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The role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

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.

Background Shortened telomeres are seen in approximately a third of patients with idiopathic aplastic anemia (AA) and they also define risk of relapse, clonal evolution and overall survival. Constitutional pathogenic mutations in the telomere gene complex (TGC) are associated with very short telomeres (typically Methods We report results using telomere length (TL) measurement by quantitative-real time PCR (qPCR) as a screening tool to identify patients for further mutation analysis on a customised panel of 10 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1 and WRAP) using deeply parallel sequencing in a cohort of patients with AA. Using a Polyphen score that predicts a variant to be possibly pathogenic, we have used telomere repeat amplification protocol (TRAP) assay to increase the robustness of classifying a variant as more likely pathogenic, where family history was unrevealing. TRAP assay was performed by introducing the variants into W138V13 cell line and comparing telomerase expression in them to wild type TERT and TERC expression of telomerase. Results From the King's College Hospital database, we screened 295 patients with AA for TL using qPCR. The median age of the cohort was 44.2 years (range18.2- 83.4) with male/female ratio of 57:43. 189 patients (64%) had TL < 10th centile and 111 (37.6%) had TL We identified a further 9 novel variants which were predicted to be possibly pathogenic, but have not been reported pathogenic in literature, as yet. All 9 patients with the novel variants had TL Conclusion Two thirds of patients with AA have TL Figure 1. Telomere length expressed as T/S ratio against age for the different cohorts Figure 1. Telomere length expressed as T/S ratio against age for the different cohorts Figure 2. Expression of telomerase activity in novel TERT and TERC variants compared to Wild-type (positive control) and Mock (negative control) Figure 2. Expression of telomerase activity in novel TERT and TERC variants compared to Wild-type (positive control) and Mock (negative control) Disclosures Kulasekararaj: Alexion: Consultancy.

Shortened telomeres are seen in approximately a quarter of patients with idiopathic pulmonary fibrosis (IPF) and aplastic anemia (AA) and may have a role in disease pathogenesis. Mutations in the telomere gene complex (TGC) are less frequently seen in IPF (1%) and 5-10% in AA. Novel mutations affecting telomere function are increasingly being reported and involve the telomerase genes, the shelterin and helicase complexes, but their prognostic significance remains undefined. We report results on telomere length (TL) using multiplex quantitative real-time PCR (qPCR) and mutation analysis on a customised panel of 10 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1 and WRAP) using deeply parallel sequencing in a cohort of 69 patients with AA and IPF and their effects on clinical variables and response rates. 69 patients (49 with AA, 14 with IPF and 6 with a bone marrow failure syndrome (BMFS) and pulmonary fibrosis; M/F =43/26) with a median age of 49.7 years (range 9-81) had TL (T/S ratio) tested using qPCR on peripheral blood mononuclear cells at diagnosis or pre-treatment. Massively parallel targeted sequencing of the TGC was performed in all cases and constitutional mutations identified using the exome variant server. Clinical variables studied included hemoglobin (Hb) level, neutrophil count (ANC), platelet count (PC), LDH and albumin levels, and response rates to immunosuppressive treatment (IST). In patients with AA and other BMFS, red cell and platelet transfusion dependency, bone marrow cellularity (1) on bone marrow (BM) staining were correlated with TL and presence of any TGC mutations. In patients with IPF, corrected transfer factor (Kco) was correlated with TL and mutation analysis. 44/69 (64%) had TL below the 1st centile and 20 (29%) of them had TL very significantly below the 1st centile compared to a normal cohort (n=180) of age-matched individuals. Median Hb was (11g/dl), ANC was 1.69 and PC was 96x109/l. There was no statistical difference between patients whose TL was below or above the 1st centile for Hb levels, reticulocyte count or PC. 24 patients (34.7%) were red cell transfusion dependent and 15(21.7%) needed platelet transfusions. 42/55 (76.3%) had BM cellularity < 30% and was not statistically significant between patients with TL above or below the 1st centile. Similarly there was no statistical difference in LDH and albumin levels between the two groups. Equal number of patients were treated with IST between the two groups and response rates were similar. Median levels of Kco (0.95) were identical in IPF patients. 62 (89.8%) of patients were alive at a median follow up of 2.15 years (range 0.3-26.8). TGC mutations were seen 16 patients (23%) in the entire cohort with 13/44 (30%) were seen where TL was below the 1st centile as compared to 3/25 (12%) where TL was above the 1st centile (p In our cohort, about two-thirds of patients had TL below the 1st centile. TL, however, does not affect blood counts, BM cellularity and p53 staining, red cell or platelet transfusion dependency or Kco in patients with IPF. A higher proportion of patients with TL below the 1st centile were found to have TGC mutations, although statistical significance was not achieved. Missense mutations in TERT were the most frequent followed by mutations in TERC. Patients with TL Table Variable TL < 1st centile TL > 1st centile Male/Female (Total) 29/15 (44) 14/11 (25) Age < 40 years at diagnosis 23 (52.2%) 7 (28%) TGC mutations % 30 12 Hb (g/dl) 11 11.2 Reticulocyte count ( x 10*9/L) 52 57 ANC ( x 10*9/L) 1.69 2.79 PC ( x 10*9/L) 90 139 Albumin g/dl 42 42 LDH (IU) 211 210 AA/BMFS 37 18 Red cell transfusion dependency % 36 44.4 Platelet transfusion dependency % 18.1 38.8% Cytogenetic abnormality % 15.6 0 BM cellularity < 30% % 77.7 72.2 P53 (Modified quick score >1) % 34.7 40 Treatment with IST Response % 23 (62.1%) 69.5% 11 (64.7%) 72.7% IPF 7 7 Kco 0.95 (0.6-1.9) 0.95 (0.8-1.2) Disclosures Kulasekararaj: Alexion Pharmaceuticals: Speakers Bureau.

