Alexander Yu

Cell-Specific Gene Expression in Langerhans Cell Histiocytosis Lesions Reveals a Distinct Profile Compared with Epidermal Langerhans Cells

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207+ Langerhans cells (LCs) and lymphocytes that can arise in almost any tissue and cause significant morbidity and mortality. After decades of research, the cause of LCH remains speculative. A prevailing model suggests that LCH arises from malignant transformation and metastasis of epidermal LCs. In this study, CD207+ cells and CD3+ T cells were isolated from LCH lesions to determine cell-specific gene expression. Compared with control epidermal CD207+ cells, the LCH CD207+ cells yielded 2113 differentially expressed genes (false discovery rate < 0.01). Surprisingly, the expression of many genes previously associated with LCH, including cell-cycle regulators, proinflammatory cytokines, and chemokines, were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly overexpressed in LCH CD207+ cells. Furthermore, several genes associated with immature myeloid dendritic cells were overexpressed in LCH CD207+ cells. Compared with the peripheral CD3+ cells from LCH patients, the LCH lesion CD3+ cells yielded only 162 differentially regulated genes (false discovery rate < 0.01), and the expression profile of the LCH lesion CD3+ cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4, and SPP1. Results from this study support a model of LCH pathogenesis in which lesions do not arise from epidermal LCs but from accumulation of bone marrow-derived immature myeloid dendritic cells that recruit activated lymphocytes.

A systems biology approach reveals common metastatic pathways in osteosarcoma

BackgroundOsteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. The survival rate of patients with metastatic disease remains very dismal. Nevertheless, metastasis is a complex process and a single-level analysis is not likely to identify its key biological determinants. In this study, we used a systems biology approach to identify common metastatic pathways that are jointly supported by both mRNA and protein expression data in two distinct human metastatic OS models.ResultsmRNA expression microarray and N-linked glycoproteomic analyses were performed on two commonly used isogenic pairs of human metastatic OS cell lines, namely HOS/143B and SaOS-2/LM7. Pathway analysis of the differentially regulated genes and glycoproteins separately revealed pathways associated to metastasis including cell cycle regulation, immune response, and epithelial-to-mesenchymal-transition. However, no common significant pathway was found at both genomic and proteomic levels between the two metastatic models, suggesting a very different biological nature of the cell lines. To address this issue, we used a topological significance analysis based on a “shortest-path” algorithm to identify topological nodes, which uncovered additional biological information with respect to the genomic and glycoproteomic profiles but remained hidden from the direct analyses. Pathway analysis of the significant topological nodes revealed a striking concordance between the models and identified significant common pathways, including “Cytoskeleton remodeling/TGF/WNT”, “Cytoskeleton remodeling/Cytoskeleton remodeling”, and “Cell adhesion/Chemokines and adhesion”. Of these, the “Cytoskeleton remodeling/TGF/WNT” was the top ranked common pathway from the topological analysis of the genomic and proteomic profiles in the two metastatic models. The up-regulation of proteins in the “Cytoskeleton remodeling/TGF/WNT” pathway in the SaOS-2/LM7 and HOS/143B models was further validated using an orthogonal Reverse Phase Protein Array platform.ConclusionsIn this study, we used a systems biology approach by integrating genomic and proteomic data to identify key and common metastatic mechanisms in OS. The use of the topological analysis revealed hidden biological pathways that are known to play critical roles in metastasis. Wnt signaling has been previously implicated in OS and other tumors, and inhibitors of Wnt signaling pathways are available for clinical testing. Further characterization of this common pathway and other topological pathways identified from this study may lead to a novel therapeutic strategy for the treatment of metastatic OS.

Elevated expression of CXC chemokines in pediatric osteosarcoma patients

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis.

Clinical utility of array comparative genomic hybridization for detection of chromosomal abnormalities in pediatric acute lymphoblastic leukemia

Accurate detection of recurrent chromosomal abnormalities is critical to assign patients to risk‐based therapeutic regimens for pediatric acute lymphoblastic leukemia (ALL).

