Clinical integration of next-generation sequencing technology.

Recent advances in next-generation sequencing (NGS) methods and technology have substantially reduced costs and operational complexity leading to production of benchtop sequencers and commercial software solutions for implementation in small research and clinical laboratories. This article addresses requirements and limitations to successful implementation of these systems, including (1) calibration and validation of the instrumentation, experimental paradigm, and primary readout, (2) secure data transfer, storage, and secondary processing, (3) implementation of software tools for targeted analysis, and (4) training of research and clinical personnel to evaluate data fidelity and interpret the molecular significance of the genomic output.

Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

Effect of Lean method implementation in the histopathology section of an anatomical pathology laboratory.

BACKGROUND: In the USA, the lack of processes standardisation in histopathology laboratories leads to less than optimal quality, errors, inefficiency and increased costs. The effectiveness of large-scale quality improvement initiatives has been evaluated rarely. AIM: To measure the effect of implementation of a Lean quality improvement process on the efficiency and quality of a histopathology laboratory section. METHODS: A non-concurrent interventional cohort study from 1 January 2003 to 31 December 2006 was performed, and the Lean process was implemented on 1 January 2004. Also compared was the productivity of the Lean histopathology section to a sister histopathology section that did not implement Lean processes. Pre- and post-Lean specimen turnaround time and productivity ratios (work units/full time equivalents) were measured. For 200 Lean interventions, a 5-part Likert scale was used to assess the impact on error, success and complexity. RESULTS: In the Lean laboratory, the mean monthly productivity ratio increased from 3439 to 4074 work units/full time equivalents (p<0.001) as the mean daily histopathology section specimen turnaround time decreased from 9.7 to 9.0 h (p = 0.01). The Lean histopathology section had a higher productivity ratio compared with a sister histopathology section (1598 work units/full time equivalents, p<0.001) that did not implement Lean processes. The mean impact, success and complexity of interventions were 2.4, 2.7 and 2.5, respectively. The mean number of specific error causes affected by individual interventions was 2.6. CONCLUSION: It is concluded that Lean process implementation improved efficiency and quality in the histopathology section.

Practical aspects of planning, building, and interpreting tissue microarrays: the Cooperative Prostate Cancer Tissue Resource experience.

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.

Cardiac fibroblasts influence cardiomyocyte phenotype in vitro.

Cardiac fibroblasts impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby fibroblasts alter cardiomyocyte behavior. Fibroblasts and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned > or =72 h by cardiac fibroblasts. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of alpha-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in fibroblast-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means +/- SD: conditioned 46.3 +/- 6.0 vs. control 5.3 +/- 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1alpha, IL-6, IL-10, IL-12p70, IL-17, and tumor necrosis factor-alpha) at or below detection levels in unconditioned medium that were significantly elevated in fibroblast-conditioned medium. Latent transforming growth factor-beta and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating factor was present above threshold levels in standard medium but decreased with fibroblast conditioning. These data indicated that under the influence of fibroblast-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions.

Microarray data mining using gene ontology.

DNA microarray technology allows scientists to study the expression of thousands of genes--potentially entire genomes--simultaneously. However the large number of genes, variety of statistical methods employed and the complexity of biologic systems complicate analysis of microarray results. We have developed a web based environment that simplifies the presentation of microarray results by combining microarray results processed for statistical significance with probe set annotation by Genbank, NCBI RefSeqs, GeneCards and the Gene Ontology. This allows rapid examination and classification of microarray experiments--annotated by NCIBI tools --by Statistical Significance and Gene Oncology Classes. By providing a simple, easily understood interface to large microarray data sets, this tool has been particularly useful for small research groups focused on a small number of related genes and for researchers who want to ask simple questions without the overhead of complex data management and analysis.

Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers.

Prostate cancer is one of the leading causes of cancer-related deaths for men in the United States. Like other malignancies, prostate cancer is underscored by a variety of aberrant genetic alterations during its development. Although loss of heterozygosity or allelic loss is frequently identified among prostate cancers, few genes have been identified thus far as critical to the development of invasive prostate cancers. In this report, we used the recently developed technology, the "differential subtraction chain," to perform a genome-wide search for sequences that are deleted in an aggressive prostate cancer. Among the deleted sequences, we found that one sequence was deleted in >50% of prostate cancers we tested. We mapped this sequence to chromosome 4q25 by screening the Genebridge 4 hamster radiation panel with primers specific to this probe, and subsequently identify a 54-kb minimal common deletion region that contains the sequence encoding myopodin. Sequence analysis indicates that myopodin shares significant homology with synaptopodin, a protein closely associated with podocyte and neuron differentiation. Further study shows that frequent complete or partial deletions of the myopodin gene occurred among invasive prostate cancer cases (25 of 31 cases, or 80%). Statistical analysis indicates that deletion of myopodin is highly correlated with the invasiveness of prostate cancers, and thus may hold promise as an important prognostic marker for prostate cancers.

