Pivotal role of BCL11B in the immune, hematopoietic and nervous systems: a review of the BCL11B-associated phenotypes from the genetic perspective.

The transcription factor BCL11B plays an essential role in the development of central nervous system and T cell differentiation by regulating the expression of numerous genes involved in several pathways. Monoallelic defects in the BCL11B gene leading to loss-of-function are associated with a wide spectrum of phenotypes, including neurological disorders with or without immunological features and susceptibility to hematological malignancies. From the genetic point of view, the landscape of BCL11B mutations reported so far does not fully explain the genotype-phenotype correlation. In this review, we sought to compile the phenotypic and genotypic variables associated with previously reported mutations in this gene in order to provide a better understanding of the consequences of deleterious variants. We also highlight the importance of a careful evaluation of the mutation type, its location and the pattern of inheritance of the variants in order to assign the most accurate pathogenicity and actionability of the genetic findings.

Impact of trisomy 19 on outcome according to genetic makeup in patients with acute myeloid leukemia.

We retrospectively studied 97 acute myeloid leukemia patients with trisomy 19 (median age at diagnosis 57 years; range, 17- 83 years) treated between 2001 and 2019 within two multicenter study groups. Trisomy 19 occurred alone in ten (10.5%) patients, with additional abnormalities being present in non-complex karyotypes in eight (8%) patients and in complex karyotypes in 79 (82%) patients. Altogether, karyotypes characterized by trisomies only were present in 27 (28%) patients. Data on response and outcome of intensively treated patients were available for 92 cases. The median follow-up was 6.4 years (95% confidence interval [95% CI]: 2.9-9.0 years). The complete remission (CR) rate after induction therapy was 52% (48 patients); the early death rate was 10% (n=9). Notably, patients with trisomy 19 as the sole abnormality had a CR rate of 89%. Allogeneic hematopoietic stem cell transplantation (allo-HCT) was performed in 34 (35%) patients (CR, n=19; active disease, n=15). Five-year relapse-free and overall survival rates were 26% (95% CI: 16-43%) and 20% (95% CI: 13-31%), respectively. Overall survival rates were significantly higher in patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time-dependent covariable on overall survival revealed that trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only was a favorable factor (hazard ratio [HR]=0.47; P=0.021); higher age at diagnosis had an adverse impact (10 years difference; HR=1.29; P=0.002), whereas allo-HCT did not have a beneficial impact (odds ratio=1.45; P=0.21). In our cohort, patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only had a high CR rate and better clinical outcome.

Evaluation of the outcomes of newly diagnosed patients with high-risk myelodysplastic syndrome according to the initial therapeutical strategies chosen in usual clinical practice.

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases without a care standard and show variability in treatment outcomes. This Spanish, observational, prospective study ERASME (CEL-SMD-2012-01) assessed the evolution of newly diagnosed and treatment-naïve high-risk MDS patients (according to IPPS-R). 204 patients were included: median age 73.0 years, 54.4% males, 69.6% 0-1 ECOG, and 94.6% with comorbidities. Active treatment was the most common strategy (52.0%) vs. stem cell transplantation (25.5%) and supportive care/watchful-waiting (22.5%). Overall (median) event-free survival was 7.9 months (9.1, 8.3, and 5.3); progression-free survival: 10.1 months (12.9, 12.8, and 4.3); and overall survival: 13.8 months (15.4, 14.9; 8.4), respectively, with significant differences among groups. Adverse events (AEs) of ≥3 grade were reported in 72.6% of patients; serious AEs reported in 60.6%. 33.1% of patients died due to AEs. Three patients developed second primary malignant neoplasms (median: 8.2 months). Our study showed better outcomes in patients receiving active therapy early after diagnosis.

Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia.

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ∼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.

Mapping the genetic features of T-ALL cases through simplified NGS approach.

