Neurological manifestations of neurofibromatosis type 1: our experience.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is a progressive multisystem disorder following an autosomal dominant inheritance pattern that presents with multiple neurological manifestations. METHODS: We reviewed medical histories of patients with NF1 followed up at our hospital's paediatric neurology department from May 1990 to 31 December 2018. We collected data on neurological symptoms. RESULTS: A total of 128 patients with NF1 were identified. Mean age (SD) at NF1 diagnosis was 4.43 (3.38) years (range, 0.5-14.5 years). There was a slight female predominance (53.1%). Macrocephaly (head circumference over 2 SDs above average for age) was present in 37.5% of cases. Attention-deficit/hyperactivity disorder was recorded in 28.9% of patients (37): combined type in 20 patients, predominantly inattentive in 15, and predominantly impulsive/hyperactive in 2. Other manifestations included headache (18.6%), cognitive impairment (7.8%), motor deficit (6.2%), and epilepsy (4.68%). Brain MRI was performed in 85 patients, revealing T2-weighted hyperintensities in the basal ganglia and/or cerebellum in 60 patients (70.5%), Chiari malformation type 1 in 4 cases, and arachnoid cysts in 3. Optic nerve gliomas were identified by MRI in 22 patients (25.8%). Other MRI findings included plexiform neurofibromas (9.3%) and central nervous system gliomas (3.1%). CONCLUSIONS: The neurological manifestations identified in our sample are consistent with those reported in the literature. Effective transfer strategies from paediatric neurology departments and subsequent clinical follow-up by adult neurology departments are needed to prevent loss to follow-up in adulthood.

Manifestaciones neurológicas en neurofibromatosis tipo 1. Nuestra experiencia / Neurological manifestations of neurofibromatosis type 1: our experience

Introducción: La neurofibromatosis tipo 1 (NF1) es un desorden progresivo multisistémico de herencia autosómica dominante que presenta numerosas manifestaciones neurológicas. Métodos: Revisión de historias clínicas de pacientes afectos de NF1 controlados en una Unidad de Neuropediatría de mayo de 1990 a 31 de diciembre de 2018 y sus manifestaciones neurológicas asociadas. Resultados: Se revisaron 128 pacientes afectos de NF1. Edad media al diagnóstico de NF1, 4,43 años ± 3,38 SDS (rango 6 meses-14,5 años) con discreto predominio femenino (53,1%). Se asocia macrocefalia (PC> 2SDS) en el 37,5% de los casos. TDAH en el 28,9% de los casos (37), subtipo combinado 20, inatento 15 casos y predominantemente hiperactivo 2 casos. Otras manifestaciones incluyen; cefalea (18,7%), déficit cognitivo (7,8%), afectación motora (6,2%) y epilepsia (4,68%). Se realizó RM cerebral a 85 pacientes, mostrando 60 (70,5%) hiperseñales en T2 en ganglios basales y/o cerebelo, junto con otras alteraciones como Chiari I (4 casos) y quistes aracnoideos (3 casos). Se identificaron gliomas de nervio óptico en 22 casos (25,8%). Otros hallazgos diagnosticados por RM incluyen neurofibromas plexiformes (9,3%) y otros gliomas localizados en sistema nervioso central (3,1%). Conclusiones: Las manifestaciones neurológicas encontradas concuerdan con lo recogido en la literatura. El seguimiento de estos pacientes se pierde en la edad adulta, siendo necesario establecer adecuadas estrategias de transferencia y posterior seguimiento de pacientes a los servicios de adultos. (AU)

