Kathy Yuen Yee Chan

TOP2A overexpression in hepatocellular carcinoma correlates with early age onset, shorter patients survival and chemoresistance.

Genomic gain represents an important mechanism in the activation of proto‐oncogenes. In many instances, induced oncogenes hold clinical implications both as prognostic markers and targets for therapeutic design. In hepatocellular carcinoma (HCC), although chromosomal gains are common, information on underlying oncogenes induced remains minimal. Here, we examined 7 causal sites of HCC for overexpressed genes by array‐based transcriptional mapping. In 22 HCC cell lines and early passages of cultures studied, clusters of up‐regulated genes were indicated, where TOP2A expression ranked the highest. Distinct TOP2A transcriptions were confirmed in an independent series of HCC tumors relative to adjacent non‐tumoral liver (p = 0.0018). By tissue microarray analysis of 172 HCC, we found TOP2A expressions correlated with advance histological grading (p < 0.001), microvascular invasion (p = 0.004) and an early age onset of the malignancy (≤40 years; p = 0.007). In conjunction with P‐gp and MRP1, TOP2A were further assessed for its association with chemotherapy responsiveness and survival in 148 patients who entered our recently reported Phase III prospective randomized study. In 73 chemoresistant and 75 nonresistant patients, only TOP2A positivity correlated with chemoresistance (p = 0.029) and shorter patients survival (p < 0.0001). The potential therapeutic value in targeting TOP2A by Etoposide, as a single agent, and in combination with Doxorubicin was also explored. In vitro cytotoxic studies suggested Etoposide at IC20 readily reduced IC50 values of Doxorubicin by a magnitude of ∼3.5 to 10‐fold compared to Doxorubicin alone (p < 0.028). Our study highlighted for the first time the prognostic value of TOP2A in HCC and the potential use of TOP2A reactive agents in therapy.

The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells

The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34(+) cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34(+)CD38(-/lo) and CD34(+)CD38(+) cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34(+) cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1-mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34(+) cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34(+)CD9(-) cells displayed lower bone marrow homing capacity compared with that of total CD34(+) cells. CD9 expression on homed CD34(+) cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.

Gut-associated biomarkers L-FABP, I-FABP, and TFF3 and LIT score for diagnosis of surgical necrotizing enterocolitis in preterm infants.

Objectives: To evaluate the use of gut barrier proteins, liver-fatty acid binding protein (L-FABP), intestinal-fatty acid binding protein (I-FABP), and trefoil factor 3 (TFF3), as biomarkers for differentiating necrotizing enterocolitis (NEC) from septicemic/control infants and to identify the most severely affected surgical NEC from nonsurgical NEC infants. Background: Clinical features and routine radiologic investigations have low diagnostic utilities in identifying surgical NEC patients. Methods: The diagnostic utilities of individual biomarkers and the combination of biomarkers, the LIT score, were assessed among the NEC (n = 20), septicemia (n = 40), and control groups (n = 40) in a case-control study for the identification of proven NEC and surgical NEC infants. Results: Plasma concentrations of all gut barrier biomarkers and the LIT score were significantly higher in the NEC than in the septicemia or control group (P < 0.01). Using median values of biomarkers and the LIT score in the NEC group as cutoff values for identifying NEC from septicemic/control cases, all had specificities of 95% or more and sensitivities of 50%. Significantly higher levels of biomarkers and the LIT score were found in infants with surgical NEC than in nonsurgical NEC cases (P ⩽ 0.02). The median LIT score of 4.5 identified surgical NEC cases with sensitivity and specificity of 83% and 100%%, respectively. A high LIT score of 6 identified nonsurvivors of NEC with sensitivity and specificity of 78% and 91%, respectively. Conclusions: The LIT score can effectively differentiate surgical NEC from nonsurgical NEC infants and nonsurvivors of NEC from survivors at the onset of clinical presentation. Frontline neonatologists and surgeons may, therefore, target NEC infants who are most in need of close monitoring and those who may benefit from early surgical intervention.

