Further definition of the proximal 19p13.3 microdeletion/microduplication syndrome and implication of PIAS4 as the major contributor.

The proximal 19p13.3 microdeletion/microduplication (prox19p13.3del/dup) syndrome is a recently described disorder with common clinical features including developmental delay, intellectual disability, speech delay, facial dysmorphic features with ear defects, anomalies of the hands and feet, umbilical hernia and hypotonia. While deletions are associated with macrocephaly, patients with duplications have microcephaly. The smallest region of overlap in multiple patients (113.5 kb) included three genes and one pseudogene, with a suggested major role of PIAS4 in determination of the phenotype and head size in these patients. Here, we refine the prox19p13.3del/dup with four additional patients: two with microdeletions, one with microduplication and one family with single-nucleotide nonsense variant in PIAS4. The patient with the PIAS4 loss of function variant displayed a phenotype quite similar to deletion patients -including the macrocephaly and many other core features of the syndrome. Patient's SNV was inherited from her mother who is similarly affected. Thus, our data indicate that PIAS4 is a major contributor to the proximal 19p13.3del/dup syndrome phenotype. In summary, we report the first patient with a pathogenic variant in PIAS4- and three additional rearrangements at the proximal 19p13.3 locus. These observations add further evidence about the molecular basis of this microdeletion/microduplication syndrome.

Aberrant DNA methylation profile of chronic and transformed classic Philadelphia-negative myeloproliferative neoplasms.

Most DNA methylation studies in classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic marker in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythemia vera, 23 essential thrombocythemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-κB pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms, we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukemic transformation of myeloproliferative neoplasms. The therapeutic modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.

Centromere-localized breaks indicate the generation of DNA damage by the mitotic spindle.

Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing.

Correction: Personalized Precision Medicine for Health Care Professionals: Development of a Competency Framework.

[This corrects the article DOI: 10.2196/43656.].

Personalized Precision Medicine for Health Care Professionals: Development of a Competency Framework.

BACKGROUND: Personalized precision medicine represents a paradigm shift and a new reality for the health care system in Spain, with training being fundamental for its full implementation and application in clinical practice. In this sense, health care professionals face educational challenges related to the acquisition of competencies to perform their professional practice optimally and efficiently in this new environment. The definition of competencies for health care professionals provides a clear guide on the level of knowledge, skills, and attitudes required to adequately carry out their professional practice. In this context, this acquisition of competencies by health care professionals can be defined as a dynamic and longitudinal process by which they use knowledge, skills, attitudes, and good judgment associated with their profession to develop it effectively in all situations corresponding to their field of practice. OBJECTIVE: This report aims to define a proposal of essential knowledge domains and common competencies for all health care professionals, which are necessary to optimally develop their professional practice within the field of personalized precision medicine as a fundamental part of the medicine of the future. METHODS: Based on a benchmark analysis and the input and expertise provided by a multidisciplinary group of experts through interviews and workshops, a new competency framework that would guarantee the optimal performance of health care professionals was defined. As a basis for the development of this report, the most relevant national and international competency frameworks and training programs were analyzed to identify aspects that are having an impact on the application of personalized precision medicine and will be considered when developing professional competencies in the future. RESULTS: This report defines a framework made up of 58 competencies structured into 5 essential domains: determinants of health, biomedical informatics, practical applications, participatory health, and bioethics, along with a cross-cutting domain that impacts the overall performance of the competencies linked to each of the above domains. Likewise, 6 professional profiles to which this proposal of a competency framework is addressed were identified according to the area where they carry out their professional activity: health care, laboratory, digital health, community health, research, and management and planning. In addition, a classification is proposed by progressive levels of training that would be advisable to acquire for each competency according to the professional profile. CONCLUSIONS: This competency framework characterizes the knowledge, skills, and attitudes required by health care professionals for the practice of personalized precision medicine. Additionally, a classification by progressive levels of training is proposed for the 6 professional profiles identified according to their professional roles.

Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics.