Introduction: Neurofibromatosis type 1 (NF1) is a progressive multisystem disorder following an autosomal dominant inheritance pattern that presents with multiple neurological manifestations. Methods: We reviewed medical histories of patients with NF1 followed up at our hospital's paediatric neurology department from May 1990 to 31 December 2018. We collected data on neurological symptoms. Results: A total of 128 patients with NF1 were identified. Mean age (SD) at NF1 diagnosis was 4.43 (3.38) years (range, 0.5-14.5 years). There was a slight female predominance (53.1%). Macrocephaly (head circumference over 2 SDs above average for age) was present in 37.5% of cases. Attention-deficit/hyperactivity disorder was recorded in 28.9% of patients (37): combined type in 20 patients, predominantly inattentive in 15, and predominantly impulsive/hyperactive in 2. Other manifestations included headache (18.6%), cognitive impairment (7.8%), motor deficit (6.2%), and epilepsy (4.68%). Brain MRI was performed in 85 patients, revealing T2-weighted hyperintensities in the basal ganglia and/or cerebellum in 60 patients (70.5%), Chiari malformation type 1 in 4 cases, and arachnoid cysts in 3. Optic nerve gliomas were identified by MRI in 22 patients (25.8%). Other MRI findings included plexiform neurofibromas (9.3%) and central nervous system gliomas (3.1%). Conclusions: The neurological manifestations identified in our sample are consistent with those reported in the literature. Effective transfer strategies from paediatric neurology departments and subsequent clinical follow-up by adult neurology departments are needed to prevent loss to follow-up in adulthood. (AU)

A novel mutation in the ZNF462 gene c.3306dup; p.(Gln1103Thrfs*10) is associated to Weiss-Kruszka syndrome. A case report.

BACKGROUND: Weiss-Kruszka syndrome (WSKA) is a rare disorder caused by mutations in the ZNF462 gene or deletion of 9p31.2 chromosome region, involving ZNF462. The prevalence of WSKA is unknown as only 24 affected individuals have been described. This syndrome should be suspected in individuals presenting mild global developmental delay and common craniofacial abnormalities. CASE PRESENTATION: We presented a case of an infant, 3 years and 4-month life who presented pondostatural and psychomotor retardation, generalized hypotonia with hypermobility, bilateral palpebral ptosis, epicanthal folds, and poorly expressive facies as the main clinical features. These characteristics lead to the realization of genetics studies that resulted in the identification of a novel mutation c.3306dup; p.(Gln1103Thrfs*10) in ZNF462. CONCLUSIONS: WSKA should be suspected in individuals presenting mild global developmental delay, ptosis, downslanting palpebral fissures, exaggerated Cupid's Bow, arched eyebrows, epicanthal folds and short upturned nose with a bulbous tip. Hypertrophy of the ventricular septum and severe OSA were described in our patient and should be considered in future reviews of the disease. This case is added to the reduced number of publications previously reported regarding WSKA and contributes to understanding the genetic characteristics, clinical features, and diagnosis of this syndrome.Abbreviations: WSKA: Weiss-Kruszka syndrome; CP: craniofacial perimeter; WES: whole-exome sequencing; RSV: respiratory syncytial virus; OSA: obstructive sleep apnoea; ACMG: American College of Medical Genetics and Genomics.

HiperCKemia persistente asintomática en edad pediátrica. Diagnóstico de miopatía central core sin biopsia muscular / Asymptomatic hyperCKemia in children. Central core disease with non-diagnostic muscle biopsy

INTRODUCCIÓN: la elevación persistente de creatinfosfoquinasa (CK) puede constituir la primera manifestación de una patología muscular subyacente. Su correcto abordaje permite un adecuado tratamiento precoz, asesoramiento familiar e información sobre su pronóstico y sus complicaciones. CASO CLÍNICO: niño de siete años, asintomático, con elevación de CK como hallazgo casual en una analítica de rutina, persistiendo en controles seriados. Exploración física normal. Tras un estudio metabólico completo normal, se solicita estudio genético dirigido a descartar distrofinopatías u otras miopatías. Se observa una mutación en el gen RYR1, c.9912C>A; p. (Cys3304*), variante probablemente patogénica compatible con miopatía congénita de cores centrales (#MIM11700). Ante un diagnóstico genético en paciente asintomático, se evita la realización de otras técnicas invasivas. CONCLUSIONES: la miopatía congénita de cores centrales es la patología neuromuscular congénita más frecuente. Se relaciona con la presencia de mutaciones en el gen RYR1 (90% de los pacientes). Pertenece a la familia de los canales liberadores de calcio iónico, cuyo papel es fundamental en el fenómeno de acoplamiento excitación-contracción muscular. Su diagnóstico clásico era la biopsia muscular. Está asociado a complicaciones como hipertermia maligna o rabdomiolisis

