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Previous data support that the inflammatory process underlying ulcerative colitis (UC) and Crohn's disease (CD) can start years before the diagnosis. The aim of this study was to determine if patients with an incidental diagnosis of UC or CD demonstrate an increase in healthcare utilization in the years preceding the symptomatic onset of the disease. We performed a multicenter, retrospective, hospital‐based, case‐control study. Patients with an incidental diagnosis of UC or CD during the colorectal cancer screening program at 9 hospitals were included. Cases were matched 1:3 and compared separately with two control populations: one including healthy non‐IBD subjects adjusted by gender, age, and date, excluding those with visits to Gastroenterology; and a second control cohort of UC/CD patients with symptomatic onset. A total of 124 patients with preclinical inflammatory bowel disease (IBD) were included (87 UC, 30 CD, 7 IBD unclassified; median age 56 years). Patients with preclinical IBD showed an increase in the number of visits to Primary Care up to 3 and 5 years before diagnosis (aIRR 1.59, 95% CI [1.37–1.86], p= 0.001; aIRR 1.43, 95% CI [1.24–1.67], p= 0.01) and more frequent use of steroids (aOR 2.84, 95% CI [1.21–6.69], p= 0.03; aOR 2.25, 95% CI [1.06–4.79], p= 0.04) compared to matched non‐IBD healthy controls, respectively. In contrast, patients with a symptomatic onset visited Primary Care less frequently, but they had an increase in the number of visits to Emergency Department, specialist care, sick‐leaves, CT/ultrasound examinations, and use of antibiotics or systemic steroids. There is an increased need for medical assistance and use of systemic steroids during the presymptomatic phase of IBD. These results will help in establishing new tools for early identification of IBD in the future.

In an effort to identify novel drugs targeting fusion-oncogene–induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANEmutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANEmRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANEpromoter TATA-box using CRISPR/Cas9D10A nickases - termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+hematopoietic stem and progenitor cells (HSPCs) in vitroand in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocation. Taken together, ex vivogene editing of ELANE-CN HSPCs using MILESTONEin the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.

Infliximab (IFX) es efectivo en la colitis ulcerosa (CU) y en obtener la curación de la mucosa (CM). Se conoce poco el papel que juega la CM en la evolución posterior de la enfermedad y qué ocurre una vez se interrumpe el tratamiento.

Most studies on leukemia focus on leukemia cells as isolated objects without considering the bone marrow niche. Pal et al. have recreated the bone marrow niche using induced pluripotent stem cells (iPSCs), identifying CDH2 as a therapeutically druggable leukemia-promoting factor.1

A unique characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSCs and potentially also in leukemia stem cells. Recently, studies have shed light on developmental molecules and evolutionarily conserved signals as regulators of stem cells in hematopoiesis and leukemia. In this study, we provide evidence that the cell fate determinant Llgl1 (lethal giant larvae homolog 1) plays an important role in regulation of HSCs. Loss of Llgl1 leads to an increase in HSC numbers that show increased repopulation capacity and competitive advantage after transplantation. This advantage increases upon serial transplantation or when stress is applied to HSCs. Llgl1−/− HSCs show increased cycling but neither exhaust nor induce leukemia in recipient mice. Llgl1 inactivation is associated with transcriptional repression of transcription factors such as KLF4 (Krüppel-like factor 4) and EGR1 (early-growth-response 1) that are known inhibitors of HSC self-renewal. Decreased Llgl1 expression in human acute myeloid leukemia (AML) cells is associated with inferior patient survival. Thus, inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.

