ALBERT

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: Abstract 1181 Poster Board I-203 MNGIE is an autosomal recessive disorder of nucleotide metabolism due to TYMP gene mutations that cause loss of activity of thymidine phosphorylase (TP). As a result, thymidine (Thd) and deoxyuridine (dUrd) plasma and tissue levels increase and cause nucleotide pool imbalances. This leads to instability of mitochondrial DNA with loss of mitochondrial respiratory chain functions. Clinical consequences manifest as a multisystemic disease with severe gastrointestinal dysmotility, most severe cachexia (BMI 10-17 kg/m2 in our patients), ptosis and/or ophthalmoparesis, peripheral neuropathy and leukencephalopathy. While TP is not expressed in all tissues, cellular and plasma Thd and dUrd levels appear to be in equilibrium among all body compartments. Mononuclear white blood cells and platelets are rich in TP activity and can transiently restore TP activity upon transfusions. Therefore allogeneic HSCT was tested as a permanent replacement therapy by several teams. A coorperative group under the auspices of the WBMT collected the global experiences. So far, 10 patients underwent an attempt for HSCT between 2005-2009. One patient stopped conditioning due to toxicity and did not proceed to transplantation. Nine patients had 12 allogeneic HSCT. They were 6 males and 3 females with a median age of 28y (range 10-41y) at transplantation. All were symptomatic at time of HSCT (5 on parenteral nutrition). A variety of different conditioning regimens and GvHD prevention strategies were used. Fludarabine was included in all conditioning regimens, combined with busulfan or cyclosphoshamide, melphalan, thiotepa or TBI. T cell depletion (TCD) with ATG, alemtuzumab or in vitro TCD was performed in 10 HSCT. GvHD prevention with sirolimus or calcineurin inhibitors was used in combination with mycophenolate mofetil or methotrexate. Graft source for first transplants was peripheral blood stem cells (PBSC) in 4, bone marrow (BM) in 3 and cord blood in 2. PBSC and BM donors were HLA-identical siblings in 2, phenotypic identical parent in 1 and unrelated donors in 4 (3 with 10/10 HLA-match, 1 with 9/10-match). Engraftment was problematic. Three primary graft failures and two late graft failures were observed. A second HSCT was performed in three patients, all engrafted but two died due to TRM. The two patients without a second HSCT died from their disease. Four patients developed acute GvHD grade I-IV. To date, five patients are alive 8-48 months posttransplant, three after related PBSC-HSCT, one after unrelated BM-HSCT (10/10-HLA identical), one after two HSCTs with BM and PBSC respectively from the same unrelated 10/10-HLA identical donor. In all of these patients metabolism normalized as measured by normal Thd and dUrd levels. All surviving patients are off parenteral nutrition without further weight loss or increase in body weight. Gastrointestinal symptoms improved and in the two patients with the longest follow up a slight improvement of neurological symptoms can be observed now. Further time is needed to determine whether other disturbed organ functions will be reversible. Conclusion: Allogeneic HSCT can restore full metabolic function and halt and restore clinical signs and symptoms in this otherwise unrelenting progressive disease. Engraftment was identified as a key obstacle. Still, the optimal transplant regimen needs to be defined to improve patients' outcome. A common consensus transplant protocol and disease specific pre- and posttransplant evaluation protocol was developed with participation of all involved teams. Disclosures: Gratwohl: Amgen: Research Funding; Roche: Research Funding; Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding. Hirano:Athena Diagnostics: Speakers Bureau; Pfizer: Research Funding.

