ALBERT

Description: Introduction Heterozygous RTEL1 mutations have recently been described in familial pulmonary fibrosis (PF) but are not known to be associated with cytopenias or bone marrow failure (BMF), in contrast to heterozygous mutations in other telomere maintenance genes TERT, TERC and TINF2. Constitutional BMF syndromes typically present with less severe pancytopenia and it is often unclear if they have hypocellular MDS (hypoMDS) or non-severe AA (NSAA) morphologically. Methods We screened 284 patients with idiopathic AA or uncharacterised BMF and 172 patients with MDS or acute myeloid leukemia (AML) for TL and RTEL1 variants, and for the other currently known telomere gene complex (TGC) mutations, after excluding patients with Fanconi anemia, DBA or other known inherited BMF syndrome. TL was measured using a monochrome multiplex quantitative PCR method on peripheral blood mononuclear cells. Illumina Nextera-amplicon sequencing was used to screen exons of the DC genes (DKC1, TERC, TERT, RTEL1, CTC1, NHP10, NOP2, USB1, WRAP53, TINF2, PARN and ACD) by MiSeq platform. Constitutional DNA was also analysed in 10 patients (skin 9, buccal swab 1) with RTEL1 variants. A targeted gene panel of 24 genes of an Illumina Tru-Seq Custom Amplicon workflow and platform was used to identify genes frequently mutated in MDS/AML. Impact of mutations was predicted based on 3D structure information from comparative modelling for the helicase domain, comprising the HD1 and HD2 subdomains, a Fe-S cluster and an ARCH domain, and for two harmonin-like (HML) domains and a RING finger domain, located in the C-terminal regulatory region of RTEL1. Results Heterozygous RTEL1 variants were identified in 20 (4.4%) patients. RTEL1 variant allele frequency (VAF) was 45-70% consistent with heterozygous inheritance in all cases. TL was short in 18 (90%) patients, being 〈 1st centile in 15 and

Description: Telomerase complex maintains telomeres and protects genomic DNA from degradation during cell divisions. Abnormal telomerase function can result in chromosomal instability predisposing to malignant transformation. Short telomere is a typical feature of inherited bone marrow failures syndromes (BMFs), especially dyskeratosis congenital (DC), caused by mutations in genes encoding components of the telomerase gene complex (TGC), shelterin proteins and DNA helicases. Telomere attrition have been associated with leukemic transformation in myelodysplastic syndromes (MDS), as well as complex cytogenetic aberrations, and also with the development of secondary MDS and acute leukemia (AML) after chemotherapy. However, the incidence of TGC mutations in de novo MDS remains largely unknown. Recurrent somatic mutations in genes involving epigenetic, spliceosome, cell signaling and proliferation pathways are common in MDS and have prognostic significance. Identifying specific associations between mutational patterns helps characterize disease biology and thereby improve the therapeutic strategies To determine the incidence of TGC mutations and study theassociation of TGC mutation patterns with recurrently mutated genes in MDS. To correlate TGC mutations with telomere length, clinical phenotypes and outcome of patients. We undertook a massively parallel targeted sequencing of all 10 TGC, (TERT, TERC, TINF2, NHP2, NOP10, RTEL1, CTC1, DKC1, USB1 and WRAP53) in a cohort of 174 MDS patients. Furthermore, we measured the telomere length (T/S ratio) by a multiple quantitative real-time PCR in bone marrow mononuclear cells. Additionally, in 151/174 MDS patients, we studied 22 recurrently mutated MDS-associated genes (MGP) by targeted sequencing. Among the whole cohort, 61% were male. The median age of patients was 63 years (range 17–87). WHO subtypes were 45 RA/RARS/isolated de5q (26%); 50 RCMD/RCMD-RS, (29%); 41 RAEB 1/2, (24%); 8 AML secondary to MDS, (5%); 8 (5%) MDS/MPD and 3 CMML (2%). IPSS cytogenetic risk groups were: 108 patients with good risk (62%), 21 intermediate (12%) and 32 poor risk, (18%) and cytogenetics failed in 10 patients (6%). IPSS categories were low risk 41(24%), intermediate-1: 54 (31%), intermediate-2: 30 (17%), high risk: 13 (7%) and 10 (6%) patients were not evaluated (proliferative CMML and MPD/MDS). Transfusion dependency was present in 80 patients (46%). Twenty nine TGC mutations were present in 26 patients (15%)(figure 1). Twenty-three patients (88%) had TERT mutations, 3 RTEL1 mutations (13%) and 1 TINF2 mutations (4%) with variant allelic frequency around 50%. Two patients presented more than one mutation in TGC genes. Most of mutations in TGC genes were previously described as germ line variants inpatients with DC and inherited aplastic anemia. All mutations found in TERT gene were missense. In patients with TGC mutations, the median T/S ratio was 1.1 (range 0.4–3.5), shorter than the T/S ratio of age-matched controls, although no statistically significant difference was seen in T/S ratio when compared to wild type. (P=0.527). TGC variations did not correlate with clinical features such as age, cytogenetic risk or IPSS, and had no impact on the overall survival (P=0.659). In 151 MDS patients, 73% (n=110) had at least one known somatic mutation in the MGP (21% TET2, 15% ASXL1, 14% TP53, 11% DNMT3A, 11% U2AF1, 9% IDH2, 9% SRSF2, 6% EZH2, 4% NRAS, 4% CEBPA, 3% SF3B1, 3% RUNX1, 2% JAK2, 2% FLT3, 1% cCBL). Among the MGP mutated patients, 13% carried also TGC mutations concurrently (Table 1). Chromatin remodeling gene mutationswere less frequent in patients with TGC mutations (P=0,001) as compared to patient with wild type TGC. We show TGC mutations are frequent in MDS patients (15%). The presence of known TERT variants seen in our cohort demonstrates a clear pathogenic association between MDS phenotype and telomerase mutations, rather than these being bystander variants. Although the heterozygous nature of these abnormalities indicates an inherited variant, the absence of telomere shortening argues against this concept and needs further evaluation. Chromatin remodeling gene mutations are less frequent in patients with TGC mutations. These findings suggest that defective telomere maintenance through TGC mutations might play an important etiological role in the multistep process in pathogenesis of a subset of MDS. Figure 1 Figure 1. Disclosures Mufti: Onconova Therapeutics, Inc: Research Funding.