Genome-wide analysis of DNA copy number alterations and loss of heterozygosity in intracranial germ cell tumors

Intracranial germ cell tumors (GCTs) are rare and heterogeneous with very little is known about their pathogenesis and underlying genetic abnormalities.

Implications of Tumor Location on Subtypes of Medulloblastoma

Medulloblastoma (MB) comprises of four molecular subtypes, Sonic hedgehog (SHH), Wingless (WNT), Groups 3 and 4. WNT‐subtype MBs were found to arise from midline of the brainstem occupying the fourth ventricle while SHH‐subtype occupied the cerebellar hemisphere in a small subset of patients.

Identification and gene expression profiling of tumor-initiating cells isolated from human osteosarcoma cell lines in an orthotopic mouse model

In the cancer stem cell model a cell hierarchy has been suggested as an explanation for intratumoral heterogeneity and tumor formation is thought to be driven by this tumor cell subpopulation. The identification of cancer stem cells in osteosarcoma (OS) and the biological processes dysregulated in this cell subpopulation, also known as tumor-initiating cells (TICs), may provide new therapeutic targets. The goal of this study, therefore, was to identify and characterize the gene expression profiles of TICs isolated from human OS cell lines. We analyzed the self-renewal capacity of OS cell lines and primary OS tumors based upon their ability to form sphere-like structures (sarcospheres) under serum-starving conditions. TICs were identify from OS cell lines using the long-term label retention dye PKH26. OS TICs and the bulk of tumor cells were isolated and used to assess their ability to initiate tumors in NOD/SCID mice. Gene expression profiles of OS TICs were obtained from fresh orthotopic tumor samples. We observed that increased sarcosphere efficiency correlated with an enhanced tumorigenic potential in OS. PKH26Hi cells were shown to constitute OS TICs based upon their capacity to form more sarcospheres, as well as to generate both primary bone tumors and lung metastases efficiently in NOD/SCID mice. Genomic profiling of OS TICs revealed that both bone development and cell migration processes were dysregulated in this tumor cell subpopulation. PKH26 labeling represents a valuable tool to identify OS TICs and gene expression analysis of this tumor cell compartment may identify potential therapeutic targets.

Coamplification of Myc/Pvt1 and homozygous deletion of Nlrp1 locus are frequent genetics changes in mouse osteosarcoma.

Osteosarcomas (OSs) are characterized by high levels of genomic instability (GI). To gain insights into the GI and its contribution toward understanding the genetic basis of OS, we characterized 19 primary and 13 metastatic mouse tumors in a genetically engineered novel mouse model of OS by a combination of genomic techniques. Through the bone‐specific deletion of the wild‐type Trp53 locus or activation of a metastatic‐promoting missense R172Hp53 allele, C57BL/6 mice developed either localized or metastatic OS. Subsequent tumors were isolated and primary cultures created from primary bone and/or distal metastatic lesions, for example, lung and liver. These tumors exhibited high levels of GI with complex chromosomal rearrangements, amplifications, and deletions comparable to human OS. The combined genomic approaches identified frequent amplification of chromosome 15D1 and loss of 11B4 by CGH and/or SKY. Both 15D1 and 11B4 have homology with frequently altered chromosomal bands 8q24 and 17p13 in human OS, respectively. Subsequent array CGH, FISH, and qRT‐PCR analysis identified coamplification and overexpression of Myc/Pvt1 transcripts from the 15D1 amplicon and loss and decreased expression of the Nlrp1b from 11B4. The Nlrp1 gene is the key mediator of apoptosis and interacts strongly with caspase 2.

Abstract 3824: Integrated analysis of array-CGH and gene expression profiling identifies HSPA5 as a prognostic marker in SHH pathway-actived Medulloblastoma.

