Ricardo J. Flores

Neurodegenerative central nervous system Langerhans cell histiocytosis and coincident hydrocephalus treated with vincristine/cytosine arabinoside.

Central nervous system (CNS) complications of Langerhans cell histiocytosis (LCH) include mass lesions and a neurodegenerative (ND) syndrome with ataxia, dysarthria, dysmetria, learning and behavior difficulties and/or characteristic changes on brain MRIs. Hydrocephalus has rarely been reported in LCH. LCH lesions of the orbit, mastoid and temporal bones (“CNS‐Risk” lesions) and diabetes insipidus predispose patients to ND‐CNS‐LCH. Treatment options have been limited and only a case series using trans‐retinoic acid (ATRA) and intravenous immunoglobulin (IVIG) have been published.

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.

A novel prognostic model for osteosarcoma using circulating CXCL10 and FLT3LG.

Osteosarcoma (OS) is the most common malignant pediatric bone tumor. The identification of novel biomarkers for early prognostication will facilitate risk‐based stratification and therapy. This study investigated the significance of circulating cytokines/chemokines for predicting the prognosis at the initial diagnosis.

Alveolar soft part sarcoma in children and young adults: A report of 69 cases

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal tumor characterized by ASPL‐TFE3 translocation. Apart from complete surgical resection, there is no standard management strategy.

The prognostic significance of circulating serum amyloid A and CXC chemokine ligand 4 in osteosarcoma

Osteosarcoma (OS) is the most common pediatric bone cancer. Despite advances in treatment regimens, the survival rate remains 60–70%. There is an urgent need to identify prognostic biomarkers, so that targeted therapies can be developed to improve the outcome.

Abstract 459: The use of circulating SAA and CXCL4 to predict outcome of osteosarcoma at diagnosis

Background: Osteosarcoma is the most common malignant bone tumor in children. The survival rate of osteosarcoma is 60-70%, which has remained stagnant for the past four decades. Identifying biomarkers that can better prognosticate osteosarcoma patients and guide therapies would improve survival of this disease. Material and Methods: In this study, we used ELISA to evaluate the expression of two previously identified circulating biomarkers, namely Serum Amyloid A (SAA) and CXC Chemokine Ligand 4 (CXCL4), in a large cohort of serum samples (n = 233) obtained at from the Children9s Oncology Group at initial diagnosis. The biomarkers’ expression was correlated with survival and evaluated for prognostic significance in the OS patients. Results: Analysis showed that patients with high SAA and low CXCL4 levels had significantly poorer outcome (p = 0.014, HR = 1.68), which was independent of initial metastasis status. The 5-year overall survival rates for the “high-risk” and “low-risk” groups were 47% (95% CI; 36% - 62%) and 64% (95% CI; 57% - 72%), respectively. In addition, low tumor expression of CXCL4 correlated with poor survival (p = 0.017) in an osteosarcoma tissue microarray. In conclusion, this study reports a novel prognostic model consisting of high SAA and low CXCL4 levels in sera that prognosticates osteosarcoma patients at initial diagnosis. This model could serve as the basis for future biomarker-guided clinical trials by incorporating targeted therapy for the poor prognostic population. Citation Format: Ricardo J. Flores. The use of circulating SAA and CXCL4 to predict outcome of osteosarcoma at diagnosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 459.

Abstract 5163: A high level of circulating CXCL10 at initial diagnosis is associated with poor prognosis of osteosarcoma patients

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Although survival rates have reached 70% over the past few decades, the prognosis of patients with metastatic disease remains very dismal. Identification of prognostic biomarkers besides clinically detectable metastasis will facilitate risk-stratification, and identify the most appropriate treatment option to improve their outcomes. Our group has previously devised a systems biology approach and identified that chemokines pathway are associated with metastasis in OS cell line models. Additionally, we reported that the plasma levels of a group of CXC chemokines were associated with a poorer patient outcome. CXC chemokines has been identified as source of potential therapeutic targets such as the CXCL12/CXCR4 and the CXCL10/CXCR3 ligand/receptor axes, which have been targeted successfully to inhibit lung metastasis in OS mouse models. Therefore, we decided to expand our findings by employing a Luminex platform to characterize circulating levels of cytokines/chemokines in OS patients, as these represent potential prognostic biomarkers and novel therapeutic targets. Materials and Methods: In this study, we employed a Luminex platform to analyze 39 chemokines/cytokines in two cohorts comprised of totally 290 OS patients. A regressive partitioning method was used to binarize the variables in the training cohort (40 samples) and the cutoff points were examined in the validation cohort (250 samples) for significance. Results: Twenty-two of the candidate biomarkers were significant with respect to overall survival and were tested in the validation cohort (250 samples). After Benjamini-Hochberg correction, CXCL10 significantly correlated with overall survival (p = 0.047) and remained significant after correcting for initial metastasis (CXCL10: p = 0.0037, Metastasis: p = 0.00012), indicating that CXCL10 is an independent prognostic factor. Therefore, we combined both prognostic factors to stratify the patients. Using this approach we identified three risk groups and the 5-year overall survival rate for the high-risk (high CXCL10 with initial metastasis), intermediate-risk (high CXCL10 with localized disease, or low CXCL10 with initial metastasis), and low-risk (low CXCL10 with localized disease) groups were 31%, 54%, and 71% respectively. Further, in patients with localized disease at diagnosis, which is conventionally considered to be low risk, a higher level of CXCL10 predicted a poorer outcome (p = 0.019). Additionally, we found that the receptor for the CXCL10 ligand, CXCR3, is expressed in OS tumors and is potentially targetable. In summary, our results suggest that circulating CXCL10 can be used as non-invasive biomarker to better prognosticate osteosarcoma patients before the treatment initiation and may lead to CXCR3-targeted therapy in selected patients in future clinical trials. Citation Format: Ricardo J. Flores, Aaron J. Kelly, Yiting Li, Manjula Nakka, Ching C. Lau, John Hicks, Tsz-Kwong Man. A high level of circulating CXCL10 at initial diagnosis is associated with poor prognosis of osteosarcoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5163. doi:10.1158/1538-7445.AM2015-5163

