Krinio Giannikou

Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndrome associated with chromosome 5q deletion

Background Lenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodysplastic syndrome and interstitial deletion of the long arm of chromosome 5 [del(5q)]. The aim of this study was to explore the effect of lenalidomide treatment on the reserves and functional characteristics of bone marrow hematopoietic progenitor/precursor cells, bone marrow stromal cells and peripheral blood lymphocytes in patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q). Design and Methods We evaluated the number and clonogenic potential of bone marrow erythroid/myeloid/megakaryocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesion molecule expression of CD34+ cells by flow cytometry, the hematopoiesis-supporting capacity of bone marrow stromal cells using long-term bone marrow cultures and the number and activation status of peripheral blood lymphocytes in ten patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q) receiving lenalidomide. Results Compared to baseline, lenalidomide treatment significantly decreased the proportion of bone marrow CD34+ cells, increased the proportion of CD36+/GlycoA+ and CD36−/GlycoA+ erythroid cells and the percentage of apoptotic cells within these cell compartments. Treatment significantly improved the clonogenic potential of bone marrow erythroid, myeloid, megakaryocytic colony-forming cells and increased the proportion of CD34+ cells expressing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48. The hematopoiesis-supporting capacity of bone marrow stroma improved significantly following treatment, as demonstrated by the number of colony-forming cells and the level of stromal-derived factor-1α and intercellular adhesion molecule-1 in long-term bone marrow culture supernatants. Lenalidomide treatment also increased the proportion of activated peripheral blood T lymphocytes. Conclusions The beneficial effect of lenalidomide in patients with lower risk myelodysplastic syndrome with del(5q) is associated with significant increases in the proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant alterations in the adhesion profile of bone marrow CD34+ cells.

De Novo Interstitial Duplication of the 15q11.2-q14 PWS/AS Region of Maternal Origin: Clinical Description, Array CGH Analysis, and Review of the Literature

The 15q11‐q13 PWS/AS critical region involves genes that are characterized by genomic imprinting. Multiple repeat elements within the region mediate rearrangements, including interstitial duplications, interstitial triplications, and supernumerary isodicentric marker chromosomes, as well as the deletions that cause Prader–Willi syndrome (PWS) and Angelman syndrome (AS). Recently, duplications of maternal origin concerning the same critical region have been implicated in autism spectrum disorders (ASD). We present a 6‐month‐old girl carrying a de novo duplication of maternal origin of the 15q11.2‐q14 PWS/AS region (17.73 Mb in size) [46,XX,dup(15)(q11.2‐q14)] detected with a high‐resolution microarray‐based comparative genomic hybridization (array‐CGH). The patient is characterized by severe hypotonia, obesity, microstomia, long eyelashes, hirsutism, microretrognathia, short nose, severe psychomotor retardation, and multiple episodes of drug‐resistant epileptic seizures, while her brain magnetic resonance imaging (MRI) documented partial corpus callosum dysplasia. In our patient the duplicated region is quite large extending beyond the Prader–Willi–Angelman critical region (PWACR), containing a number of genes that have been shown to be involved in ASD, exhibiting a severe phenotype, beyond the typical PWS/AS clinical manifestations. Reporting of similar well‐characterized clinical cases with clearly delineated breakpoints of the duplicated region will clarify the contribution of specific genes to the phenotype.

Microdeletion and microduplication 17q21.31 plus an additional CNV, in patients with intellectual disability, identified by array-CGH.

The recognition of the 17q21.31 microdeletion and microduplication syndrome has been facilitated by high resolution oligonucleotide array comparative genome hybridization technology (aCGH). Molecular analysis of the 17q21.31 microdeletion/duplication syndrome demonstrated a critical region involving at least six genes, including STH and MAPT. The 17q21.31 microdeletion syndrome has an incidence of 1 in 16,000 births, while the microduplication 17q21.31 has been reported so far in only five patients. In general, phenotypes associated with 17q21.31 microduplication seem to be milder than those associated with the microdeletion. Here, we present four patients who have been referred for genetic evaluation by clinical geneticists due to developmental delay and minor congenital abnormalities. Previous standard karyotypes were negative, while aCGH analysis revealed three patients with 17q21.31 microdeletion and one with the respective microduplication, being the sixth reported case so far. Most importantly one of the microdeletion cases involves only partial MAPT gene deletion while leaving the STH gene intact. Two of our patients, one with the 17q21.31 microdeletion and another with the respective microduplication, carried additional clinically relevant microdeletions (del Xq21.31 and del 15q11.2, respectively), possibly modifying their phenotype.

