Diagnostic Utility of TSSC3 and RB1 Immunohistochemistry in Hydatidiform Mole.

The general notion of complete hydatidiform moles is that most of them consist entirely of paternal DNA; hence, they do not express p57, a paternally imprinted gene. This forms the basis for the diagnosis of hydatidiform moles. There are about 38 paternally imprinted genes. The aim of this study is to determine whether other paternally imprinted genes could also assist in the diagnostic approach of hydatidiform moles. This study comprised of 29 complete moles, 15 partial moles and 17 non-molar abortuses. Immunohistochemical study using the antibodies of paternal-imprinted (RB1, TSSC3 and DOG1) and maternal-imprinted (DNMT1 and GATA3) genes were performed. The antibodies' immunoreactivity was evaluated on various placental cell types, namely cytotrophoblasts, syncytiotrophoblasts, villous stromal cells, extravillous intermediate trophoblasts and decidual cells. TSSC3 and RB1 expression were observed in all cases of partial moles and non-molar abortuses. In contrast, their expression in complete moles was identified in 31% (TSSC3) and 10.3% (RB1), respectively (p < 0.0001). DOG1 was consistently negative in all cell types in all cases. The expressions of maternally imprinted genes were seen in all cases, except for one case of complete mole where GATA3 was negative. Both TSSC3 and RB1 could serve as a useful adjunct to p57 for the discrimination of complete moles from partial moles and non-molar abortuses, especially in laboratories that lack comprehensive molecular service and in cases where p57 staining is equivocal.

Copy number profiling in von Hippel-Lindau disease renal cell carcinoma.

Germline mutations in the VHL tumor suppressor gene cause von Hippel-Lindau (VHL) disease and somatic VHL mutations occur in the majority of clear cell renal cell carcinoma (cRCC). To compare copy number abnormalities (CNAs) between cRCC from VHL patients and sporadic cRCC cases without detectable somatic VHL mutations, we analyzed 34 cRCC with Affymetrix 250K arrays. To increase the power of the study, we then combined our results with those of a previously published study and compared CNAs in VHL and non-VHL related cRCC using the genomic identification of significant targets in cancer (GISTIC) program. In VHL, cRCC GISTIC analysis identified four statistically significant regions of copy number gain and four statistically significant regions of deletion that occurred in >10% of tumors analyzed. Sporadic cRCC without detectable VHL mutations had, on average, more copy number abnormalities than VHL cRCC though the most common regions of loss/gain (e.g., 3p and 14q loss and 5q gain) were present in both tumor sets. However, CNAs on chromosome arms 7p (gain) and 8p (loss) were only detected in VHL RCC. Although individual copy number abnormality peaks contained clear candidate cancer genes in some cases (e.g., the 3p loss peak in VHL cRCC contained only six genes including VHL), most peaks contained many genes. To date, only a minority of the candidate genes included in these peaks have been analyzed for mutation or epigenetic inactivation in cRCC but TNFRSF10C and DUSP4 map to the 8p region deleted in VHL cRCC and TP53 and HIF2A (EPAS1) mapped to CNA loss and gain peaks (chromosomes 17 and 2, respectively) detected in sporadic VHL wild-type cRCC.

Rare but Potentially Fatal Presentations of Diffuse Large B-cell Lymphoma: Leukemic Phase or Hemophagocytic Syndrome in Bone Marrow.

Diffuse large B-cell lymphoma (DLBCL) is a type of non-Hodgkin Lymphoma commonly presenting as a solid tumor either by nodal or extra-nodal manifestations. Here we describe two atypical presentations of lymphoma, finally resulting in the diagnosis of DLBCL. Case 1: A 53-year-old man with a previous history of nasopharyngeal carcinoma presented with a two-week history of B-symptoms and hyperleukocytosis. Peripheral blood film showed 78% abnormal mononuclear cells. Immunohistochemical stain showing Ki-67 of 90%, negative c-myc, BCL2 and BCL6, and negative c-MYC with fluorescence in-situ hybridization studies on the trephine biopsy, concluded the diagnosis of CD5+ DLBCL of ABC subtype. He received intravenous cyclophosphamide and oral prednisolone for cytoreduction, followed by 6 cycles of chemo-immunotherapy. However, he succumbed due to severe sepsis after the completion of therapy. Case 2: A 56-year-old lady who was initially investigated for pyrexia of unknown origin was noted to have hemophagocytosis upon bone marrow aspirate examination. The bone marrow trephine biopsy revealed some atypical clusters of B-cells positive for CD20 which was inconclusive. PET-CT scan noted an enlarged hypermetabolic spleen without lymphadenopathy. Splenic biopsy with immunohistochemical studies revealed DLBCL of ABC subtype. The diagnosis was consistent with primary splenic DLBCL. She became unwell post splenic biopsy and was admitted to the intensive care unit where she passed away 2 weeks later from Candida and Sternotrophomonas septicemia. These cases highlight the atypical presentations of a common subtype of NHL in our center. Arriving at the definitive diagnosis can be difficult especially when patients are acutely ill, hampering the necessary invasive procedures for diagnosis. The outcomes of both cases are briefly discussed hoping to spread awareness among clinicians on the rare and acutely critical presentations of DLBCL.