INTRODUCTION: persistently elevated serum creatine kinase levels may lean the first manifestation of an underlying neuromuscular disease. Its appropriate approach allows an adequate early treatment, a genetic counselling and information concerning complications and prognosis. CASE DESCRIPTION: our patient was an asymptomatic 7-year-old boy with persistent serum CK elevation. He had a normal physical examination. After a normal metabolic study, a specific genetic study for dystrophinopaties or other myopathies was requested. A variant of uncertain significance mutation [RYR1, c.9912C>A; p. (Cys3304*)] associated with central core disease (#MIM11700) was obtained. Before this genetic diagnosis the invasive testing was rejected. DISCUSSION: central core disease is the most frequent congenital neuromuscular disease. About 90% of cases are linked to RYR1 gene mutations. RYR1 protein is a part of macromolecular complex deputed to excitation-contraction coupling through Ca2+ channels. Its diagnosis is confirmed by histological examination. CCD is associated to malignant hyperthermia and rabdomiolisis susceptibility

Neurological manifestations of neurofibromatosis type 1: our experience. / Manifestaciones neurológicas en neurofibromatosis tipo 1. Nuestra experiencia.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is a progressive multisystem disorder following an autosomal dominant inheritance pattern that presents with multiple neurological manifestations. METHODS: We reviewed medical histories of patients with NF1 followed up at our hospital's paediatric neurology department from May 1990 to 31 December 2018. We collected data on neurological symptoms. RESULTS: A total of 128 patients with NF1 were identified. Mean age (SD) at NF1 diagnosis was 4.43 (3.38) years (range, 0.5-14.5 years). There was a slight female predominance (53.1%). Macrocephaly (head circumference over 2 SDs above average for age) was present in 37.5% of cases. Attention-deficit/hyperactivity disorder was recorded in 28.9% of patients (37): combined type in 20 patients, predominantly inattentive in 15, and predominantly impulsive/hyperactive in 2. Other manifestations included headache (18.6%), cognitive impairment (7.8%), motor deficit (6.2%), and epilepsy (4.68%). Brain MRI was performed in 85 patients, revealing T2-weighted hyperintensities in the basal ganglia and/or cerebellum in 60 patients (70.5%), Chiari malformation type 1 in 4 cases, and arachnoid cysts in 3. Optic nerve gliomas were identified by MRI in 22 patients (25.8%). Other MRI findings included plexiform neurofibromas (9.3%) and central nervous system gliomas (3.1%). CONCLUSIONS: The neurological manifestations identified in our sample are consistent with those reported in the literature. Effective transfer strategies from paediatric neurology departments and subsequent clinical follow-up by adult neurology departments are needed to prevent loss to follow-up in adulthood.

Declaración de Sant Joan d’Alacant en defensa del acceso abierto a las publicaciones científicas, del Grupo de Editores de Revistas Españolas sobre Ciencias de la Salud (GERECS) / Declaration of Sant Joan d'Alacant in defense of open access to the scientific publications, from the publishers of Spanish journals group on (GERECS) health sciences

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Phenotype in patients with intellectual disability and pathological results in array CGH. / Fenotipo en pacientes con discapacidad intelectual y array-CGH patológico.

INTRODUCTION: Global developmental delay (GDD) and intellectual disability (ID) are frequent reasons for consultation in paediatric neurology departments. Nowadays, array comparative genomic hybridisation (array-CGH) is one of the most widely used techniques for diagnosing these disorders. Our purpose was to determine the phenotypic features associated with pathological results in this genetic test. METHODS: We conducted a blind study of the epidemiological, clinical, anthropometric, and morphological features of 80 patients with unexplained ID to determine which features were associated with pathological results in array-CGH. RESULTS: Pathological results were found in 27.5% of the patients. Factors associated with pathological results in array-CGH were a family history of GDD/ID (OR = 12.1), congenital malformations (OR = 5.33), having more than 3 facial dysmorphic features (OR = 20.9), and hypotonia (OR = 3.25). CONCLUSIONS: Our findings are consistent with those reported by other published series. We therefore conclude that the probability of having pathological results in array-CGH increases with the presence of any of the features mentioned above in patients with ID/GDD.