Description: The diagnosis of myelodysplastic syndrome, especially in the absence of ring sideroblasts, excess blasts or cytogenetic abnormalities, is challenging in daily clinical practise and is biased by poor inter-observer concordance. The array of genetic abnormalities identified in MDS by targeted sequencing might aid in diagnosis, especially in cases with mild cytopenias and subtle dysplastic features. However, the presence of such somatic mutations in 'normal' elderly population leading to clonal haematopoiesis of indeterminate potential (CHIP) further confounds use of somatic mutations to diagnose MDS. We set out to prospectively evaluate 647 unselected, consecutive patients with variable degrees of cytopenia referred to our molecular diagnostics laboratory at Kings College Hospital with a 24 gene targeted mutation panel initially using Roche 454 amplicon sequencing panel and subsequently an Illumina MiSeq platform. The average read depth was 200 x and all known pathogenetic variants 〉10% variant allele frequency (VAF) were reported if present in the Cosmic database.. The median age was 57 years (range 16-92) and sequencing was performed on peripheral blood DNA in 58% (374/647) and bone marrow in the remainder. One third (36%, 234/647) harboured a somatic abnormality, with 51% (120/235) having more than one mutation. 443 mutations were detected in 235 patients, with 1.9 mutations/patient. The median VAF was 38% (9-99%) and the most frequently mutated genes were TET2 (N=67, 12%), ASXL1 (N=63, 10%) and SRSF2 (N=45, 7%). All 24 genes, except KDM6A, was mutated at least in one patient. Comprehensive clinical information was available for patients seen in our institution (n=422) and revealed a similar overall frequency of mutation detection (30%, 128/422). Mutations were extremely rare in patients (8%, N=102) presenting with single cytopenia (especially neutropenia) and a non-diagnostic bone marrow with no morphological features of MDS. None of patients with aplastic anaemia (23%, n=99) at diagnosis had mutations detected by our panel, although 3 acquired abnormalities before or at the time of MDS transformation. Of the 186 patients with MDS, 56% (N=105) harboured mutations, with a relatively low frequency in cases with RCMD/RA (26%, 26/98) compared to published literature. Contrarily mutation frequency was higher in patients with RARS, RAEB and AML with somatic aberrations detected in 100%, 70%, 90% cases respectively. No cases of hypoplastic MDS had detectable mutations. Further analysis, by including variants with VAF 5-10% (N=9) and variants of unknown significance (VUS) (N=31) increased the mutation frequency, i.e. mutations were present in 40% (39/98) of RCMD/RA patients and 17% (17/102) of cytopenic patients with no evidence of morphological MDS. Overall the ability to detect mutations only in a third of RCMD/RA patients shows the considerable drawback in using mutations alone for diagnosis in RCMD which is currently based purely on morphology which is subjective and with poor concordance between morphologists. The cautious/conservative reporting of only pathogenetic variants with VAF 〉10% in our routine clinical practice led to decreased frequency of detectable mutations in MDS compared to published literature. This also avoided the reporting of variants of unknown clinical significance in the context of isolated or mild cytopenia in the absence of overt dysplasia in our cohort of unselected cytopenias. The reported incidence of mutations in MDS is influenced by the chosen VAF cut-off, dynamic evolution of the COSMIC database, inclusion of mutations that have not yet been reported in the literature as pathogenic and inclusion of stop or missense mutations predicted to be pathogenic. This appears to differ in this real-life referral population compared with well-defined and curated MDS cohorts. Our current diagnostic panel has been expanded to include a repertoire of 35 genes and we are planning to combine this with SNP-A karyotyping to better characterise patients with cytopenia, early MDS and cases with subtle dysplasia to improve certainty of diagnosis. Disclosures Marsh: Alexion pharmaceuticals: Honoraria.

