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There is no standard or widely effective treatment of patients with moderate aplastic anemia (MAA) or hypo-productive uni-lineage cytopenias (UC). Eltrombopag (EPAG), a small molecule thrombopoietin mimetic, has previously been shown to result in durable multi-lineage hematologic responses with low toxicity in patients with refractory severe aplastic anemia (SAA). Its safety and efficacy in MAA are unknown. This prospective phase 2 study enrolled previously untreated and treated MAA and UC patients with clinically relevant cytopenias. EPAG was administered at doses escalating from 50 to 300 mg/d. Hematologic responses were assessed at 16 to 20 weeks. Responding patients were continued on EPAG until reaching defined robust or stable blood counts. EPAG was reinstituted for relapse. Thirty-four patients were enrolled between 2012 and 2017, including 31 with MAA and 3 with UC. Seventeen patients responded in at least 1 eligible lineage by the primary end point. A striking improvement in anemia was observed in a patient with Diamond-Blackfan anemia. EPAG was well tolerated, and it was discontinued for robust or stable blood counts in 12 of 17 patients after a median of 8 months. A majority required re-initiation of EPAG for declining counts, and all regained response. Two of 34 patients developed non–chromosome 7 bone marrow cytogenetic abnormalities while taking EPAG, without dysplasia or increased blasts. Somatic mutation allele frequencies in cancer genes did not increase overall on EPAG. EPAG is a well-tolerated oral treatment of cytopenias in patients with MAA/UC. This trial was registered at www.clinicaltrials.gov as #NCT01328587.

Acute lower gastrointestinal bleeding is a significant problem, a rare and life threatening situation, that has a mortality rate situated between 2 and 4% (1). According to recent studies up to 15% of them present as massive bleeding and 5% require surgery (2). Acute lower gastrointestinal bleeding can be divided according to their location in the small or large bowel. The small intestine is less interested and is known to be the commonest cause of obscure bleeding. About 5% of total lower gastrointestinal bleeding comes from the small intestine (3). When conservative medical treatment associated with interventional endoscopy can not handle the bleeding, endovascular therapy can be salutary. Also in some rare cases of acute lower gastrointestinal bleeding with hemodynamic instability the last therapeutic resource remains surgery. Thus, we will present two cases of acute lower gastrointestinal bleeding which were managed different, we will then summarize the different variants of available treatment and finally, in the conclusions, we will underscore the most important steps of the management algorithm and highlight the crossing point in acute lower gastrointestinal bleeding.

Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3 probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

The confidentiality clause and the service secret are two means coming from different branches of law, public and private, but they have the same goal – to protect information, the component of a person’s patrimony, which is a more and more important issue in the world we live in. The protections provided by the two ways are different in terms of the gravity of the penalty they may involve and for this reason they may be used with discrimination, proportionally with the importance of the protected object. But in the present conditions when the information is sancta sanctorum, only this responsibility in punishment tends to dim, those interested in providing the protection of information seek for most effective and efficient punishment.

Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4+ and CD8+ T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8+ TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8+ TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8+ TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8+ and CD4+ T cell subsets in AA patients, including CD8+ and CD4+ TSCMs. CD8+ TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8+ TSCM subset is a novel biomarker and a potential therapeutic target for AA.

Summary Background T-cell large granular lymphocytic leukaemia (T-LGL) is a lymphoproliferative disease that presents with immune-mediated cytopenias and is characterised by clonal expansion of cytotoxic CD3+ CD8+ lymphocytes. Use of methotrexate, ciclosporin, or cyclophosphamide as first therapy improves cytopenias in 50% of patients, but long-term use of these can lead to toxicity. We aimed to explore the activity and safety of alemtuzumab, an anti-CD52 monoclonal antibody, in patients with T-LGL. Methods We did this single-arm, phase 2 trial in consecutively enrolled adults with T-LGL referred to the National Institutes of Health in Bethesda, MD, USA. Alemtuzumab was given intravenously at 10 mg per day for 10 days. The primary endpoint was haematological response at 3 months after infusion. A complete response was defined as normalisation of all affected lineages, and a partial response was defined in neutropenic patients as 100% increase in the absolute neutrophil count to more than 5 × 10 8 cells per L, and in those with anaemia, as any increase in haemoglobin of 20 g/L or higher observed in at least two serial measurements 1 week apart and sustained for 1 month or longer without exogenous growth factors support or transfusions. Analysis was by intention to treat. We report results from the first stage of this Simon two-stage design trial; enrolment into the second stage is continuing. This study is registered with ClinicalTrials.gov, number NCT00345345. Findings From Oct 1, 2006, to March 1, 2015, we enrolled 25 patients with T-LGL. 14 patients (56%; 95% CI 35–76) had a haematological response at 3 months. Four patients with associated myelodysplastic syndrome and two who had received haemopoietic stem cell transplantation had either no response or were not evaluable, meaning 14 (74% [49–91]) of the 19 patients with classic T-LGL responded. All patients had an infusion reaction (24 [96%] patients grade 1–2, one [4%] patient grade 3), which improved with symptomatic therapy. All patients developed lymphopenia, with 22 (88%) patients having grade 3 or 4 lymphopenia. The other most common grade 3 and 4 adverse events were leukopenia (eight [32%]) and neutropenic infections (five [20%]). Seven patients died; all were non-responders. Interpretation This is the largest and only prospective study of alemtuzumab in patients with T-LGL. The activity reported with a single course of a lymphocytotoxic drug in patients with mainly relapsed and refractory disease suggests that haematological response can be achieved without continued use of oral immunosuppression. Funding National Heart, Lung, and Blood Institute.

Emerging evidence indicates that microRNA control and modulate immunity. MicroRNA have not been investigated in acquired aplastic anemia, a T-cell-mediated immune disease. Analysis of 84 microRNA expression levels in CD4(+) and CD8(+) T cells of patients with aplastic anemia revealed concurrent down-regulation of miR-126-3p, miR-145-5p, miR-223-3p, and miR-199a-5p (3-fold change, P0.05) in both T-cell populations, which were unique in aplastic anemia compared to other hematologic disorders. MiR-126-3p and miR-223-3p were down-regulated in CD4(+) T effector memory cells, and miR-126-3p, miR-145-5p, and miR-223-3p were down-regulated in CD8(+) T effector memory and terminal effector cells. Successful immunosuppressive therapy was associated with restoration to normal expression levels of miR-126-3p, miR-145-5p, and miR-223-3p (2-fold change, P0.05). In CD4(+) and CD8(+) T cells in aplastic anemia patients, MYC and PIK3R2 were up-regulated and proved to be targets of miR-145-5p and miR-126-3p, respectively. MiR-126-3p and miR-145-5p knockdown promoted proliferation and increased interferon-γ and granzyme B production in both CD4(+) and CD8(+) T cells. Our work describes previously unknown regulatory roles of microRNA in T-cell activation in aplastic anemia, which may open a new perspective for development of effective therapy. Clinicaltrials.gov identifier: NCT 01623167.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase complex that regulates telomerase activity to maintain telomere length for all animals with linear chromosomes. As the Mus musculus (MM) laboratory mouse has very long telomeres compared to humans, a potential alternative animal model for telomere research is the Peromyscus leucopus (PL) mouse that has telomere lengths close to the human range and has the wild counterparts for comparison. We report the full TERT coding sequence (pTERT) from PL mice to use in the telomere research. Comparative analysis with eight other mammalian TERTs revealed a pTERT protein considerably homologous to other TERTs and preserved all TERT specific-sequence signatures, yet with some distinctive features. pTERT displayed the highest nucleotide and amino acid sequence homology with hamster TERT. Unlike human but similar to MM mice, pTERT expression was detected in various adult somatic tissues of PL mice, with the highest expression in testes. Four different captive stocks of PL mice and wild-captured PL mice each displayed group-specific average telomere lengths, with the longest and shortest telomeres in inbred and outbred stock mice, respectively. pTERT showed considerable numbers of synonymous and nonsynonymous mutations. A pTERT proximal promoter region cloned was homologous among PL and MM mice and rat, but with species-specific features. From PL mice, we further cloned and characterized ribosomal protein, large, P0 (pRPLP0) to use as an internal control for various assays. Peromyscus mice have been extensively used for various studies, including human diseases, for which pTERT and pRPLP0 would be useful tools.

Neurocysticercosis, an infection of the central nervous system with the larval stage of the cestode Taenia solium, is common in developing countries but its occurrence and management in allogeneic hematopoietic stem cell transplantation (HSCT) has not been reported previously, to our knowledge. We report the case of an immigrant female patient who underwent a matched-related allogeneic HSCT for acute lymphoblastic leukemia and was incidentally found to have a solitary viable neurocysticercosis lesion. However, despite severe immunosuppression, the size of the cyst did not increase. More importantly, restoration of the immune system did not induce significant inflammation or seizures. Subsequent follow-up demonstrated complete resolution of the neurocysticercosis lesion. Thus, in the setting of HSCT, an asymptomatic patient with a single neurocysticercosis lesion was successfully managed without the use of anthelmintics, steroids, or anti-epileptics.

Although hematopoietic growth factors are at high levels in aplastic anemia (AA) patients, little is known about their dynamic change over time post treatment. We examined plasma concentrations of hematopoietic growth factors sequentially in 55 severe AA patients (45 treatment-naïve patients who had received immunosuppressive therapy (IST) or IST and eltrombopag; 10 IST-refractory patients who had received eltrombopag only), focusing on thrombopoietin (TPO). TPO concentrations were much higher than normal range in patients before treatment, then decreased in responders but not in non-responders. We followed up a cohort of 9 patients who obtained stable complete remission for up to 7 years post immunosuppressive therapy, and found that TPO levels declined gradually by 3 months post treatment accompanying an increase in platelet counts, but stabilized at the levels higher than normal range. An inverse correlation was noted between TPO levels and platelet counts. The increased plasma TPO levels could be required to maintain normal platelet counts in remission, as well as attributed to the reduced consumption by circulating platelets.