BACKGROUND: Despite the irruption of massive sequencing technologies in clinical routine, the genetic diagnosis of T-cell acute lymphoblastic leukemia (T-ALL) continues to be based on traditional techniques. The integration of old and new technologies with diagnostic and prognostic purposes represents a major challenge. METHODS: A High-Throughput Sequencing (HTS) approach was applied to analyze the genetic landscape of two patients diagnosed with T-ALL and one early T cell precursor acute leukemia. Orthogonal standard techniques were used to confirm the findings of NGS analysis. RESULTS: By using a single test, a complete genetic map including 2 previously unreported missense mutations in BCL11B gene are reported. Cooperating oncogenic lesions including CDKN2A/B deletions, SIL-TAL1 rearrangement and FLT3 amplification were also captured by using a single test. CONCLUSIONS: HTS is a useful approach that allows simultaneously analyzing mutations, CNVs and the clonal repertoire in T-ALL patients. This approach may simplify the genetic assessment of ALL.

Healthcare resource utilization trends in patients with acute myeloid leukemia ineligible for intensive chemotherapy receiving first-line systemic treatment or best supportive care: A multicenter international study.

OBJECTIVES: This retrospective chart review examined real-world healthcare resource utilization (HRU) in patients with AML ineligible for intensive therapy who received first-line systemic therapy or best supportive care (BSC). METHODS: Data were collected anonymously on patients with AML who initiated first-line hypomethylating agents (HMA), low-dose cytarabine (LDAC), other systemic therapy, or BSC. HRU endpoints included hospitalizations, outpatient consultations, transfusions, and supportive care. RESULTS: Of 1762 patients included, 46% received HMA, 11% received LDAC, 17% received other systemic therapy, 26% received BSC; median treatment durations were 118, 35, 33, and 57 days, respectively. Most patients were hospitalized, most commonly for treatment administration, transfusion, or infection (HMA 82%, LDAC 93%, other systemic therapy 83%, BSC 83%). A median number of hospitalizations were 2-6 across systemic groups and two for BSC, with median durations of 8-18 days. Transfusion rates and outpatient consultations were highest for HMA (80% and 79%) versus LDAC (57% and 53%), other systemic therapy (57% and 63%), and BSC (71% and 66%). Antivirals/antibiotics and antifungals were used more frequently than growth factors (72-92%, 34-63%, and 7-27%, respectively). CONCLUSION: Patients with AML ineligible for intensive therapy have high HRU; novel therapies are needed to alleviate this burden.

Characteristics, clinical outcomes, and risk factors of SARS-COV-2 infection in adult acute myeloid leukemia patients: experience of the PETHEMA group.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces higher morbidity and mortality in hematological malignancies, but evidence in acute myeloid leukemia (AML) is scarce. A multicenter observational study was conducted to determine the clinical outcomes and assess the impact of therapeutic approaches in adult AML patients with SARS-CoV-2 infection in the first wave (March-May 2020). Overall, 108 patients were included: 51.9% with active leukemia and 70.4% under therapeutic schedules for AML. Signs and symptoms of SARS-CoV-2 were present in 96.3% of patients and 82.4% received specific treatment for SARS-CoV-2. The mortality rate was 43.5% and was correlated with age, gender, active leukemia, dyspnea, severe SARS-CoV-2, intensive care measures, neutrophil count, and D-dimer levels. A protective effect was found with azithromycin, lopinavir/ritonavir, and normal liver enzyme levels. During the SARS-CoV-2 first wave, our findings suggested an increased mortality in AML in a short period. SARS-CoV-2 management could be guided by risk factors in AML patients.

Long-Term Outcomes After Autologous Versus Allogeneic Stem Cell Transplantation in Molecularly-Stratified Patients With Intermediate Cytogenetic Risk Acute Myeloid Leukemia: A PETHEMA Study.