Description: Introduction Alemtuzumab is a monoclonal anti-CD52 antibody, a pan-lymphodepleting immunosuppressive agent in common use as part of conditioning for allogeneic stem cell transplantation (Allo-HSCT) in United Kingdom and many other centres across the globe, with benefits related to reduced graft versus Host disease (GVHD) and lower non-relapse mortality (NRM). However, evidence for effective dose schedule in Allo-HSCT remains debatable with some concerns related to delayed immune reconstitution and increased relapses with higher dosages; but increased risk of acute and chronic GVHD observed with lower doses. We present a large single-centre UK experience evaluating differential dosage effect of Alemtuzumab on HSCT outcomes. Methods: We retrospectively evaluated 330 patients undergoing Allo-HSCTs for myeloid malignancies (AML/MDS/MPNs) during a 10-year period (Jan 2010 to April 2019) at King's College Hospital, London. Two dosage schedules of Alemtuzumab based T-cell deplete conditioning regimen using 100mg (n-96) were compared to those receiving 60mg (n-234;

Description: Introduction Telomere length is shortened in patients with idiopathic aplastic anemia (AA) and other bone marrow failure disorders (BMFD) and predicts risk of clonal evolution (CE), relapse and overall survival (OS). Telomereopathies predominantly cause bone marrow failure, are multi-systemic disorders with variable penetrance, and may involve inter-play of other factors in disease manifestation and organ affliction. Telomere length (TL) in AA and other hypocellular BMFD, independent of mutations in telomere genes (TGC), has not been studied as a scoring tool, as well predicting the risk of affliction of other organ/systems in these disorders. We systematically review a large cohort of 472 patients in a single centre with AA/BMFD using TL and TGC analysis as a discriminator, to study risk of CE and OS, manifesting with liver/lung and skin complications, cancer predisposition and likelihood of a family member presenting with cytopenias. Methods We screened 1060 consecutive patients at a single centre from the years 2011-18, with AA or unexplained cytopenias for telomere length (TL) analysis using a multiplex qPCR methodology as described by Cawthon et al. 472 (44.5%) patients had TL less than the 25th centile, of whom 243 had

Description: Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Description: Abstract 3826 Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders characterized by ineffective haematopoiesis. Age, gender, mutagen exposure and telomere length have been linked to MDS. Previous studies have demonstrated that patients with MDS have shortened telomeres compared to healthy controls, based on measurements obtained from either peripheral blood mononuclear cells, peripheral blood granulocytes, bone marrow, and/or CD34+ stem cells, indicating that individuals with shorter telomeres may be at increased risk of developing MDS. To investigate the association between telomere length and pathogenesis of MDS, we measured the telomere length (T/S ratio) by a multiplex quantitative real-time PCR in bone marrow mononuclear cells of 307 MDS patients and PBMCs of 182 healthy controls. In the assay, the relative telomere length is measured by the fluorescence signal of telomere amplification normalized to the signal obtained from a single copy gene, albumin. The median age of patients was 64 years (range 17–89 years). The median haemoglobin levels was 9.9 g/dl(IQR 8.6–11.6), neutrophil 1.4 × 109 /l (IQR 0.6–2.99) and platelet 98x 109 /l (IQR 39–187).The WHO subtypes were RA/RARS/Isolated de5q; 50 (16%), RCMD/RCMD-RS; 116 (38%), RAEB 1/2; 70 (23%), AML secondary to MDS; 29(9%),therapy related MDS; 18 (6%) and MDS/MPD; 24 (8%). IPSS cytogenetic risk groups were; good risk-199(65%), intermediate -34 (11%) and poor risk-55(18%) and cytogenetics failed in 19 patients (6%). The IPSS categories were, low risk: 80(26%), intermediate-1:97(32%), intermediate-2:50 (16%), high risk: 26 (9%) and 54(18%) patients were not evaluable (proliferative CMML and MPD/MDS).Transfusion dependency was present in 141(46%) patients. Progression to AML occurred in 68 patients (20%). In healthy controls (n=182; age range: 2–90 years), the T/S ratios measured in PBMCs demonstrated a progressive decline with ageing (Y=2.3–0.014X; R2=0.2417; P 〈 0.0001). The median telomere length was 0.97(range 0.3–2.8).In patients with MDS, T/S values did not show a correlation with age (P=0.327). Neither statistically difference in T/S values was observed between male and female patients (P=0.976). However, compared to PBMCs of the age-matched healthy controls, the mean T/S value obtained from BMNC of the MDS patients was significantly lower (P