Purpose: The primary objective of this study was to evaluate with venography the rate of thrombus regression after a fixed dose of low-molecular weight heparin (LMWH) per day for 3 months compared with oral anticoagulant therapy for deep venous thrombosis (DVT). Secondary endpoints were the comparisons of the efficacy and safety of both treatments. Methods: This study was designed as an open randomized clinical study in a university hospital setting. Of the 165 patients finally enrolled in the study, 85 were assigned LMWH therapy and 80 were assigned oral anticoagulant therapy. In the group randomized to oral anticoagulant therapy, the patients first underwent treatment in the hospital with standard unfractionated heparin and then coumarin for 3 months. Doses were adjusted with laboratory monitoring to maintain the international normalized ratio between 2.0 and 3.0. Patients in the LMWH group were administered subcutaneous injections of fixed doses of 40 mg enoxaparin (4000 anti-Xa units) every 12 hours for 7 days, and after discharge from the hospital, they were administered 40 mg enoxaparin once daily at fixed doses for 3 months without a laboratory control assay. A quantitative venographic score (Marder score) was used to assess the extent of the venous thrombosis, with 0 points indicating no DVT and 40 points indicating total occlusion of all deep veins. The rate of thrombus reduction was defined as the difference in quantitative venographic scores after termination of LMWH or coumarin therapy as compared with the scores obtained on the initial venographic results. The efficacy was defined as the ability to prevent symptomatic extension or recurrence of venous thromboembolism (documented with venograms or serial lung scans). The safety was defined as the occurrence of hemorrhages. Results: After 3 months of treatment, the mean Marder score was significantly decreased in both groups in comparison with the baseline score, although the effect of therapy was significantly better after LMWH therapy (49.4% reduction) than after coumarin therapy (24.5% reduction; P < .001). LMWH therapy and male gender were independently associated with an enhanced resolution of the thrombus. A lower frequency of symptomatic recurrent venous thromboembolism was also shown in patients who underwent treatment with LMWH therapy (9.5%) than with oral anticoagulant therapy (23.7%; P < .05), although this difference was entirely a result of recurrence of DVT. Bleeding complications were significantly fewer in the LMWH group than in the coumarin group (1.1% vs 10%; P < .05). This difference was caused by minor hemorrhages. Coumarin therapy and cancer were independently associated with an enhanced risk of complications. Subcutaneous heparin therapy was well tolerated by all patients. Conclusion: The patients who were allocated to undergo enoxaparin therapy had a significantly greater improvement in their quantitative venographic score, a significantly lower recurrence rate of symptomatic venous thromboembolism, and a significantly lower incidence of bleeding than patients who underwent treatment with coumarin. LMWH can be used on an outpatient basis as a safer and more effective alternative to classical oral anticoagulant therapy for the secondary prophylaxis of selected patients with DVT. (J Vasc Surg 1999;30:283-92.)

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3Rand RUNX1are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1mutant BAALC(hi) de novoAML.

Introduction: Relapse remains the major cause of mortality in acute myeloid leukemia (AML). Prior work indicates that a rare subset of leukemic stem cells (LSCs) self-renew and propagate AML. However, characterizing LSCs is complicated by their scarcity, the lack of universal markers and the heterogeneity across patients. Here, we aim to define a transcriptional program associated with LSCs in patient samples.

No relevant conflicts of interest to declare.

Döhner: Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bullinger:Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Abbvie: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Bayer: Other: Financing of scientific research; Pfizer: Honoraria.

No relevant conflicts of interest to declare.

Heidel: Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

No relevant conflicts of interest to declare.

Recent publications suggest that mutated FLT3 kinase (FLT3-ITD) is a valid therapeutic target in acute myeloid leukemia (AML). Small molecule FLT3 tyrosine kinase inhibitors (TKIs) of the first and second generation such as midostaurin (PKC412) or quizartinib (AC220) are currently being investigated in advanced clinical trials. Previously, we described that ITD mutations are not always located in the juxtamembrane domain (JMD) of FLT3 but can also be found in the beta1-sheet of tyrosine kinase domain 1 (TKD1) in more than 25% of cases. Moreover, patients harboring ITD mutations in both locations revealed distinct response to chemotherapy: multivariable analyses with regard to achievement of CR, relapse-free survival as well as overall survival revealed consistently similar results, with FLT3-ITD insertion sites in the beta1-sheet of the TKD1 being an unfavorable variable that even outweighed the impact of FLT3-ITD mutant to wildtype allelic ratio.