BACKGROUND: Malignant lymphomas are classified based on morphology, immunophenotype, genetics and clinical features. The pathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in specific entities, their detection is an important adjunct for increasing the reliability of the diagnosis. Recently, split-signal fluorescence in situ hybridization has become available as a robust method to detect chromosomal breaks in paraffin-embedded formalin-fixed tissues. A bright field approach would bring this technology within the reach of every pathology laboratory. DESIGN AND METHODS: Our study was initiated to determine the consistency between chromogenic in situ hybridization and fluorescence in situ hybridization, both using split-signal probes developed for the detection of chromosomal breaks. Five hundred and forty cases of 11 lymphoma entities and reactive, benign lymphoid tissues, collected from eight different pathology laboratories, placed on 15 fluorescence in situ hybridization pre-stained tissue microarray slides, were double stained for the chromogenic hybridization. For each core morphology and actual signal were compared to the original fluorescence hybridization results. In addition, hematoxylin background staining intensity and signal intensity of the double-staining chromogenic in situ hybridization procedure were analyzed. RESULTS: With respect to the presence or absence of chromosomal breaks, 97% concordance was found between the results of the two techniques. Hematoxylin background staining intensity and signal intensity were found to correspond. The overall morphology after double-staining chromogenic in situ hybridization had decreased compared to the initial morphology scored after split-signal fluorescence in situ hybridization staining. CONCLUSIONS: We conclude that double-staining chromogenic in situ hybridization is equally reliable as fluorescence in situ hybridization in detecting chromosomal breaks in lymphoid tissue. Although differences in morphology, hematoxylin staining and chromogenic signal intensity vary between the tumor entities none of the entities appeared more easy or difficult to score.

Novel genomic alterations and mechanisms associated with tumor progression in oligodendroglioma and mixed oligoastrocytoma.

Combined 1p/19q deletions are very prevalent in oligodendrogliomas (OGs) and, to a lesser extent, in oligoastrocytomas (OAs). These losses are associated with responsiveness to therapy. Using array-based comparative genomic hybridization, we screened for recurrent genomic alterations in OG and oligoastrocytoma subtypes on chromosome 19. Concomitant 1p/19q loss was detected in most of the tumors with allelic loss, but array-based comparative genomic hybridization revealed some tumors to have unrelated 1p/19q arm losses, suggesting alternative mechanisms of loss to that related to the reported t(1;19) translocation. Analyses of 1p/19q loss by fluorescence in situ hybridization and loss of heterozygosity assays and correlations of genomic data with the Ki-67 proliferation marker were also performed. Four 1q (or 19p) and 2 1p (or 19q) fluorescence in situ hybridization probe signals together with homozygosity of the 1p/19q microsatellites suggested a hypothetical mechanism of genome duplication consecutive to the loss of the derivative chromosome der(1p;19q) from the t(1;19)(1q;19p) translocation. This genome duplication was frequent in high-grade OGs and was strongly correlated with Ki-67 expression; thus, it could be related to tumor progression. Finally, in addition to the frequent 1p/19q loss, we report a novel 17q amplified region in OGs with BIRC5 as one of the possible candidate target genes of the amplicon.

Primary Ewing sarcoma of the tentorium presenting with intracranial hemorrhage in a child.

Spontaneous intracerebral hemorrhage in children is usually related to cerebrovascular conditions. Brain tumors presenting with spontaneous bleeding account for approximately 10% of intracranial hemorrhages in children. The occurrence of primitive central nervous system lesions in the Ewing sarcoma family of tumors (ESFT) not related to bone or metastatic disease is a rare condition. The authors report on a child who presented with intracranial bleeding secondary to a nonmetastatic tentorial ESFT confirmed by detection of the fusion gene EWS-ERG. A detailed review of the literature reveals that most primary intracranial ESFT had a meningeal attachment, and that almost half of them presented at diagnosis with hemorrhage. Distinguishing between ESFT and other intracranial neoplasms is essential because the treatment and prognosis differ remarkably from that of other tumors, namely central primitive neuroectodermal tumors (PNETs). Whereas adjuvant treatment for ESFT consists of local or regional radiotherapy and chemotherapy containing alkylating agents, central PNETs are generally treated with whole neuraxis radiation and platinum-based chemotherapy. Additionally, the prognosis for intracranial ESFT might be better than the one for nonpineal central PNETs.