Almost 2% of Spanish breast cancer families are associated to germline pathogenic mutations in the ATM gene.

PURPOSE: There is still a considerable percentage of hereditary breast and ovarian cancer (HBOC) cases not explained by BRCA1 and BRCA2 genes. In this report, next-generation sequencing (NGS) techniques were applied to identify novel variants and/or genes involved in HBOC susceptibility. METHODS: Using whole exome sequencing, we identified a novel germline mutation in the moderate-risk gene ATM (c.5441delT; p.Leu1814Trpfs*14) in a family negative for mutations in BRCA1/2 (BRCAX). A case-control association study was performed to establish its prevalence in Spanish population, in a series of 1477 BRCAX families and 589 controls further screened, and NGS panels were used for ATM mutational screening in a cohort of 392 HBOC Spanish BRCAX families and 350 patients affected with diseases not related to breast cancer. RESULTS: Although the interrogated mutation was not prevalent in case-control association study, a comprehensive mutational analysis of the ATM gene revealed 1.78% prevalence of mutations in the ATM gene in HBOC and 1.94% in breast cancer-only BRCAX families in Spanish population, where data about ATM mutations were very limited. CONCLUSION: ATM mutation prevalence in Spanish population highlights the importance of considering ATM pathogenic variants linked to breast cancer susceptibility.

Localized states in advanced dielectrics from the vantage of spin- and symmetry-polarized tunnelling across MgO.

Research on advanced materials such as multiferroic perovskites underscores promising applications, yet studies on these materials rarely address the impact of defects on the nominally expected materials property. Here, we revisit the comparatively simple oxide MgO as the model material system for spin-polarized solid-state tunnelling studies. We present a defect-mediated tunnelling potential landscape of localized states owing to explicitly identified defect species, against which we examine the bias and temperature dependence of magnetotransport. By mixing symmetry-resolved transport channels, a localized state may alter the effective barrier height for symmetry-resolved charge carriers, such that tunnelling magnetoresistance decreases most with increasing temperature when that state is addressed electrically. Thermal excitation promotes an occupancy switchover from the ground to the excited state of a defect, which impacts these magnetotransport characteristics. We thus resolve contradictions between experiment and theory in this otherwise canonical spintronics system, and propose a new perspective on defects in dielectrics.

Colonisation by Faecalibacterium prausnitzii and maintenance of clinical remission in patients with ulcerative colitis.

BACKGROUND: Although incrimination of the intestinal microbiota in the pathogenesis of IBD is widely accepted, few data are available about the role of specific bacteria. Potentially, Faecalibacterium prausnitzii, bacteria with anti-inflammatory properties, might be deficient in ulcerative colitis (UC). AIM: To quantify F. prausnitzii in the faecal microbiota of UC patients in remission and determine its relationship with relapse. METHODS: A cross-sectional study included 116 UC patients in remission, 29 first-degree relatives and 31 healthy controls. A subset of eighteen patients, recruited during the first month of remission, underwent a 1-year follow-up. Total bacteria and F. prausnitzii were measured by quantitative Real Time PCR (qPCR, copies/g). Calprotectin was determined as inflammatory index (µg/g). RESULTS: We found that F. prausnitzii was reduced in patients (median, IQR: 1.4 × 108 , 5.1 × 107-4.5 × 108) and relatives (1.7 × 108, 9.3 × 107-5.1 × 108) vs. controls (6.5 × 108, 3.7 × 108-1.6 × 109, P < 0.0001). Moreover, low counts of F. prausnitzii were associated with less than 12 months of remission (8.0 × 107, 2.0 × 107-3.5 × 108 vs. 2.1 × 108, 1.0 × 108-7.9 × 108, P < 0.001) and more than 1 relapse/year (8.0 × 107, 3.2 × 107-3.8 × 108 vs. 1.9 × 108, 6.8 × 107-6.0 × 108, P < 0.01). When patients were followed up, F. prausnitzii increased steadily until reaching similar levels to those of controls if remission persisted (2.9 × 108, 9.3 × 106-1.2 × 109; calprotectin: 76, 19-212), whereas it remained low if patients relapsed (2.2 × 108, 1.4 × 106-3.3 × 108; calprotectin: 1760, 844-3662 P < 0.05 vs. controls). CONCLUSIONS: Defective gut colonisation by F. prausnitzii occurred in UC patients during remission and in their unaffected relatives. The recovery of the F. prausnitzii population after relapse is associated with maintenance of clinical remission.