Description: BCR-ABL negative myeloproliferative neoplasms (MPNs), such as polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are chronic myeloid malignancies characterized by overproduction of hematopoietic cells. JAK2 mutations are found in most patients with PV, and in only 50-60% of patients with ET and MF. JAK2 mutation testing has greatly simplified MPN diagnosis, but distinguishing JAK2-wildtype ET from reactive thrombocytosis remains a diagnostic challenge. Mutations in signalling pathways (MPL, LNK) and epigenetic regulators (TET2, DNMT3A, IDH1/2, EXH2, ASXL1) have been found in a minority of MPNs. However genome-wide data are lacking and the pathogenesis of MPNs that do not harbor JAK2 or MPLmutations remains obscure. Methods Exome sequencing was performed in 151 MPN patients on matched tumor and constitutional samples. CALR status was assessed in 3412 samples using Sanger sequencing and analysis of exome/genome sequencing data. Presence of CALR mutations in hematopoietic stem and progenitor cells was assessed by flow sorting and sequencing. Phylogenetic trees were established using hematopoietic colonies. Calreticulin cellular localisation was assessed in patient samples and cell lines expressing CALR variants by flow cytometry and immunofluorescence. Results Exome sequencing identified 1498 somatic mutations with a median of 6.5 mutations in PV and ET, and 13 in MF (MF vs ET, P=0.0002; MF vs PV, P=0.008). JAK2V617F was found in all cases of PV (n=48), 56% of ET (35/62), and 69% of MF (27/39), and MPL mutations in 7 ET and MF cases.  Mutations in epigenetic regulators TET2, DNMT3A, ASXL1, EZH2, and IDH1/2 were identified in 22, 12, 12, 4, 3 patients respectively, and components of the splicing machinery (U2AF1, SF3B1 or SRSF2) were mutated in 9 patients.  Mutations in rare genes reported to be mutated in MPNs were found in four patients (1 CBL; 2 NFE2; 1 SH2B3/LNK). We found novel somatic mutations in CHEK2 (1 PV, 1 ET and 1 MF) which have not been previously reported in MPNs.  The mutation spectrum showed a predominance of C〉T transitions. Pairwise associations between MPN genes demonstrated that ASXL1 and SRSF2 mutations were positively correlated with mutations in epigenetic modifiers. Novel somatic mutations in calreticulin (CALR) were identified by exome sequencing in the majority (26/31) of JAK2 or MPL unmutated patients. CALR and JAK2/MPL mutations were mutually exclusive, and 97% of patients harbored a mutation in 1 of these 3 genes. In an extended follow up screen of 1345 hematological malignancies, 1517 other cancers and 550 controls we found CALR mutations in 71% of ET (80/112), 56% of idiopathic MF (18/32), 86% of post ET-MF (12/14) and 8% of myelodysplasia (10/115), but not in other myeloid, lymphoid or solid cancers. Compared to JAK2-mutated MPNs, those with CALR mutations presented with higher platelet counts (Wilcoxon rank-sum, P=0.0003), lower hemoglobin levels (Student’s t test, P=0.02) and showed a higher incidence of transformation to MF (Fishers exact, P=0.03). All CALR mutations were insertions or deletions affecting exon 9, with 2 common variants L367fs*46 (52 bp deletion) and K385fs*47 (5 bp insertion). Loss of heterozygosity over CALR was seen in a minority of patients. Of 148 CALR mutations identified, there were 19 distinct variants. Remarkably, all generated a +1 basepair frameshift, which results in loss of most of the C-terminal acidic domain of the protein as well as the KDEL Golgi-to-endoplasmic reticulum (ER) retrieval signal, raising the possibility of compromised ER retention. Mutant proteins were readily detected in transfected cell lines and localised to the ER in the same manner as wildtype CALR, without Golgi or cell surface accumulation. These results are consistent with studies reporting KDEL-independent mechanisms of ER retention. Mutation of CALR was detected in highly purified hematopoietic stem/progenitor cells. Clonal analyses demonstrated CALR mutations in the earliest phylogenetic node in 5/5 patients, consistent with it being an initiating mutation in these individuals. Conclusions We describe the mutational landscape of BCR-ABL negative MPNs and demonstrate that somatic mutations in the endoplasmic reticulum chaperone CALR are found in the majority of patients with JAK2-unmutated MPNs. These results reveal a novel biological pathway as a target for tumorigenic mutations and will simplify diagnosis of MPN patients. Disclosures: Bowen: Celgene: Honoraria. Harrison:S Bio: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Shire: Speakers Bureau; Celgene: Honoraria; YM Bioscience: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Vannucchi:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Description: This retrospective study of diffuse mixed (DM) cell lymphoma was undertaken as a collaborative study between the Repository Center for Lymphoma Clinical Studies and four cooperative oncology groups (CALGB, ECOG, SECSG, SWOG), and was based on 62 patients from the files of the Repository Center. We wanted to ascertain whether there were any significant clinical differences among the various morphological subtypes of this lymphoma. All patients were treated according to different protocols of the Cooperative Oncology Groups sponsored by the National Cancer Institute. In 16 patients (26%), the malignant lymphoma (ML) had morphological features consistent with follicular center cell origin (FCC); in 34 patients (55%), the ML did not have features of follicular center cell type (non-FCC), but had morphology described for peripheral T-cell-derived ML. In 8 of the patients (13%), no agreement could be reached by the 7 histopathologists who participated in the study, and these were classified as unresolved; the remaining 4 (6%) were unclassifiable. We compared the survival times of the 16 patients having the morphological features of the FCC subtype with the survival times of the 34 patients with the non-FCC subtype and found that patients with FCC lived longer (p = 0.07 Cox's regression). In the FCC group, all patients who had complete remissions (CR) were alive; however, their survival times were similar to those who had a partial or no response (p = 0.32). In contrast, in the non-FCC group, the median survival was 20 mo, and patients with a CR had a significantly longer survival than did incomplete responders (p = 0.003). According to these results the non-FCC diffuse mixed cell lymphoma appears to be a high-grade malignant lymphoma, whereas the FCC type is not.