New miRNA Profiles Accurately Distinguish Renal Cell Carcinomas and Upper Tract Urothelial Carcinomas from the Normal Kidney

Background Upper tract urothelial carcinomas (UT-UC) can invade the pelvicalyceal system making differential diagnosis of the various histologically distinct renal cell carcinoma (RCC) subtypes and UT-UC, difficult. Correct diagnosis is critical for determining appropriate surgery and post-surgical treatments. We aimed to identify microRNA (miRNA) signatures that can accurately distinguish the most prevalent RCC subtypes and UT-UC form the normal kidney. Methods and Findings miRNA profiling was performed on FFPE tissue sections from RCC and UT-UC and normal kidney and 434 miRNAs were significantly deregulated in cancerous vs. the normal tissue. Hierarchical clustering distinguished UT-UCs from RCCs and classified the various RCC subtypes among them. qRT-PCR validated the deregulated expression profile for the majority of the miRNAs and ROC analysis revealed their capability to discriminate between tumour and normal kidney. An independent cohort of freshly frozen RCC and UT-UC samples was used to validate the deregulated miRNAs with the best discriminatory ability (AUC>0.8, p<0.001). Many of them were located within cytogenetic regions that were previously reported to be significantly aberrated. miRNA targets were predicted using the miRWalk algorithm and ingenuity pathway analysis identified the canonical pathways and curated networks of the deregulated miRNAs. Using the miRWalk algorithm, we further identified the top anti-correlated mRNA/miRNA pairs, between the deregulated miRNAs from our study and the top co-deregulated mRNAs among 5 independent ccRCC GEO datasets. The AB8/13 undifferentiated podocyte cells were used for functional assays using luciferase reporter constructs and the developmental transcription factor TFCP2L1 was proved to be a true target of miR-489, which was the second most upregulated miRNA in ccRCC. Conclusions We identified novel miRNAs specific for each RCC subtype and UT-UC, we investigated their putative targets, the networks and pathways in which they participate and we functionally verified the true targets of the top deregulated miRNAs.

Array comparative genomic hybridization as a clinical diagnostic tool in syndromic and nonsyndromic congenital heart disease.

Background:Congenital heart diseases (CHDs) are often associated with other congenital anomalies, dysmorphic features, and developmental delay, and only a few cases of chromosomal abnormalities are detected by conventional cytogenetic techniques. The microarray comparative genomic hybridization (CGH) analysis allows the identification of submicroscopic genomic rearrangements.Methods:During the past 3 y, 55 of 330 patients referred for array CGH had CHD of unknown etiology plus at least one additional indication of abnormal chromosomal phenotype. High-resolution 1 × 244K or 4 × 180K Agilent arrays were used in this study (average resolution 7–13 kb).Results:Copy-number variations were detected in 37 of 55 patients, and in 29 of 37 patients there were genes that have been associated with CHD. All 37 patients had at least one additional phenotypic abnormality: 30 of 37 had one or more other congenital anomalies, 23 of 37 had dysmorphic features, 16 of 37 had intellectual disability, 13 of 37 had abnormal magnetic resonance imaging, 10 of 37 had hypotonia, and 7 of 37 had seizures. In 9 of 55 patients, unexpected genomic rearrangements in relation to their phenotype were identified.Conclusion:In patients with CHD and at least one additional indication of abnormal chromosomal phenotype, array CGH analysis could detect possible submicroscopic chromosomal abnormalities and provide proper genetic counseling.