Acute myeloid leukemia (AML) with intermediate risk cytogenetics (IRcyto) comprises a variety of biological entities with distinct mutational landscapes that translate into differential risks of relapse and prognosis. Optimal postremission therapy choice in this heterogeneous patient population is currently unsettled. In the current study, we compared outcomes in IRcyto AML recipients of autologous (autoSCT) (n = 312) or allogeneic stem cell transplantation (alloSCT) (n = 279) in first complete remission (CR1). Molecular risk was defined based on CEBPA, NPM1, and FLT3-ITD mutational status, per European LeukemiaNet 2017 criteria. Five-year overall survival (OS) in patients with favorable molecular risk (FRmol) was 62% (95% confidence interval [CI], 50-72) after autoSCT and 66% (95% CI, 41-83) after matched sibling donor (MSD) alloSCT (P = .68). For patients of intermediate molecular risk (IRmol), MSD alloSCT was associated with lower cumulative incidence of relapse (P < .001), as well as with increased nonrelapse mortality (P = .01), as compared to autoSCT. The 5-year OS was 47% (95% CI, 34-58) after autoSCT and 70% (95% CI, 59-79) after MSD alloSCT (P = .02) in this patient subgroup. In a propensity-score matched IRmol subcohort (n = 106), MSD alloSCT was associated with superior leukemia-free survival (hazard ratio [HR] 0.33, P = .004) and increased OS in patients alive 1 year after transplantation (HR 0.20, P = .004). These results indicate that, within IRcyto AML in CR1, autoSCT may be a valid option for FRmol patients, whereas MSD alloSCT should be the preferred postremission strategy in IRmol patients.

Outcomes of patients with chronic myelomonocytic leukaemia treated with non-curative therapies: a retrospective cohort study.

BACKGROUND: Approval of hypomethylating agents in patients with chronic myelomonocytic leukaemia is based on trials done in patients with myelodysplastic syndromes. We aimed to investigate whether hypomethylating agents provide a benefit in subgroups of patients with chronic myelomonocytic leukaemia compared with other treatments. METHODS: For this retrospective cohort study, data were retrieved between Nov 30, 2017, and Jan 5, 2019, from 38 centres in the USA and Europe. We included non-selected, consecutive patients diagnosed with chronic myelomonocytic leukaemia, who received chronic myelomonocytic leukaemia-directed therapy. Patients with acute myeloid leukaemia according to 2016 WHO criteria at initial diagnosis (ie, ≥20% blasts in the bone marrow or peripheral blood) or with unavailability of treatment data were excluded. Outcomes assessed included overall survival, time to next treatment, and time to transformation to acute myeloid leukaemia. Analyses were adjusted by age, sex, platelet count, and Chronic myelomonocytic leukaemia-Specific Prognostic Scoring System (CPSS). Patients were grouped by first received treatment with either hydroxyurea, hypomethylating agents, or intensive chemotherapy, and stratified by risk according to blast count, French-American-British subtype, CPSS, WHO 2016 subtype, and the eligibility criteria of the DACOTA trial (NCT02214407). FINDINGS: 949 patients diagnosed with chronic myelomonocytic leukaemia between April 13, 1981, and Oct 26, 2018, were included. Median follow-up was 23·4 months (IQR 11·5-42·3) from diagnosis and 16·2 months (6·6-31·6) from start of first-line treatment. 412 (43%) of 949 patients received hypomethylating agents as first treatment, 391 (41%) hydroxyurea, and 83 (9%) intensive chemotherapy. Adjusted median overall survival for patients treated with hydroxyurea versus hypomethylating agents was 15·6 months (95% CI 13·1-17·3) versus 20·7 months (17·9-23·4); hazard ratio (HR) 1·39 (1·17-1·65; p=0·0002) and 14·0 months (9·8-17·2) versus 20·7 months (17·9-23·4; HR 1·55 [1·16-2·05]; p=0·0027) for those treated with intensive chemotherapy versus hypomethylating agents. In patients with myeloproliferative chronic myelomonocytic leukaemia (myeloproliferative CMML), median overall survival was 12·6 months (10·7-15·0) versus 17·6 months (14·8-21·5; HR 1·38 [1·12-1·70]; p=0·0027) for patients treated with hydroxyurea versus hypomethylating agents, and 12·3 months (8·4-16·6) versus 17·6 months (14·8-21·5; HR 1·44 [1·02-2·03]; p=0·040) for intensive chemotherapy versus hypomethylating agents. Hypomethylating agents did not confer an overall survival advantage for patients classified as having lower-risk disease (ie, myelodysplastic chronic myelomonocytic leukaemia with <10% blasts, CMML-0, or lower-risk CPSS). INTERPRETATION: These data suggest hypomethylating agents as the preferred therapy for patients with higher-risk chronic myelomonocytic leukaemia and those with myeloproliferative CMML. Our findings also suggest that CPSS is a valuable tool to identify patients who are most likely to benefit from hypomethylating agents. Further evidence from prospective cohorts would be desirable. FUNDING: The Austrian Group for Medical Tumor Therapy.