Description: We grouped 162 patients wtih advanced, diffuse histiocytic lymphoma (DHL) into various morphological subtypes to ascertain whether there were any significant differences in survival among them. These patients were staged and treated from 1972 to 1977 according to the protocols of the Southwest Oncology Group. Of the 159 patients on whom a consensus on the diagnosis was reached, 115 were classified morphologically as having large non-cleaved, 26 as B-immunoblastic, 9 as large cleaved, and 6 as T-immunoblastic. The 3 remaining patients did not fit any of these subtypes, but each had a single prominent nucleolus in most tumor cells (“prominent nucleolus” type). Morphological subdivision of DHL did not identify any subgroup of patients with a significantly longer survival, but clinical parameters such as stage, symptoms, and type of treatment significantly influenced survival times.

Description: Background: Toxicities following allogeneic hematopoietic stem cell transplantation are specific to the conditioning regimen used and its intensity. Therefore, we hypothesized that the hazard of individual comorbidities is dependent on the conditioning agents and their dose. The aim of this analysis was to study, in a regimen specific manner, the impact of individual comorbidities (cardiac disease, severe pulmonary disease, and diabetes mellitus) on mortality. Methods: We included a multi-center cohort of 3,338 patients from the registry of the Acute Leukemia Working Party of the European Society of Blood and Marrow Transplantation (EBMT), who underwent first allogeneic HSCT for treatment of AML in all disease stages, transplanted between 2005 and 2016. Patients included received grafts from a matched sibling or 10/10 HLA-allele matched unrelated donor, and who were conditioned using one of 7 common regimens (Busulfan [BU[/Cyclophposphamide [Cy], Cy/Total Body Irradiation [TBI], Flu [Fludarabine]/Bu at reduced-intensity [RIC] dosage, Flu/Bu at myeloablative [MAC] dosage, Flu/Melphalan [Mel], Flu/Treosulfan [Treo], Flu/TBI). Regimens were excluded from a given analysis if fewer than ten overall mortality events occurred among patients with or without the studied condition. We constructed a multivariable Cox model for the outcome of overall survival, adjusted for key transplantation variables, including patient age, disease status, and donor type among others. For each comorbidity, separate models were constructed within each of the seven regimens, and the comorbidity's hazard ratio was extracted. Using the same method, the hazard ratio across all regimens, as well as in the myeloablative and the reduced-intensity setting, was obtained. Results: The median age was 55 years; 70% of patients were in first complete remission (CR), and 54% of patients received grafts from matched sibling donors. The most common regimens studied were Flu/Bu at a reduced-intensity dose (22%), Flu/Mel (20%) and Bu/Cy (16%, Table 1). We find that the studied comorbidities were associated with different degrees of added risk for overall mortality in each regimen studied. For cardiac disease, hazard ratios (HR) ranged from 1.00 (95% CI: 0.63-1.60) in Flu/Treo to 1.65 (1.15-2.36) in Flu/Bu with myeloablative dose (Figure 1). Among patients with severe pulmonary disease, significant increases in hazard were seen only in patients treated with myeloablative Flu/Bu and Flu/Mel. In diabetes mellitus, no regimens were clearly associated with increased risk of overall mortality. However, Flu+TBI trended strongly toward increased risk with an HR of 1.55 (1.00-2.41, p = 0.051). Greater comorbidity-associated risk was not consistently associated with increasing conditioning, and the hazards associated with each comorbidity in the MAC and RIC settings were broadly overlapping. Similar trends were seen for the outcome of non-relapse mortality, although statistical significance (p 〈 0.05) was not observed in the setting of low event numbers. Conclusions: These results confirm our hypothesis that comorbidities exert an effect on transplantation outcome in a conditioning regimen-specific manner. Additional study, both retrospective, and prospective, may eventually allow for the precision selection of a conditioning regimen based on the individual patient's physiological status. Disclosures Finke: Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding. Malladi:Roche: Membership on an entity's Board of Directors or advisory committees. Mohty:MaaT Pharma: Consultancy, Honoraria.

