLI, 22% pDLI, p=ns). Median time to GvHD after last DLI was 46 days (range 17-249). There was no significant impact of post-DLI GvHD on RI (RI at 1y post DLI 18% in GvHD cohort vs 21% with no GvHD, p=ns), but GvHD increased TRM at 1y post DLI (17% in the GvHD cohort vs 3% with no GvHD, p=0.05). This is the largest series to date with long-term follow-up examining outcomes after Alemtuzumab-based RIC HSCT for mature lymphoid malignancy. Our data indicate that allo-HSCT is effective and is associated with good long term outcomes. Use of pDLI for was associated with excellent outcomes and supports use of DLI in patients developing mixed chimerism following allo-HSCT for lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Background: Over the last decade, there has been a significant increase in the number of patients with Myelofibrosis (MF) undergoing allogeneic stem cell transplantation (SCT). However, scarce information exists on the outcome and management of those patients who relapse following SCT. Moreover, the management of relapse occurring post-SCT is often heterogeneous and ranges from palliation to intensive salvage approaches. We therefore conducted a retrospective EBMT registry analysis of adult MF patients who relapsed following first SCT episode. Results: A total of 1216 adult patients (997 (82%) with Primary MF (PMF) and 219 (18%) with secondary MF (sMF)) underwent 1st allogeneic SCT between 2000 and 2010. A total of 251 patients from this cohort (206 with PMF and 45 with sMF) had conformed relapse ≥ day 30 after HSCT and were included in the analysis. Within this relapse cohort, there were 163 males and 88 females; median age was 55 years old (range 21.5-70 years). A total of 84 patients (33%) had received Myeloablative Conditioning (MAC) and 167 patients (67%) Reduced Intensity Conditioning (RIC). Regarding donor type, there were 123 matched siblings (49%) and 128 unrelated donors (51%). Acute GVHD (aGVHD) status was available for 243/251 (97%) patients; no aGVHD was evident in 143 patients, Grade I-II aGVHD 76 patients, Grade III-IV aGVHD 22 patients and 2 patients with aGVHD ungraded. The median time to relapse after SCT was 7.1 months (range 1-111 months). The median Overall Survival (OS) from the time of relapse was 17.7 months (95% Confidence Intervals 11-24). Collectively, there was a significant difference in survival outcome for those relapsing 〉 7.1 months post-SCT (median survival 30.3 months post relapse) compared to those relapsing within 7.1 months following the initial SCT episode (median survival 7.9 months post relapse; p