Rapid identification and differentiation of Gram-negative and Gram-positive bacterial bloodstream infections by quantitative polymerase chain reaction in preterm infants

Objective:To evaluate the usefulness of the Gram-specific probe-based quantitative polymerase chain reaction test for rapid detection and differentiation of Gram-negative and Gram-positive bacterial bloodstream infection in preterm infants. Design:Cross-sectional study. Setting:University-affiliated Level III neonatal intensive care unit. Patients:Preterm infants with clinical features suggestive of late-onset infection. Interventions:None. Measurements and Main Results:In addition to the full sepsis screen, 0.5 mL of EDTA blood was collected aseptically for Gram-specific quantitative polymerase chain reaction evaluation. The results were analyzed with respect to outcomes of bacterial culture in blood and other body fluids, including peritoneal and cerebrospinal fluids. The diagnostic utilities of the quantitative polymerase chain reaction were determined. A total of 218 suspected infection episodes were investigated, of which 42 episodes were culture positive and 176 were culture negative. For Gram-negative infection, the quantitative polymerase chain reaction test correctly identified 19 of 22 episodes, and the sensitivity and specificity were 86.4% and 99.0%, respectively. For Gram-positive infection, the test correctly identified 14/19 episodes, and the sensitivity and specificity were 73.7% and 98.5%. The remaining one episode was Candida albicans septicemia. None of the episodes with positive quantitative polymerase chain reaction test were classified into the wrong Gram stain category. More importantly, despite negative blood culture in five infants suffering from intra-abdominal sepsis (peritonitis [n = 4] and hepatosplenic abscess [n = 1]), the quantitative polymerase chain reaction test could detect the Gram-specific category of causative organisms in blood. Conclusions:The Gram-specific quantitative polymerase chain reaction test is reliable and highly specific for rapid identification and differentiation of Gram-negative and Gram-positive bloodstream and intra-abdominal infections. The result could be made available within 5 hrs after the specimen reaches the laboratory. A positive test is able to “rule in” bacterial bloodstream infection before blood culture results become available, and serves as a guide to predict the virulence of the causative organism according to its Gram-specific category so that critical patients can be targeted for intensive treatment.

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients.

Genome-wide expression profiles of necrotizing enterocolitis versus spontaneous intestinal perforation in human intestinal tissues: dysregulation of functional pathways.

Objective:To provide a comprehensive database of gene regulation and compare differentially regulated molecular networks in human tissues of necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP). Background:Both NEC and SIP are devastating surgical emergencies associated with high morbidity and mortality in preterm infants. Their pathophysiology and molecular mechanisms remain unclear. Methods:Differential whole genome microarray analysis was performed on intestinal tissues collected from NEC (n = 15) and SIP (n = 12) infants and compared with tissues collected from surgical-control patients with noninflammatory intestinal conditions (n = 14). Validation of 52 target gene expressions was performed by quantitative polymerase chain reaction. Regulatory networks of significantly affected genes were constructed according to functional pathways. Results:Extensive and significant changes of gene expression were observed in NEC tissues, which comprised multiple pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia and oxidative stress, inflammation, and muscle contraction. These dysregulated genes could be networked downstream of key receptors, TLR2, TLR4, and TREM1, and mediated via NF-&kgr;B, AP-1, and HIF1A transcription factor pathways, indicating predominant microbial and inflammatory involvement. In contrast, SIP tissues exhibited much milder and less diversified expressional changes, with target genes significantly associated with G-protein–mediated muscle contraction and extracellular matrix remodeling. Conclusions:The molecular evidence suggests that NEC and SIP are likely 2 different diseases caused by distinct etiology and pathophysiology. This first comprehensive database on differential gene expression profiles of human NEC and SIP tissues could lead to development of disease-specific diagnostic and prognostic biomarkers and new therapeutic strategies for improving outcomes.