Description: This retrospective study of diffuse mixed (DM) cell lymphoma was undertaken as a collaborative study between the Repository Center for Lymphoma Clinical Studies and four cooperative oncology groups (CALGB, ECOG, SECSG, SWOG), and was based on 62 patients from the files of the Repository Center. We wanted to ascertain whether there were any significant clinical differences among the various morphological subtypes of this lymphoma. All patients were treated according to different protocols of the Cooperative Oncology Groups sponsored by the National Cancer Institute. In 16 patients (26%), the malignant lymphoma (ML) had morphological features consistent with follicular center cell origin (FCC); in 34 patients (55%), the ML did not have features of follicular center cell type (non-FCC), but had morphology described for peripheral T-cell-derived ML. In 8 of the patients (13%), no agreement could be reached by the 7 histopathologists who participated in the study, and these were classified as unresolved; the remaining 4 (6%) were unclassifiable. We compared the survival times of the 16 patients having the morphological features of the FCC subtype with the survival times of the 34 patients with the non-FCC subtype and found that patients with FCC lived longer (p = 0.07 Cox's regression). In the FCC group, all patients who had complete remissions (CR) were alive; however, their survival times were similar to those who had a partial or no response (p = 0.32). In contrast, in the non-FCC group, the median survival was 20 mo, and patients with a CR had a significantly longer survival than did incomplete responders (p = 0.003). According to these results the non-FCC diffuse mixed cell lymphoma appears to be a high-grade malignant lymphoma, whereas the FCC type is not.

Description: We grouped 162 patients wtih advanced, diffuse histiocytic lymphoma (DHL) into various morphological subtypes to ascertain whether there were any significant differences in survival among them. These patients were staged and treated from 1972 to 1977 according to the protocols of the Southwest Oncology Group. Of the 159 patients on whom a consensus on the diagnosis was reached, 115 were classified morphologically as having large non-cleaved, 26 as B-immunoblastic, 9 as large cleaved, and 6 as T-immunoblastic. The 3 remaining patients did not fit any of these subtypes, but each had a single prominent nucleolus in most tumor cells (“prominent nucleolus” type). Morphological subdivision of DHL did not identify any subgroup of patients with a significantly longer survival, but clinical parameters such as stage, symptoms, and type of treatment significantly influenced survival times.

Description: Abstract 4489 Introduction: The speed of immune reconstitution post haematopoietic stem cell transplant (HSCT) is important both in terms of post transplant infectious complications and the risk of disease relapse or development of secondary malignancies. It is, therefore, important that immune reconstitution is assessed following the introduction of a new conditioning protocol. Yttrium-90 labelled anti-CD66 has been used in our institution as part of phase I and II clinical trials in both the autologous and allogeneic setting. There is currently, to our knowledge, no published data looking at immune reconstitution post targeted radiotherapy. Patients and methods: 19 patients in our institution have undergone allogeneic HSCT with reduced intensity conditioning and the addition of targeted radiotherapy (both sibling and volunteer unrelated donor). In 18 patients conditioning was with Fludarabine, Melphalan and Campath-1H (one Fludarabine, Cyclophosphamide and Campath-1H). The Yttrium-90 labelled anti-CD66 was shown to add a mean of 35Gy radiation dose to the bone marrow at the highest radiation dose level. 19 control patients were matched as closely as possible for conditioning chemotherapy, underlying disease, prior treatment, age and donor age. Immune reconstitution data are routinely recorded in our institution every three months for the first two years post allogeneic stem cell transplantation. These data were retrospectively collected and analysed for all 38 patients. The p values were calculated using Mann-Whitney tests. Patient characteristics for the two groups are shown below (Table 1). Results: There was no statistically significant difference in the engraftment data between the two groups (Table 2). CD3+, CD4+ and CD19+ results for the two groups are shown in the graphs below: Differences were statistically significant only for CD3+ cells at three and six months post transplant, where levels were higher in the targeted radiotherapy group (p=0.02 and p=0.01 respectively) and at six months for CD4+ cells, where levels were significantly higher in the targeted radiotherapy group (p=0.03). Beyond six months post