E-cadherin expression and PSA secretion in human prostate epithelial cells.

Prostate-specific antigen (PSA) is the most widely used marker for the diagnosis of prostate cancer and is an independent predictor of prostatic capsular invasion. A number of studies have identified E-cadherin, a cell adhesion protein, as a potential invasion suppressor which is decreased in prostate adenocarcinoma. Our goal in the present study was to evaluate E-cadherin expression in primary cultures and determine the relationship between E-cadherin expression and PSA secretion in both primary cultures and the prostate tumor cell line, LNCaP. Immunohistochemical studies and Western blot analysis confirmed greater expression of E-cadherin in normal epithelial cells than tumor-derived prostate cells. This is the first report that the incubation of normal prostate epithelial cells with E-cadherin antibody increases the amount of PSA detected in the media of normal cells as well as in LNCaP. Since E-cadherin may function as an invasion suppressor, an understanding of the decreased expression of this adhesion factor and the impact on PSA secretion may aid in understanding epithelial tumorigenesis.

Expression patterns of vitamin D receptor in human prostate.

UV exposure and serum levels of vitamin D have been linked in several studies with prostate cancer risk. At the cellular level, the principal action of vitamin D is mediated though vitamin D receptors (VDR). Since prostate cancer is a disease strongly associated with age, we examined the presence of VDR in normal prostate from donors of various ages to determine if the VDR expression pattern changed with age. We also compared the VDR expression in the peripheral and central zones of the prostate to determine if the expression pattern varied by location. Immunohistochemical studies were performed on paraffin-embedded tissue from cases selected by the following age decades; 10-19, 20-29, 30-39, 40-49, 50-59, and 60-69. Both the central and peripheral zones were examined for VDR expression. The intensity of VDR expression in prostate was compared with expression in different types of human tissues. Mean VDR expression was lowest in the 10-19 years of age group. The intensity of the nuclear VDR was higher though the fifth decade, and then declined in cases of ages 60-70. When multiple sections of the same donor prostate were compared, VDR expression was greater in the peripheral zone compared to the central zone.

Identification of a novel gene with increasing rate of suppression in high grade prostate cancers.

Prostate cancer is the second leading cause of cancer-related deaths in the United States. However, the underlying molecular events for prostate cancer development are not clear. In this study, we applied the recently developed technology known as differential subtraction chain (DSC) to identify a novel gene whose expression is inactivated in high grade prostate cancer. This gene, designated as SAPC, is expressed in normal prostate acinar cells. Its expression is dramatically down-regulated in high grade prostate cancers (4/4) but is unaltered in low grade prostate cancers. It encodes a 7.7-kd protein. Its sequence shares some homology with the cysteine-rich domain of 2-5A-dependent RNase L, which is a critical component of the interferon-induced apoptosis cascade. The selective inactivation in the more aggressive prostate cancers holds promise for SAPC as a potential prognostic marker for high grade prostate cancer.

Expression of cadherins and catenins in paired tumor and non-neoplastic primary prostate cultures and corresponding prostatectomy specimens.

Cadherins are a family of transmembrane proteins that play a crucial role in cell differentiation, cell migration, and intercellular adhesion. Cadherins are associated with catenins through their highly conserved cytoplasmic domain. Down-regulation of E-cadherin protein has been shown in various human cancers. This study examined the expression of cadherins and associated catenins at the mRNA level. Paired tumor and nonneoplastic primary prostate cultures were obtained from surgical specimens. Quantitative multiplex fluorescence reverse transcriptase-polymerase chain reaction (QMF RT-PCR) and quantitative analysis were performed and correlated with immunostain results. Six of seven cases of neoplastic cultures showed moderately-to-markedly decreased levels of E-cadherin and P-cadherin mRNA. Similar losses of alpha-catenin and beta-catenin mRNA were also observed. The results of QMF RT-PCR showed good correlation with the results of immunohistochemical studies based on corresponding formalin-fixed sections. In conclusion, this paper presents a coordinated down-regulation in the expression of E-cadherin and associated catenins at the mRNA and protein level in most of the cases studied. This down-regulation may play an important role in the pathogenesis of prostate cancer.

