ALBERT

Description: ABSTRACT The exact pattern, process and timing of the human re-colonization of northern Europe after the end of the last Ice Age remain controversial. Recent research has provided increasingly early dates for at least pioneer explorations of latitudes above 54°N in many regions, yet the far north-west of the European landmass, Scotland, has remained an unexplained exception to this pattern. Although the recently described Hamburgian artefacts from Howburn and an assemblage belonging to the arch-backed point complex from Kilmelfort Cave have established at least a sporadic human presence during earlier stages of the Lateglacial Interstadial, we currently lack evidence for Younger Dryas/Greenland Stadial 1 (GS-1) activity other than rare stray finds that have been claimed to be of Ahrensburgian affiliation but are difficult to interpret in isolation. We here report the discovery of chipped stone artefacts with technological and typological characteristics similar to those of the continental Ahrensburgian at a locality in western Scotland. A preliminary analysis of associated tephra, pollen and phytoliths, along with microstratigraphic analysis, suggest the artefacts represent one or more episodes of human activity that fall within the second half of GS-1 and the Preboreal period. © 2015 The Authors. Journal of Quaternary Science published by John Wiley & Sons, Ltd.

Description: Abstract 731 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure. Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure. To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders. In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: In past 22 years, we have identified using flow cytometry 705 patients with detectable PNH (GPI deficient) populations of granulocytes, monocytes and red cells in the peripheral blood in samples sent for diagnosis. We undertook an analysis of presenting clinical features, blood count data and PNH clone sizes in order to better understand the natural history and provide a more objective classification of disease. Based on serial flow cytometry measurements of PNH clone sizes, we also studied disease stability, frequency of recovery and progression with an aim to guiding future management of individual patients. Clinical classification of patients at presentation was as follows; aplastic anemia (58%), hemolytic anemia (36.1%); myelodysplasia (2.5%); thrombosis (2.4%); hemolysis & thrombosis (0.6%), myeloproliferative neoplasm (0.3%); Fanconi anemia (0.1%). Median age at presentation was 45 years (observed range 0.5 – 90 years) and the Male:Female ratio was 1.05. Descriptive statistical analysis of presenting blood count data revealed novel gender related features not previously described in PNH. At presentation, pancytopenia was found in 61% of male and 47% of female patients; a normal blood count was present in only 0.3% of males and 4% of females. A combined low red blood cell count (RBC) and white cell count (WBC) was the most frequent bicytopenia affecting 19% males and 22% females. Leucopenia as a sole abnormality did not occur in males and was present in