Description: Abstract 1304 Disease relapse following allogeneic haematopoietic stem cell transplant (HSCT) for MDS/AML remains one of the primary causes of treatment failure, particularly in the setting of T-cell depleted reduced-intensity conditioning (RIC). Pre-emptive immunotherapy such as the use of donor lymphocyte infusions (DLI) is one strategy utilized to prevent relapse, although this approach may be associated with an increased risk of GvHD. We present our experience, evaluating toxicity and efficacy of pre-emptive DLI for the treatment of mixed donor chimerism (MDC) in a cohort of high risk MDS/AML patients who received a uniform alemtuzumab-based RIC HSCT protocol. 37 patients received a RIC protocol using fludarabine, busulphan and alemtuzumab. The stem cell source was BM (n=8) and PBSC (n=29) with a median cell dose of 2.40 and 5.34×106̂ CD34/kg respectively. The donor source was from HLA-matched sibling (n=20), HLA-matched unrelated (n=14) with 3 1-allele mismatched unrelated donors. Peripheral blood CD3 chimerism was monitored at day 30, 60, 100, 180 and 1 year post-HSCT. Patients with MDC (defined as CD3

Description: The use of IV busulphan (Bu) has reported improved bioavailability with lower variability of busulphan plasma levels, and may reduce the effects of transplant related toxicity particularly in the context of myeloablative regimens. In contrast, there is limited data avaliable on the use of IV Bu in the setting of reduced conditioning (RIC) regimens. We retrospectively analysed data from 163 consecutive patients treated at our centre for myeloid malignancies (AML n=78, MDS/MPD n=73, CML n= 12) using RIC HSCT, and evaluated the engraftment and chimerism kinetics as well as the early transplant outcomes between patients receiving either IV or oral Bu. Patients received fludarabine (30mg/m2 × 5 days), alemtuzumab (20mg × 5 days) and either oral Bu (4mg/kg × 2 days) or IV Bu (3.2mg/kg × 2 days). 84 consecutive patients received oral Bu up to Jan 2004. Thereafter, 79 consecutive patients received IV Bu. The median age of the cohort was 53 years(range: 19–72). 50 patients received stem cells from HLA-matched sibling donor, and 113 from a volunteer unrelated donor. 128 patients received PBSC and 35 received BM stem cells. 64 patients had early disease vs 99 advanced disease (advance disease defined as AML 〉 CR1, CML 〉 CP1, MDS with RAEB or AML with multilineage dysplasia). Median follow-up was 1531 days (range: 853–1987) for the oral Bu group and 551 days (range: 228–1072) for the IV Bu group. There was no difference between groups in terms of recipient age, stem cell dose/source, donor type, prior therapies or disease type. However, patients receiving IV Bu had more advanced disease compared with oral Bu (70% vs 52%, p=0.03). There was no significant difference in the median time to neutrophil and platelet regeneration between groups. In contrast lymphoid(CD3) engraftment was significantly delayed in the IV Bu group, with 35% vs 53%(p=0.04) recipients achieving full donor chimerism(FDC) at day 30, and 32% vs 51%(p=0.03) at day 100. The cumulative incidence of acute GvHD was lower in the IV Bu group 18% vs 29%(p=0.04), with no difference in the cummulative incidence of chronic GvHD between groups. Early(1-year) transplant-related mortality (TRM) was higher in the oral Bu group(TRM:: 25%vs13%, p=0.07) with a significantly lower overall survival(60%vs79%, p=0.02) primarily as a result of death from GvHD and related infection complications. There was no significant difference in relapse incidence at 1 year between cohorts(IV Bu 27% vs oral Bu 23%, p=0.84). On multivariate analysis, the type of conditioning regimen had no effect on the overall transplant outcomes. Attainment of CD3 FDC at day 30(HR: 2.14, 95%CI: 1.26–3.63, p