Defining the role of anatomic pathology images in the multimedia electronic medical record--a preliminary report.

The development of the Multimedia Electronic Medical Record System (MEMRS) offers new opportunities for integrating medical imaging data with text-based clinical data. The effective integration of pathology images into the patient's medical record poses some significant technical and organizational challenges. Before these challenges can be met, it is imperative that we investigate the value and utility of providing these images to clinicians. In this study we examined attitudes towards use of pathology images in Image Engine, a MEMRS under development at the University of Pittsburgh Cancer Institute (UPCI). We conducted semi-structured standardized interviews with a cohort of practicing oncologists, all of whom had significant experience with Image Engine. This study is a first step towards elucidating the potential barriers, uses, and value of anatomic pathology images in the MEMRS.

Prototype Web-based continuing medical education using FlashPix images.

Continuing Medical Education (CME) is a requirement among practicing physicians to promote continuous enhancement of clinical knowledge to reflect new developments in medical care. Previous research has harnessed the Web to disseminate complete pathology CME case studies including history, images, diagnoses, and discussions to the medical community. Users submit real-time diagnoses and receive instantaneous feedback, eliminating the need for hard copies of case material and case evaluation forms. This project extends the Web-based CME paradigm with the incorporation of multi-resolution FlashPix images and an intuitive, interactive user interface. The FlashPix file format combines a high-resolution version of an image with a hierarchy of several lower resolution copies, providing real-time magnification via a single image file. The Web interface was designed specifically to simulate microscopic analysis, using the latest Javascript, Java and Common Gateway Interface tools. As the project progresses to the evaluation stage, it is hoped that this active learning format will provide a practical and efficacious environment for continuing medical education with additional application potential in classroom demonstrations, proficiency testing, and telepathology. Using Microsoft Internet Explorer 4.0 and above, the working prototype Web-based CME environment is accessible at http://telepathology.upmc.edu/WebInterface/NewInterface/welcome.html.

Characterizing "blue blobs". Immunohistochemical staining and ultrastructural study.

OBJECTIVE: To investigate the nature and origin of "blue blobs" (Bbs) in atrophic Pap smears in postmenopausal women and to study their clinical significance. STUDY DESIGN: A retrospective study of 412 atrophic Pap smears from postmenopausal women was done to detect the presence of Bbs. The smears from 24 cases showing Bbs were further studied to evaluate the nature of the Bbs with special stains, immunohistochemistry and electron microscopy. RESULTS: Bbs showed a heterogeneous morphology, with variable numbers and staining intensity. The diameter of Bbs was approximately equivalent to that of a parabasal/intermediate squamous cell. Special stains showed Bbs to be positive for periodic acid-Schiff and methyl green pyronin and negative for mucicarmine and calcium. Immunohistochemistry revealed Bbs to be positive for cytokeratin, epithelial membrane antigen and carcinoembryonic antigen and negative for vimentin and muscle-specific actin. Some Bbs had residual ghost nuclear shadows. Electron microscopy revealed cellular skeletons with residual tonofilaments enmeshed within a loose cytoskeleton matrix and nuclei with variable degrees of degeneration. CONCLUSION: Special stains, immunohistochemistry and electron microscopy indicated that Bbs represent parabasal/intermediate squamous cells exhibiting various degree of degeneration. In general, Bbs appear to be of no clinical significance except as a source of potential diagnostic error.

Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4.

We have identified previously six nuclear matrix proteins (NMPs) that are bladder cancer specific. In this study, we analyzed the expression of one of these proteins, BLCA-4, in bladder tumors and normal bladder tissue. We also examined the appearance of BLCA-4 in the urine as a biomarker for bladder cancer. BLCA-4 was isolated from nuclear matrix preparations of bladder tumors, and its peptide sequence was determined. The antibodies generated against the resulting BLCA-4 peptides were then used to detect its presence in immunoblots and in urine samples by immunoassay. We analyzed tissue samples of bladder tumor and normal donor bladders and urine obtained from 51 normal individuals and 54 patients with pathologically confirmed bladder cancer. The BLCA-4 peptide sequences do not resemble any known human protein sequences. On immunoblot analysis, BLCA-4 expression was detectable in tumor and normal tissues from patients with bladder cancer but not in any of the normal bladder tissue obtained from organ donors. Using a prospectively determined cutoff level of 13 A (absorbance) units/microg protein, all 51 normal individuals tested were negative for BLCA-4 expression, whereas 53 of 55 samples from patients with bladder cancer were positive. These results suggest that BLCA-4 is present throughout the bladder in both the tumor and morphologically normal areas in bladder cancer patients. BLCA-4 is a very sensitive (96.4%) and specific (100%) marker for bladder cancer. BLCA-4 is a bladder cancer-specific marker that can be detected using a urine-based assay and can be used in the diagnosis of bladder cancer.