Strategy for identification of a potential inherited leukemia predisposition in a 299 patient's cohort with tumor-only sequencing data.

Myeloid neoplasms (MN) are usually sporadic late-onset cancers; nevertheless, growing evidence suggests that ∼5% of the cases could emerge as a consequence of inherited predisposition. Distinguishing somatic from germline variants is of vital importance, in order to establish an appropriate individualized management and counsel the patients and their relatives. Since many of the genes associated with myeloid neoplasm germline predisposition (MNGP) are also affected in sporadic MN, we intended to design a strategy to identify potentially inherited variants in a tumor only NGS panel in a cohort of 299 patients with a variety of MN. We considered as indicative of potential inherited origin, variants detected in BM sample at a ∼50% VAF classified as pathogenic, likely pathogenic or of unknown significance detected in MNGP-related genes. A total of 104 suspicious variants from 90 patients were filtered-in in tumor samples. Mutational patterns, follow-up data, and sequencing of a range of non-myeloid tissues were used for narrowing down the list of suspicious variants, and ultimately discriminate their nature. Our data supports the importance of considering variants found upon tumor-only sequencing as potentially of germline origin, and we offer a pipeline to define the nature of the variants.

Performance of prognostic scoring systems in elderly patients with acute myeloid leukaemia on intensive chemotherapy: A PETHEMA registry study.

Selection of elderly patients (aged ≥60 years) for intensive chemotherapy treatment of acute myeloblastic leukaemia (AML) remains challenging. Several cooperative groups such as Acute Leukaemia French Association (ALFA), Haematological Oncology Clinical Studies Group (HOCSG) and MD Anderson Cancer Center (MDACC) have developed predictive models to select those patients who can benefit from intensive chemotherapy. Our purpose is to validate and compare these three models in a cohort of patients treated in real-life setting. For this, a total of 1724 elderly AML patients and treated with intensive chemotherapy regimens were identified in the PETHEMA registry. Median age was 67.2 years (range, 60-84,9) and median overall survival [OS] 9 months (95 % confidence interval [CI], 8.2-9.7). Taking into account the ALFA group's model, patients likely to benefit from intensive chemotherapy had longer OS (14 months, 95 % CI 12.3-15.7) than those unlikely to benefit (5 months, 95 % CI 4.1-5.9; p < 0.001). Significant differences in OS were observed between patients with favourable risk (17 months, 95 % CI 13.2-20.7), intermediate risk (11 months, 95 % CI 9.3-12.6) and adverse risk (6 months, 95 % CI 5.1-6.4; p < 0.001) according to the HOCSG model. No significant differences in OS were observed between patients with 0, 1, 2 or ≥3 points according to the MDACC model. However, when patients with ≥1 point were compared with those with 0 points, median OS was significantly longer in the latter [15 months (95 % CI 12.1-17.8) vs 7 (95 % CI 5.7-8.5)]. This retrospective study validates predictive models proposed by the ALFA, HOCSG and MDACC groups in this real-life cohort.

Iniciación a la investigación en el Grado en Medicina mediante la realización de proyectos tutorados / Introduction to research in the Medical Degree through supervised projects

Objetivo: Presentar la experiencia de iniciación a la investigación con estudiantes de medicina mediante asignaturas optativas: 'Proyectos de investigación de grado' (1, 2 y 3). Sujetos y métodos: Han participado estudiantes de segundo, tercero y cuarto año de los cursos académicos 2011-2012 a 2017-2018 que se matricularon sucesivamente en 'Proyectos 1' (identifi cación del tema, documentación y elaboración de hipótesis y objetivos), 'Proyectos 2' (diseño metodológico y adquisición de datos) y 'Proyectos 3' (divulgación científi ca). Cada grupo, de 2-4 estudiantes, trabajó bajo la dirección de un tutor, realizó presentaciones y elaboró un manuscrito sobre su actividad. En 'Proyectos 3', presentaron un póster con la actividad de los tres cursos que se expuso en una jornada monográfi ca. La evaluación de la satisfacción de los estudiantes se realizó mediante una encuesta. Resultados: En total, 546 estudiantes han iniciado la experiencia en 'Proyectos 1', de los que el 43,9% la completan. El número de tutores participantes ha sido de 83. Se han abordado 195 temas de investigación, de los que el 59% son de investigación clínica, el 31% de investigación fundamental y el 10% de otros. Las califi caciones obtenidas han sido superiores a 9 puntos sobre 10. La encuesta resalta que los estudiantes valoran el papel de los tutores y el potencial formativo de la actividad. Conclusión: El diseño de los cursos permite que el estudiante tome contacto con diferentes aspectos del proceso de investigación, actividad que consideran gratifi cante y apropiada a su formación como médico