Our understanding of the pathophysiology of aplastic anemia is undergoing significant revision, with implications for diagnosis and treatment. Constitutional and acquired disease is poorly delineated, as lesions in some genetic pathways cause stereotypical childhood syndromes and also act as risk factors for clinical manifestations in adult life. Telomere diseases are a prominent example of this relationship. Accelerated telomere attrition is the result of mutations in telomere repair genes and genes encoding components of the shelterin complex and related proteins. Genotype-phenotype correlations show genes responsible for X-linked (DKC1) and severe recessive childhood dyskeratosis congenita, typically with associated mucocutaneous features, and others (TERC and TERT) for more subtle presentation as telomeropathy in adults, in which multiorgan failure may be prominent. Telomerase mutations also are etiologic in familial pulmonary fibrosis and cryptic liver disease. Detection of a telomere disease requires awareness in the clinic, appropriate laboratory testing of telomere content, and genetic sequencing. In treatment decisions, genetic screening of related donors for hematopoietic stem cell transplantation is critical, and androgen therapy may be helpful. Telomeres shorten normally with aging, as well as under environmental circumstances, with regenerative stress and oxidative damage. Telomere biology is complexly related to oncogenesis: telomere attrition is protective by enforcing senescence or apoptosis in cells with a long mitotic history, but telomere loss also can destabilize the genome by chromosome rearrangement and aneuploidy.

Beware of T cells that don't know how to stop During an infection, T cells divide extensively and secrete proteins that can severely damage tissues. But T cells know when to stop—they express proteins on their surface such as CTLA4, which put on the brakes. Kuehn et al. now report genetic evidence of the importance of CTLA4 in humans (see the Perspective by Rieux-Laucat and Casanova). They identified six patients with mutations in one copy of CTLA4 . Patients presented with symptoms of an overzealous immune response, with immune cells infiltrating their organs. The findings support the idea that CTLA4 tells the immune system when enough is enough. Science , this issue p. 1623 ; see also p. 1560

About a quarter of patients with severe aplastic anemia remain pancytopenic despite immunosuppressive therapy. We have previously demonstrated that eltrombopag has efficacy in this setting with 44% (11/25) of patients having clinically significant hematologic responses. We now report safety and efficacy data on a further 18 patients and long-term follow-up on the entire cohort of 43 patients. The overall response rate was 17 of 43 patients (40%) at 3 to 4 months, including tri- and bilineage responses. The majority of patients who remained on eltrombopag in an extension study (14/17) continued to show improvement, and 7 eventually had significant increases in neutrophil, red cell, and platelet lineages. Five patients with robust near-normalization of blood counts had drug discontinued at a median of 28.5 months after entry (range, 9-37 months), and all maintained stable counts a median of 13 months (range, 1-15 months) off eltrombopag. Eight patients, including 6 nonresponders and 2 responders, developed new cytogenetic abnormalities on eltrombopag, including 5 with chromosome 7 loss or partial deletion. None evolved to acute myeloid leukemia to date. Eltrombopag is efficacious in a subset of patients with aplastic anemia refractory to immunosuppressive therapy, with frequent multilineage responses and maintenance of normalized hematopoiesis off treatment. This study is registered at www.clinicaltrials.gov as #NCT00922883.

Telomeres are the capping ends of chromosomes that protect the loss of genetic material and prevent chromosomal instability. In human tissue-specific stem/progenitor cells, telomere length (TL) is maintained by the telomerase complex, which consists of a reverse transcriptase catalytic subunit (TERT) and an RNA template (TERC). Very short telomeres and loss-of-function mutations in the TERT and TERC genes have been reported in acute myeloid leukemia, but the role of telomeres in acute promyelocytic leukemia (APL) has not been well established. We report the results for a large cohort of 187 PML/RARα-positive APL patients. No germline mutations in the TERT or TERC genes were identified. Codon 279 and 1062 TERT polymorphisms were present at a frequency similar to that in the general population. TL measured in blood or marrow mononuclear cells at diagnosis was significantly shorter in the APL patients than in healthy volunteers, and shorter telomeres at diagnosis were significantly associated with high-risk disease. For patients who achieved complete remission, the median increase in TL from diagnosis to remission (delta TL) was 2.0 kilobase (kb), and we found delta TL to be the most powerful predictor of overall survival when compared with well-established risk factors for poor outcomes in APL.

Although recent observations implicate the importance of telomerase activity in acute myeloid leukaemia (AML), the roles of epigenetic regulations of the TERT gene in leukaemogenesis, drug resistance and clinical prognosis in AML are not fully understood. We developed a quantitative pyrosequencing-based methylation assay covering the TERT proximal promoter and a partial exon 1 (TERTpro/Ex1) region and tested both cell lines and primary leukaemia cells derived from AML and AML with preceding myelodysplastic syndrome (AML/MDS) patients (n = 43). Prognostic impact of methylation status of the upstream TERT promoter region was assessed by the Kaplan-Meier method. The activity of the telomerase inhibitor, imetelstat, was measured using leukaemia cell lines. The TERTpro/Ex1 region was highly methylated in all cell lines and primary leukaemia cells showed diverse methylation profiles. Most cases showed hypermethylated regions at the upstream TERTpro/Ex1 region, which were associated with inferior patient survival. TERTpro/Ex1 methylation status was correlated with the cytotoxicity to imetelstat and its combination with hypomethylating agent enhanced the cytotoxicity of imetelstat. AML cell lines and primary blasts harbour distinct TERTpro/Ex1 methylation profiles that could serve as a prognostic biomarker of AML. However, validation in a large cohort of patients is necessary to confirm our findings.

The effectiveness of salvage therapy for aplastic anemia patients unresponsive to initial rabbit antithymocyte globulin (r-ATG) or cyclophosphamide is not known. We investigated the administration of standard horse ATG (h-ATG) plus cyclosporine (CsA) in patients who were refractory to initial r-ATG/CsA (n = 19) or cyclophosphamide/CsA (n = 6) (registered at clinicaltrials.gov as NCT00944749). The primary endpoint was hematologic response at 3 months and was defined as no longer meeting the criteria for severe aplastic anemia. Of the 19 patients who received r-ATG as initial therapy, 4 (21%) achieved a hematologic response by 3 months, and of the 6 patients who received cyclophosphamide, only 1 (17%) responded by 6 months. Among the responders there were no cases of relapse, and in nonresponders 2 patients evolved to monosomy 7. The overall survival for the cohort at 3 years was 68% (95% CI, 50-91%). These results suggest that only a minority can be successfully salvaged after receiving as first therapy either r-ATG or cyclophosphamide. Although h-ATG may be utilized in the salvage setting, the overall response rate probably will be lower than when h-ATG is used as initial treatment.

Eltrombopag (EPAG) received FDA approval for treatment of refractory severe aplastic anemia (rSAA) in 2014, based on our phase I/II dose escalation trial of single agent EPAG for patients failing one or more treatment cycles with ATG/cyclosporine (Olnes NEJM 2012; Desmond Blood 2014). There is no standard treatment for patients with moderate aplastic anemia (MAA) or hypo-productive uni-lineage cytopenias (MAA/UC), conditions that can also impact on morbidity, mortality and quality of life. To explore the safety and effectiveness of EPAG in MAA/UC, we conducted a phase II study of EPAG given at escalating doses from 50-300mg/day (25-150mg/day for East Asians) through a primary hematologic response endpoint at 16-20 weeks (NCT 01328587). 34 patients enrolled between February 2012 and March 2017. 27 had never been treated with ATG/CSA IST. Responding patients could continue EPAG treatment on an extension arm. The drug was well-tolerated in 33/34 patients, with 1 patient coming off study at 10 weeks for nausea and vomiting. 25 patients reached the maximal dose. The median duration of follow-up in all patients was 16 months, and 27 months in responding patients. 17 of 34 (50%) of patients met criteria for response at the primary endpoint in at least one initially protocol-qualifying lineage (Hb 1.5 gr/dL increase in Hb or > 50% reduction in transfusions), including a patient with RSP19-mutated Diamond-Blackfan anemia. 7/24 with severe thrombocytopenia had a platelet response (>20,000/ul or transfusion-independence). 2/13 with both severe anemia and thrombocytopenia had bi-lineage responses at the primary endpoint, and an additional 5 went on to bi-lineage responses during the extension period. 3 patients without a response to EPAG were later treated with ATG/CSA and responded. EPAG was discontinued in 11/17 (65%) responding patients upon achievement of robust blood counts (Hb > 10mg/dL, platelets > 50,000/uL, and ANC > 1000) or stable blood counts for 6 months in 1 patient (6%) after a median duration of drug administration of 8 months (2-14 months) (Fig 1). In contrast to our prior experience in rSAA, the majority of patients still being followed on study after drug discontinuation (8/10) needed to have EPAG re-initiated for declining counts at a median of 6 months (2-38) later. All 8 responded again, with 4/8 able to discontinue EPAG after a second robust or stable response. 2/34 patients (6%) developed marrow cytogenetic abnormalities while on drug in contrast to 16/83 (16%) rSAA patients treated with EPAG in our prior studies. Both MAA patients were responders and neither had dysplastic changes or increased blasts. Patient # 5 had +8 in 7/20 metaphases at 22m, went off drug and relapsed, and EPAG was restarted off protocol. Repeat cytogenetics on EPAG were normal. Patient #20 had del13q in 5/20 metaphases at 9.5m, EPAG was stopped, and repeat cytogenetics off drug were normal. Of note, patient #12 had a robust response and had been off drug for 25m when counts declined and BM revealed dysplastic changes with no increase in blasts and normal cytogenetics. The acquisition and selection of somatic mutations, particularly in myeloid candidate genes (MCG) recurrently mutated in MDS/AMLhas been proposed to be an initiating step in clonal evolution. We performed targeted next generation exome sequencing (NGS) of 66 MCG and additional genes previously found to be somatically-mutated in SAA (Yoshizato, NEJM, 2015) pre-EPAG treatment and at the primary response endpoint of 16-20 weeks. Only 5 patients showed somatic mutations in these genes at baseline (SETBP1, CBL, SF3B1, PPMID, EP300). There were no significant changes in VAF. 2 mutations became detectable on EPAG (BCOR, and DNMT3A transiently), and 1 disappeared (SF3B1). In summary, administration of EPAG at escalating doses up to of 300mg/day was well-tolerated and 50% of patients with MAA/UC had clinically-meaningful responses, including those not previously treated with IST. The responses were durable, often robust, although frequently required ongoing EPAG treatment, in contrast to the experience in rSAA. Clonal cytogenetic evolution was rare, with no instances of chromosome 7 abnormalities, and there was no consistent expansion of MCG somatically-mutated clone size in patients during EPAG treatment. Figure. Figure. Disclosures Winkler: National Institute of Health: Research Funding. Young:CRADA with Novartis: Research Funding; GlaxoSmithKline: Research Funding; National Institute of Health: Research Funding. Dunbar:National Institute of Health: Research Funding.