Karyotypic imbalances and differential gene expressions in the acquired doxorubicin resistance of hepatocellular carcinoma cells

Administration of doxorubicin has been shown to prolong survival of patients with hepatocellular carcinoma (HCC). However, treatment regimen is often complicated by the emergence of drug resistance. The goal of our study is to enhance our understanding on the genetic changes that confer cellular chemoresistance to doxorubicin. To model this insensitive response, we established five doxorubicin-resistant (DOR) sublines through repeated exposure of escalating doses of doxorubicin to HCC cell lines (HKCI-2, -3, -4, -C1 and -C2). The DOR sublines developed displayed an average ∼17-fold higher IC50 value than their sensitive parental cell lines. The resistant phenotype displayed was investigated by the genome-wide analyses of comparative genomic hybridization (CGH) and complementary DNA microarray for the affected genomic anomalies and deregulated genes expressed, respectively. Over-representations of regional chr. 7q11–q21, 8q22–q23 and 10p13–pter were indicated in the DOR sublines from CGH analysis. Of particular interest was the finding of amplicon augmentations from regional or whole chromosome gains during the clonal expansion of resistant sublines. Most notably, recurring amplicon 7q11.2–q21 identified coincided with the location of the multi-drug-resistant gene, MDR1. The potential involvement of MDR1 was examined by quantitative reverse transcription-polymerase chain reaction RT-PCR (qRT-PCR), which indicated an upregulation in all DOR sublines (P=0.015). Consistent overexpression of the translated MDR1 gene, P-glycoprotein, in all five DOR sublines was further confirmed in Western blot analysis. Two distinct cluster dendrograms were achieved between the DOR sublines and their sensitive parental counterparts in expression profiling. Within the doxorubicin-resistant group, distinct features of candidate genes overexpressions including ABC transporting proteins, solute carriers and TOP2A were suggested. Further assessment of TOP2A messenger RNA levels by qRT-PCR confirmed array findings and pinpointed to a common up-regulation of TOP2A in DOR sublines. Our present study highlighted areas of genomic imbalances and candidate genes in the acquired doxorubicin-resistance behavior of HCC cells.

Positional expression profiling indicates candidate genes in deletion hotspots of hepatocellular carcinoma

Molecular characterizations of hepatocellular carcinoma have indicated frequent allelic losses on chromosomes 4q, 8p, 16q and 17p, where the minimal deleted regions have been further defined on 4q12–q23, 4q31–q35, 8p21–p22, 16q12.1–q23.1 and 17p13. Despite these regions are now well-recognized in early liver carcinogenesis, few underlying candidate genes have been identified. In an effort to define affected genes within common deleted loci of hepatocellular carcinoma, we conducted transcriptional mapping by high-resolution cDNA microarray analysis. In 20 hepatocellular carcinoma cell lines and 20 primary tumors studied, consistent downregulations of novel transcripts were highlighted throughout the entire genome and within sites of frequent losses. The array-derived candidates including fibrinogen gamma peptide (FGG, at 4q31.3), vitamin D binding protein (at 4q13.3), fibrinogen-like 1 (FGL1, at 8p22), metallothionein 1G (MT1G, at 16q12.2) and alpha-2-plasmin inhibitor (SERPINF2, at 17p13) were confirmed by quantitative reverse transcription–polymerase chain reaction, which also indicated a more profound downregulation of FGL1, MT1G and SERPINF2 relative to reported tumor-suppressor genes, such as DLC1 (8p22), E-cadherin (16q22.1) and TP53 (17p13.1). In primary hepatocellular carcinoma examined, a significant repression of MT1G by more than 100-fold was indicated in 63% of tumors compared to the adjacent nonmalignant liver (P=0.0001). Significant downregulations of FGG, FGL1 and SERPINF2 were also suggested in 30, 23 and 33% of cases, respectively, compared to their nonmalignant counterparts (P<0.016). In summary, transcriptional mapping by microarray indicated a number of previously undescribed downregulated genes in hepatocellular carcinoma, and highlighted potential candidates within common deleted regions.