Acute experimental stress evokes a differential gender-determined increase in human intestinal macromolecular permeability.

BACKGROUND: Intestinal epithelial dysfunction is a common pathophysiologic feature in irritable bowel syndrome (IBS) patients and might be the link to its clinical manifestations. We previously showed that chronic psychosocial stress induces jejunal epithelial barrier dysfunction; however, whether this epithelial response is gender-specific and might thus explain the enhanced female susceptibility to IBS remains unknown. METHODS: Intestinal responses to acute stress were compared in age-matched groups of healthy women and men (n = 10 each) experiencing low background stress. A 20-cm jejunal segment, was perfused with an isosmotic solution, and intestinal effluents were collected under basal conditions, for 15 min during cold pain stress and for a 45-min recovery period. Epithelial function (net water flux and albumin output), changes in stress hormones, and cardiovascular and psychologic responses to cold stress were measured. KEY RESULTS: Heart rate and blood pressure significantly increased during cold pain stress with no differences between men and women. Adrenocorticotropic hormone and cortisol levels during cold pain stress were significantly higher in men. Basal net water flux and epithelial permeability were similar in men and women. Cold pain stress increased water flux in both groups (72 ± 23 and 107 ± 18 µL min(-1) cm(-1) , respectively; F(5, 90) = 5.5; P = 0.003 for Time) and, interestingly, this was associated with a marked increase of albumin permeability in women but not in men (0.8 ± 0.2 vs.-0.7 ± 0.2 mg/15 min; P < 0.0001). CONCLUSIONS & INFERENCES: Intestinal macromolecular permeability in response to acute experimental stress is increased in healthy women, a mechanism that may contribute to female oversusceptibility to IBS.

[Prader-Willi and Angelman syndromes: 21 years of experience]. / Síndromes de Prader-Willi y de Angelman. Expriencia de 21 años.

INTRODUCTION: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) were the first syndromes in humans that were known to originate from the phenomenon of the genomic imprinting. We review our experience of 21 years with PWS and AS that were confirmed with the genetically. RESULTS: Of the 13,875 patients recorded during the study period, 11 were diagnosed with PWS (18%), 7 males (63.6%) and 4 females (36.4%), with a mean age of 9.06 years (+/- 6.92, range: 0.68-21.6). The time of the follow up of this group was 3.83 years (+/- 4.03, range: 0.49-15.3), and the age at diagnosis was 4.4 years (+/- 6.84, range: 0.03-19.38). Almost three quarters (72.7% of the PWS patients had a uniparental dysomy and 27.3% a paternal deletion. Six patients (8%) were diagnosed with AS, 4 females (66.6%) and 2 males (33.4%), with a mean age of 14.65 years (+/- 11.89, range: 1.3-30.7). The time of follow up was 6.76 years (+/- 5.89,range: 0.16-15), and the age at diagnosis was 8.84 years (+/- 9.11, range: 1.10-23). A maternal deletion was present in 83.3% of the AS patients and 16.7% had a maternal dysomy. DISCUSSION: As genetic advances are made these pathologies are confirmed before. Unlike the data in the literature, in our series most patients diagnosed with PWS (72'3%) had uniparental disomy. Recent studies correlation genotype with phenotype, in PWS is more serious if it occurs a deletion and in SA is milder in the case of uniparental disomy. CONCLUSIONS: Genetic studies must be performed in view of the established clinical symptoms: neonatal hypotonia of unknown cause in PWS and psychomotor deficits with autism features, particularly associated with epilepsy, must be evaluated in AS to prevent diagnostic uncertainties, unnecessary complementary examinations and to provide early genetic counselling.

