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Introduction. Gastric cancer remains among the top three digestive diseases with the highest mortality rates in the world. Treatment of gastric cancer is multidisciplinary, gastric resection being essential for the best result. Anemia is one of the most common comorbidities present in patients diagnosed with gastric cancer. Materials and Methods. This is a retrospective analytical study over a period of 6 years (2014-2019). It is based on 114 consecutive gastric resections for cancer performed by a single team using exclusively resection and reconstruction stapling methods. The study aims to investigate a correlation between the presence of preoperative anemia and the incidence of postoperative morbidity and mortality. Results. Preoperative anemia was found in 70% of patients, with about half of these patients presenting with mild anemia. Most postoperative complications were grade I and II according to the Clavien Dindo scale. Anemia was correlated with an increase in infectious complications, anastomotic leaks and secondary peritoneal abscesses, pancreatic complications after multivisceral resection and length of hospital stay. Conclusions. Preoperative anemia is a risk factor that exposes the cancer patient to an increased incidence of life-threatening postoperative complications. In addition, it also extends the length of hospital stay and costs. Therefore, special attention should be paid to the identification and reduction of anemia before extensive gastric surgery in order to obtain the best possible therapeutic result.

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.

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.

Acquired aplastic anemia results from immune-mediated destruction of bone marrow. Immunosuppressive therapies are effective, but reduced numbers of residual stem cells may limit their efficacy. In patients with aplastic anemia that was refractory to immunosuppression, eltrombopag, a synthetic thrombopoietin-receptor agonist, led to clinically significant increases in blood counts in almost half the patients. We combined standard immunosuppressive therapy with eltrombopag in previously untreated patients with severe aplastic anemia.We enrolled 92 consecutive patients in a prospective phase 1-2 study of immunosuppressive therapy plus eltrombopag. The three consecutively enrolled cohorts differed with regard to the timing of initiation and the duration of the eltrombopag regimen (cohort 1 received eltrombopag from day 14 to 6 months, cohort 2 from day 14 to 3 months, and cohort 3 from day 1 to 6 months). The cohorts were analyzed separately. The primary outcome was complete hematologic response at 6 months. Secondary end points included overall response, survival, relapse, and clonal evolution to myeloid cancer.The rate of complete response at 6 months was 33% in cohort 1, 26% in cohort 2, and 58% in cohort 3. The overall response rates at 6 months were 80%, 87%, and 94%, respectively. The complete and overall response rates in the combined cohorts were higher than in our historical cohort, in which the rate of complete response was 10% and the overall response rate was 66%. At a median follow-up of 2 years, the survival rate was 97%; one patient died during the study from a nonhematologic cause. Marked increases in bone marrow cellularity, CD34+ cell number, and frequency of early hematopoietic progenitors were noted. Rates of relapse and clonal evolution were similar to our historical experience. Severe rashes occurred in two patients, resulting in the early discontinuation of eltrombopag.The addition of eltrombopag to immunosuppressive therapy was associated with markedly higher rates of hematologic response among patients with severe aplastic anemia than in a historical cohort. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT01623167 .).

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.

The pathophysiology of severe aplastic anemia (SAA) is immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). Most patients respond to immunosuppressive therapies, but a minority transform to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), frequently associated with monosomy 7 (-7). Thirteen SAA patients were analyzed for acquired mutations in myeloid cells at the time of evolution to -7, and all had a dominant HSPC clone bearing specific acquired mutations. However, mutations in genes associated with MDS/AML were present in only 4 cases. Patients who evolved to MDS and AML showed marked progressive telomere attrition before the emergence of -7. Single telomere length analysis confirmed accumulation of short telomere fragments of individual chromosomes. Our results indicate that accelerated telomere attrition in the setting of a decreased HSPC pool is characteristic of early myeloid oncogenesis, specifically chromosome 7 loss, in MDS/AML after SAA, and provides a possible mechanism for development of aneuploidy.

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM–derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

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: Definitive erythropoiesis is a vital process throughout life. Both its basal activity under physiological conditions and its increased activity under anemia-induced stress conditions are highly stimulated by the hormone erythropoietin. The transcription factor Sox6 was previously shown to enhance fetal erythropoiesis together and beyond erythropoietin signaling, but its importance in adulthood and mechanisms of action remain unknown. We used here Sox6 conditional null mice and molecular assays to address these questions. Methodology/Principal Findings: Sox6 fl/fl ErGFPCre adult mice, which lacked Sox6 in erythroid cells, exhibited compensated anemia, erythroid cell developmental defects, and anisocytotic, short-lived red cells under physiological conditions, proving that Sox6 promotes basal erythropoiesis. Tamoxifen treatment of Sox6 fl/fl CaggCreER mice induced widespread inactivation of Sox6 in a timely controlled manner and resulted in erythroblast defects before reticulocytosis, demonstrating that impaired erythropoiesis is a primary cause rather than consequence of anemia in the absence of Sox6. Twenty five percent of Sox6 fl/fl ErGFPCre mice died 4 or 5 days after induction of acute anemia with phenylhydrazine. The others recovered slowly. They promptly increased their erythropoietin level and amplified their erythroid progenitor pool, but then exhibited severe erythroblast and reticulocyte defects. Sox6 is thus essential in the maturation phase of stress erythropoiesis that follows the erythropoietin-dependent amplification phase. Sox6 inactivation resulted in upregulation of embryonic globin genes, but embryonic globin chains remained scarce and apparently inconsequential. Sox6 inactivation also resulted in downregulation of erythroid terminal markers, including the Bcl2l1 gene for the anti-apoptotic factor Bcl-xL, and in vitro assays indicated that Sox6 directly upregulates Bcl2l1 downstream of and beyond erythropoietin signaling. Conclusions/Significance: This study demonstrates that Sox6 is necessary for efficient erythropoiesis in adult mice under both basal and stress conditions. It is primarily involved in enhancing the survival rate and maturation process of erythroid cells and acts at least in part by upregulating Bcl2l1. Citation: Dumitriu B, Bhattaram P, Dy P, Huang Y, Quayum N, et al. (2010) Sox6 Is Necessary for Efficient Erythropoiesis in Adult Mice under Physiological and

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.