Description: Monoclonal chronic lymphocytic leukemia (CLL)–phenotype cells are detectable in 3.5% of otherwise healthy persons using flow cytometric analysis of CD5/CD20/CD79b expression on CD19-gated B cells. To determine whether detection of such CLL-phenotype cells is indicative of an inherited predisposition, we examined 59 healthy, first-degree relatives of patients from 21 families with CLL. CLL-phenotype cells were detected in 8 of 59 (13.5%) relatives, representing a highly significant increase in risk (P = .00002). CLL-phenotype cell levels were stable with time and had the characteristics of indolent CLL. Indolent and aggressive clinical forms were found in family members, suggesting that initiation and proliferation involves distinct factors. The detection of CLL-phenotype cells provides a surrogate marker of carrier status, potentially facilitating gene identification through mapping in families and direct analysis of isolated CLL-phenotype cells.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by intravascular hemolysis, often resulting in the need for red blood cell (RBC) transfusions. PNH RBCs lack two complement regulatory molecules - CD59, a terminal complement inhibitor, and CD55, a C3 convertase inhibitor. Eculizumab, a humanized monoclonal antibody that inhibits terminal complement by binding to C5, effectively controls intravascular hemolysis as determined by a dramatic reduction in lactate dehydrogenase (LDH) to levels in or just above the normal range. Control of intravascular hemolysis in these patients led to a reduction in, or cessation of, RBC transfusions. During eculizumab treatment, a majority of patients demonstrate evidence of residual, low-level hemolysis; LDH levels remain slightly elevated, haptoglobin levels are low or undetectable, and bilirubin levels are above normal. We hypothesized that this low-level residual hemolysis may be due to clearance of PNH RBCs through a C3b-mediated mechanism. Therefore we investigated C3 deposition on RBC in PNH patients before and on eculizumab. A direct antiglobulin test (DAT) using monoclonal anti-C3d was positive in 29 out of 39 PNH patients on eculizumab. Of these 29 DAT-positive patients, who were all receiving transfusions, 25 had DAT testing prior to eculizumab therapy and only one of these was positive. DAT was negative in all of 8 normal volunteers. By two-color flow cytometric analysis with anti-CD59 and anti-C3, the majority of patients on eculizumab demonstrated three distinct RBC populations: CD59+/C3− (normal RBCs); CD59-/C3− (PNH RBCs without C3 coating); and CD59-/C3+ (PNH RBCs coated by C3). No CD59+/C3+ RBCs were observed. Of 21 DAT positive eculizumab treated patients tested, the median proportion of total RBCs that were C3b positive was 17.6%. 18 of 29 [62%] eculizumab patients with a positive DAT received at least one transfusion during eculizumab therapy compared with 1 of 10 [10%] for DAT negative patients (p=0.01), although even patients who did not become transfusion independent during eculizumab treatment showed a marked reduction in transfusion requirement. The median hemoglobin value for the 29 DAT positive eculizumab patients was 9.8 g/dL compared with 11.3 g/dL in the 10 DAT negative eculizumab patients (p= 0.08). No apparent relationship between LDH and DAT positivity was observed. It is proposed that resolution of intravascular hemolysis in PNH patients on eculizumab results in deposition of C3b on the surface of PNH RBCs which may explain, at least in part, the residual low level hemolysis occurring in some patients. This appears to be a previously undescribed mechanism of RBC clearance in PNH, most likely obscured by the rapidity of intravascular hemolysis in the absence of eculizumab therapy. Despite the low-level residual hemolysis, patients continue to receive significant benefit from eculizumab treatment.

Description: Flow cytometric analysis of GPI-linked antigens has had a major impact on the diagnosis of PNH. Significant numbers of patients with aplastic anemia have small PNH clones, and due to the precision in clone size measurement, reliable serial monitoring can now be undertaken although the clinical value of this is not proven. From our series of 234 PNH patients, we analysed clinical correlates between disease type and red cell and granulocyte peripheral blood clone sizes as determined by flow cytometry at presentation. For hemolytic patients (n = 99) the mean PNH clone sizes were: granulocytes 84.8%; red cells 45.3% (type III cells 33.6%). For aplastic patients (no macroscopic hemolysis) the mean clone sizes were: granulocytes 18.5%; red cells 6.4% (type III cells 4.5%). The two groups were statistically different (Mann Whitney U; P90% granulocyte clones (n = 30; mean follow up 48 months) with virtually all their hematopoiesis maintained from PNH stem cells have clone sizes that remain stable and their clinical behavior suggests that their PNH can persist for up to 40 years. The second group of patients (n = 16) were those with hemolytic PNH with granulocyte clones of 90% granulocyte clone group (2/30) and developed as a terminal event, one with GPI-MDS, and the second with a rapid emergence of GPI+MDS. One patient in the aplastic group showed progression to AML (1/34). 27% of patients had an improvement in cytopenias with concurrent decrease in PNH clone size. For hemolytic patients with granulocyte clones of