Phospholipase C beta 1 deficiency is associated with early-onset epileptic encephalopathy.

The epileptic encephalopathies of infancy and childhood are a collection of epilepsy disorders characterized by refractory, severe seizures and poor neurological outcome, in which the mechanism of disease is poorly understood. We report the clinical presentation and evolution of epileptic encephalopathy in a patient, associated with a loss-of-function mutation in the phospholipase C-ß 1 gene. We ascertained a consanguineous family containing a male infant who presented with early-onset epileptic encephalopathy for detailed clinical phenotyping and molecular genetic investigation. In addition, a cohort of 12 consanguineous families of children with infantile spasms were analysed for linkage to the phospholipase C-ß 1 gene locus. The male infant presented with tonic seizures in early infancy and subsequently developed infantile spasms. Over time, he developed drug-resistant epilepsy associated with severe neurological regression and failure to thrive. Molecular genetic investigation revealed a homozygous loss-of-function 0.5-Mb deletion, encompassing the promoter element and exons 1, 2 and 3 of phospholipase C-ß 1 in the index case. Linkage to the phospholipase C-ß 1 locus was excluded in the 12 other consanguineous families, consistent with genetic heterogeneity in this disorder. Although phospholipase C-ß 1 deficiency has not previously been reported in humans, the Plcb1 homozygote knockout mouse displays early-onset severe tonic seizures and growth retardation, thus recapitulating the human phenotype. Phospholipase C-ß 1 has important functions in both hippocampal muscarinic acetylcholine receptor signalling and in cortical development. Thus, the discovery of a phospholipase C-ß 1 mutation allows us to propose a novel potential underlying mechanism in early-onset epileptic encephalopathy.

Clastogenicity and Aneugenicity of 1,4-Benzoquinone in Different Lineages of Mouse Hematopoietic Stem/Progenitor Cells.

Previous reports on hematotoxicity and leukemogenicity related to benzene exposure highlighted its adverse effects on hematopoiesis. Despite the reported findings, studies concerning the mechanism of benzene affecting chromosomal integrity in lineage-committed hematopoietic stem/progenitor cells (HSPCs) remain unclear. Here, we studied the clastogenicity and aneugenicity of benzene in lineage-committed HSPCs via karyotyping. Isolated mouse bone marrow cells (MBMCs) were exposed to the benzene metabolite 1,4-benzoquinone (1,4-BQ) at 1.25, 2.5, 5, 7, and 12 µM for 24 h, followed by karyotyping. Then, the chromosomal aberration (CA) in 1,4-BQ-exposed hematopoietic progenitor cells (HPCs) comprising myeloid, Pre-B lymphoid, and erythroid lineages were evaluated following colony-forming cell (CFC) assay. Percentage of CA, predominantly via Robertsonian translocation (Rb), was increased significantly (p < 0.05) in MBMCs and all progenitors at all concentrations. As a comparison, Pre-B lymphoid progenitor demonstrated a significantly higher percentage of CA (p < 0.05) than erythroid progenitor at 1.25, 2.5, and 7 µM as well as a significantly higher percentage (p < 0.05) than myeloid progenitor at 7 µM of 1,4-BQ. In conclusion, 1,4-BQ induced CA, particularly via Rb in both MBMCs and HPCs, notably via a lineage-dependent response. The role of lineage specificity in governing the clastogenicity and aneugenicity of 1,4-BQ deserves further investigation.