The mechanisms underlying synovial joint development remain poorly understood. Here we use complete and cell-specific gene inactivation to identify the roles of the redundant chondrogenic transcription factors Sox5 and Sox6 in this process. We show that joint development aborts early in complete mutants (Sox5(-/-)6(-/-)). Gdf5 and Wnt9a expression is punctual in articular progenitor cells, but Sox9 downregulation and cell condensation in joint interzones are late. Joint cell differentiation is unsuccessful, regardless of lineage, and cavitation fails. Sox5 and Sox6 restricted expression to chondrocytes in wild-type embryos and continued Erg expression and weak Ihh expression in Sox5(-/-)6(-/-) growth plates suggest that growth plate failure contribute to this Sox5(-/-)6(-/-) joint morphogenesis block. Sox5/6 inactivation in specified joint cells and chondrocytes (Sox5(fl/fl)6(fl/fl)Col2Cre) also results in a joint morphogenesis block, whereas Sox5/6 inactivation in specified joint cells only (Sox5(fl/fl)6(fl/fl)Gdf5Cre) results in milder joint defects and normal growth plates. Sox5(fl/fl)6(fl/fl)Gdf5Cre articular chondrocytes remain undifferentiated, as shown by continued Gdf5 expression and pancartilaginous gene downregulation. Along with Prg4 downregulation, these defects likely account for joint tissue overgrowth and incomplete cavitation in adult mice. Together, these data suggest that synovial joint morphogenesis relies on essential roles for Sox5/6 in promoting both growth plate and articular chondrocyte differentiation.

International audience; Background - Genetic defects in telomere maintenance and repair cause bone marrow failure, liver cirrhosis, and pulmonary fibrosis, and they increase susceptibility to cancer. Historically, androgens have been useful as treatment for marrow failure syndromes. In tissue culture and animal models, sex hormones regulate expression of the telomerase gene. Methods - In a phase 1-2 prospective study involving patients with telomere diseases, we administered the synthetic sex hormone danazol orally at a dose of 800 mg per day for a total of 24 months. The goal of treatment was the attenuation of accelerated telomere attrition, and the primary efficacy end point was a 20% reduction in the annual rate of telomere attrition measured at 24 months. The occurrence of toxic effects of treatment was the primary safety end point. Hematologic response to treatment at various time points was the secondary efficacy end point. Results - After 27 patients were enrolled, the study was halted early, because telomere attrition was reduced in all 12 patients who could be evaluated for the primary end point; in the intention-to-treat analysis, 12 of 27 patients (44%; 95% confidence interval [CI], 26 to 64) met the primary efficacy end point. Unexpectedly, almost all the patients (11 of 12, 92%) had a gain in telomere length at 24 months as compared with baseline (mean increase, 386 bp [95% CI, 178 to 593]); in exploratory analyses, similar increases were observed at 6 months (16 of 21 patients; mean increase, 175 bp [95% CI, 79 to 271]) and 12 months (16 of 18 patients; mean increase, 360 bp [95% CI, 209 to 512]). Hematologic responses occurred in 19 of 24 patients (79%) who could be evaluated at 3 months and in 10 of 12 patients (83%) who could be evaluated at 24 months. Known adverse effects of danazol--elevated liver-enzyme levels and muscle cramps--of grade 2 or less occurred in 41% and 33% of the patients, respectively. Conclusions - In our study, treatment with danazol led to telomere elongation in patients with telomere diseases. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT01441037.).

Maintain stemness, commit to a specific lineage, differentiate, proliferate, or die. These are essential decisions that every cell is constantly challenged to make in multi-cellular organisms to ensure proper development, adult maintenance, and adaptability. SRY-related high-mobility-group box (Sox) transcription factors have emerged in the animal kingdom to help cells effect such decisions. They are encoded by twenty genes in humans and mice. They share a highly conserved high-mobility-group box domain that was originally identified in SRY, the sex-determining gene on the Y chromosome, and that has derived from a canonical high-mobility-group domain characteristic of chromatin-associated proteins. The high-mobility-group box domain binds DNA in the minor groove and increases its DNA binding affinity and specificity by interacting with many types of transcription factors. It also bends DNA and may thereby confer on Sox proteins a unique and critical role in the assembly of transcriptional enhanceosomes. Sox proteins fall into eight groups. Most feature a transactivation or transrepression domain and thereby also act as typical transcription factors. Each gene has distinct expression pattern and molecular properties, often redundant with those in the same group and overlapping with those in other groups. As a whole the Sox family controls cell fate and differentiation in a multitude of processes, such as male differentiation, stemness, neurogenesis, and skeletogenesis. We review their specific molecular properties and in vivo roles, stress recent advances in the field, and suggest directions for future investigations.

Sox4 belongs to the family of Sry-related HMG box transcription factors, which specify cell fate and differentiation in many lineages. Sox4 is widely expressed in the embryo and controls such processes as neuronal tissue, lymphocyte, heart, and bone development. Sox4-null mice die at embryonic day 14 from heart malformation. This early lethality has therefore limited studies on Sox4 functions. We show here that we have generated mice harboring a Sox4 conditional null allele (Sox4fl+) by flanking the entire coding region with loxP sites. Sox4fl+/fl+ mice are indistinguishable from wildtype mice and produce the wildtype Sox4 protein at a normal level. Sox4fl+ is efficiently converted into a null allele (Sox4fl-) by Cre recombinase in somatic and germ-line cells, and Sox4fl-/fl- embryos die from the same heart defects as Sox4-/- mice. This Sox4 conditional null allele will thus be a valuable tool to further uncovering Sox4 functions in various processes in vivo.

[BACKGROUND]In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. [METHODS]We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. [RESULTS]Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- andBCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. [CONCLUSIONS]Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.), 再生不良性貧血における遺伝子変異の解明 -白血病発症にいたる過程を初めて解明-. 京都大学プレスリリース. 2015-07-09.

Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 μM 5-HT was unaffected by 100 μM spiperone, whereas it was effectively blocked by 100 μM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 μM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

First-line therapy of severe aplastic anemia (SAA) with high-dose cyclophosphamide causes toxicity and increased short-term mortality. We investigated cyclophosphamide at a lower, more moderate dose in combination with aggressive supportive care to determine whether severe infections might be avoided and hematologic outcomes defined for this regimen. From 2010 to 2012, 22 patients received cyclophosphamide at 120 mg/kg plus cyclosporine and antibacterial, antiviral, and antifungal prophylaxis. Toxicity was considerable, mainly due to prolonged absolute neutropenia, which occurred regardless of pretherapy blood counts, and persisted an average of 2 months. Granulocyte transfusions for uncontrolled infection were required in 5 patients, confirmed fungal infections were documented in 6, and 9 patients died. Nine patients (41%) responded at 6 months. After a median follow-up of 2.2 years, relapse occurred in 2 patients, and cytogenetic abnormalities (including monosomy 7) were observed in 4 patients. Although cyclophosphamide has activity in SAA, its toxicity is not justified when far less dangerous alternatives are available. This trial was registered at www.clinicaltrials.gov as #NCT01193283.

The effectiveness of salvage therapy for aplastic anemia patients unresponsive to initial rabbit antithymocyte globulin (r-ATG) or cyclophosphamide is not known. We investigated the administration of standard horse ATG (h-ATG) plus cyclosporine (CsA) in patients who were refractory to initial r-ATG/CsA (n = 19) or cyclophosphamide/CsA (n = 6) (registered at clinicaltrials.gov as NCT00944749). The primary endpoint was hematologic response at 3 months and was defined as no longer meeting the criteria for severe aplastic anemia. Of the 19 patients who received r-ATG as initial therapy, 4 (21%) achieved a hematologic response by 3 months, and of the 6 patients who received cyclophosphamide, only 1 (17%) responded by 6 months. Among the responders there were no cases of relapse, and in nonresponders 2 patients evolved to monosomy 7. The overall survival for the cohort at 3 years was 68% (95% CI, 50-91%). These results suggest that only a minority can be successfully salvaged after receiving as first therapy either r-ATG or cyclophosphamide. Although h-ATG may be utilized in the salvage setting, the overall response rate probably will be lower than when h-ATG is used as initial treatment.

We present the case of a 93-year-old patient with intestinal occlusion due to a descending colon tumor, with carcinomatous ascites and secondary liver and lung determinations. Considering the risks associated with a surgical act in such a patient and the impossibility of performing a curative intervention, a self-expanding metallic colonic stent was mounted. The post-intervention evolution was favorable, the patient being discharged 48 hours later. Left colon cancer is diagnosed in the occlusive phase in 8 to 26% of cases (1). It often requires an immediate surgical resolution due to the potential risk of death. Emergency surgery involves increased rates of morbidity and mortality (2). Thus, other ways of resolving these surgical emergencies have been developed. Colonic stents were first reported in the literature by Dohmoto (3). Initially, the use of stents was as the final method of palletising (4). Later, they were used as a bridge to minimally invasive programmed surgery (5).