In AML, the recently described tyrosine kinase domain-1 (TKD1)-ITDs of FLT3 (Breitenbuecher et al., Blood 2009, Kayser-S et al., Blood 2009) are located within the beta1-sheet, nucleotide binding loop and beta2-sheet of tyrosine kinase domain 1 (TKD1), respectively. Multivariate analysis of clinical data revealed that location of FLT3-ITDs within the beta1-sheet of TKD1 is an unfavorable prognostic factor (Kayser-S et al., Blood 2009). Recently, we uncovered a novel mechanism of primary resistance to FLT3 tyrosine kinase inhibitors (TKIs) in a patient displaying an atypical localization within the beta2-sheet-ITD (A627E). Here, we characterized in-vitro sensitivity to FLT3-TKI in growth factor dependent hematopoietic cell lines expressing a representative panel of FLT3-ITDs isolated from patient material. In particular, we compared sensitivity of beta1-sheet ITDs with typical ITDs located in the juxtamembrane domain of FLT3.FLT3-ITDs isolated from patient material, were sequenced, subcloned in parallel into two expression vectors (pAL and pMSCV-Puromycin-IRES-GFP), and stably expressed in growth-factor dependent hematopoietic Ba/F3 cells and in parallel in 32D cells. Constitutive phosphorylation of FLT3-ITD receptors and of downstream signaling pathways was analyzed by Western-blotting. Transformation of Ba/F3 and 32D cells was investigated by colony formation assays and by withdrawal of IL-3. Induction of apoptosis in response to various concentrations of FLT3-kinase inhibitors midostaurin (PKC412) and quizartinib (AC220) was measured by flow-cytometry at 24h and 48h, respectively. For statistical analysis of replicates t-test was employed.Biological characteristics of FLT3-ITD mutations located in two different structural domains of FLT3-kinase were characterized: (1) beta1-sheet-ITDs E611V(96nt) and Q613E(99nt) and (2) nucleotide binding loop-ITD A620V(84nt). (Our nomenclature used for description of ITDs indicates position of amino-acid where ITD is located, possible exchange of amino-acid residues at this position and nucleotide length of ITD) Hematopoietic cells (32D and Ba/F3) expressing these ITD mutations showed colony formation in methylcellulose medium, growth-factor independent proliferation upon IL-3 withdrawal and constitutive phosphorylation of FLT3 signaling. Three ‘typical’ juxtamembrane domain (JMD) ITDs were used as controls; ITD598/599(36nt), ITD598/599(66nt) and ITDK602R(21nt). As compared to these JM-ITDs, we observed significantly less apoptosis of beta1-sheet-ITDs at all concentrations of FLT3-kinase inhibitors applied. However, this difference in sensitivity gradually decreased when incubating with midostaurin or quizartinib for longer periods of time as 48h. Of note, length of FLT3-ITD mutations did not appear to influence sensitivity to TKI treatment.Investigating representative FLT3-ITD-mutations located within the TKD1 revealed that beta1-sheet-ITDs mediate constitutive activation of FLT3 receptors leading to transformation of hematopoietic cell lines 32D and Ba/F3. In comparison to typical JMD-ITDs, beta1-sheet-ITDs analyzed here revealed resistance to FLT3-inhibitors as midostaurin and quizartinib across two cellular reconstitution models (32D and Ba/F3) and using two different expression vectors (pAL and pMSCV). Our results indicate that differential sensitivity is rather an effect of ITD-localization within a functional domain of FLT3 and not necessarily conferred by the length of ITDs. Taken together, our data provide a rationale to prospectively analyze not only the FLT3-ITD mutation status or FLT3-ITD allelic ratio but also location of ITD-mutations in ongoing clinical trials as this may have direct impact on response to therapy (tyrosine kinase inhibitors, chemotherapy, allogeneic stem cell transplantation) in FLT3-ITD positive AML.Heidel: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fischer:Novartis: Honoraria.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.