Description: Introduction Aplastic anaemia (AA) affects 1-2 per million of the UK population. At least 50% of patients have a Paroxysmal Nocturnal Hemoglobinuria (PNH) clone, which may either require monitoring or concomitant management with the aplasia if clinical indications for eculizumab are fulfilled. There is a paucity of data available to guide concurrent treatment for AA and PNH. Method The UK PNH National Service (Leeds and London) database was reviewed retrospectively. Patients commencing eculizumab within a year of AA treatment, or those treated for aplasia who were already established on eculizumab were selected. Response to AA therapy was assessed according to aplastic anaemia guidelines. Results Twenty six patients were identified who were treated with eculizumab and immunosuppressive therapy (IST) concurrently. Median age 39.5 years (range 7-75). Median granulocyte clone immediately prior to eculizumab was 82%. Ten patients had severe AA, 15 non severe and one had hypoplastic MDS. Treatment varied as per national guidelines dependant on patient's age, patient choice, prior treatment and co-morbidities. Eight patients received ATG and ciclosporin (median follow-up 21 months post ATG treatment), 14 patients received ciclosporin monotherapy (median follow-up from commencement of ciclosporin 29 months). One patient received androgens as a single agent achieving a partial response (PR),3 patients underwent haematopoietic stem cell transplant (HSCT) as initial treatment at the time of eculizumab and IST overlap (5 other patients received HSCT as discussed below). Six of the 8 (75%) patients receiving ATG+Ciclosporin responded, one patient achieved complete remission (CR) and five PR, one of whom subsequently achieved a CR with androgen therapy. Two patients did not respond and both achieved a CR after HSCT. In five patients who had data available six months post ATG there was no change in the median granulocyte or monocyte PNH clone size. Of 14 patients treated with single agent ciclosporin, 1 patient achieved CR; 7 PR, 2 of whom achieved a subsequent CR with further therapy ( androgens N=1, HSCT N=1); 6 had no response, 3 of whom received subsequent treatment, two HSCT (one achieved a CR and one died), and one eltrombopag (response awaited; follow-up 12 weeks) . Three patients underwent HSCT during the defined entry criteria above. 2 had frontline HSCT, and 1 had a transplant due to late relapse following ATG and ciclosporin 6 years prior. Five other patients underwent HSCT as discussed above for second or third line treatment (ATG and ciclosporin N=2, ciclosporin single agent N=3). All transplant patients achieved a CR except 1 who died during the procedure. All 7 patients who survived transplant stopped eculizumab due to resolution of PNH. Six of 26 (9.8%) patients died,1 who achieved a CR with HSCT and died 2 years later of GVHD , two patients who had achieved a partial response to treatment one of whom died of infection, two had not responded to treatment, and one HSCT recipient died during the procedure. Fourteen age matched controls not on eculizumab received similar therapies, 9 of whom received ATG and ciclosporin, median follow-up from ATG commencement 37 months. Four of the 9 had a CR, 1 had a CR then a relapse with no response to the re-introduction of ciclosporin, 3 had a partial response one of whom achieved a CR with androgens and 1 patient had no response achieving a CR with HSCT. 5 matched controls were treated with ciclosporin single agent, median follow-up from ciclosporin commencement 115 months. Two patients had a CR, 1 had a PR then relapsed, 2 had no response. Conclusion This is the largest reported cohort of patients receiving concurrent treatment for both AA and PNH. The presence of symptomatic PNH requiring complement inhibition should not influence AA treatment decisions. The response rates for IST in patients on eculizumab compared with age matched controls were similar, with similar numbers of patients achieving CR or PR with immunosuppressive therapy, suggesting no detriment to response to IST with concurrent eculizumab therapy. Therefore, patients with concurrent AA and PNH should be treated as per AA guidelines and PNH can be managed concurrently if required. This strategy will increasingly be required in the future, especially with improved life expectancy for PNH patients receiving complement inhibition therapy. Eculizumab therapy does not appear to affect response to IST for AA patients Disclosures Griffin: Alexion Pharmaceuticals: Honoraria, Other: Conference support. Kulasekararaj:Alexion pharmaceuticals: Honoraria, Other: conference support. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding. Munir:Alexion pharmaceuticals: Honoraria. Richards:Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding. Arnold:Alexion Pharmaceuticals: Honoraria. Riley:Alexion pharmaceuticals: Other: Travel for conference. Marsh:Alexion pharmaceuticals: Honoraria. Hill:Alnylam Pharmaceuticals: Consultancy, Honoraria.