DBA is a rare bone marrow failure syndrome clinically defined by onset of hypoproliferative anemia in early childhood accompanied by a grossly normocellular bone marrow. With age, a significant fraction of patients progress to pancytopenia and a hypocellular bone marrow, with some increased risk of progression to leukemia. Autosomal dominant or sporadic heterozygous mutations in genes encoding for ribosomal subunit proteins have been etiologically linked to DBA. How loss of function mutations in ribosomal proteins results in selective loss of erythroid progenitors with variable severity is incompletely understood. Chronic transfusion therapy and corticosteroid treatment can alleviate anemia, but both cause severe long term toxicities. Hematopoietic stem cell transplantation, when available, is the only definitive treatment modality for the hematological manifestations, and the only known treatment for DBA patients once pancytopenia develops. Here we report on a 28 year old female patient diagnosed with DBA at age of one month based on clinical criteria. Recent mutational analysis identified a novel mutation in intron 4(c.356+3>C) of the RPS19 gene affecting a known splice donor site that has previously reported in DBA patients. She did not respond to corticosteroids and was maintained on transfusions. She failed various experimental therapies including danazol, hematopoietic growth factors including IL-3 and GM-CSF, and anti-thymocyte globulin/cyclosporine without sustained clinical response. After treatment with daclizumab in 2006 her transfusion requirements decreased for about 4 years. In 2012 she presented with progressive pancytopenia and hypocellular bone marrow without overt dysplasia, and normal cytogenetic analysis. She required red cell transfusions. The patient was enrolled in a clinical research protocol investigating the efficiency and safety of the thrombopoietin receptor agonist eltrombopag (EPAG) in patients with moderate aplastic anemia or those with bone marrow failure with unilineage cytopenia (ClinicalTrials.gov: NCT01328587). The study is designed as a non-randomized, phase II, dose modification study with the primary endpoint hematological response at 16 weeks. EPAG was administered at 50mg daily and escalated every 2 weeks by 25 mg to the maximal dose of 300mg daily. At response assessment the patient's hemoglobin (Hgb) improved from 7.0g/dl to 8.9g/dl and platelets from 112K/ul to 171k/ul. She required no further transfusions, and was deemed a responder. EPAG was subsequently continued at the same dose level for an additional 6 months when the peripheral cells counts peaked at Hgb 11.9g/dl, platelets 251K/ul, ANC 3.19 K/ul, and EPAG was discontinued for protocol-defined robust response. After discontinuation of EPAG the patient's hemoglobin steadily declined over a period of 8 months. EPAG was reinitiated at 300mg per protocol. Peripheral blood cell counts rapidly improved and the EPAG dose was slowly tapered. The patient is currently on EPAG 50mg daily with normal blood cell counts. We have not observed any EPAG related toxicities in our patient, and repeated cytogenetic studies on bone marrow documented a normal karyotype. To the best of our knowledge this is the first report documenting EPAG efficacy in a DBA patient. Trautman et al. (2012) previously reported a DBA patient that failed treatment with low dose EPAG after six months. Our observation warrants further systematic investigation of EPAG treatment for DBA patients within a clinical research study setting. Figure Figure. Disclosures Winkler: GSK/Novartis: Research Funding. Townsley:Novartis: Research Funding. Desmond:Novartis: Honoraria. Dumitriu:GSK/Novartis: Research Funding. Grasmeder:GSK/Novartis: Research Funding. Young:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding.

BACKGROUND Severe aplastic anemia, which is characterized by immune-mediated bone marrow hypoplasia and pancytopenia, can be treated effectively with immunosuppressive therapy or allogeneic transplantation. One third of patients have disease that is refractory to immunosuppression, with persistent, severe cytopenia and a profound deficit in hematopoietic stem cells and progenitor cells. Thrombopoietin may increase the number of hematopoietic stem cells and progenitor cells. METHODS We conducted a phase 2 study involving patients with aplastic anemia that was refractory to immunosuppression to determine whether the oral thrombopoietin mimetic eltrombopag (Promacta) can improve blood counts. Twenty-five patients received eltrombopag at a dose of 50 mg, which could be increased, as needed, to a maximum dose of 150 mg daily, for a total of 12 weeks. Primary end points were clinically significant changes in blood counts or transfusion independence. Patients with a response continued to receive eltrombopag. RESULTS Eleven of 25 patients (44%) had a hematologic response in at least one lineage at 12 weeks, with minimal toxic effects. Nine patients no longer needed platelet transfusions (median increase in platelet count, 44,000 per cubic millimeter). Six patients had improved hemoglobin levels (median increase, 4.4 g per deciliter); 3 of them were previously dependent on red-cell transfusions and no longer needed transfusions. Nine patients had increased neutrophil counts (median increase, 1350 per cubic millimeter). Serial bone marrow biopsies showed normalization of trilineage hematopoiesis in patients who had a response, without increased fibrosis. Monitoring of immune function revealed no consistent changes. CONCLUSIONS Treatment with eltrombopag was associated with multilineage clinical responses in some patients with refractory severe aplastic anemia. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00922883.).

Background. Aplastic anemia (AA), the prototypical bone marrow (BM) failure syndrome, is caused by immune-mediated destruction of hematopoietic stem/progenitor cells (HSPCs). CD8+ cytotoxic T cells with restricted TCR diversity (oligoclonal T cells) are expanded in AA, leading to production of proinflammatory cytokines, such as IFN-γ, which induce apoptosis of HSPCs. Recent studies have identified a new subset of memory T cells with stem cell-like properties, TSCM, which are the least differentiated cells of all distinct memory populations. Functionally, TSCM possess an enhanced capacity for self-renewal and can generate multiple memory T cell populations, and they likely have an important role in controlling immunity. In autoimmune diseases, there is abnormal CD4+ and CD8+ T cell activation. We evaluated TSCM frequency in AA and its association with severity, treatment response, relapse, and changes after immunosuppressive therapy (IST). Further, to evaluate the TSCM in other autoimmune diseases, we examined CD4+ and CD8+ TSCM frequencies in uveitis, systemic lupus erythematosus (SLE), and sickle cell disease (SCD), as compared with healthy controls. Method. We retrospectively analyzed CD4+ and CD8+ TSCM populations by flow cytometry. PB specimens were collected from 55 AA samples and 41 age-matched healthy donor samples. Among 55 AA samples, 21 samples were analyzed at diagnosis and 34 after IST. For comparison, blood samples were obtained from 34 uveitis patients (27 inactive or 7 active cases), 43 SLE patients who met the American College of rheumatology (ACR) criteria for the disease [19 inactive SLE (SLE disease activity index-2K (SLEDAI-2K) score < 3; and 24 active SLE (SLEDAI-2K score > 3)], and 5 SCD patients who were receiving frequent transfusions. TSCM was defined as CD3+ CD4 (CD8)+ CD45RO- CD45RA+ CCR7+ CD27+ CD95+ population. Results and Discussion. In healthy controls, TSCM represented a relatively small percentage of circulating CD4+ or CD8+ T cells (median 2.4% CD4+ TSCM and 2.1% CD8+ TSCM, Fig. 1A). A significantly higher CD8+ TSCM frequency was detected in AA patients (4.2% vs. 2.1%, p < 0.05) while there was no difference in the CD4+ TSCM frequency (p > 0.05), compared to controls (Fig. 1B-C). In AA, high CD8+ TSCM frequency at diagnosis correlated with complete (CR) or partial response (PR) to IST [5.0 % in CR and PR vs 2.8 % in non-responders (NR), p < 0.05). In AA patients prior to IST (n=21), CD8+ TSCM frequency was not correlated with age, sex, absolute neutrophil count, platelet count, time from diagnosis to therapy, and serum ferritin levels. CD8+ TSCM were significantly increased in the two AA cohorts (with or without IST), relative to controls (p < 0.05, respectively). Higher CD8+ TSCM frequency after IST associated with treatment-failure (3.5 % in responders vs 5.5 % in NR or relapse, p < 0.05). Stimulation with anti-CD3/CD28 beads successfully induced cytokine production in CD4+ and CD8+ T cells from AA and healthy controls. Elevated IFN- γ and IL-2 levels were seen in CD4+ and CD8+ TSCM in AA compared to healthy controls. We next compared CD4+ or CD8+ TSCM frequency between each patient group (AA, uveitis, SLE, or SCD) and a healthy control group. Among the four patient groups, the uveitis group alone displayed a reduction in CD4+ TSCM frequency (1.8%) relative to the healthy controls (2.4 %; p < 0.05). An elevated CD8+ TSCM frequency was observed in AA (4.2 %), uveitis (3.6 %), and SCD (4.3 %), but not in SLE, compared to controls (2.1%; p < 0.05) (Fig. 2A). Positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and SLE (Fig. 2B). Evaluation of PD-1 expression revealed that TSCM were the least exhausted T cell compartment, as compared to other types of memory T cells. Immune therapies appeared to have negative effects on the TSCM population both in uveitis and SLE patient cohorts, as well as in AA. Conclusion. We provide evidence for increased circulating CD8+ TSCM in AA, underscoring the importance of this novel subset in regulation of immune responses and pathogenesis of autoimmunity. Our work described previously unknown potential roles of TSCM in AA, such as cytokine secretion correlated with effector functions. Understanding the CD8+ TSCM population may offer new therapeutic strategies and novel mechanistic insight into the various autoimmune diseases. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