A Review of Placenta and Umbilical Cord-Derived Stem Cells and the Immunomodulatory Basis of Their Therapeutic Potential in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a devastating lung disorder of preterm infants as a result of an aberrant reparative response following exposures to various antenatal and postnatal insults. Despite sophisticated medical treatment in this modern era, the incidence of BPD remains unabated. The current strategies to prevent and treat BPD have met with limited success. The emergence of stem cell therapy may be a potential breakthrough in mitigating this complex chronic lung disorder. Over the last two decades, the human placenta and umbilical cord have gained increasing attention as a highly potential source of stem cells. Placenta-derived stem cells (PDSCs) and umbilical cord-derived stem cells (UCDSCs) display several advantages such as immune tolerance and are generally devoid of ethical constraints, in addition to their stemness qualities. They possess the characteristics of both embryonic and mesenchymal stromal/stem cells. Recently, there are many preclinical studies investigating the use of these cells as therapeutic agents in neonatal disease models for clinical applications. In this review, we describe the preclinical and clinical studies using PDSCs and UCDSCs as treatment in animal models of BPD. The source of these stem cells, routes of administration, and effects on immunomodulation, inflammation and regeneration in the injured lung are also discussed. Lastly, a brief description summarized the completed and ongoing clinical trials using PDSCs and UCDSCs as therapeutic agents in preventing or treating BPD. Due to the complexity of BPD, the development of a safe and efficient therapeutic agent remains a major challenge to both clinicians and researchers.

Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone.

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25-12 µM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7-14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 µM and tail moment at 12 µM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

Microarray based analysis of 3p25-p26 deletions (3p- syndrome).

Distal deletion of chromosome 3p25-pter (3p- syndrome) produces a distinct clinical syndrome characterized by low birth weight, mental retardation, telecanthus, ptosis, and micrognathia. Congenital heart disease (CHD), typically atrioventricular septal defect (AVSD) occurs in about a third of patients. Previously we reported on an association between the presence of CHD and the proximal extent of the deletion such that a CHD susceptibility gene was mapped between D3S1263 and D3S3594. In addition, we and others have suggested several candidate genes for the psychomotor retardation usually seen with constitutional 3p25 deletions. In order to further investigate genotype-phenotype correlations in 3p- syndrome we analyzed 14 patients with cytogenetically detectable deletions of 3p25 (including one patient with a normal phenotype) using Affymetrix 250K SNP microarrays. Deletion size varied from approximately 6 to 12 Mb. Assuming complete penetrance, a candidate critical region for a CHD susceptibility gene was refined to approximately 200 kb and a candidate critical region for mental retardation was mapped to an approximately 1 Mb interval containing SRGAP3 but other 3p neurodevelopmental genes including CHL1, CNTN4, LRRN1, and ITPR1 mapped outside the candidate critical interval. We suggest that current evidence suggests that SRGAP3 is the major determinant of mental retardation in distal 3p deletions.

Inherited t(9;22) as the cause of DiGeorge syndrome: a case report.

DiGeorge syndrome is associated with microdeletion of chromosome 22q11.2. Most cases occur sporadically although vertical transmission has been documented. We report a rare case of DiGeorge syndrome in an 8-year-old girl. Blood sample of the patient was cultured and harvested following standard procedure. All of the 20 cells analysed showed a karyotype of 45, XX, -22, t (9;22) (p23; q11.2). Cytogenetic investigation done on the patient's mother revealed that she was the carrier for the translocation. Her karyotype was 46, XX, t (9;22) (p23; q11.2). Fluorescence in situ hybridisation (FISH) analysis using TUPLE1 and N25 (Vysis, USA) probes showed deletion of the 22q11.2 region in the patient, confirming the diagnosis of DiGeorge syndrome. FISH analysis showed no deletion of the region in the mother.

Multiplexed automated digital quantification of fusion transcripts: comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients.

BACKGROUND: The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL. METHODS: "nCounter® Leukemia fusion gene expression assay" by NanoString was employed to detect various fusion transcripts in a large set samples (n = 94) utilizing RNA from formalin fixed paraffin embedded (FFPE) diagnostic bone marrow biopsy specimens. This series included AL patients with various recurrent translocations (n = 49), normal karyotype (n = 19), or complex karyotype (n = 21), as well as normal bone marrow samples (n = 5). Fusion gene expression data were compared with results obtained by conventional karyotype and FISH technology to determine sensitivity/specificity, as well as positive /negative predictive values. RESULTS: Junction probes for PML/RARA; RUNX1-RUNX1T1; BCR/ABL1 showed 100 % sensitivity/specificity. A high degree of correlation was noted for MLL/AF4 (85 sensitivity/100 specificity) and TCF3-PBX1 (75 % sensitivity/100 % specificity) probes. CBFB-MYH11 fusion probes showed moderate sensitivity (57 %) but high specificity (100 %). ETV6/RUNX1 displayed discordance between fusion transcript assay and FISH results as well as rare non-specific binding in AL samples with normal or complex cytogenetics. CONCLUSIONS: Our study presents preliminary data with high correlation between fusion transcript detection by a throughput automated multiplexed platform, compared to conventional karyotype/FISH technique for detection of chromosomal translocations in AL patients. Our preliminary observations, mandates further vast validation studies to explore automated molecular platforms in diagnostic pathology.

