Wusheng Yan

Identification of evidence-based biospecimen quality-control tools: a report of the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group.

Control of biospecimen quality that is linked to processing is one of the goals of biospecimen science. Consensus is lacking, however, regarding optimal sample quality-control (QC) tools (ie, markers and assays). The aim of this review was to identify QC tools, both for fluid and solid-tissue samples, based on a comprehensive and critical literature review. The most readily applicable tools are those with a known threshold for the preanalytical variation and a known reference range for the QC analyte. Only a few meaningful markers were identified that meet these criteria, such as CD40L for assessing serum exposure at high temperatures and VEGF for assessing serum freeze-thawing. To fully assess biospecimen quality, multiple QC markers are needed. Here we present the most promising biospecimen QC tools that were identified.

Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.MethodsProtein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).ResultsGelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.ConclusionsIncreased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.

SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling

Introduction: Laser capture microdissection (LCM) facilitates procurement of defined cell populations for study in the context of histopathology. The morphologic assessment step in the LCM procedure is time consuming and tedious, thus restricting the utility of the technology for large applications. Results: Here, we describe the use of Spatially Invariant Vector Quantization (SIVQ) for histological analysis and LCM. Using SIVQ, we selected vectors as morphologic predicates that were representative of normal epithelial or cancer cells and then searched for phenotypically similar cells across entire tissue sections. The selected cells were subsequently auto-microdissected and the recovered RNA was analyzed by expression microarray. Gene expression profiles from SIVQ-LCM and standard LCM-derived samples demonstrated highly congruous signatures, confirming the equivalence of the differing microdissection methods. Conclusion: SIVQ-LCM improves the work-flow of microdissection in two significant ways. First, the process is transformative in that it shifts the pathologist′s role from technical execution of the entire microdissection to a limited-contact supervisory role, enabling large-scale extraction of tissue by expediting subsequent semi-autonomous identification of target cell populations. Second, this work-flow model provides an opportunity to systematically identify highly constrained cell populations and morphologically consistent regions within tissue sections. Integrating SIVQ with LCM in a single environment provides advanced capabilities for efficient and high-throughput histological-based molecular studies.

Short-Term Stability Study of RNA at Room Temperature

The quality of RNA preserved in different stabilization matrices was investigated after 2 weeks of storage at room temperature. RNA samples in RNAstable (Biomatrica), GenTegra (IntegenX), and RNAshell (Imagene) were compared to RNA stored at -80°C (the current gold standard for RNA preservation) and with liquid or dried RNA stored at room temperature without additives in this multi-center study. One center prepared all of the RNA samples, and five participating laboratories applied the samples to the matrices and stored them for 2 weeks at room temperature. Samples were shipped to three testing laboratories, where the 336 RNA samples were rehydrated and then analyzed for RNA recovery, purity, and integrity. Parallel RNA quality analyses and real-time PCR analyses were performed at each of the three testing laboratories. Each of the RNA matrices tested was shown to be fit-for-purpose for short-term room temperature storage in terms of total RNA recovery and rRNA integrity. All but one of the matrices was judged to be fit-for-purpose for mRNA integrity when assessed by real-time PCR analysis. In a follow-up study, RNase-contaminated samples were shown to provide accurate real-time PCR results when stored for up to 3.5 months in either RNAshell or RNAstable.

Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets.ResultsAs a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels.ConclusionsThese data reveal insight into genes and molecular pathways mediating ESCC development and provide information potentially useful in designing novel therapeutic interventions for this tumor type.

Biorepository proficiency testing for the quality control of biospecimens for the global biobanking community

Until now, proficiency testing programs have not existed for the quality assessment of biospecimens that are used in basic, translational, and clinical research studies, yet the discovery, validation, and clinical evaluation of biomarkers necessary for the advancement of global health depend upon the availability of a large number of standardized specimens. To meet this void, ISBER has launched a biorepository Proficiency Testing (PT) Program in partnership with the Integrated Biobank of Luxembourg (IBBL). The ISBER PT Program belongs to the category of interlaboratory comparison ‘‘schemes’’ involving simultaneous participation of biorepository laboratories located in different countries. Randomly selected aliquots from a source material prepared at IBBL (the test items) are being distributed and tested concurrently by all registered participants. After the completion of the testing, the participants’ results will be returned to the Proficiency Testing provider (ISBER) and compared with the assigned value(s) derived from the reference laboratories to give an indication of the performance of the individual participants and of the group as a whole. The ISBER PT Program allows biorepositories performing quality control assays and/or characterization of the biospecimens to assess the accuracy of their testing and to compare their results with those obtained in other laboratories around the world. It has been designed to include four schemes: DNA quantification and purity, RNA integrity, cell viability, and tissue antigenicity. The first two schemes were open for participation in the latter part of 2011. The cell viability and tissue antigenicity schemes will be added to the program in 2012.

Menin immunoreactivity in secretory granules of human pancreatic islet cells.