In bone marrow failure, levels of circulating hematopoietic growth factors (HGFs) are extremely elevated, in compensation for low blood counts. In general, administration of recombinant HGFs is not effective in aplastic anemia (AA) and related syndromes. We have established the utility of eltrombopag, a thrombopoietin (Tpo) mimetic, in severe AA (SAA) refractory to immunosuppression (IST) (Olnes, N Engl J Med, 2012; Desmond, Blood, 2014). Eltrombopag in combination with standard IST also appears to increase the rate and speed of recovery in treatment-naive SAA (Townsley, EHA. 2015). These clinical results are surprising, as plasma Tpo is markedly increased in SAA (Feng, Haematologica, 2011). In investigating possible mechanisms of action of eltrombopag, we studied HGF dynamics over time in SAA patients undergoing various treatment regimens and manifesting a range of hematologic responses. In a cohort of 37 treatment-naive SAA patients who were treated with either standard IST (horse antithymocyte globulin/cyclosporine A:ATG/CsA, n = 10) alone or with the addition of eltrombopag (n = 27), among whom 8 were non-responders and 29 responders at 6 months, we measured plasma Tpo, granulocyte-colony stimulating factor (G-CSF) and erythropoietin (Epo) using magnetic multiplex assays. Concentrations of these HGFs were greatly elevated before treatment. In a majority of patients, G-CSF declined rapidly to the normal range by 6 months, with no difference between responders and non-responders, and no correlation between G-CSF and absolute neutrophil counts. Epo concentrations decreased to the normal range at 6, 12, and 24 months post treatment in responders, to a significantly greater degree than in non-responders (p = 0.001, 0.0012, 0.038, respectively). There was a negative correlation between Epo and hemoglobin (r2 = 0.3126, p Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Winkler:Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

Immunosuppressive treatment (IST) with horse antithymocyte globulin (hATG) and cyclosporine (CsA) for severe aplastic anemia (SAA) has a 60-65% rate of overall hematologic response (OR) and approximately 10% complete response (CR). These rates have remained stable over the last three decades despite efforts to improve upon this regimen (Scheinberg Blood 2012). Eltrombopag (EPAG), an oral thrombopoietin receptor agonist has activity in refractory SAA producing hematologic responses, the majority multilineage, in about 40% of patients when administered as a single agent (Olnes NEJM 2012; Desmond Blood 2014). We conducted an investigator-initiated, phase II, single-center trial to test the efficacy of adding EPAG to h-ATG/CsA (clinicaltrials.gov, NCT01623167), using a triple cohort design to optimize the regimen. Eighty-eight patients with treatment-naive SAA have been enrolled from July 2012 to October 2015. All received standard h-ATG and CsA. EPAG was administered at 150 mg daily to three consecutive cohorts: in the first (n=30) and second (n=31) cohorts, EPAG was introduced after hATG, from day 14 until 6 months (cohort 1) or 3 months (cohort 2), due to initial concern of hepatotoxicity. In the third cohort, EPAG was concurrent with hATG on day 1 and continued for 6 months (n=27). As analysis of our previous IST trials suggested that CR correlates with better survival and less evolution to MDS/AML, the primary endpoint in the current protocol was CR at 6 months. Baseline enrollment characteristics were similar across cohorts: median age was 31 years (range 3 – 82; 20% were 1%. EPAG was well tolerated when combined with CsA and hATG; 2 patients discontinued EPAG for severe cutaneous reactions, and 6 patients withdrew before 6 months due to refractoriness (n=4) or evolution to MDS (n=2). OR and CR were higher at 3 months and 6 months for all cohorts compared to historic rates (Table): OR at 6 months was 80%, 87% and 92%, respectively for the three cohorts; CR at 6 months was 33%, 26%, and 54%, respectively. For all patients evaluable to date, OR at 3 and 6 months of 80% and 85% and CR rates of 28 and 34% were higher when compared to our historical rates (p Improvements in blood counts among partial responders were robust by 3 months in all cohorts: median ANC 1790/ul; hemoglobin 10 gm/dL; and platelets 60,500/ul. For patients with very severe neutropenia (ANC500/uL was 47 days for all cohorts (n=23), and 35 days for cohort 3 (n=7). Among responders, median time to transfusion-independence for platelets was 32 days and for red cells 42 days. Serial BM biopsies showed improved cellularity in 80% without increased fibrosis. Median increase from baseline in BM CD34+ cells, as measured by flow cytometry, was 17-fold and 4-fold at 3 months for cohorts 1 and 2 respectively (p At median follow up 15 months (range 1 – 42 m), cytogenetic abnormalities have occurred in 7 patients: 4 monosomy or partial deletion of chromosome 7, 1 complex (t(3;3)(q21;q26), -7), 1 deletion 13q that later normalized, and 1 subject with trisomy 6 and trisomy 15 in 2 metaphases. We sequenced 54 candidate genes, recurrently mutated in MDS/AML: somatic mutations were not identified in 4 of 7 subjects prior to treatment or at the time of cytogenetic evolution. Clonal evolution to MDS occurred at a similar frequency compared to our historic experience with standard IST. Addition of EPAG to IST markedly increases overall and complete hematologic response rates in treatment-naive SAA. These results suggest that immediate institution of EPAG with IST may salvage and expand residual hematopoietic stem cells in aplastic anemia, accelerating the rate and quality of hematopoietic recovery. Rapid blood count improvement and reduced transfusion burden support early use of EPAG for patients with newly diagnosed severe aplastic anemia. Disclosures: Townsley: Novartis: Research Funding ; GSK: Research Funding. Off Label Use: Eltrombopag (Promacta) is not FDA approved for treatment naive aplastic anemia. Dumitriu:Novartis: Research Funding ; GSK: Research Funding. Winkler:GSK: Research Funding ; Novartis: Research Funding. Larochelle:Novartis: Research Funding ; GSK: Research Funding. Dunbar:Novartis: Research Funding ; GSK: Research Funding. Young:Novartis: Research Funding ; GSK: Research Funding.

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder that arises from hematopoietic stem cells (HSCs). PNH is caused by a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A), responsible for a deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). PNH is a clonal disease that originates from HSCs, as the originating PIGA mutation is present in cells of multiple lineages, including myeloid, erythroid, and lymphoid cells. However, a critical question regarding PNH that has yet to be fully explained despite several decades of research is the mechanism responsible for clonal expansion of PIGA -mutant cells in bone marrow failure. Using RNA-seq, we identify pathways, coding and non-coding RNA transcripts, splice variants, or single nucleotide variants and other alterations that may relate to the selective advantage of PNH clone. Method. Blood samples were obtained after informed consent from patients with 14 PNH and 18 age-matched healthy donors. From PNH patients and healthy donors, 4 samples were used for RNA sequencing 6 samples were used for validation by flow cytometry. The liquid FLAER method was used for the detection of PNH-type granulocytes. For RNA extraction, granulocytes were sorted for CD11b+ FLAER+ granulocytes, CD11b+ FLAER- granulocytes. For bone marrow staining, cells not expressing lineage markers were separated into five subpopulations: Long-term hematopoietic stem cells (LT-HSC; Lin- CD34+ CD38- CD90+), short-term hematopoietic stem cells (ST-HSC; Lin- CD34+ CD38- CD90-), common myeloid progenitors (CMP; Lin- CD34+ CD38+ CD123+ CD45RA-), granulocyte-monocyte progenitors (GMP; Lin- CD34+ CD38+ CD123+ CD45RA+) and megakaryocyte-erythrocyte progenitors (MEP; Lin- CD34+ CD38+ CD123- CD45RA-). Results and Discussion. First, RNA expression levels in CD11b+ FLAER+ and CD11b+ FLAER- populations of PNH patients were analyzed using RNA sequencing. Expression levels of 7 mRNAs (CSF2RB, ACSL1, FCGR3B, IL1RN, CXCR2, TREM1, and TNFRSR10C) were significantly upregulated (> 3 FC, P < 0.01) in CD11b+ FLAER- cells of PNH patients compared with CD11b+ FLAER+ cells. To validate the differential expression observed in GPI-AP- granulocytes from PNH patients, protein expression levels of CSF2RB, FCGR3B, CXCR2, TREM1, and TNFRSF10C were assessed by flow cytometry. In CD11b+ FLAER- granulocytes of 6 PNH patients, increased expression of CXCR2 was validated, whereas decreased expression of FCGRB and TNFRSF10C were validated compared with CD11b+ FLAER+ granulocytes and that of healthy controls. Low expression FCGRB and TNFRSR10C in CD11b+ FLAER- granulocytes were considered to be reasonable, as these were GPI-APs. Next, we examined whether increased CXCR2 expression in PNH-type cells was validated in different peripheral blood cell populations. Increased CXCR2 expression in PNH-type cells was confirmed in granulocyte and monocyte populations, not in T cell or B cell population. We checked the expression levels of CXCR1 and CXCR2, which are closely related receptors that recognize CXC chemokines. CXCR2 expression was significantly different between normal and PNH-type cells in granulocytes and monocytes, and CXCR1 expression was significant only for granulocytes. To address the difference of CXCR2 expression levels between normal and PNH-type cells in more undifferentiated cells, we next examined the CXCR2 expression levels in bone marrow hematopoietic stem cells. Expression of CXCR2 was weakly expressed in hematopoietic stem cells and progenitors, both in normal and PNH-type cells, suggesting that difference of CXCR2 expression between normal and PNH-type cells is evident only in differentiated myeloid cells, not in hematopoietic stem cells or lymphoid cells. Conclusion. We provide evidence for increased expression of CXCR2 in PNH-type granulcoytes and monocytes by RNA-seq and flow cytometry. The differential expression of CXCR2 might partly explain the dominance of PNH clones in myeloid cells in patients. CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML (Schinke C, et al, Blood, 2015). Understanding the mechanism of increased CXCR2 expression in PNH-type cells may offer new therapeutic strategies and novel mechanistic insight into the pathophysiology of PNH. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

Sox6 belongs to the family of Sry-related HMG box transcription factors, which determine cell fate and differentiation in various lineages. Sox6 is expressed in several tissues, including cartilage, testis, neuronal, and erythropoietic tissues. Mice lacking Sox6 have revealed critical roles for Sox6 in several of these tissues, but their multiple defects and early lethality has limited studies in specific cell types and in postnatal mice. We show here that we have generated mice harboring a Sox6 conditional null allele (Sox6(fl+)) by flanking the second coding exon with loxP sites. This allele encodes wildtype Sox6 protein, is expressed normally, and is efficiently converted into a null allele (Sox6(fl-)) by Cre-mediated recombination in somatic and germ cells. Sox6(fl+/fl+) mice are indistinguishable from wildtype mice, and Sox6(fl-/fl-) mice from Sox6(-/-) mice. These Sox6 conditional null mice will thus be valuable for further uncovering the roles of Sox6 in various processes in vivo.