Aim: To present the experience of an initiation in to research for medical students through optional courses: 'Research Projects of Degree' (1, 2, and 3). Subjects and methods: Medical students in their second, third, and fourth year from the academic years 2011-2012 to 2017-2018, enrolled consecutively in the subjects: 'Projects 1', identifi cation of the topic, documentation and elaboration of hypotheses and objectives; 'Projects 2', methodological design and acquisition of data; and 'Projects 3', scientifi c disclosure. Each group, of 2-4 students, did a research project under the supervision of a tutor; the research project was presented orally and on paper. In 'Projects 3', students made a poster about the activities performed during the three courses, posters were shown in a monographic exhibition. Evaluation of student satisfaction was carried out through a survey. Results: A total of 546 students started the experience in 'Projects 1', of which 43.9% completed it. The number of participating tutors was 83, addressing 195 research topics, of which 59% were clinical research, 31% fundamental research and 10% were termed others. The scores obtained in these courses were higher than 9 points out of 10. The survey highlights the important role of advisers and the training potential of the activity. Conclusion: The design of the course allows the students to be in contact with diff erent aspects of the biomedical research processes. Students considered the activity rewarding, useful, and appropriate to their training as a physician

Cytogenetics and gene mutations influence survival in older patients with acute myeloid leukemia treated with azacitidine or conventional care.

Older patients with newly diagnosed acute myeloid leukemia (AML) in the phase 3 AZA-AML-001 study were evaluated at entry for cytogenetic abnormalities, and a subgroup of patients was assessed for gene mutations. Patients received azacitidine 75 mg/m2/day x7 days (n = 240) or conventional care regimens (CCR; n = 245): intensive chemotherapy, low-dose cytarabine, or best supportive care only. Overall survival (OS) was assessed for patients with common (occurring in ≥10% of patients) cytogenetic abnormalities and karyotypes, and for patients with recurring gene mutations. There was a significant OS improvement with azacitidine vs CCR for patients with European LeukemiaNet-defined Adverse karyotype (HR 0.71 [95%CI 0.51-0.99]; P = 0.046). Azacitidine-treated patients with -5/5q-, -7/7q-, or 17p abnormalities, or with monosomal or complex karyotypes, had a 31-46% reduced risk of death vs CCR. The most frequent gene mutations were DNMT3A (27%), TET2 (25%), IDH2 (23% [R140, 15%; R172, 8%]), and TP53 (21%). Compared with wild-type, OS was significantly reduced among CCR-treated patients with TP53 or NRAS mutations and azacitidine-treated patients with FLT3 or TET2 mutations. Azacitidine may be a preferred treatment for older patients with AML with Adverse-risk cytogenetics, particularly those with chromosome 5, 7, and/or 17 abnormalities and complex or monosomal karyotypes. The influence of gene mutations in azacitidine-treated patients warrants further study.

Azacitidine improves clinical outcomes in older patients with acute myeloid leukaemia with myelodysplasia-related changes compared with conventional care regimens.