Methylation profiling and evaluation of demethylating therapy in renal cell carcinoma.

BACKGROUND: Despite therapeutic advances in targeted therapy, metastatic renal cell carcinoma (RCC) remains incurable for the vast majority of patients. Key molecular events in the pathogenesis of RCC include inactivation of the VHL tumour suppressor gene (TSG), inactivation of chromosome 3p TSGs implicated in chromatin modification and remodelling and de novo tumour-specific promoter methylation of renal TSGs. In the light of these observations it can be proposed that, as in some haematological malignancies, demethylating agents such as azacitidine might be beneficial for the treatment of advanced RCC. RESULTS: Here we report that the treatment of RCC cell lines with azacitidine suppressed cell proliferation in all 15 lines tested. A marked response to azacitidine therapy (>50% reduction in colony formation assay) was detected in the three cell lines with VHL promoter methylation but some RCC cell lines without VHL TSG methylation also demonstrated a similar response suggesting that multiple methylated TSGs might determine the response to demethylating therapies. To identify novel candidate methylated TSGs implicated in RCC we undertook a combined analysis of copy number and CpG methylation array data. Candidate novel epigenetically inactivated TSGs were further prioritised by expression analysis of RCC cell lines pre and post-azacitidine therapy and comparative expression analysis of tumour/normal pairs. Thus, with subsequent investigation two candidate genes were found to be methylated in more than 25% of our series and in the TCGA methylation dataset for 199 RCC samples: RGS7 (25.6% and 35.2% of tumours respectively) and NEFM in (25.6% and 30.2%). In addition three candidate genes were methylated in >10% of both datasets (TMEM74 (15.4% and 14.6%), GCM2 (41.0% and 14.6%) and AEBP1 (30.8% and 13.1%)). Methylation of GCM2 (P = 0.0324), NEFM (P = 0.0024) and RGS7 (P = 0.0067) was associated with prognosis. CONCLUSIONS: These findings provide preclinical evidence that treatment with demethylating agents such as azacitidine might be useful for the treatment of advanced RCC and further insights into the role of epigenetic changes in the pathogenesis of RCC.

Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility.

Perlman syndrome is a congenital overgrowth syndrome inherited in an autosomal recessive manner that is associated with Wilms tumor susceptibility. We mapped a previously unknown susceptibility locus to 2q37.1 and identified germline mutations in DIS3L2, a homolog of the Schizosaccharomyces pombe dis3 gene, in individuals with Perlman syndrome. Yeast dis3 mutant strains have mitotic abnormalities. Yeast Dis3 and its human homologs, DIS3 and DIS3L1, have exoribonuclease activity and bind to the core RNA exosome complex. DIS3L2 has a different intracellular localization and lacks the PIN domain found in DIS3 and DIS3L1; nevertheless, we show that DIS3L2 has exonuclease activity. DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins. DIS3L2 overexpression suppressed the growth of human cancer cell lines, and knockdown enhanced the growth of these cells. We also detected evidence of DIS3L2 mutations in sporadic Wilms tumor. These observations suggest that DIS3L2 has a critical role in RNA metabolism and is essential for the regulation of cell growth and division.

Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell carcinoma.

BACKGROUND: Inactivation of the Von Hippel-Lindau (VHL) tumour suppressor gene leading to overexpression of hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha is a critical event in the pathogenesis of most clear cell renal cell carcinomas (RCC). HIF-1alpha and HIF-2alpha share significant homology and regulate overlapping repertoires of hypoxia-inducible target genes but may have differing effects on RCC cell growth. Loss of HIF-1alpha expression has been described in RCC cell lines and primary tumours. Whether mutations in the alpha-subunits of HIF-1alpha and HIF-2alpha contribute to renal tumourigenesis was investigated here. MATERIALS AND METHODS: Mutation analysis of the complete coding sequence of HIF-1alpha and HIF-2alpha was carried out in primary RCC (n=40). RESULTS: The analysis revealed a somatic HIF1A missense substitution, p.Val116Glu, in a single RCC. Functional studies demonstrated that p.Val116Glu impaired HIF-1alpha transcriptional activity. Genotyping of HIF1A variants p.Pro582Ser and p.Ala588Thr demonstrated no significant differences between RCC patients and controls. CONCLUSION: The detection of a loss-of-function HIF1A mutation in a primary RCC is consistent with HIF-1 and HIF-2 having different roles in renal tumourigenesis, However, somatic mutations of HIF1A are not frequently implicated in the pathogenesis of RCC.