Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders.

INTRODUCTION: Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost. AIM: The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs. METHODS: A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology. RESULTS: In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant. CONCLUSION: Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.

ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-update on methodological approaches and results interpretation.

In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.

Immunoglobulin lambda isotype gene rearrangements in B cell malignancies.

The human immunoglobulin lambda (IGL) locus contains seven J-Clambda gene regions of which only J-Clambda1, J-Clambda2 J-CA3 and J-Clambda7 encode the four Iglambda isotypes, ie Mcg, Ke-Oz-, Ke-Oz+, and Mcp, respectively. We used isotype-specific DNA probes for detection of IGL gene rearrangements in 212 B cell malignancies: 76 precursor B cell acute lymphoblastic leukemias (precursor B-ALL), 74 Iglambda+ chronic B cell leukemias (CBL), 34 Iglambda+ non-Hodgkin lymphomas (B-NHL), and 28 Iglambda+ multiple myelomas (MM). The J-Clambda3 gene region was most frequently involved (50%), followed by J-Clambda2 (38%) and J-Clambda1 (9%). There was no involvement of the J-Clambda4 and J-Clambda5 gene regions. Rearrangements to J-Clambda6 (n= 4) were exclusively found in precursor B-ALL (19% of all IGL rearrangements in precursor B-ALL) and only a single J-Clambda7 recombination was detected in an Iglambda+ B-NHL. In the group of Iglambda+ malignancies, a significant shift was observed from predominant J-Clambda3 usage (54%) in mature surface Iglambda+ malignancies (CBL and B-NHL) to 60% J-Clambda2 usage in Iglambda+ secreting MM. The distribution of IGL isotype rearrangements found in MM resembled the Iglambda isotype protein expression reported in MM patients. Based on these extensive Southern blot data, we suggest that a rapid and efficient detection of clonal IGL gene rearrangements can be obtained when a single Bg/II digest is used in combination with the IGLJ2 probe, which detects clonality in >95% of cases with an Iglambda+ malignancy. Higher percentages (>98%) can be reached by including a second digest (HindIII) that reduces the chance of comigration of rearranged and germline bands. In case of precursor B-ALL we recommend including the IGLJ6 probe for the detection of rearrangements to J-Clambda6.

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

Multiple cranial neuropathy and intracranial hypertension associated with all-trans retinoic acid treatment in a young adult patient with acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) induces complete remission in 64-100 % of patients with acute promyelocytic leukemia (APL), and is considered to be a safe agent. Pseudotumor cerebri is a neurological side effect of ATRA reported in pediatric patients, and which is characterized by raised cerebrospinal fluid pressure in the absence of any intracranial pathology or secondary causes of intracranial hypertension. Involvement of cranial nerves other than II and VI is very uncommon in idiopathic intracranial hypertension (IIH); peripheral facial nerve palsy is exceptional and has rarely been described in the context of treatment with ATRA. We describe the case of a 15-year-old female patient with APL who developed an IIH and involvement of cranial nerves (bilateral papilledema, left facial and right sixth nerves) after receiving induction therapy including ATRA. Viral infections and other causes of secondary cranial nerve lesions were excluded. Symptoms completely subsided with the temporary withdrawal of ATRA and did not recur after reintroducing the drug. To date, the patient has managed to receive the treatment as per protocol. In conclusion, we report an atypical presentation of IIH that merits consideration, especially with respect to young patients with APL receiving ATRA; our most important observation is that the drug could be safely reintroduced once the symptoms had resolved.

Value of the bone scintigraphy in multiple osteochrondromatosis with sarcomatous degeneration.

Multiple osteochondromatosis can become malignant in 20% of the cases, this being more common when the lesion is multiple than when it is solitary. A male patient with multiple osteochondromatosis who had several local recurrences of secondary chondrosarcoma and who is still under follow-up by the Nuclear Medicine Department is presented. The bone scintigraphy findings were compared with the histopathologic results, and the importance of the patient's symptoms was verified when a sarcomatous transformation is suspected. The bone scintigraphy has the potential to detect malignization of the benign bone lesions. It also makes it possible to obtain whole-body images in a single examination, this being very useful to detect the presence of new bone lesions.