BACKGROUND: Compared with World Health Organization-defined acute myeloid leukaemia (AML) not otherwise specified, patients with AML with myelodysplasia-related changes (AML-MRC) are generally older and more likely to have poor-risk cytogenetics, leading to poor response and prognosis. More than one-half of all older (≥65 years) patients in the phase 3 AZA-AML-001 trial had newly diagnosed AML-MRC. METHODS: We compared clinical outcomes for patients with AML-MRC treated with azacitidine or conventional care regimens (CCR; induction chemotherapy, low-dose cytarabine, or supportive care only) overall and within patient subgroups defined by cytogenetic risk (intermediate or poor) and age (65-74 years or ≥75 years). The same analyses were used to compare azacitidine with low-dose cytarabine in patients who had been preselected to low-dose cytarabine before they were randomized to receive azacitidine or CCR (ie, low-dose cytarabine). RESULTS: Median overall survival was significantly prolonged with azacitidine (n = 129) versus CCR (n = 133): 8.9 versus 4.9 months (hazard ratio 0.74, [95%CI 0.57, 0.97]). Among patients with intermediate-risk cytogenetics, median overall survival with azacitidine was 16.4 months, and with CCR was 8.9 months (hazard ratio 0.73 [95%CI 0.48, 1.10]). Median overall survival was significantly improved for patients ages 65-74 years treated with azacitidine compared with those who received CCR (14.2 versus 7.3 months, respectively; hazard ratio 0.64 [95%CI 0.42, 0.97]). Within the subgroup of patients preselected to low-dose cytarabine before randomization, median overall survival with azacitidine was 9.5 months versus 4.6 months with low-dose cytarabine (hazard ratio 0.77 [95%CI 0.55, 1.09]). Within the low-dose cytarabine preselection group, patients with intermediate-risk cytogenetics who received azacitidine had a median overall survival of 14.1 months versus 6.4 months with low-dose cytarabine, and patients aged 65-74 years had median survival of 14.9 months versus 5.2 months, respectively. Overall response rates were similar with azacitidine and CCR (24.8% and 17.3%, respectively), but higher with azacitidine versus low-dose cytarabine (27.2% and 13.9%). Adverse events were generally comparable between the treatment arms. CONCLUSIONS: Azacitidine may be the preferred treatment for patients with AML-MRC who are not candidates for intensive chemotherapy, particularly patients ages 65-74 years and those with intermediate-risk cytogenetics. TRIAL REGISTRATION: This study was registered at clinicalTrials.gov on February 16, 2010 ( NCT01074047 ).

International phase 3 study of azacitidine vs conventional care regimens in older patients with newly diagnosed AML with >30% blasts.

This multicenter, randomized, open-label, phase 3 trial evaluated azacitidine efficacy and safety vs conventional care regimens (CCRs) in 488 patients age ≥65 years with newly diagnosed acute myeloid leukemia (AML) with >30% bone marrow blasts. Before randomization, a CCR (standard induction chemotherapy, low-dose ara-c, or supportive care only) was preselected for each patient. Patients then were assigned 1:1 to azacitidine (n = 241) or CCR (n = 247). Patients assigned to CCR received their preselected treatment. Median overall survival (OS) was increased with azacitidine vs CCR: 10.4 months (95% confidence interval [CI], 8.0-12.7 months) vs 6.5 months (95% CI, 5.0-8.6 months), respectively (hazard ratio [HR] was 0.85; 95% CI, 0.69-1.03; stratified log-rank P = .1009). One-year survival rates with azacitidine and CCR were 46.5% and 34.2%, respectively (difference, 12.3%; 95% CI, 3.5%-21.0%). A prespecified analysis censoring patients who received AML treatment after discontinuing study drug showed median OS with azacitidine vs CCR was 12.1 months (95% CI, 9.2-14.2 months) vs 6.9 months (95% CI, 5.1-9.6 months; HR, 0.76; 95% CI, 0.60-0.96; stratified log-rank P = .0190). Univariate analysis showed favorable trends for azacitidine compared with CCR across all subgroups defined by baseline demographic and disease features. Adverse events were consistent with the well-established safety profile of azacitidine. Azacitidine may be an important treatment option for this difficult-to-treat AML population. This trial was registered at www.clinicaltrials.gov as #NCT01074047.

Nanobody conjugated PLGA nanoparticles for active targeting of African Trypanosomiasis.