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms

BackgroundAlthough, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy.MethodsOverall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients’ clinical characteristics and pathological measures.ResultsIn several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied.ConclusionsDeeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

Disruptions in asymmetric centrosome inheritance and WDR62-Aurora kinase B interactions in primary microcephaly

Recessive mutations in WD repeat domain 62 (WDR62) cause microcephaly and a wide spectrum of severe brain malformations. Disruption of the mouse ortholog results in microcephaly underlain by reduced proliferation of neocortical progenitors during late neurogenesis, abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. Spindle pole localization of WDR62 and mitotic progression are defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Expression of WDR62 is closely correlated with components of the chromosome passenger complex (CPC), a key regulator of mitosis. Wild type WDR62, but not disease-associated mutant forms, interacts with the CPC core enzyme Aurora kinase B and staining of CPC components at centromeres is altered in patient-derived fibroblasts. Our findings demonstrate critical and diverse functions of WDR62 in neocortical development and provide insight into the mechanisms by which its disruption leads to a plethora of structural abnormalities.

Rapid clinical-scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105 cells

Current clinical protocols used for isolation and purification of mesenchymal stem cells (MSC) are based on long‐term cultures starting with bone marrow (BM) mononuclear cells. Using a commercially available immunoselection kit for enrichment of MSC, we investigated whether culture of enriched BM‐CD105+ cells could provide an adequate number of pure MSC in a short time for clinical use in the context of graft versus host disease and graft failure/rejection. We isolated a mean of 5.4 × 105 ± 0.9 × 105 CD105+ cells from 10 small volume (10–25 ml) BM samples achieving an enrichment >100‐fold in MSC. Seeding 2 × 103 immunoselected cells/cm2 we were able to produce 2.5 × 108 ± 0.7 × 108 MSC from cultures with autologous serum enriched medium within 3 weeks. Neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product. Expanded cells fulfilled both phenotypic and functional current criteria for MSC; they were CD29+, CD90+, CD73+, CD105+, CD45−; they suppressed allogeneic T‐cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential. Moreover, comparative genomic hybridization analysis revealed chromosomal stability of the cultured MSC. Our data indicate that adequate numbers of pure MSC suitable for clinical applications can be generated within a short time using enriched BM‐CD105+ cells.

miR-15a and miR-24-1 as putative prognostic microRNA signatures for pediatric pilocytic astrocytomas and ependymomas

In the current setting, we attempted to verify and validate miRNA candidates relevant to pediatric primary brain tumor progression and outcome, in order to provide data regarding the identification of novel prognostic biomarkers. Overall, 26 resected brain tumors were studied from children diagnosed with pilocytic astrocytomas (PAs) (n = 19) and ependymomas (EPs) (n = 7). As controls, deceased children who underwent autopsy and were not present with any brain malignancy were used. The experimental approach included microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the expression profiles of miR-15a and miR-24-1. The multiparameter analyses were performed with MATLAB. Matching differentially expressed miRNAs were detected in both PAs and EPs, following distinct comparisons with the control cohort; however, in several cases, they exhibited tissue-specific expression profiles. On correlations between miRNA expression and EP progression or outcome, miR-15a and miR-24-1 were found upregulated in EP relapsed and EP deceased cases when compared to EP clinical remission cases and EP survivors, respectively. Taken together, following several distinct associations between miRNA expression and diverse clinical parameters, the current study repeatedly highlighted miR-15a and miR-24-1 as candidate oncogenic molecules associated with inferior prognosis in children diagnosed with ependymoma.

Combined microdeletions and CHD7 mutation causing severe CHARGE/DiGeorge syndrome: clinical presentation and molecular investigation by array-CGH.

Phenotypic variation in CHARGE syndrome remains unexplained. A subcategory of CHARGE patients show overlapping phenotypic characteristics with DiGeorge syndrome (thymic hypo/aplasia, hypocalcemia, T-cell immunodeficiency). Very few have been tested or reported to carry a mutation of the CHD7 (chromodomain helicase DNA-binding domain) gene detected in two-thirds of CHARGE patients. In an attempt to explore the genetic background of a severe CHARGE/DiGeorge phenotype, we performed comparative genomic array hybridization in an infant carrier of a CHD7 mutation. The high-resolution comparative genomic array hybridization revealed interesting findings, including a deletion distal to the DiGeorge region and disruptions in other chromosomal regions of genes implicated in immunological and other functions possibly contributing to the patients severe phenotype and early death.