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia.

There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.

Efectividad de la psicoterapia breve en los centros de salud mental II: resultados / Effectiveness of brief psychotherapy in community mental health centers II: results

Introducción: El objetivo de la presente investigación ha sido comparar mediante un ensayo clínico la efectividad de una Psicoterapia Breve con el tratamiento habitual de los Trastornos Mentales Comunes en los Centros de Salud Mental (CSM) de Asturias. La introducción general a este estudio se ha descrito por Fernández-Méndez y cols. (2010) y se remite a los lectores a ese artículo para más detalles respecto al fundamento, diseño, tratamientos y procedimientos de la investigación. Sujetos y método: Se seleccionaron al azar 216 personas mayores de 14 años que consultaban por primera vez en seis CSM y que fueron diagnosticadas de trastornos depresivos, de ansiedad y/o de adaptación. Ciento cuarenta y una cumplían los criterios de inclusión y aceptaron participar en la investigación, siendo asignadas al azar a dos grupos: Psicoterapia breve integradora-ecléctica (n = 76) o Tratamiento habitual en los CSM (n = 65). Se han comparado sus resultados a los 6, 12, 24 y 36 meses en diversos índices de mejoría clínica, funcionamiento psicosocial e indicadores sanitarios indirectos. Los datos se han obtenido del Registro de Casos Psiquiátricos, de la Historia Clínica y del propio paciente. Se han usado los siguientes instrumentos: Impresión Clínica Global (ICG, Guy, 1976), Inventario de Discapacidad de Sheehan (SDI, Sheehan, 1996) y Cuestionario de Satisfacción (Moré y Muñoz, 2000). Resultados: El programa de Psicoterapia breve ha sido más efectivo que el tratamiento habitual: Obtiene una mayor tasa de altas y los sujetos muestran mayores mejorías en estado clínico y discapacidad y mayor satisfacción. Estas diferencias se dan tanto a los seis meses como al año y a los dos años de iniciarse los tratamientos. Además, aunque el número medio de sesiones es igual en ambos grupos (en torno a seis), el tratamiento experimental duró mucho menos tiempo. Conclusiones: La Psicoterapia ha resultado ser un tratamiento viable y efectivo para la mayoría de los casos que consultan en los CSM (AU)

Background: The aim of the current research has been comparing the effectiveness of a brief psychotherapy with the usual treatment of Common Mental Disorders in Mental Health Centers (MHC) of Asturias. The general background of this study has already been described by Fernández-Méndez et al (2010) and readers are referred to that article for details regarding rationale, design, treatments and procedures of the study. Subjects and method: Two hundred and sixteen patients over the age of 14 were selected at random among those who consulted for the first time in six MHC and were diagnosed of depressive, anxiety or adjustment disorders. One hundred and forty-one fulfilled the inclusion criteria and agreed to take part in the study; they were assigned at random into two groups: brief integrative-eclectic psychotherapy (n = 76) or usual treatment in the MHC (n = 65). Their results have been compared at 6, 12, 24 and 36 months against diverse indexes of clinical improvement, psycho-social functioning and sanitary indirect indicators. Information has been obtained from the Psychiatric Cases Record, the Clinical History and from the patients. The following instruments have been used: the Clinical Global Impression of Improvement scale, the Sheehan Disability Inventory and a Satisfaction Survey. Results: Brief psychotherapy was more effective than usual treatment: it resulted in a higher number of discharges and subjects show greater improvements in clinical status and disability, and greater satisfaction. These differences occur both at 6 months to one year and two years of starting treatment. Furthermore, although the average number of sessions is equal in both groups (about six), time-wise the psychological treatment lasted much less (AU)

Efectividad de la psicoterapia breve en los centros de salud mental I: Diseño, tratamientos y procedimiento / Effectiveness of Brief Psychotherapy in Community Mental Health Centers I: design, treatments and procedures