Positive predictive value (PPV) of prostate specific antigen (> ng/ml) and abnormal digital rectal exam for prostate cancer on the Caribbean island of Tobago

OBJECTIVE: To establish and compare the positive predictive values (PPV) for elevated (4 ng/ml) prostate specific antigen (PSA) and abnormal digital rectal exam (DRE) in an Afro-Caribbean population. DESIGN AND METHODS: We screened 728 men aged 40-79 years, recruited from the general population on the Caribbean island of Tobago. Ninety-five percent reported African ancestry. This population had not previously undergone screening for prostate cancer. RESULTS: PSA was elevated (> or = 4 ng/ml) and/or DRE was abnormal in 291 (40 percent) men. Pathological diagnosis of random sextant biopsies was completed in 191 (66 percent) of men. Ninety-two (13 percent) of the screened men were diagnosed with prostate cancer. Among men biopsied for abnormal DRE in the presence of normal PSA, PPV for abnormal DRE was 26 percent (11/43), range 9-50 percent across age groups. Among men with elevated PSA and normal DRE, the PPV for PSA was 46 percent (29/63), range 42-54 percent (no men aged 40-49 years (n=105) fell into this category). When all men with elevated PSA were considered, ignoring DRE status, PPV for PSA was 55 percent (79/144), range 50-60 percent. If both PSA and DRE were abnormal, the PPV was 63 percent. CONCLUSIONS: The PPV of abnormal DRE was similar to that observed in other populations undergoing screening for the first time. We speculate that a lower PSA cut-off point may be appropriate for optima ascertainment of cases in this high-risk population.(Au)

The role of the pathologist as tissue refiner and data miner: the impact of functional genomics on the modern pathology laboratory and the critical roles of pathology informatics and bioinformatics.

This article provides an overview of how functional genomics is likely to impact on the pathology laboratory and highlights how informatics and tissue banking will greatly facilitate the molecular age of medicine. Important aspects of functional genomics in the post-genome era, including the roles of laser capture microdissection, DNA- and complementary DNA-based microarrays, proteomic methods, collaborative human tissue banking, tissue microarrays, and pathobioinformatics in the modern pathology laboratory are discussed. The role of mass spectroscopy in the analysis of RNA, DNA, and protein and its impact on the clinical laboratory, particularly in cost-effectiveness and time savings, are evaluated. This article explores how laboratory information systems (LISs) and the devices that feed them information may need to be modified to adapt to greater volumes of data for the new testing modalities that require understanding sophisticated fluorescence detection methods and image processing. Emerging genomic testing methods and their impact on pathology laboratory testing, especially in the area of molecular classification of neoplasms, are examined. The role of the tissue bank in the modern pathology laboratory as an archive of control normal tissues, as well as subsamples of the spectrum of progressive neoplastic states, is discussed in light of its critical importance to the molecular classification of cancer. Establishing a database that combines structured reports in pathology LISs and construction of tissue banking information systems will provide a rich resource for pathology departments. The article discusses a hypothetical resource, such as the Shared Tumor Expression Profiler, that would provide access to well-characterized tissue-based research resources for clinicians and researchers. Last, the article emphasizes how LISs can prepare for these changes, and how training pathologists in pathology informatics and bioinformatics (pathobioinformatics) is critical to ensure pathology's overall leadership role in the post-genome era.

Pathology imaging on the Web. Extending the role of the pathologist as educator to patients.

The increasing role of patients in medical decision making and the growing availability of medical information are changing the physician-patient relationship. One aspect of this change is that patients are searching for medical information on the Internet and are visiting sites, including pathology sites, that were not originally intended for patient use. This article describes evidence for this activity and how the University of Pittsburgh Medical Center Department of Pathology has evolved mechanisms to handle patient use of its web site.