Introduction Eculizumab, humanized monoclonal antibody directed against complement component C5, has changed the treatment paradigm of patients with haemolytic paroxysmal nocturnal haemoglobinuria (hPNH). It stabilizes the hemoglobin by inhibiting complement mediated intravascular haemolysis, prevents thrombotic complications and significantly improves the quality of life. The data for hematopoietic stem cell transplant (HSCT) in eculizumab era is lacking, although patients with large PNH clone and severe aplastic anaemia (SAA) should be treated as dictated by their predominant clinical manifestation. HSCT should be the preferred option for PNH patients on eculizumab who develop overt bone marrow failure (BMF) during their clinical course or presenting concomitantly with SAA and PNH. The optimal use of eculizumab in the peri-transplant setting to prevent haemolytic/thrombotic complications and also the ideal conditioning regimen to eradicate the PNH clone is yet undetermined. Patients and Methods We analyzed 7 patients with PNH and SAA who underwent HSCT at our Centre for their BMF. Of these 5 patients were on eculizumab for a median of 8 months (range 4-12) for PNH prior to worsening of their aplasia necessitating regular blood product (red cells and platelet) support. Two patients were initiated eculizumab during the pre-transplant period for their coexisting haemolytic PNH and SAA, whilst donor search was undertaken. The median age 32 years (range 18-56 yrs) and M/F was 4;3. The median disease duration was 48 months (range 4-252 months). All patients had evidence of haemolysis, elevated LDH and a large granulocyte clone (median 95%, range 57-100). The red cell clone was The conditioning received was a fludarabine/BU-based RIC HSCT, of these 4 received T-cell depletion using ATG (anti-thymocyte globulin) and 1 with alemtuzumab (100% CD3+ cells co-expressed CD52). Two patients received a haploidentical transplant using the Seattle protocol with post-transplantation cyclophosphamide. The source of stem cells in all 7 was GCSF mobilized peripheral blood stem cells. Eculizumab was continued until the transplant and the last dose was given on the start of the conditioning protocol, i.e 7 days prior to cell infusion. Results Pre-transplant 3 patients successfully underwent embryo (n=1) and ova (n=2) cryopreservation with ovarian hyper stimulation protocol (supraphysiological doses of estrogen) with eculizumab used prophylactically to cover thrombotic complication, as they were severely thrombocytopenic and were unable to receive low molecular weight heparin. Eculizumab given during the immediate pre-transplant period did not increase the infectious complications. Neutrophil and platelet engraftment occurred at 16 and 21 days respectively. The PNH clone was undetectable on D+14 and none was seen after engraftment with normalization of LDH. No clinical evidence of venoocclusive disease (VOD) was seen. Acute GVHD (grade 2) and Chronic GvHD (skin and gut) were observed in one patient each. Two patients failed to engraft, of which one was successfully rescued with a haploidentical HSCT although the PNH clone disappeared following the conditioning used in the first transplant for both patients. Full donor chimerism in unfractionated, CD3 and CD15 lineages was achieved at D100 post HSCT. Conclusion This data demonstrated the feasibility of using eculizumab in patients with PNH and SAA, during the peri-transplant setting. The use of myeloablation in conditioning facilitates the eradication of the PNH clone. The successful use of eculizumab in preventing the thrombotic risks associated with ovarian hyper stimulation protocol is also evident. It is theoretically possible that VOD post HSCT could be exaggerated in the presence of another thrombotic condition (PNH), but we did not observe VOD to warrant eculizumab in the immediate post-transplant period. Larger studies and uniform strategies are needed to evaluate the optimal use of anti-complement therapy for SAA/PNH patients undergoing HSCT. Disclosures: Kulasekararaj: Alexion: Consultancy, Honoraria, Speakers Bureau. Large:Alexion: Speakers Bureau. Marsh:Alexion: Honoraria, Speakers Bureau.

Abstract 1340 We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response. Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures: No relevant conflicts of interest to declare.