The protein product of the Multiple Endocrine Neoplasia Type I (MEN1) gene is thought to be involved in predominantly nuclear functions; however, immunohistochemical (IHC) analysis data on cellular localization are conflicting. To further investigate menin expression, we analyzed human pancreas (an MEN1 target organ) using IHC analyses and 6 antibodies raised against full-length menin or its peptides. In 10 normal pancreas specimens, 2 independently raised antibodies showed unexpected cytoplasmic immunoreactivity in peripheral cells in each islet examined (over 100 total across all 10 patients). The staining exhibited a distinct punctate pattern and subsequent immunoelectron microscopy indicated the target antigen was in secretory granules. Exocrine pancreas and pancreatic stroma were not immunoreactive. In MEN1 patients, unaffected islets stained similar to those in normal samples but with a more peripheral location of positive cells, whereas hyperplastic islets and tumorlets showed increased and diffuse cytoplasmic staining, respectively. Endocrine tumors from MEN1 patients were negative for menin, consistent with a 2-hit loss of a tumor suppressor gene. Secretory granule localization of menin in a subset of islet cells suggests a function of the protein unique to a target organ of familial endocrine neoplasia, although the IHC data must be interpreted with some caution because of the possibility of antibody cross-reaction. The identity, cellular trafficking, and role of this putative secretory granule-form of menin warrant additional investigation.

Abstract 794: Targeted-therapy related molecular biomarker characterization in NSCLC smokers and never-smokers by a novel qRT-PCR for microdissected tissues methodology

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Several studies have focused on the identification of biologic markers that predict early recurrence and death in patients with non-small cell lung cancer (NSCLC); however, few studies have focused on indicators of outcome in ever-smoker and never-smoker lung cancer patients. We hypothesize that NSCLC primary tumors from smokers and never-smokers differ significantly in the expression of molecular abnormalities and their corresponding biomarkers. We also believe that the different pattern of molecular pathway abnormalities in NSCLCs based on smoking status may explain different response to targeted therapy in both patients’ populations. To test our hypotheses, and better understand the results of biomarker analysis in molecularly targeted clinical trials, we developed a novel qRT-PCR pre-amplification methodology for gene expression analysis of microdissected (MD) tissues to determine and compare the gene expression patterns of targeted therapy related molecular biomarkers and pathways from surgically resected tissue specimens obtained from NSCLC patients. Previously only 9 genes of interest could be analyzed from a typical MD sample (Erickson et al. 2009 Nature Protocols). Our new methodology allows for the use of 8 ng of total RNA for the analysis of up to 24 genes of interest or 24 ng of total RNA for the analysis of up to 72 genes of interest. We then applied this methodology to 40 MD stage I/II NSCLC adenocarcinoma tissues from tumor epithelium and matched normal bronchial epithelium from 20 smokers and 20 never-smokers for EGFR, KRAS, HER2N, BRAF, PIK-3, IGFR-1, LKB-1, mTOR, and AKT analysis. An average of 77.3 ng of total RNA (average RIN = 5.0) was recovered from ∼5000 MD epithelial cells per tissue sample. Gene expression was analyzed by relative quantitation using the 2−ΔΔCt method. Results show that analysis of the expression of multiple genes using a pre-amplification step is a feasible approach for small tissue specimens obtained from clinical trials with advanced lung cancer patients. Preliminary analysis revealed distinct gene expression patterns in smokers compared to never-smokers. Future plans include correlating gene expression with gene mutation, copy number variation, and immunohistochemistry analysis of these tissue specimens. This novel qRT-PCR methodology for gene expression analysis of MD tissues will be used to analyze fine needle aspiration and core needle biopsy tissue specimens from biomarker-driven clinical trial patients (e.g. BATTLE) for validation of profiles and analysis of multiple molecular pathway targets that can predict response to chemotherapy and targeted therapy. This work was funded by Department of Defense BATTLE Lung Cancer Program Initiative W81XWH-06-1-0303, Cohen-Reinauch BATTLE-2 Fund, and National Cancer Institute Intramural Program. 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 794.

Abstract 2168: Molecular pathway analysis of esophageal squamous cell differentiation versus carcinoma formation

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer and is characterized by high mortality. Our previous work analyzing candidate tumor associated genes in ESCC highlighted the important differences in mRNA expression between the normal esophageal and tumor epithelial compartments. However, to date there are limited studies examining expression changes that occur during esophageal squamous cell differentiation versus those associated with ESCC tumorigenesis. To address this issue, we microdissected the basal cell layer and differentiated cell layer from normal epithelium and compared these expression profiles with matched tumor samples in twelve patients using gene expression microarrays (Hu133A 2.0, Affymetrix, Inc.) and qRT-PCR for selected miRNAs. Genes and pathways of interest were determined using class comparison and pathway analyses. Pathway expression of BRCA1 in the DNA damage response pathway presented the most significant difference (P Overall, the preliminary results indicate that tumor profiles were closer to basal cells than differentiated cells, consistent with their rapid growth rate and less well differentiated morphology. Investigation of gene expression patterns in normal cell types and ESCC provides novel insight into the transcript profiles associated with normal development versus carcinogenesis. 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 2168.