Síndrome de inversión duplicación del cromosoma 15 (INVDUP15) «de novo»: presentación de tres casos clínicos / Chromosome 15 inversion duplication syndrome (INVDUP15) “de novo”: presentation of three clinical cases

Introducción. El síndrome de inversión duplicación del cromosoma 15 se refiere a un conjunto de características clínicas entre las que se encuentran hipotonía central desde el nacimiento, retraso psicomotor, epilepsia o trastorno del espectro autista. Una invdup(15) resulta de la tetrasomía parcial de 15q y generalmente está implicada la región del Síndrome de Prader-Willi (SPW). Se evalúan tres casos remitidos a Genética por hipotonía y retraso psicomotor. Material y métodos. Cultivo de linfocitos de sangre periférica, cariotipo de alta resolución, FISH, extracción de ADN de linfocitos de sangre periférica, MS-MLPA de SPW y estudio de microsatélites. Resultados. El primer caso presentó un cariotipo 47,XY+der(15)(q13;p11.2)(pter->q13::p11.2->pter) y un cariotipo molecular arr 15q12.1q13(18,432,358-26,658,490)x3∼4 con ganancia de 8,23Mb implicando a genes sometidos a imprinting de la región causante de los síndromes de PWS y Angelman (SA). En el segundo caso se obtuvo una fórmula cromosómica 47, XX, + mar.ish idic (15)(q13)(Acro p-arm ++, D15Z1 ++, D15S10 ++, PML-) y cariotipo molecular arr 15q11.2q13.3(18,432,358-30,230,511)x3, con duplicación de aproximadamente 12Mb. En el tercer caso la paciente resultó ser portadora de una doble línea celular en mosaico 47,XX+ der (15) inv (15)(q11;p11.2) [40%] / 46,XX [60%]. En los tres casos se analizó mediante MLPA la región del SPW encontrándose un patrón de metilación alterado y la causa genética resultó ser un síndrome invdup(15) «de novo». Discusión. A pesar de la dificultad para establecer una correlación fenotipo-genotipo en los casos con invdup(15) las técnicas genéticas más recientes pueden aportar información para el diagnóstico clínico de estos pacientes (AU)

Introduction. The chromosome 15 inversion duplication syndrome refers to distinctive clinical findings, such as, early central hypotonia, developmental delay, epilepsy and autistic behaviour. Invdup(15) results from partial 15q tetrasomy and the Prader-Willi syndrome(PWS) region is generally involved. We have analyzed three clinical cases in a Genetics Unit diagnosed with hypotonia and developmental delay. Material and methods. Lymphocyte cultures from peripheral blood samples, high resolution karyotype, FISH, DNA isolation from peripheral blood leukocytes, PWS MS-MLPA and microsatellites study. Results. The first case showed a karyotype 47,XY+der(15)(q13;p11.2)(pter->q13::p11.2->pter) and a molecular karyotype arr 15q12.1q13(18,432,358-26,658,490)x3 ∼ 4 with an extra 8.23Mb genetic material involving imprinted genes from SPW and Angelman (SA) syndromes region. In the second case there was a karyotype 47, XX, + mar.ish idic (15)(q13)(Acro p-arm ++, D15Z1 ++, D15S10 ++, PML-) and a molecular karyotype arr 15q11.2q13.3(18,432,358-30,230,511)x3 with an approximately 12Mb duplication. The third patient was a carrier of a mosaic double line cell with karyotype 47,XX+ der (15) inv (15)(q11;p11.2) [40%] / 46,XX [60%]. In the three cases the SPW region was analysed using a modified methylation pattern and all resulted from a invdup(15) «de novo» genetic defect. Discussion. Although it is difficult to establish a phenotype-genotype correlation in invdup (15) cases, most recent genetic techniques should provide information for the clinical diagnosis in these patients (AU)

Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size.

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)].

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ ABL x 10(4) also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with < or = 0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels < or = 10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Adverse prognostic impact of CD36 and CD2 expression in adult de novo acute myeloid leukemia patients.