transplantation no statistically significant difference was seen. No statistically significant difference was seen for B cells at any time point in the first two years. Mean IgG and IgM values were within normal reference ranges for both groups at all time point measured. Donor lymphocyte infusion (DLI) may influence the rate of immunological reconstitution, therefore data regarding DLIs given in the first two years were collected. The control group received slightly more DLIs in the first two years post transplant (a mean of 4.1 compared to 2.8 in the targeted radiotherapy group, p= 0.17) with a mean total CD3+ cell dose of 5.4×106/Kg in the targeted radiotherapy group and 8.3×106/Kg in the control group, p= 0.50. Early Chimerism results (Day 30, 3 months and 6 months post transplant) were also compared, and no statistically significant differences were found. Conclusions: Targeted radiotherapy has great potential for delivering significant doses of radiation to sites of disease without additional toxicity. It appears that the addition of targeted radiotherapy to a reduced intensity conditioning regimen does not adversely affect immune reconstitution post allogeneic stem cell transplantation. Larger patient numbers are required to confirm these findings. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4511 Introduction: The outcome for patients with primary refractory or refractory relapse of acute leukaemia, particularly those with disease present at the time of transplant, remains very poor even using full intensity conditioning schedules. With the aim of improving outcome, we have employed the use of pre-conditioning chemotherapy prior to the delivery of both reduced toxicity (RTC) and full intensity transplants during the subsequent period of cytopenia. Initial results indicate feasibility and efficacy in this poor risk disease. The bi-halogen purine analogue Clofarabine has significant activity as a single agent for the treatment of ALL and AML and is well tolerated, characteristics suggesting Clofarabine is an ideal agent to use in pre-conditioning. Patients and methods: We have treated 11 patients with high-risk, refractory acute leukaemia or advanced myelodysplasia (MDS) using Clofarabine pre-conditioning to reduce disease burden before full intensity or RTC allogeneic transplantation. Patient characteristics for the two groups are shown below (Table 1). Clofarabine was administered as a single agent (40mg/m2/day for 5 days), five received RTC using Busulphan or Melphalan-containing transplant schedules; six full intensity total body irradiation (TBI) based conditioning. Indications for using Clofarabine included severe allergy to Cytarabine, heavy prior exposure to anthracycline or lack of response to other salvage agents. 10 of 11 patients had significant marrow involvement prior to administration of Clofarabine. Results: In this cohort we have not seen additional unexpected complications. One patient who received a RTC regimen, was treated for VOD on clinical suspicion but liver biopsy indicated grade 3–4 haemosiderosis only. Two patients who received TBI-based transplants suffered grade 3–4 mucositis. The extended period of pancytopenia compared with conventional transplant schedules was manageable, with no unexpected infectious complications. The median time to a neutrophil count of 〉 0.5 × 109/l was 18 days post stem cell infusion (range 12–33 days) and to a platelet count 〉 20 × 109/l was 13 days (range 11–32 days). All patients achieved ≥ 98% donor chimerism by day +30. 2 of 11 patients (18%) experienced grade 3–4 acute GvHD (1 cutaneous, 1 gut) which responded to additional immunosuppression. 9 of 10 patients with disease present at the time of Clofarabine achieved substantial bulk reduction or eradication of marrow leukaemic blasts following the Clofarabine. 5 of 11 patients died from disease relapse post allograft at 2–13 months post transplant, including the patient with Clofarabine-refractory disease. 6 patients are currently in CR at 1–77 months follow up. Overall survival for the 11 patients, using Kaplan-Meier analysis, is 48% with a median follow up of survivors of 26 months (range 1–77), shown below in figure 1: Conclusions: The use of Clofarabine as a pre-conditioning agent prior to the administration of the transplant conditioning schedule is well tolerated with both full intensity and reduced toxicity protocols. This approach to allogeneic transplantation shows promise for the therapy of patients with high risk refractory acute leukaemia. Disclosures: Richardson: Genzyme: Advisory board Other, Research Funding. Off Label Use: Clofarabine is licensed for the treatment of relapsed/refractory ALL in paediatric and young adult patients. This paper includes discussion of use in the treatment of AML/high risk MDS for which the drug has orphan drug status in the EU.