Transcriptional profiling on chromosome 19p indicated frequent downregulation of ACP5 expression in hepatocellular carcinoma

Chromosomal rearrangements unraveled by spectral karyotyping (SKY) indicated frequent chromosome 19 translocations in hepatocellular carcinoma (HCC). In an effort to characterize the aberrant 19 rearrangements in HCC, we performed positional mapping by fluorescence in‐situ hybridization (FISH) in 10 HCC cell lines. SKY analysis indicated structural rearrangements of chromosome 19 in 6 cell lines, 4 of which demonstrated recurring 19p translocations with different partner chromosomes. Using fluorescence‐labeled BAC probes, physical mapping indicated a breakpoint cluster between 19p13.12 and 19p12. A corresponding transcriptional mapping by cDNA array on 19p suggested the differential expression of a single downregulated gene ACP5 (tartrate‐resistant acid phosphatase type 5). Quantitative RT‐PCR confirmed the reduced expression of ACP5 and indicated a strong correlation of its repressed expression only in cell lines that contain a 19p rearrangement (p = 0.004). We further examined the expression of ACP5 in a cohort of 82 primary tumors and 74 matching nonmalignant liver tissues. In the primary HCC examined, a reduction of ACP5 transcripts by 2 to as much as 1,000‐fold was suggested in 67% of tumors (55/82 cases). When compared to adjacent nonmalignant tissues, 46% of tumors (34/74 cases) demonstrated a lower expression level (p = 0.015). On closer examination, a high significance of ACP5 repression was suggested in the cirrhotic HCC subgroup that was derived from chronic hepatitis B infected patients (55%; 30/54 cases; p = 0.001). Functional examination of ACP5 ectopic expression in HCC cells further demonstrated a significant growth inhibitory effect of ACP5 on tumor cell survival (p < 0.001). In our study, the novel finding of common ACP5 downregulation in HCC may provide basis for further investigations on the role of acid phosphatase in hepatocarcinogenesis.

Comparative MiRNA Expressional Profiles and Molecular Networks in Human Small Bowel Tissues of Necrotizing Enterocolitis and Spontaneous Intestinal Perforation

Background Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are acute intestinal conditions which could result in mortality and severe morbidity in preterm infants. Our objective was to identify dysregulated micro-RNAs (miRNAs) in small bowel tissues of NEC and SIP, and their possible roles in disease pathophysiology. Methods We performed differential miRNA arrays on tissues of NEC (n = 4), SIP (n = 4) and surgical-control (Surg-CTL; n = 4), and validated target miRNAs by qPCR (n = 10 each group). The association of target miRNAs with 52 dysregulated mRNAs was investigated by bioinformatics on functional and base-pair sequence algorithms, and correlation in same tissue samples. Results We presented the first miRNA profiles of NEC, SIP and Surg-CTL intestinal tissues in preterm infants. Of 28 validated miRNAs, 21 were significantly different between NEC or SIP and Surg-CTL. Limited overlapping in the aberrant expression of miRNAs between NEC and SIP indicated their distinct molecular mechanisms. A proposed network of dysregulated miRNA/mRNA pairs in NEC suggested interaction at bacterial receptor TLR4 (miR-31, miR-451, miR-203, miR-4793-3p), mediated via key transcription factors NFKB2 (miR-203), AP-1/FOSL1 (miR-194-3p), FOXA1 (miR-21-3p, miR-431 and miR-1290) and HIF1A (miR-31), and extended downstream to pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia/oxidative stress, inflammation and muscle contraction. In contrast, upregulation of miR-451 and miR-223 in SIP suggested modulation of G-protein-mediated muscle contraction. Conclusions The robust response of miRNA dysregulation in NEC and SIP, and concerted involvement of specific miRNAs in the molecular networks indicated their crucial roles in mucosa integrity and disease pathophysiology.