Targeted delivery of therapeutics is an alternative approach for the selective treatment of infectious diseases. The surface of African trypanosomes, the causative agents of African trypanosomiasis, is covered by a surface coat consisting of a single variant surface glycoprotein, termed VSG. This coat is recycled by endocytosis at a very high speed, making the trypanosome surface an excellent target for the delivery of trypanocidal drugs. Here, we report the design of a drug nanocarrier based on poly ethylen glycol (PEG) covalently attached (PEGylated) to poly(D,L-lactide-co-glycolide acid) (PLGA) to generate PEGylated PLGA nanoparticles. This nanocarrier was coupled to a single domain heavy chain antibody fragment (nanobody) that specifically recognizes the surface of the protozoan pathogen Trypanosoma brucei. Nanoparticles were loaded with pentamidine, the first-line drug for T. b. gambiense acute infection. An in vitro effectiveness assay showed a 7-fold decrease in the half-inhibitory concentration (IC50) of the formulation relative to free drug. Furthermore, in vivo therapy using a murine model of African trypanosomiasis demonstrated that the formulation cured all infected mice at a 10-fold lower dose than the minimal full curative dose of free pentamidine and 60% of mice at a 100-fold lower dose. This nanocarrier has been designed with components approved for use in humans and loaded with a drug that is currently in use to treat the disease. Moreover, this flexible nanobody-based system can be adapted to load any compound, opening a range of new potential therapies with application to other diseases.

Monovinyl sulfone ß-cyclodextrin. A flexible drug carrier system.

Cyclodextrins have been conjugated to target various receptors and have also been functionalized with carbohydrates for targeting specific organs. However, this approach is based on a rigid design that implies the ad hoc synthesis of each cyclodextrin-targeting agent conjugate. We hypothesized that: 1)a modular design that decouples the carrier function from the targeting function leads to a flexible system, 2) combining the reactivity of the vinyl sulfone group toward biomolecules that act as targeting agents with the ability of cyclodextrin to form complexes with a wide range of drugs may yield a versatile system that allows the targeting of different organs with different drugs, and 3) the higher reactivity of histidine residues toward the vinyl sulfone group can be exploited to couple the cyclodextrin to the targeting system with a degree of regioselectivity. As a proof of concept, we synthesized a monovinyl sulfone ß-cyclodextrin (module responsible for the payload), which, after coupling to recombinant antibody fragments raised against Trypanosoma brucei (module responsible for targeting) and loading with nitrofurazone (module responsible for therapeutic action) resulted in an effective delivery system that targets the surface of the parasites and shows trypanocidal activity.

Novel therapy based on camelid nanobodies.

Nanobodies (Nbs) are small antibody fragments derived from camelid heavy chain antibodies through recombinant gene technology. Their exceptional physicochemical properties, possibility of humanization and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components. Several different therapeutic approaches based on the novel camelid Nbs have been developed to treat a wide range of diseases ranging from immune, bone, blood and neurological disorders; infectious diseases and cancer. This review provides a comprehensive overview of the current state of the use of camelid-derived Nbs as novel therapeutic agents against multiple diseases.

A set of activators and repressors control peripheral glucose pathways in Pseudomonas putida to yield a common central intermediate.

Pseudomonas putida KT2440 channels glucose to the central Entner-Doudoroff intermediate 6-phosphogluconate through three convergent pathways. The genes for these convergent pathways are clustered in three independent regions on the host chromosome. A number of monocistronic units and operons coexist within each of these clusters, favoring coexpression of catabolic enzymes and transport systems. Expression of the three pathways is mediated by three transcriptional repressors, HexR, GnuR, and PtxS, and by a positive transcriptional regulator, GltR-2. In this study, we generated mutants in each of the regulators and carried out transcriptional assays using microarrays and transcriptional fusions. These studies revealed that HexR controls the genes that encode glucokinase/glucose 6-phosphate dehydrogenase that yield 6-phosphogluconate; the genes for the Entner-Doudoroff enzymes that yield glyceraldehyde-3-phosphate and pyruvate; and gap-1, which encodes glyceraldehyde-3-phosphate dehydrogenase. GltR-2 is the transcriptional regulator that controls specific porins for the entry of glucose into the periplasmic space, as well as the gtsABCD operon for glucose transport through the inner membrane. GnuR is the repressor of gluconate transport and gluconokinase responsible for the conversion of gluconate into 6-phosphogluconate. PtxS, however, controls the enzymes for oxidation of gluconate to 2-ketogluconate, its transport and metabolism, and a set of genes unrelated to glucose metabolism.