Immortalization of primary human prostate epithelial cells by c-Myc.

A significant percentage of prostate tumors have amplifications of the c-Myc gene, but the precise role of c-Myc in prostate cancer is not fully understood. Immortalization of human epithelial cells involves both inactivation of the Rb/p16INK4a pathway and telomere maintenance, and it has been recapitulated in culture by expression of the catalytic subunit of telomerase, hTERT, in combination with viral oncoproteins. Here, we show the immortalization of human prostate epithelial cells (HPrEC) by a single genetic event, the expression of the c-Myc oncogene. Myc stabilizes telomere length in HPrEC through up-regulation of hTERT expression and overrides the accumulation of cell cycle inhibitory proteins, such as p16INK4a. Overall, HPrECs expressing c-Myc retain many characteristics of normal cells, such as the induction of a senescence-like growth arrest in response to oncogenic Ras, an intact p53 response, and an absence of gross karyotypic abnormalities. However, HPrECs expressing c-Myc lack a Rb/p16INK4a checkpoint and can be transformed without the need for additional genetic lesions in that pathway. These results give a partial explanation for the physiologic role of c-Myc overexpression in prostate cancer.

Recommendations for the use of microarrays in prenatal diagnosis. / Recomendaciones para el uso de microarrays en el diagnóstico prenatal.

Microarray technology, recently implemented in international prenatal diagnosis systems, has become one of the main techniques in this field in terms of detection rate and objectivity of the results. This guideline attempts to provide background information on this technology, including technical and diagnostic aspects to be considered. Specifically, this guideline defines: the different prenatal sample types to be used, as well as their characteristics (chorionic villi samples, amniotic fluid, fetal cord blood or miscarriage tissue material); variant reporting policies (including variants of uncertain significance) to be considered in informed consents and prenatal microarray reports; microarray limitations inherent to the technique and which must be taken into account when recommending microarray testing for diagnosis; a detailed clinical algorithm recommending the use of microarray testing and its introduction into routine clinical practice within the context of other genetic tests, including pregnancies in families with a genetic history or specific syndrome suspicion, first trimester increased nuchal translucency or second trimester heart malformation and ultrasound findings not related to a known or specific syndrome. This guideline has been coordinated by the Spanish Association for Prenatal Diagnosis (AEDP, «Asociación Española de Diagnóstico Prenatal¼), the Spanish Human Genetics Association (AEGH, «Asociación Española de Genética Humana¼) and the Spanish Society of Clinical Genetics and Dysmorphology (SEGCyD, «Sociedad Española de Genética Clínica y Dismorfología¼).

Genomic imbalances and patterns of karyotypic variability in mantle-cell lymphoma cell lines.

Mantle-cell lymphoma (MCL) is genetically characterized by 11q13 chromosomal translocations involving the CCND1 gene. We have characterized five MCL cell lines, JVM-2, GRANTA-519, REC-1, JEKO-1, and NCEB-1, combining metaphase and array comparative genomic hybridization, multicolor-FISH, and molecular analysis. Our results revealed common gained regions at 2p14, 9q31.2-qter, 11q13.1-q21, 13q14-q21.2, 13q34-qter and 18q21.1-q22.1, and losses at 1p21.2-p31.1, 2p11.2, 8p21.2-pter, 9p21.3-pter, 11q23.3-qter, 17p11.2-pter, and 17q21.2-q22.2. All cell lines except JVM-2, displayed moderate or high numerical chromosome instability. In addition, an ongoing level of chromosome rearrangements was observed in REC-1. Surprisingly, NCEB-1 carried several stable mouse chromosomes and showed expression of both human and murine bcl-2 protein. Our findings indicate that these cell lines represent three patterns of chromosome evolution in MCL and may be useful to understand the pathogenesis of this neoplasm.