Description: Abstract 639 PNH is an acquired clonal hemolytic anemia associated with severe symptoms, life-threatening thrombosis, pulmonary hypertension (PH), chronic renal impairment (CRI) and bone marrow failure resulting in reduced quality of life (QoL) and survival (median: 10 – 15 years). Eculizumab is a monoclonal humanized antibody that inhibits terminal complement activity and thereby prevents intravascular hemolysis, reduces transfusions, improves QoL and protects against PH, CRI and thromboses. Eculizumab was approved for PNH in 2007 as a result of studies with short follow-up and with no information on it's impact on survival. We present data on 79 patients treated with eculizumab from the Leeds PNH Center between May 2002 – July 2010 including 34 patients from the clinical trials and a further 45 treated since, funded in England by the National Commissioning Group (NCG) or locally in Scotland and Wales. The NCG indications for treatment include transfusion-dependent hemolysis (4 or more transfusions in 12 months) or significant PNH-related complications (i.e. thrombosis or renal failure) regardless of transfusion history. Three patients did not fulfil these criteria but were treated for profound symptoms as agreed with the NCG. Forty men and 39 women were treated for a mean of 39 months (1-98). Median age at diagnosis was 37yo (12-79) and at initiation of eculizumab was 46yo (14-84). Median granulocyte clone size at the start of eculizumab was 96.38% (41.78-99.98). Patient survival on eculizumab was compared with age and sex matched controls obtained using data from the UK Office of National Statistics. We previously published that there was a significantly worse survival for PNH patients compared to matched controls with ~ 50% of patients dying as a result of PNH (Hillmen et al., NEJM 1995). In the current series there was no difference in mortality between patients on eculizumab and the normal population (P=0.46; Fig 1). Patients over 70yo had worse survival (P=0.0042) with none of the 45 patients under the age of 50yo dying. Three patients died: a 55yo man from metastatic caecal carcinoma diagnosed prior to eculizumab, a 76yo woman who died from pneumionia following a long history of recurrent bronchopneumonia prior to eculizumab and a 79yo man with a preceding history of ischaemic heart disease who died from cardiac failure. Three patients have had a clonal evolution of their PNH, 1 to acute myeloid leukemia and 2 to myelodysplasia. Two patients have stopped eculizumab: - 1 due to pre-existing aplastic anemia and 1 with spontaneous remission of his PNH. Seventy four patients remain on eculizumab to date.Figure 1Kaplan-Meier survival plots depicting PNH patients on eculizumab compared to age and sex matched controlsFigure 1. Kaplan-Meier survival plots depicting PNH patients on eculizumab compared to age and sex matched controls There were 34 thrombotic episodes in 21/79 patients (27%) prior to eculizumab and only 2 thromboses since. Importantly primary prophylaxis with warfarin has now been stopped in 21 patients (mean duration of 8.4 months (range: 1–25) and total of 〉 14 years) with no thromboses occurring. Seven patients had thrombosis within 12 months prior to starting eculizumab and they had no further thromboses on therapy. Therefore eculizumab is effective in preventing new, or evolution of pre-existing thrombosis, and appears to remove the need for primary prophylactic anticoagulation. We have not systematically stopped the anticoagulation of patients with a previous history of thrombosis. Patients had a mean of 19.9 units transfused (0-156) in the 12 months before eculizumab. Of the 64 patients on eculizumab for at least a year, 43 (67%) have been transfusion independent for 〉12 months. Twenty one patients still needing transfusions had a significant (P=0.028) reduction in their mean requirement of 24.6 (4-52) units in the 12 months before eculizumab compared to a mean of 14.6 (2-50) units in the last 12 months. Eculizumab is well tolerated long-term (〉 8 years of therapy) with continued improvement in PNH associated symptoms, reduction in transfusion requirements and a higher proportion of transfusion independent patients than previously seen. There is a significant reduction in the development of thromboembolism on eculizumab therapy and importantly we have been able to stop primary prophylaxis with warfarin in 21 patients without any thrombotic complications. We demonstrate for the first time that eculizumab has a major impact on survival in PNH so that survival is comparable to an age- and sex-matched control population. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Richards:Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Arnold:Alexion Pharmaceuticals: Honoraria. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.