Medulloblastoma (MB) is the most malignant brain tumor in children and is characterized by marked biological and clinical heterogeneity. It includes four distinct molecular subtypes [wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4]. Previous attempts to identify prognostic markers applicable to all MBs were unsuccessful and we therefore hypothesize that each molecular subtype of MB may be associated with its unique prognostic markers. The aim of this study is to identify prognostic markers specific for SHH-active MB tumors. We analyzed SNP-array CGH data and gene expression profiling for 60 pediatric MBs and compared the molecular results with clinical outcome data. Using unsupervised hierarchical clustering, we identified 18 MBs that have activated SHH signaling. This subtype of SHH-active tumors also have frequent chromosome 9q loss which are associated with more favorable outcome. In an attempt to identify candidate genes on chromosome 9q with survival impact, we found that low expression of Heat Shock 70kDa Protein 5 (HSPA5) was significantly associated with better event free survival (EFS) in SHH MB patients independent of other clinical variables. This finding had been validated in two other independent MB cohorts. By immuohistochemistry and western blotting, we confirmed the negative correlation between patient survival and HSPA5 protein expression in SHH tumors. Furthermore, in vitro study indicated that knockdown of HSPA5 by siRNA caused dramatic growth suppression in SHH active MB cells. These findings suggest that HSPA5 is a potential prognostic marker in predicting survival of SHH active MB patients and this protein may also be exploited as therapeutic target for SHH MBs that do not harbor chromosome 9q loss. Citation Format: Angela Kwok-Fung Lo, Jian Wang, Jianhe Shen, Alexander Yu, Wing-Yuk Chow, Jack Su, Adekunle Adesina, Robert Dauser, William Whitehead, Murali Chintagumpala, Rudy Guerra, Tsz-Kwong Man, Ching C. Lau. Integrated analysis of array-CGH and gene expression profiling identifies HSPA5 as a prognostic marker in SHH pathway-actived Medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3824. doi:10.1158/1538-7445.AM2013-3824

Abstract 5571: Identification of autoantibody biomarkers in metastatic osteosarcoma using a high-density protein microarray approach

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. A leading cause of death in OS is the development of metastasis. Therefore, it is important to detect metastatic patients as early as possible, so these patients can be stratified and treated while the disease is still in its early stages and potentially curable. Autoantibodies represent a promising class of plasma proteins that can be used for the early detection of cancer. These immunoglobulins can recognize aberrant proteins in the tumor cell during the development of metastasis. We have used a new high-density protein microarray platform, ProtoArray Human Protein Array (HPA), to identify significant autoantibody biomarkers that are associated with metastatic OS at the time of diagnosis. ProtoArray HPA consists of over 8,000 purified human proteins from different functional classes. Plasma samples from metastatic and localized OS patients, and children with non-cancerous diseases were analyzed by the microarrays. The results showed that the amounts of 136 distinct autoantibodies were significantly different between the plasma samples of metastatic and localized OS cases (p < 0.01). Among these autoantibodies, 66 were higher while 70 were lower in metastatic patients. Two of these autoantibodies, which target CTTN and PFKFB1, were selected for further investigation. We found that the phosphorylated form (p85) of the CTTN protein in two metastatic OS cell lines was expressed higher relative to the non-metastatic parental cells. This result is consistent with the previous finding that increased phosphorylation of CTTN is important for the metastatic phenotype of cancer cells. The PFKFB1 autoantibody in the localized patients was higher than the metastatic patients; however, both groups of OS patients had a higher PFKFB1 level relative to the control group. When compared to normal osteoblasts, Western blotting showed that PFKFB1 protein was overexpressed in seven of the eight OS cell lines. PFKFB1 is involved in the Warburg effect, which is important to the energy metabolism of cancer cells. Our results suggest that the high-density protein microarray approach is useful for identifying metastasis-associated autoantibodies in cancer patients. Further validation and investigation of the candidate autoantibody biomarkers may lead to discovery of novel biomarkers and unravel the underlying mechanism in metastatic OS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5571.