A predictor based on the somatic genomic changes of the BRCA1/BRCA2 breast cancer tumors identifies the non-BRCA1/BRCA2 tumors with BRCA1 promoter hypermethylation.

The genetic changes underlying in the development and progression of familial breast cancer are poorly understood. To identify a somatic genetic signature of tumor progression for each familial group, BRCA1, BRCA2, and non-BRCA1/BRCA2 (BRCAX) tumors, by high-resolution comparative genomic hybridization, we have analyzed 77 tumors previously characterized for BRCA1 and BRCA2 germ line mutations. Based on a combination of the somatic genetic changes observed at the six most different chromosomal regions and the status of the estrogen receptor, we developed using random forests a molecular classifier, which assigns to a given tumor a probability to belong either to the BRCA1 or to the BRCA2 class. Because 76.5% (26 of 34) of the BRCAX cases were classified with our predictor to the BRCA1 class with a probability of >50%, we analyzed the BRCA1 promoter region for aberrant methylation in all the BRCAX cases. We found that 15 of the 34 BRCAX analyzed tumors had hypermethylation of the BRCA1 gene. When we considered the predictor, we observed that all the cases with this epigenetic event were assigned to the BRCA1 class with a probability of >50%. Interestingly, 84.6% of the cases (11 of 13) assigned to the BRCA1 class with a probability >80% had an aberrant methylation of the BRCA1 promoter. This fact suggests that somatic BRCA1 inactivation could modify the profile of tumor progression in most of the BRCAX cases.

Identification of novel cytogenetic markers with prognostic significance in a series of 968 patients with primary myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: The main prognostic factors in myelodysplastic syndromes (MDS) are chromosomal abnormalities, the proportion of blasts in bone marrow and number and degree of cytopenias. A consensus-defined International Prognostic Scoring System (IPSS) for predicting outcome and planning therapy in MDS has been developed, but its prognostic value in a large and independent series remains unproven. Furthermore, the intermediate-risk cytogenetic subgroup defined by the IPSS includes a miscellaneous number of different single abnormalities of uncertain prognostic significance at present. The main aim of the present study was to identify chromosomal abnormalities with a previously unrecognized good or poor prognosis in order to find new cytogenetic markers with predictive value. DESIGN AND METHODS: We report the cytogenetic findings in a series of 968 patients with primary MDS from the Spanish Cytogenetics Working Group, Grupo Cooperativo Español de Citogenética Hematológica (GCECGH). RESULTS: In this series of 968 MDS patients, we found various cytogenetic aberrations with a new prognostic impact. Complex karyotype, -7/7q- and i(17q) had a poor prognosis; normal karyotype, loss of Y chromosome, deletion 11q, deletion 12p and deletion 20q as single alterations had a good prognosis. Intermediate prognosis aberrations were rearrangements of 3q21q26, trisomy 8, trisomy 9, translocations of 11q and del(17p). Finally, a new group of single or double cytogenetic abnormalities, most of which are considered rare cytogenetic events and are usually included in the intermediate category of the IPSS, showed a trend to poor prognosis. INTERPRETATION AND CONCLUSIONS: This study suggests that some specific chromosomal abnormalities could be segregated from the IPSS intermediate-risk cytogenetic prognostic subgroup and included in the low risk or in the poor risk groups.

Chronic neutrophilic leukemia: a clinical perspective.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) that includes only 150 patients described to date meeting the latest World Health Organization (WHO) criteria and the recently reported CSF3R mutations. The diagnosis is based on morphological criteria of granulocytic cells and the exclusion of genetic drivers that are known to occur in others MPNs, such as BCR-ABL1, PDGFRA/B, or FGFR1 rearrangements. However, this scenario changed with the identification of oncogenic mutations in the CSF3R gene in approximately 83% of WHO-defined and no monoclonal gammopathy-associated CNL patients. CSF3R T618I is a highly specific molecular marker for CNL that is sensitive to inhibition in vitro and in vivo by currently approved protein kinase inhibitors. In addition to CSF3R mutations, other genetic alterations have been found, notably mutations in SETBP1, which may be used as prognostic markers to guide therapeutic decisions. These findings will help to understand the pathogenesis of CNL and greatly impact the clinical management of this disease. In this review, we discuss the new genetic alterations recently found in CNL and the clinical perspectives in its diagnosis and treatment. Fortunately, since the diagnosis of CNL is not based on exclusion anymore, the molecular characterization of the CSF3R gene must be included in the WHO criteria for CNL diagnosis.