Erythropoiesis, the essential process of hematopoietic stem cell development into erythrocytes, is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver, neonatal spleen, and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen, short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype, demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation, including hemoglobinization, cell condensation, and enucleation, and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells.

Despite having well-characterized disease-associated mutations, the mechanisms underlying the progressive bone marrow failure and cancer susceptibility of Fanconi anemia have been unclear. In this issue of Cell Stem Cell, Ceccaldi et al. identify an overactive p53/p21 stress response and cell cycle arrest as an underlying cause that starts during fetal development.

Telomeres, the terminal complex of hexameric repeats and shelterin protein of linear chromosomes, shorten with every mitosis. Telomere attrition is accelerated in patients with mutations in telomerase complex genes (Calado and Young, NEJM 2009) and with replicative stress, as in chronic bone marrow failure. Historically, male hormones were effective in some patients with aplastic anemia (AA), and case reports and retrospective observations have suggested hematologic improvement in patients with telomeropathies treated with male hormones. Exposure of normal lymphocytes and CD34+ cells to androgens increased telomerase activity in vitro, and in cells from individuals carrying loss-of-function TERT mutations to normal levels (Calado et al. Blood 2009). We have conducted a phase I/II single-center trial (www.clinicaltrials.gov NCT01441037) assessing the safety and the effect of male hormones on telomere attrition in patients with telomere disease. Entry criteria included age-adjusted mean telomere content ≤1%ile, ± identified mutations in telomerase complex genes, and low blood counts (hemoglobin 1.5 g/dL or platelets >20,000/uL or neutrophils >500/uL). Twenty seven patients were enrolled, accrual commencing August 2011. Most patients had moderate (n=20) or severe (n=4) AA, one had myelodysplasia, and two pulmonary fibrosis. Median age was 41 years (range 17-66); 15 patients were females. There was only one severe adverse event possibly related to drug. Frequent reported symptoms were muscle cramping with dehydration and exacerbation of headaches. Changes in serum lipid profiles were observed in all patients, with increased serum LDL and decreased HDL. Severe elevation in liver enzymes was not observed. One death occurred on study, not treatment related (pneumonia in a pulmonary fibrosis case). Mean telomere content of leukocytes at enrollment was compared with mean telomere content at 6, 12, and 24 months on drug as well as available samples before starting danazol. Telomere attrition prior to protocol entry, determined by q-pcr, was estimated at loss of 227 bp/year (95% CI, 58-368bp; p=0.009). Androgen administration appeared to elongate telomeres: the average increase in telomere length at 6 months was 205 bp (95% CI, 82-329 bp; p=0.002), at 12 months 441 bp (95% CI, 263-620 bp; p=0.0001), and at 24 months 347 bp (95% CI, 87-607 bp; p=0.01). A similar trend of increase in mean telomere content with danazol was confirmed in flow-sorted lymphocytes. Hematologic response rate, as defined by protocol, was 67% at 3 months and 60% at 6 months. Nine of eleven patients who required RBCs became transfusion-independent; two of them also became platelet transfusion independent. Liver cirrhosis was present in 6 patients at enrollment; worsening of liver disease in one occurred with continued alcohol abuse. To date 8 patients have completed two years of danazol, all of them responders; 10 patients remain on danazol, and 9 patients stopped drug prior to 2 years. Blood counts in all patients have been stable with drug discontinuation, with median follow up of 258 days (range 31-335). In conclusion, male hormones are safe and efficacious in patients with inherited bone marrow failure associated with telomeropathies, producing clinically meaningful hematologic improvement. Increased mean telomere content in patients, suggests that in vitro demonstration of up-regulation by sex hormones of TERT and of telomerase enzymatic activity is the mechanism of action of androgens in vivo. To our knowledge, this is the first successful prospective effort to favorably modulate telomere length by pharmacologic intervention in humans. Sex hormones may be useful in other conditions of accelerated telomere attrition, as for example after chemotherapy, and other drugs and small molecules may be usefully screened for their effects on telomerase in vitro. Disclosures Off Label Use: we want to determine if treatment with male hormone danazol is safe and improves hematologic response as first-line treatment in patients with AA and telomere disease(www.clinicaltrials.gov NCT01441037)..

Telomeres are the ends of linear chromosomes composed of tandem hexameric nucleotide repeats, maintained by a telomerase enzyme complex, made of reverse transcriptase catalytic subunit (TERT) and an RNA template (TERC). Present in very specific cells, such as embryonic and adult stem cells, the telomerase complex functions to elongate and slow the attrition of telomeric repeats associated with cellular division during replicative cellular age, as well as conferring stability to the ends of the chromosomes. In humans, both telomere length (TL) and loss-of-function mutations in the telomerase complex genes have been linked to malignant transformation and alteration of normal hematopoiesis. Though very short telomeres and germline mutations in TERT and TERC are associated with acute myeloid leukemia (Calado et al. PNAS 2009), the association between TL or telomerase complex integrity and outcome in patients (pts) with acute promyelocytic leukemia (APL) has not been thoroughly analyzed. We present the largest analysis to date of mean telomere content, as a function of TL, and germline mutations in TERT and TERC in peripheral blood leukemic promyelocytes or total mononuclear cells (remission) from 164 APL pts at diagnosis, complete remission (CR) and relapse, enrolled in the intergroup trials, E2491 and C9710. The goal was to evaluate the correlation of TL in APL blasts at diagnosis and at relapse, in CR in normal total mononuclear cells, as well as delta TL (the difference between TL at diagnosis and CR) with clinical and laboratory parameters, including disease risk status by presenting white blood cell count (WBC ≤ or > 10,000/μl), early mortality, achievement of CR and overall survival (OS). Peripheral blood leukocytes from healthy subjects (ages 0-90 yrs) were collected and analyzed as controls. TL was measured by real-time quantitative PCR, and was dichotomized into short or long by the third quartile. Patient demographics and outcomes were compared using Fisher’s exact test, the Kruskal-Wallis test, and the Wilcoxon signed rank test. To evaluate the effect of TL on clinical outcomes, the Cochran-Mantel-Haenszel statistic was used in the association test on early mortality and CR. OS were compared using stratified log-rank tests. Hazard ratios were computed using stratified univariate and multivariate Cox models. Median TL at presentation was significantly shorter in the APL than in the control healthy volunteer group (5.1 vs. 7.8 kb, p In the largest study of APL pts analyzed to date, we demonstrate that APL blasts consistently harbor shorter TL at presentation compared to healthy people, and that TL at presentation correlates with higher risk disease. TL significantly recovers by the time of disease remission, and among those pts who achieve CR, delta TL is the most powerful predictor of OS. These findings warrant confirmation in a prospective fashion, and if corroborated, may offer TL measurement as a simple and inexpensive parameter in disease risk stratification and as meaningful predictor of OS in pts with APL. Disclosures Dumitriu: GSK: Research Funding. Stein:Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Larson:Novartis: Consultancy, Research Funding.

Background. Emerging evidence indicates that microRNAs (miRNAs) may regulate immunity. Their role in acquired aplastic anemia (AA), a T cell-mediated autoimmune disease, has not been investigated. We characterized miRNA signatures in patient T cells to identify novel genes involved in the regulation of immune responses of CD4+ and CD8+ T cells in AA. Cells from age-matched healthy donors and patients with myelodysplastic syndrome (MDS) and transfusion-dependent sickle cell disease (SCD) were used for comparison. Method. MiRNAs and mRNAs from flow cytometrically sorted T cells from patients and controls were extracted and subjected to profiling using miRNA PCR array platform (miScript miRNA PCR array, MIHS-111Z, Qiagen Inc). Real-time PCR was employed for validation of specific miRNA expression changes. To define identified miRNA functions, their target genes were predicted using bioinformatic software (TargetScan, miRBase, DIANA-miRPath). MiRNA target gene profilings were performed using custom PCR array platform (Custom PCR array, Qiagen Inc) in CD4+ and CD8+ T cells in AA. MiRNA knockdown (miR-126-3p and/or miR-145-5p) on healthy CD4+ and CD8+ T cells was performed using the Human T Cell Nucleofector kit (Lonza AG). Cell proliferation was estimated by CFSE assays. Effects of miRNA knockdown on the target genes were evaluated by qPCR and immunoblot. MiRNA expression profilings were examined in CD4+ and CD8+ T cells in a mouse model of lymph node cell infusion-induced bone marrow failure (BMF). Results and Discussion. As compared to healthy controls, miRNA expression profiling revealed significant down-regulation of miRNAs in a disease-specific pattern. MiR-199a-5p expression was reduced in CD4+ and CD8+ T cells from patients with all diseases that were tested (AA, MDS, SCD). Reduced expression of miR-223-3p (more than 3 fold change (FC), P Conclusion. This work describes previously unknown potentially regulatory role of miRNAs 145-5p and 126-3p in T cell activation in AA. Further investigation is warranted to determine the role of these miRNAs in the more general pathogenesis of autoimmune diseases. Understanding these novel pathways might offer new therapeutic strategies and a novel mechanistic insight into the epigenetic control of human T cell effector responses. Figure 1 Figure 1. Disclosures Townsley: GSK: Research Funding.