Introducción. El objetivo de la presente investigación ha sido comparar mediante un ensayo clínico la efectividad de una Psicoterapia Breve con el tratamiento habitual de los Trastornos Mentales Comunes en los Centros de Salud Mental (CSM) de Asturias. En este artículo se describen la justificación, el diseño y los procedimientos de ese estudio. Los primeros resultados se publican en Fernández- Méndez y cols. (en prensa). Sujetos y método. Se seleccionaron al azar 216 personas mayores de 14 años que consultaban por primera vez en seis CSM y que fueron diagnosticadas de trastornos depresivos, de ansiedad y/o de adaptación. Ciento cuarenta y una cumplían los criterios de inclusión y aceptaron participar en la investigación, siendo asignadas al azar a dos grupos: Psicoterapia breve integradora- ecléctica (n = 76) o Tratamiento habitual en los CSM (n = 65). Se han comparado sus resultados a los 6, 12, 24 y 36 meses en diversos índices de mejoría clínica, funcionamiento psicosocial e indicadores sanitarios indirectos. Los datos se han obtenido del Registro de Casos Psiquiátricos, de la Historia Clínica y del propio paciente. Se han usado los siguientes instrumentos: Impresión Clínica Global (ICG, Guy, 1976), Inventario de Discapacidad de Sheehan (SDI, Sheehan, 1996) y Cuestionario de Satisfacción (Moré y Muñoz, 2000) (AU)

Background. The aim of the current research has been comparing the effectiveness of a brief psychotherapy with the usual treatment of Common Mental Disorders in Mental Health Centers (MHC) of Asturias. The present article describes the rationale, design and procedures of the study. Initial outcome findings are reported by Fernández- Méndez et al (in press). Subjects and method. Two hundred and sixteen patients over the age of 14 were selected at random among those who consulted for the first time in six MHC and were diagnosed of depressive, anxiety or adjustment disorders. One hundred and forty-one fulfilled the inclusion criteria and agreed to take part in the study; they were assigned at random into two groups: brief integrative-eclectic psychotherapy (n = 76) or usual treatment in the MHC (n = 65). Their results have been compared at 6, 12, 24 and 36 months against diverse indexes of clinical improvement, psycho-social functioning and sanitary indirect indicators. Information has been obtained from the Psychiatric Cases Record, the Clinical History and from the patients. The following instruments have been used: the Clinical Global Impression of Improvement scale, the Sheehan Disability Inventory and a Satisfaction Survey (AU)

[Undergraduate teaching of primary health care. A Delphi study]. / La docencia de atención primaria de salud en el pregrado. Un estudio Delphi.

OBJECTIVE: Given both the limited experience of Undergraduate Teaching in Primary Health Care (PHC) and the lack of contact between different teaching centres, we sought to identify the minimum contents of a potential Syllabus. We made a distinction between items of knowledge required and skills students would have to develop. DESIGN: We undertook a Delphi study, which was longitudinal, prospective and observational. PARTICIPANTS: We defined three groups of experts among groups with PHC teaching experience. 20 Associate Lecturers at Undergraduate level, attached to Health centres; 20 postgraduate PHC teachers, and 7 University staff members involved with Public Health and Preventive and Community Medicine teaching. MAIN MEASUREMENTS AND RESULTS: The reply rate from the three questionnaires was about 60%. There was a high level of agreement. Of the items of knowledge which scored highest, we noted: the doctor-patients relationship, the Promotion of Health and Health education, Integrated Health Programmes, Prevention of Disease and Care of Risk-Groups. Of the skills, we pointed to the Clinical Interview, the construction of a Clinical History at the PHC level, Detection of Risk factors and Physical Examination. CONCLUSIONS: The most valued items of knowledge are those related to the overall Conception of Health, Promotion and Prevention. The most highly-scored skills are those concerning the Doctor-Patient relationship. Questions relating to the Individual are prioritised over those concerning Family and Community. There is a high level of agreement that the acquisition of the necessary theoretical knowledge must be accompanied by the ability to put them into practice.