Cytogenetic profile of myelodysplastic syndromes with complex karyotypes: an analysis using spectral karyotyping.

We have performed a cytogenetic analysis of 23 myelodysplastic syndromes (MDS) with complex karyotypes (CK) using GTG-banding and spectral karyotyping techniques. Fifty-five percent of cases were hypodiploid, 34% were hyperdiploid, and 11% were pseudodiploid. The most recurrent alterations were monosomy of chromosomes 18, 5, and 7; trisomy of chromosome 8; and deletion of 5q, 11q, and 12p. Ninety-two structural alterations were mostly identified as unbalanced. The chromosomes and regions more frequently affected were 16q12, 17p11, and 20q11. Eight of 92 structural alterations were reciprocal translocations. Two translocations were recurrent, t(X;20)(p11.4;q11.2) and der(17)t(5;17)(?;p11.2); each one was present in about 10% of cases (2 cases, t[X:20] and 3 cases, t[5:17]). Mutations of TP53 were observed in five cases (22%), all with rearrangements affecting 17p. Total or partial inactivation of TP53 was detected in six cases (26%) as a result of loss of either both copies (four cases) or just one copy (two cases). Fluorescence in situ hybridization analysis showed amplification of genes previously identified in myeloid and/or hematological processes, such as HER2neu, MLL, and AML1, which could represent frequent events in MDS with CK.

A new case of acute nonlymphocytic leukemia (French-American-British subtype M1) with double minutes and c-MYC amplification.

Herein, we present a new case of acute nonlymphocytic leukemia (ANLL) French-American-British M1 subtype with presence of multiple double minutes (dmin) derived from the amplification of the c-MYC oncogene. A review of dmins in ANLL is presented.

Overexpression and activation of EGFR and VEGFR2 in medullary thyroid carcinomas is related to metastasis.

Therapeutic options for patients with metastatic medullary thyroid carcinoma (MTC) are limited due to lack of effective treatments. Thus, there is a need to thoroughly characterize the pathways of molecular pathogenesis and to identify potential targets for therapy in MTC. Since epidermal growth factor receptor (EGFR) seems to play a crucial role for RET activation, a key feature of MTCs, and several promising EGFR/vascular endothelial growth factor receptor 2 (VEGFR2)-targeted drugs have been developed, the present study was designed to investigate whether these proteins are altered in MTCs. We used a well-characterized series of 153 MTCs to evaluate EGFR activation by sequencing and FISH analysis, and to perform EGFR and VEGFR2 immunohistochemistry. EGFR tyrosine kinase domain mutations were not a feature of MTCs; however, EGFR polysomy and a strong EGFR expression were detected in 15 and 13% of the tumors respectively. Interestingly, EGFR was significantly overexpressed in metastases compared with primary tumors (35 vs 9%, P=0.002). We also studied whether specific RET mutations were associated with EGFR status, and found a decrease in EGFR polysomies (P=0.006) and a tendency towards lower EGFR expression for the most aggressive RET mutations (918, 883). Concerning VEGFR2, metastasis showed a higher expression than primary tumors (P=2.8 x 10(-8)). In this first study investigating the relationship between EGFR, RET, and VEGFR2 in a large MTC series, we found an activation of EGFR and VEGFR2 in metastasis, using both independent and matched primary/metastasis samples. This suggests that some MTC patients may benefit from existing anti-EGFR/VEFGR2 therapies, although additional preclinical and clinical evidence is needed.

Epigenetic signatures associated with different levels of differentiation potential in human stem cells.

BACKGROUND: The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles. METHODOLOGY/PRINCIPAL FINDINGS: to address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes. CONCLUSIONS/SIGNIFICANCE: Our study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.