Background: Acquired aplastic anemia (AA), the prototypical bone marrow failure syndrome, is inferred to result from immune-mediated destruction of hematopoietic progenitors, as most patients respond to immunosuppressive therapies. Clonal hematopoiesis in AA is evident in the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells in as many as half of patients and by identification of uniparental disomies involving 6p (6pUPD) chromosome in 13% of cases. In addition, "clonal transformation", as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML) is a serious long-term complication in 10-15% AA patients. Methods: We performed targeted deep sequencing and SNP array-based copy number (CN) analysis of peripheral blood- or granulocyte-derived DNA from 439 patients with AA (280 from US and 159 from Japanese cohorts) for a panel of 103 candidate genes, chosen because they are known to be frequently mutated in myeloid neoplasms. Germline DNA was available for 288 out of 439 patients and was used to confirm the somatic origin of mutations. Whole exome sequencing (WES) was performed in 52 cases. Where serial samples were available, the chronology of detected mutations was also investigated. Results: Targeted deep sequencing provided highly concordant results between the US and Japanese cohorts; approximately one third of AA patients had mutations in genes commonly affected in myeloid neoplasms, and about one third of patients in whom mutations were identified had multiple mutations. Multi-lineage involvement of mutations was confirmed in 6 cases using flow-sorted bone marrow samples. However, compared to myeloid neoplasms, mutations in AA were at much lower variant allele frequencies (VAFs) ( Conclusions: Clonal hematopoiesis in AA was prevalent, associated in about half of cases with mutations in genes recurrently mutated in myeloid neoplasms. The highly biased set of mutated genes associated with clonal hematopoiesis in AA is evidence for Darwinian selection of particular cell clones under in the bone marrow failure environment. Mutations could be used to better predict prognosis of AA patients. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

It is unknown if telomere length (TL) is associated with clinical outcome or molecular profile in acute myeloid leukemia (AML). We collected tumor samples from 67 AML patients treated at Memorial Sloan Kettering Cancer Center. DNA extraction was performed using viably frozen peripheral blood and bone marrow mononuclear cells. RainDance was used to amplify all exons of a set of 30 genes commonly mutated in AML. Capture was followed by next-generation sequencing; mutations were called if the variant was supported by >5% of the total number of reads (minimum >10 reads). TL was measured as mean telomere content by qPCR. Patients were assessed for FLT3 and NPM1 mutations, cytogenetics, and outcomes by chart review. Results In our 67 patient AML cohort, median TL was 5.22 kb (range 3.73-8.76). Median age was 64.1 years (range 26.2-84.4). While in healthy individuals TL shortens with age, in our cohort there was no association (R2=0.043). There was no difference in TL between newly diagnosed (ND) and relapsed/refractory (RR) patients or between de novo and secondary AML patients. In the 45 ND patients, there was a trend of early improved survival following sample collection in the longest TL tertile compared to the middle and shortest TL tertiles (86.7% vs. 60.0% and 33.3% at 6 months); however, this association was not statistically significant (p=0.662) (Fig 1). In the 22 RR patients, there was also a trend toward improved OS in the longest TL tertile (60.0% vs. 11.1% and 25.0%, p=0.284). In ND patients, there was no association between TL and primary induction failure or relapse-free survival. Targeted sequencing data for 30 myeloid genes were available in 62 of the 67 patients. Analysis of single mutation correlation with TL showed that patients with IDH1 mutations had significantly longer TL than those without (p=0.02) (Table 1). Moreover, mutations in a set of genes associated with epigenetic functions ( IDH1 / 2 , ASXL1 , DNMT3A , and TET2 ) also correlated with longer TL when examined together as a group (p=0.073) . FLT3 -ITD mutations were associated with shorter TL (p=0.084). The median ages of patients with IDH1 or FLT3 mutations were not different from the rest of the cohort. Of note, FLT3 -mutated patients did have a higher WBC than FLT3 wild-type patients (p

Background Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. Methods Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. Results Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p

The pathophysiology of human aplastic anemia (AA) is immune-mediated destruction of bone marrow stem and progenitor cells. Most patients respond to immunosuppressive therapies (IST), which markedly improved survival in this disease. However, a minority of patients undergoes transformation to malignant hematologic disease, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), usually accompanied by the cytogenetic abnormality monosomy 7. Clonal evolution in AA confers a poor prognosis in the clinic but is an opportunity to assess early events in oncogenesis in the setting of inflammation and tissue regeneration. We previously reported that low mean telomere length of leukocytes at the time of diagnosis of AA (Scheinberg et al., JAMA 2010) was associated with increased risk of progression to MDS. In the current study, we directly compared acquired mutations in candidate genes and chromosomal instability, as measured by telomere length, in a cohort of AA patients that had progressed to MDS. Thirteen AA patients who developed monosomy 7 were compared with 30 AA patients who had received similar treatments but did not progress to MDS. Leukocytes telomere content was measured by qPCR in samples obtained at different time points from the diagnosis of AA and chromosome-specific telomere length was assessed by single telomere length assay (STELA) for Xp, Yp, 12q, and 17p. In the AA patients who had evolved to MDS and AML, analysis of acquired mutations in myeloid-specific genes was performed by comparison with control “germline” DNA from purified CD3 lymphocytes by exome sequencing. Cells from AA patients with clonal evolution showed marked progressive telomere attrition, 419 bp/year during the period preceding development of monosomy 7. Telomere attrition was progressive from the time of diagnosis of AA. By STELA, accumulation of very short telomere fragments was apparent at 6 months after IST. In contrast, for the AA control group, patients whose disease was stable, telomere attrition was not accelerated in serial qPCR determinations, nor was there increased accumulation of short telomere fragments by STELA. A similar pattern of increased telomere attrition was reproduced in vitro by cultivation of bone marrow cells obtained six months after IST in all AA patients who developed MDS, while none of the AA control bone marrow cultured cells developed shorter telomeres. We examined bone marrow myeloid cells at the time of monosomy 7 for acquired mutations in 125 candidate genes reported to be recurrently mutated in AML and MDS. Exome sequencing was performed using Agilent SureSelect Target Enrichment System. The raw reads were mapped to UCSC Human Genome hg19 by BWA software with default setting. With an average of 111-fold coverage on selected exon regions, somatic mutations were identified between paired samples using SAMtools and Shimmer software for SNP detection. Acquired mutations in myeloid cells were found in two cases. One patient had a heterozygous mutation in DNMT3A (K829T) present since diagnosis of AA. The other patient also had a heterozygous mutation in DNMT3A (P904S) as well as mutations in DOTL1, ASXL1, SETBP1, and STAT3. All these mutations were identifiable after IST as neutrophils recovered. Despite the presence of multiple mutations, this patient had shown a good hematologic response to IST; evolution was manifest as recurrent pancytopenia and stable marrow myeloblasts at about 5% for over 2 years after first detection of monosomy 7. The remaining 11 patients, all of whom lacked candidate gene mutations, had progressive increase in bone marrow myeloblast numbers; the only other three survivors in this cohort had received hematopoietic stem cell transplant. In conclusion, telomere shortening rather than accumulation of point mutations in hematopoietic cells preceded aneuploidy and malignant transformation at an early stage of oncogenesis in this group of patients. These results from AA may be generalizable to other cancers arising in the setting of inflammation and tissue regeneration in other organs. Identification of critically short telomeres before the development of cytogenetic abnormalities may allow for improved management of patients at risk of clonal evolution, and pharmacologic strategies to increase telomerase activity might mitigate the risk of cancer in these settings Disclosures: No relevant conflicts of interest to declare.

Translocation of 8;21 (q22;q22), generating a fusion of RUNX1 and AML1 genes is considered leukemia-defining and typically presents as an unequivocal acute myeloid leukemia (AML) with peripheral blood blasts and a hypercellular marrow, although it has been reported in patients with Fanconi anemia and myelodysplastic syndrome (MDS) (Quentin S et al. Blood 2011 Apr 14;117(15)). Aplastic anemia is a rare disease characterized by severe pancytopenia and a hypocellular marrow. A few cytogenetic abnormalities, namely trisomy 8 and monosomy 7, are associated with particularly refractory aplastic anemia, and monosomy 7 is associated with clonal evolution to MDS and rapid progression to AML. We describe a case of newly-acquired severe aplastic anemia in a 23 year old woman. Laboratory studies at presentation showed white blood cells 1.38 k/uL, absolute neutrophil count 0 k/uL, hemoglobin 7.4 g/dL, absolute reticulocyte count 5 k/uL, and platelets 38 k/uL. Bone marrow biopsy was 5% cellular with trilineage hematopoiesis and absolutely no dysplasia, even on repeated review. Initial cytogenetic analysis performed outside the NIH at presentation was normal. The patient was transferred to our institution and promptly received standard immunosuppressive therapy given the severity of neutropenia. However, a repeat bone marrow analysis performed immediately prior to immunosuppression showed t(8:21) (q22;q22) by standard cytogenetics in 3 out of 20 metaphases, with confirmation by fluorescence in situ hybridization (FISH). Blasts were not identified despite multiple repeat bone marrow aspirations utilizing immunohistochemistry and flow cytometry. Testing for Fanconi anemia was negative and leukocyte telomere length was normal for age. She remained severely pancytopenic and transfusion dependent for many months. Chemotherapy for AML was withheld given the severe pancytopenia and absence of blasts, and a search for a bone marrow transplant donor was initiated. Progression to frank leukemia with circulating blasts occurred 8 months following initial presentation, just prior to unrelated donor allogeneic stem cell transplantation. To our knowledge, this is the first reported case of acquired severe aplastic anemia, profound marrow hypocellularity, hypocellular MDS or hypocellular AML occurring in association with the t(8;21)(q22;q22). This unusual case prompted us to perform comparative genomic hybridization (CGH) using the single nucleotide polymorphism (SNP) based CytoScan high density microarrays on DNA from the patient’s bone marrow mononuclear cells. We detected multiple, large regions of copy neutral loss of heterozygosity (also referred to as uniparental disomy) in the patient’s marrow, ranging in size from 3 to 29 Mbp on multiple chromosomes. We hypothesized that the copy-neutral loss of heterozygosity observed in this case would not be present in other patients with acquired aplastic anemia at diagnosis or in normal controls. CGH did not demonstrate any large regions of copy neutral loss of heterozygosity in 10 patients with acquired severe aplastic anemia and normal cytogenetics at diagnosis, nor in 35 healthy controls. Emerging data show that SNP arrays can detect abundant copy neutral loss of heterozygosity amongst select hematologic malignancies and are associated with the duplication of oncogenic mutations. In our patient, copy neutral loss of heterozygosity possibly provided a second lesion, in addition to the RUNX1/AML1 abnormality, that facilitated initiation or progression to leukemia. These results suggest SNP based CGH arrays may be useful in distinguishing hypocellular AML from aplastic anemia and further studies utilizing this technology are warranted. Disclosures: No relevant conflicts of interest to declare.