BACKGROUND AND OBJECTIVES: A consecutive series of acute myeloid leukemias (AML) patients was analyzed in conditions which reduce the inter-assay variations (the same flow cytometer, the same observers and the same panel of monoclonal antibodies) in order to investigate the prognostic information provided by flow cytometry. DESIGN AND METHODS: Two hundred and sixty-six bone marrow (BM) samples from 326 patients enrolled in the LMA-99 protocol from the CETLAM group were studied by multiparametric flow cytometry. Immunophenotyping studies were performed on erythrocyte-lysed BM samples. Antigen expression of leukemic cells was analyzed using triple stainings with fluorochrome-conjugated combinations of monoclonal antibodies. RESULTS: CD2 was positive in 21 cases (8%); an associated inv(16) was detected in eight CD2+ cases (38%). Two-year overall survival (OS) rate for CD2+/inv(16)+ patients was 75%, whereas it was 0% for CD2+/inv(16)- patients and 47% for CD2- patients (p=0.0001). CD36 was expressed in 37% of patients (n=98). Two-year leukemia-free survival (LFS) rate was 34% for CD36+ patients and 55% for CD36- patients (p=0.001). In the multivariate analysis, CD2+ (RR=8.4; p=0.0001) and adverse karyotype (RR=10.2; p=0.0001) were associated with a lower CR rate, CD36+ (RR=1.5; p=0.03), CD2+ (RR=2; p=0.04) and adverse karyotype (RR=4; p=0.0001) were associated with a lower OS and CD36+ (RR=2; p=0.002) and adverse karyotype (RR=3.5; p=0.005) predicted a lower LFS. CONCLUSIONS: CD2+ patients had a very poor OS when CD2/inv(16)+ cases were excluded. CD36 and CD2 expression at diagnosis can provide prognostically important information in adult de novo AML.

Quantification of foetomaternal haemorrhage. An analysis of two cytometric techniques and a semiquantitative gel agglutination test.

Traditional tests to screen for foetomaternal haemorrhage are time-consuming and difficult to perform. The Kleihauer test is widely used but difficult to standardize. We evaluated three techniques for quantifying foetomaternal haemorrhage: a semiquantitative gel agglutination test and two flow cytometric techniques. The gel agglutination test is based on the consumption of anti-D reagent by D+ cells, analysing the reaction of the supernatant against indicator cells in a Coombs-gel card. In the two colour direct immunofluorescent technique, the sample is incubated with Per-CP labelled anti CD45 antibody, fixed with glutaraldehyde and permeabilized by exposure to Triton X-100. An aliquot is stained with an antibody to foetal haemoglobin, conjugated with fluorescein isothiocyanate or phycoerythrin. The indirect immunofluorescent technique is based on the labelling of Rh (D) antigen with an anti D reagent, followed by the addition of an anti IgG antibody conjugated with phycoerythrin. Foetomaternal haemorrhage was not detected in 75 of the 85 samples analysed by the direct immunofluorescent technique. In the remaining 10 samples, the volume was very low. Thirty-five samples with Rh (D) antigen incompatibility were analysed in parallel by the indirect immunofluorescent technique and in 15 of the 35 samples, the gel agglutination technique was also carried out. The three techniques gave similar results. The gel agglutination test can be used to screen for foetomaternal haemorrhage, while greater volumes should be quantified by flow cytometric techniques.

Residual low-level viral replication could explain discrepancies between viral load and CD4+ cell response in human immunodeficiency virus-infected patients receiving antiretroviral therapy.

We report the evolution of chronic infection with human immunodeficiency virus type 1 (HIV-1) in a patient treated with stavudine plus didanosine, whose CD4+ lymphocyte count progressively decreased, despite a sustained plasma viral load <20 copies/mL. After 12 months of therapy, treatment was switched to zidovudine plus lamivudine plus nelfinavir. CD4+ T cell count decreased from 559 x 10(6)/L at month 0 to 259 x 10(6)/L at month 12. Plasma viral load decreased from 21,665 HIV-1 RNA copies/mL at baseline (month 0) to <20 copies/mL after 1 month of therapy with stavudine plus didanosine, and remained below 20 copies/mL until month 12, but always >5 copies/mL. Viral load in tonsilar tissue at month 12 was 125,000 copies/mg of tissue. After the change to triple-drug therapy, the plasma viral load decreased to 5 copies/mL, the CD4+ T cell count increased to 705 x 10(6)/L, and the viral load in tonsilar tissue decreased to <40 copies/mg of tissue at month 24. A low level of HIV-1 replication could explain the lack of immunologic response in patients with apparent virological response.