Abstract 2869: A three-protein cytokine signature that correlates with the prognosis of osteosarcoma

Osteosarcoma (OS) is the most common malignant bone tumor in pediatrics. Despite the use of surgery and chemotherapy, the survival rate of patients who develop a metastatic disease or a poor response towards the chemotherapy remains dismal. Identification of novel biomarkers for early prognostication will facilitate risk-based stratification before the treatment is initiated, so that the high-risk patients can be treated more appropriately to increase their chance of survival. Others and we have previously reported that cytokines and chemokines were implicated in OS. In this study, we employed highly sensitive Luminex beads assays to investigate 90 cytokines/chemokines and receptors on a cohort of 40 OS plasma samples collected at diagnosis from the Texas Children9s Hospital. The protein expression data were analysed to identify candidate biomarkers that correlate with the chemotherapy response, initial metastatic status, and overall survival. Then, the candidates were validated in a large set of 250 serum samples obtained from the Children9s Oncology Group. Our results show that a three-protein signature consisting of CXCL9, CXCL10 and CXCL11, all ligands of the CXCR3 receptor, is significantly associated with survival. Interestingly, the CXCR3-axis has been previously implicated in an OS mouse model, but its role in human is still not known. Hence, the next step is to validate these prognostic biomarkers in an independent cohort of OS patient samples. In addition, we will test the expression of these molecules and their receptor in tumor samples to identify potential therapeutic targets. In summary, we characterized the expression of circulating cytokines/chemokines in osteosarcoma samples and identified candidate prognostic biomarkers that are linked to the CXCR3-axis. A further study of these molecules may allow us to develop a biomarker approach for early prognostication and a novel therapeutic approach for treating high-risk OS. Citation Format: Ricardo J. Flores, Yiting Li, Aaron Kelly, Ching Lau, Tsz-Kwon Man. A three-protein cytokine signature that correlates with the prognosis of osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2869. doi:10.1158/1538-7445.AM2014-2869

Abstract 5305: Phosphorylations of Serine-10 and Tyrosine-198 residues in the cytoplasmic mislocalized p27 are critical to migration and invasion of osteosarcoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Osteosarcoma is the most common malignant bone tumor in adolescents and children. Metastasis is the major cause of death among osteosarcoma patients even with the advent of chemotherapy. Therefore, it is an urgent need to identify the key determinants of metastasis in osteosarcoma, so that new and effective therapeutic approaches can be developed to combat this deadly subtype of osteosarcoma. Recent studies have shown that the tumor suppressor protein p27 promotes metastasis when it is mislocalized from the nucleus to the cytoplasm in some solid tumors. However, the subcellular localization of the p27 protein in osteosarcoma and its role in metastasis are not known. In this study, we found that 82% human osteosarcoma cases harbored cytoplasmic staining of p27 using a tissue microarray. An immunohistochemistry analysis revealed that the p27 protein was localized in the nuclei of three pairs of non-metastatic osteosarcoma cell lines, but mislocalized in the cytoplasm of their metastatic sublines. Overexpression of a stable fusion construct of nuclear exporting sequence and p27 in a non-metastatic osteosarcoma cell line significantly increased tumor cell migration and invasion in vitro, suggesting that cytoplasmic p27 can promote metastatic potential in osteosarcoma cells. Site-directed mutagenesis on key phosphorylation sites of cytoplasmic p27 indicated that a mutation on Serine-10 or Tyrosine-198 residue abolished the promoting effects of p27 on cell motility and invasiveness, while a mutation on Tyrosine-157 had no effect. This result suggests that phosphorylations of these two residues are essential for tumor cell migration and invasion. Since mTOR signaling has been implicated in metastatic osteosarcoma, we tested if rapamycin, an mTOR inhibitor, can affect the subcellular localization of p27. We found that exogenous addition of the drug reversed the protein trafficking of p27 from the cytoplasm to the nuclei of metastatic osteosarcoma cells, suggesting that p27 may be a yet undiscovered player in the mTOR signaling of metastatic osteosarcoma. In summary, our results indicate that a novel relationship between p27 mislocalization and the metastatic potential of osteosarcoma cells. Therefore, further characterizations of the role of cytoplasmic p27 and its phosphorylations may lead to the development of a new therapeutic approach for metastatic osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5305. doi:1538-7445.AM2012-5305