Donor-cell derived leukemia (DCL) is a rare but serious complication of hematopoietic stem cell transplantation (HSCT), reported in 5% of all relapses following allogeneic HSCT. Only 12 cases of DCL after umbilical cord blood (UCB) HSCT are reported in the literature. Multiple mechanisms have been proposed for DCL, including occult donor leukemia, impaired immunity, drug toxicity, or a bone marrow “leukemogenic milieu”. We propose a novel mechanism of leukemogenesis, mediated by very short telomeres of donor cells with subsequent severe telomere attrition in vivo, genomic instability, and progression to complex cytogenetics acute myeloid leukemia (AML), based on study of a patient who underwent UCB HSCT for myelodysplasia (MDS). The patient was a 44-year-old woman, subsequently shown to have a T354M mutation in GATA2, who presented at age 19 years with multiple infections and MDS. At age 41, due to progression of MDS to AML, she received induction chemotherapy and underwent a single 4/6 HLA-matched UCB HSCT. She had delayed engraftment. achieving an absolute neutrophil count of 500/ul more than 100 days post-transplant. Chimerism studies demonstrated 100% donor cell at all time-points post-transplant. Two years and eight months after HSCT she presented with pancytopenia, circulating myeloblasts, and 50% myeloblasts in bone marrow, indicating recurrence of with AML. However, cytogenetics revealed complex abnormalities, t(2p;3q) and an interstitial deletion of 5q, male cells, indicating donor origin of AML. Telomere length of the transplanted cord blood cells measured by Q-PCR showed a severe decrease in length to 7.7 kb compared with an average length of 13 kb in control UCB (n=12). Moreover the telomere length decreased precipitously to 6.6 kb 2 years after transplantation and 5.6 kb at the time of diagnosis of AML. Single telomere length assay (STELA) was used to assess chromosome-specific telomere length. Very short telomeres ( Telomere attrition occurs in the first year after HSCT, presumably due to proliferative stress on the stem cell compartment. In comparison to bone marrow, UCB have longer telomeres, but HSC number may be lower than in a BM inoculum, especially for adult recipients. In our case, initial telomere length of UCB was extremely short, for unclear reasons but possibly related to low cord blood cell viability. The long time that was needed for engraftment generated additional telomere attrition with accumulation of very short telomere and genomic instability leading to development of leukemia. Screening UCB for telomere length prior to HSCT might prove useful in identifying situations at risk for graft failure or malignant transformation. Disclosures: No relevant conflicts of interest to declare.

Background T-large granular lymphocytosis (T-LGL) is a rare lymphoproliferative disease characterized by clonal expansion of cytotoxic CD3+CD8+ lymphocytes, presenting with immune mediated cytopenias and associated with autoimmune syndromes. Immunosuppressive therapy (IST) with methotrexate, cyclosporine, or cyclophosphamide can improve the cytopenias in about half the patients but can lead to significant toxicity in older patients. The anti CD52 antibody alemtuzumab is a potent immunosuppressive agent with a good safety profile. We therefore initiated a pilot phase II study to evaluate alemtuzumab as a treatment for T-LGL. Methods 20 consecutive patients with T-LGL were enrolled from October 2006 to August 2012 at National Institutes of Health (www.clinicaltrials.gov - NCT00345345). After a 1 mg test dose, alemtuzumab was administered at 10 mg/dose/day intravenously for 10 days. Peripheral blood, bone marrow, and plasma samples were collected from patients before and at 3 or 6 months after treatment. Blood was analyzed for 1) lymphocytes subsets, T-cell receptor V-beta repertoire and CD57 and CD52 expression by flow cytometry (LSR II, BD, San Jose, California), 2) plasma cytokines using a a magnetic bead based Luminex assay (Affymetrix, CA, USA), 3) STAT3 mutation by direct Sanger sequencing and 4) expression level of 84 genes of the JAK-STAT signaling pathway quantified by PCR array 384 well from SABiosciences (Frederick, MD, USA). Results We report here the results of treatment with alemtuzumab in 20 T-LGL patients enrolled in the first stage of the protocol. Three had associated myelodysplasia (MDS) and two had T-LGL following hematopoietic stem cell transplantation (HSCT). The median age was 61 years (range, 26-82). The median number of prior therapeutic interventions for T-LGL leukemia was 3 (range, 0-7) and the median time from diagnosis to alemtuzumab therapy was 1096 days (range, 18-6054). The median follow-up for all patients is 508 days (range, 99-1481) and for surviving patients 650 days (range, 120-1481). One patient was lost to follow-up 4 months after alemtuzumab therapy. Alemtuzumab was generally well tolerated. Labeled infusion related reactions were common and managed symptomatically. Prolonged and subclinical EBV and CMV reactivations were common but there were no cases of EBV or CMV disease without instituting prophylactic or pre-emptive therapy. Hematological response as defined by protocol was observed in 11 of 20 patients by 3 months after treatment. No patient with MDS or post HSCT responded to alemtuzumab. Four patients relapsed and received a second round of immunosuppression. One patient achieved stable blood counts on cyclosporine, three received alemtuzumab with one patient responding but relapsing 1 year later. STAT3 mutations in the SH2 domain identified in 10 of 20 patients did not correlate with response to alemtuzumab (5 responders and 5 non-responders). Treatment with alemtuzumab reduced significantly the absolute clonal population of T-cytotoxic lymphocytes, as identified by their V-beta receptor phenotype, but they tended to persist in frequency in the peripheral blood of responders. The expanded V-beta clone expressed both CD52 positive and negative cells and both compartments reduced in size after the treatment. When compared with healthy volunteers T-LGL patients had a distinct plasma cytokine signature (IL-12p40, TRAIL, IL22, IP10, MCP1, M-CSF, PDGF-AA, LIF, SCF) as well as JAK-STAT pathway activation prior to treatment but neither was correlated to clinical responses to alemtuzumab, likely due to the various prior IST regimens. Conclusion This is the largest cohort of T-LGL patients treated with alemtuzumab yet reported. Treatment was well tolerated and at this dose minimal side effects were observed. Alemtuzumab treatment in previously heavily treated T-LGL patients results in over 50% response rate and represents a good treatment option for this condition. Disclosures: Off Label Use: Alemtuzumab for T-LGL.

Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors. However, its natural course could be more complicated than expected for a simple immune-mediated disorder, as in the development of apparently acquired (somatic) clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), during its course. Although these evidences suggest a pathogeneic link between these disorders, the clonal architecture in AA has not been fully explored. In order to genetically define the origin of clonal hematopoiesis in patients with acquired AA, we sought gene mutations, by targeted deep sequencing of peripheral blood DNA from 192 Japanese patients with AA for mutations, using a panel of 51 genes including common mutational targets in myeloid malignancies using a SureSelect custom kit. An extended cohort of 293 American AA patients was further analyzed for targeted sequencing of granulocyte-derived DNA; for these cases, multiple sequential specimens with germline controls and complete clinical follow-up data were available. Exome sequencing was also performed for selected cases. In the Japanese cohort, about 40% were severe or very severe diseases with an excellent response to immunosuppressive therapies (IST). In total, 43 somatic mutations were detected in 18% of the cases with the mean allelic burden of 18%. Mutations were most frequent in DNMT3A (3.6%), followed by ZRSR2 (3.1%), ASXL1 (2.6%), BCOR (2.0%) and more biased to nonsense (25.6%), frameshift (14.0%), splice site changes (7.0%) and non-frameshift indel (11.6%), indicating driver roles of these mutations in many cases. Mutations were associated with older age (p=0.014) and a better response to IST (p=0.040). We next examined an extended cohort of 293 AA cases from the United States, for which samples were collected at 6 months after treatment in the patients of severe or very severe disease. Except for 12 cases, CD3(+) cells were available and used to confirm the somatic origin of mutations by comparison with CD3 cells representing germline sequence. All patients had received IST, with overall response about 65%. As of the date of submission, data analysis was completed for 86 of the 293 cases, in whom we confirmed somatic mutations detected in BM samples from 45 cases (53%) 6 months after IST, with the mean number of mutations and the mean allelic burden were 1.08 and 15.3%, respectively. Similar to the finding in the Japanese cohort, BCOR (13.8%), DNMT3A (11.5%), and ASXL1 (10.3%) were most frequently mutated. PIGA (6.9%) and CSMD1 (4.6%) were also mutated in 6.9% and 4.6%, respectively. Again, mutations were associated with older age. Although there was no significant difference in response to IST (p=0.133) between mutation (+) and (-) cases, responders showed significantly higher numbers of mutations compared with non-responders (p=0.033). Evolution to MDS/AML occurred in 12 out of the 45 cases with mutations, while 13 out of the 62 cases without mutations developed MDS/AML. Therefore, candidate genes associated with some but not most evolution events. We further performed whole exome sequencing in 6 cases, for whom sequential samples were available: in 5 of 6 cases, somatic mutations were detected and the mean number of mutations was 9. There was evidence over time of clonal selection with or without progression to MDS or AML. Small clones of cells containing RUNX1 and U2AF1-mutated clones present in the initial specimen showed expansion in size with progression to MDS (0.003 to 0.46 and 0.013 to 0.097, respectively). In conclusion, mutations in common target genes in myeloid malignancies can drive clonal evolution during the course of AA. However, overall there was no correlation between the presence of mutations and clinical evolution to MDS/AML, as many patients with evidence of clones containing mutations remained stable. Clonal expansion and the appearance and disappearance of clones occurred in some cases without clinical changes. The marrow failure environment may favor selection of mutant clones. In addition, other genetic/epigenetic alterations, including chromosomal instability induced by telomere shortening (accompanying abstract by Dumitriu and Feng) provide a mechanism of oncogenesis. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding.