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Concept: Morphoproteomics combines the disciplines of histopathology, molecular biology and protein chemistry to paint a portrait of the protein circuitry in diseased cells for the purpose of uncovering molecular targets amenable to specific intervention, thereby customizing therapy for individual patients. Methods: Morphoproteomic analysis involves the visual detection of protein analytes through an immunohistochemical signal using bright-field microscopy, quantification of said chromogenic signal noting its distribution throughout the tumor, an assessment of the state of activation of the expressed proteins (translocation within cellular compartments and phosphorylation) and correlative expression of the proteins along signal transduction pathways. In the process, it detects those proteins that may be essential to the growth, integrity, and migratory potential of an individual tumor and its resistance to conventional chemoradiation therapy. Proofs of Concept: The following aspects supportive of morphoproteomics as a viable concept include: preclinical and clinical outcomes data that validate the methodology and its predictive value; applications in targeted therapy; anecdotal successes using morphoproteomics as a guide to therapy; and the predictive value of morphoproteomic markers in clinical trials. Furthermore, it is possible to combine morphoproteomic data with genomic and protein function microarray data to design specific therapies. An algorithmic approach has been developed to allow the application of morphoproteomics to the selection of compatible conventional cytotoxic therapies and small molecule inhibitors based on the following: the state of constitutive activation of appropriate signal transduction pathways; cell cycle analysis; the historical efficacy of cytotoxic agents against a specific tumoral phenotype, and the site of action of such cytotoxic agents within the cell cycle. Conclusion: Morphoproteomics is a concept that has the potential to fulfill the promise of individualized therapy and personalized medicine.
[2] Genomic and Proteomic Markers of Response in Clinical Investigation of Molecularly Targeted Therapy. Carter Van Waes, National Institutes of Health, Bethesda, MD.
Genomic and proteomic methods can provide expression profiles, and bioinformatic analysis of these data can reveal alterations in molecular pathways and target gene programs, in cancers, and cancer subsets, for development of molecularly targeted therapy. Kinase, immunohistochemical (IHC), and reverse phase protein array (RPPA) analysis of signal activation and quantitative RT-PCR and immunoassay of target expression can be used to validate expression data and effects of specific agents in preclinical models and post-treatment biopsies and serum from patients. Expression profiling data from head and neck squamous cell carcinoma (HNSCC) reveals an increased prevalence of genes regulated by NF-kB, TP53, MAPK/AP-1, and STAT3 pathways. Distinct subsets expressing genes enriched for NF-kB or TP53 regulatory motifs are detected in association with TP53 mutation status and protein expression in cell lines and tumor specimens. NF-kB and regulated genes were most strongly activated in HNSCC with low expression of TP53. In a phase I study of proteasome inhibitor bortezomib, inhibition of NF-kB and increased expression of TP53 with corresponding effects on target molecules, proliferation and apoptosis, were detected in tumor biopsies and serum 24–48 hr post-treatment in a subset of patients. In a phase I study of EGFR inhibitor gefitinib, inhibition of EGFR, NF-kB, MAPK, and STAT3 phosphorylation with decreased proliferation and increased apoptosis was observed, but only in 1/7 patients, consistent with the low response rate observed with EGFR inhibitors in SCCHN in larger clinical trials. These and wider effects on signal molecules were observed using RPPA. In conclusion, genomic and proteomic methods can be used to generate hypotheses about molecular pathways and functionally important markers in cancer, which can be validated in clinical pathologic studies and trials using molecularly targeted agents. (Supported by NIDCD intramural project Z01-DC-00016)
[3] Reverse Phase Protein Microarrays and Protein Detection Arrays: Tools for the Next Generation of Quantitative Protein Network Analysis. Christian Loebke and Ulrike Korf, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Substantial bottlenecks for the time-resolved and quanitative description of signaling networks are the limited throughput and the lack of sensitivity of currently established methods. Protein microarrays are anticipated to guarantee improved sensitivity and higher throughput compared to standard technologies. Two different microarray-based approaches were explored for quantitative biology. Both approaches rely on signal detection in the near-infrared range (NIR). First, a multiplexed microspot immunoassay was developed. In this case, the target protein is captured from a complex mixture via a specific monoclonal antibody immobilized on the microarray surface. The visualization of captured proteins is performed in a three-step procedure: (1) a protein-specific rabbit antibody binds to a different epitope of the same protein; (2) the portion of microarray surface-bound rabbit antibody is visualized with NIR-dye labeled secondary antibodies; and (3) the signal intensity is determined by NIR-imaging. The quantitative read-out is based on a serial dilution of suitable recombinant protein standards. A tailored program based on the Bioconductor software R was developed for the quantitative analysis, the identification of suitable antibody pairs, and the analysis of cross reactivity between different antibody pairs in a multiplexed setting. The major current focus involves the systems biology of ERK, JAK/STAT and SMAD mediated signaling. Second, an improved lysate array approach was developed that permits a specific antibody-based quantification of proteins derived from as little as only 20,000 cells with a standard deviation of 5% or less. The capacity is limited to the analysis of up to 500 different samples per microarray. Protein abundance is determined comparatively, and absolutely, if recombinant protein is available. A detailed analysis of activated signaling networks in tumor biopsy samples is possible, and data from the analysis of GIST tumors will be presented. Lysate arrays are also useful for proteome profiling to analyze the crosstalk between signaling modules, for example after RNA-based silencing experiments.
[4] Using Oncogenomic and Oncoproteomic Data to Predict Therapeutic Responses. Mansoor M. Ahmed, Weis Center for Research, Geisinger Clinic, Danville, PA.
A major goal of cancer biology is the development of a successful predictive assay to measure therapeutic response in solid tumors. We have developed a classifier that predicts the clinical response to chemotherapy and radiation therapy. This classifier is based on targeted signaling pathway gene expression profiles and surviving fraction values from tumor cell lines treated with radiation therapy or chemotherapy or combined with both chemo- and radiotherapy. In this study, we developed two signaling pathway based classifiers in two different tumor backgrounds. We developed (1) a NF-kappaB targeted gene classifier using four SCCHN tumor cell lines and (2) a TGF-beta signaling effector gene classifier using six pancreatic cancer cell lines. In this approach, we consider therapy-based killing of tumor cells as a continuous variable; significance analysis of targeted gene arrays is used for gene selection; and a multivariate linear regression model is used for response prediction. Using SCCHN cell lines, the gene selection step identified eight novel NF-kappaB dependent genes of which expression values correlated with resistance to radiation and chemotherapy. Using pancreas cancer cell lines, the gene selection step identified eleven novel TGF-beta signaling targets of which expression values correlated with sensitivity to radiation and chemotherapy. We are currently validating the use of these two classifiers clinically by using tumor specimens from both SCCHN and pancreas cancer group of patients. In particular, from these tumor specimens we isolated mRNA to test the expression levels of identified novel genes in NF-kappaB and TGF-beta signaling pathways. Furthermore, we performed immunohistochemistry to confirm the mRNA gene expression findings. These data will be stratified using the classifier and response will be predicted. The predicted response will be correlated with actual clinical outcome data of these patients using Epic database. These analyses are under current investigation. If the classifier predicts truly the clinical outcome in these patients, then the implications of our findings will be significant. In conclusion, we establish that therapeutic sensitivity can be predicted based on gene expression profiles and we introduce a genomic and proteomic approach to the identification of novel molecular markers of treatment sensitivity.
[5] Signal Transduction Inhibitors in the Management of Hematologic Malignancies. David F Claxton, Penn State Cancer Institute, Penn State College of Medicine, Hershey, PA.
Over the last two decades, remarkable advances have been made in the understanding of the genetic basis of the pathogenesis of a variety of hematological malignancies. In the most recent five to ten years, specific inhibitors have become available for individual cell regulatory pathways. The advent of imatinib in the therapy of chronic myelogenous leukemia represented a breakthrough in therapy of leukemias in particular and cancer in general. This remarkable agent, developed and targeted specifically to inhibit the abnormal bcr-abl gene product resulting from the Philadelphia chromosome found in chronic myelogenous leukemia, represents superb therapy for this previously very difficult-to-treat disease. Second generation tyrosine kinase inhibitors are now available that address the development of imatinib resistance in these and other leukemia patients. Recent data show that these agents, through global inhibition of the ubiquitously expressed enzyme abl, have unexpected toxicities. This finding and its potential impact on therapy will be examined.
[6] Heterogeneous Knowledge Modeling for Morphoproteomics. M Sriram Iyengar, Robert E. Brown, and Mary F. McGuire, University of Texas Health Science Center, Houston TX.
This presentation describes computer-based mathematical and statistical modeling to support the emerging discipline of morphoproteomics. Signal transduction processes are fundamental to the regulation of cellular processes including cell division, proliferation, apoptosis, and others. The search for chemotherapeutic agents often involves identification of target biomolecules within oncogenic or apoptotic branches of these pathways. Recent research has shown in detail the progress of signal transduction pathways across multiple locations within the cell, from receptors on the plasma membrane through subcellular structures including the nucleus, where genetic factors can mediate the progress of the pathway. Further, the temporal aspect, expressed by the cell cycle, can also play an important role. The morphoproteomic paradigm takes into account these spatio-temporal dimensions, and others, thus providing more complete and detailed characterizations of signal transduction pathways. While morphoproteomic-based modeling of signal transduction pathways is complex because it involves the integration of heterogeneous data and multilevel knowledge, it can yield useful insights including identification of cross-talk between pathways. These insights, which can have significant therapeutic implications, are obtained by in-silico simulations including knock-out experimentation, and other ‘what if’ queries.
[7] Gene Expression Array Validation of First-Generation Guidelines for NCI-Supported Biorepositories. Jeffrey W. Prichard, Geisinger Medical Center, Danville, PA.
Currently biorepositories serve as critical resources for the research community in the performance of basic, epidemiologic, translational, and clinical postgenomics cancer research. It is becoming increasingly important that biorepositories strive to achieve the best possible biospecimen quality, which would necessarily call for the adoption of consistent documentation, collection, processing, storage, and retrieval guidelines. The NCI initiated a multiyear process that began in 2002 and that showed substantial heterogeneity in biorepository management practices across the Institute (NCAB 2004). This study showed that NCI-supported biorepositories are not optimized in terms of operational, legal, and ethical policies and procedures, nor are they coordinated to provide a unique resource value. In April 2006, the NCI released the "First-Generation Guidelines for NCI Supported Biorepositories" intended as a first step toward unifying policies and procedures for NCI-supported biorepositories. We proposed to undertake a study validating that these tissue and data banking guidelines result in tissue that can be used in gene expression microarray analysis of RNA, and that software can accurately manage, de-identify, and transfer de-identified data to a researcher. In this study, 15 matched frozen specimens of normal breast tissue and diseased breast tissue from excess tissue of resected breast specimens were collected. RNA was extracted and the quality verified by ribosomal banding. Amersham gene chips gene expression array data of the specimens compared the transcriptional activity findings from these specimens to findings from previously studied normal and diseased breast tissues. Associated clinical data were collected, de-identified by GML-PORT software, and uploaded to the PCABC Statewide Biospecimen network and audited for de-identification and accuracy. Our tissue banking technique resulted in excellent RNA yield and quality in 30 of 31 specimens (one fatty benign breast specimen produced low yield) for the performance of gene expression microarray analysis. GML-PORT software accurately managed, de-identified, and transferred de-identified data. These results confirm the Biospecimen Banking Standards promoted by the NCI for tissue handling procedures maintain RNA integrity and patient confidentiality and ensure the greatest research value from banked specimens. The NCI has developed several initiatives to assist its Cancer Centers in implementation of these guidelines. These initiatives include caBIGTM (https://cabig.nci.nih.gov), an infrastructure project designed to facilitate the exchange of data and programs among NCI-supported Cancer Centers. An associated Tissue Banking and Pathology Tools Workspace provides free informatics tools for biorepositories. These tools include: (1) caTISSUE Core. An intranet/Internet-based application for managing a biorepository. The caTISSUE Core also provides an object model through which existing biorepository systems may be used to share biospecimen data. (2) caTISSUE Clinical Annotation. An application for handling the annotation of biospecimens with clinical data. (3) Cancer Text Information Extraction System (caTIES). A system for extracting concepts from free text pathology reports into a structured data model.
[8] Clinical Evaluation of a Novel OncologicTissue of Origin Assay Based on Gene Expression Microarray. Iris Schrijver, J. Rodriguez-Paris, J.L. Zehnder, and J.R. Pollack. Pathology Department, Stanford University School of Medicine, Stanford, CA.
Based on histopathology and ancillary methods alone, the correct recognition of the tissue of origin in metastatic cancers can be very challenging. The PathworkTM Diagnostics Tissue of Origin test is a complementary molecular signature assay that analyzes the expression pattern of more than 1550 genes on PathchipTM microarrays. The arrays are analyzed by a proprietary prediction algorithm that determines the molecular similarity between oncologic tissue samples and 15 well-characterized malignancies from breast, lymph node, thyroid, lung, pancreas, kidney, liver, germ cell, soft tissue, colon, ovary, skin, stomach, bladder, and prostate (60 morphologies). The goal of our ongoing evaluation is to determine assay performance prior to application as an FDA-cleared diagnostic test. Thus far, 12 different samples have been examined at Stanford, covering 7/15 tissues on the Pathwork panel. Eight (two non-Hodgkin’s lymphomas, two metastatic thyroid carcinomas, one renal cell carcinoma, one poorly-differentiated gastric adenocarcinoma, one high grade sarcoma, and one endometrioid adenocarcinoma) were scanned at two sites with identical results. These malignancies were correctly identified by the test, except the uterine cancer, which was predicted to be of ovarian origin. Uterine carcinoma, however, is not one of the 15 tissues on the panel. The poorly differentiated gastric cancer was classified as indeterminate. We evaluated four additional metastatic tumors, including one carcinoma of unknown primary (CUP), one hepatocellular metastasis obtained from abdominal soft tissue, one medullary thyroid carcinoma biopsied from pharyngeal soft tissue, and one melanoma obtained from the parotid gland. Three were unequivocally identified. The CUP remained indeterminate, although nine tissue types could be ruled out with high confidence. In summary, 9/12 samples were identified correctly (75%) in this preliminary study, with two indeterminates and one misclassification. We will present the results of our completed study and report on our findings regarding sensitivity, specificity, robustness, and ease of assay use.
[9] Utility of HepPar-1 and MOC 31 for the Distinction of Hepatocellular Carcinoma and Adenocarcinoma. S Kuruvilla, J Liu, D Zander, and S Krishnamurthy. Department of Pathology and Lab Medicine, University of Texas Medical School, and Department of Pathology, M. D. Anderson Cancer Center, Houston, TX.
Several immunomarkers have been described for the distinction of hepatocellular carcinoma (HCC) from cholangiocarcinoma and metastatic adenocarcinoma [AC] in the liver. These markers are needed not for the investigation of well differentiated tumors but mainly for poorly differentiated (PD) and sometimes moderately differentiated (MD) tumors in the liver. We evaluated the utility of a limited panel of HepPar-1 and MOC31 for distinguishing MD and PD HCC from AC. We studied formalin-fixed, paraffin-embedded surgical tissue sections from 25 cases of HCC (12 MD, 13 PD) and 25 cases of AC (16 MD, 9 PD). These included 28 needle core biopsies and 22 surgical resections. Sections were stained with primary antibodies against HepPar-1 and MOC31, and blocked with Avidin X0590 (DAKO); EnVision+[HRP] system (DAKO) was used to detect HepPar-1 and PowerVision (ImmunoVision Technologies) was used to detect MOC31. Of the HCC cases, 15/25 (60%) were HepPar-1+/MOC31–; 5/25 (20%) HCC were HepPar-1+/MOC31+; 4/25 (16%) were HepPar-1–/MOC31–; and 1/25 (4%) were HepPar-1–/MOC31+. Of the AC cases, 25/25 (100%) stained HepPar-1 negative/MOC31 positive. Used by itself, MOC31 was 100% sensitive and 76% specific for the detection of AC. Used by itself, HepPar-1 showed 80% sensitivity and 100% specificity for the detection of HCC. The sensitivity and specificity of the combined HepPar-1/MOC31 panel in the detection of HCC were 96% and 100% respectively. The sensitivity and specificity of the same panel in the detection of AC were 100% and 96% respectively. In conclusion: (1) HepPar-1 was highly specific (100%) and sensitive (80%) for the diagnosis of HCC. (2) MOC-31 was highly sensitive (100%) and specific (76%) for the diagnosis of AC. (3) Using a limited panel of only HepPar-1 and MOC31, we were able to statistically distinguish the majority of moderately and poorly differentiated HCC from AC in the liver, so that a HepPar-1–/MOC31+ immuno-phenotype represented AC and HepPar1+/MOC31- immuno-phenotype represented HCC. (4) Simultaneous positivity or negativity for both markers was seen only in HCC.
[10] The Warburg Effect: Role in Cancer-Induced Abnormal Tubulogenesis. Egil Fosslien, College of Medicine, University of Illinois at Chicago, Chicago, IL.
The objective of this paper is to examine the role of altered mitochondrial function in morphogen signaling that determines formation of abnormal ducts and vascular channels (tubulogenesis) in cancer. Reports of morphogen regulation of in vitro and developmental tubulogenesis and involvement of mitochondria and cyclooxygenase (COX) in morphogen signaling were compared and analyzed. The findings were as follows: Bimodal morphogens of the transforming growth factor (TGF)-beta family, nitric oxide (NO), thrombin, and hydrogen peroxide (H2O) inhibit and stimulate cell proliferation at high and low concentrations respectively. TGF-beta1 modulates basic cell proliferation and differentiation along its diffusion/perfusion gradient of declining concentration. Inhibition of cell proliferation gradually decreases and stimulation gradually increases with increasing distance from the morphogen source. The morphogen concentration at the source and the slope of the gradient determines the morphology of concave structures (morphe concavus) as viewed from the source ( Med Hypotheses 2002;59:233–238[Medline] ). Basic cell proliferation is ultimately controlled by available adenosine triphosphate (ATP), normally mainly supplied by mitochondria. Inherited or functional defects of Krebs cycle or oxidative phosphorylation enzymes are associated with neoplastic proliferation. TGF-beta1 can regulate the mitochondrial synthesis of ATP via regulation of adenine nucleotide translocator (ANT)-2, or via regulation of COX-2, which is significantly induced in many malignancies. In conclusion, an energetics shift from ATP production by oxidative phosphorylation to increased glycolysis and lactate production (the Warburg effect), derails normal dynamic post-natal morphostat regulation of mitochondrial ATP generation, causes abnormal spatial variation in ATP generation, and disrupts proper bimodal morphogen signaling responses that lead to abnormal tubulogenesis. These findings indicate that detection of structural (by genomics and proteomics) or functional (by MRI) defects of mitochondrial ATP generation and their repair can help to restore normal tissue morphology.
[11] Multianalyte Chip-Based RNA Nanobiosensor for Circulating Tumor cells and Cancer Diagnosis. Dana Kausmeyer, Rustom Bhiladvala, James Sioss, Susan Patrick, Weihua Pan, Mingwei Li, Tom Morrow, Ping Xin, Susan Trolier-McKinstry, Chris Keating, Theresa Mayer, and Gary Clawson, Gittlen Cancer Research Foundation, Hershey Medical Center, Hershey, PA, and Pennsylvania State University, University Park, PA.
Improvements in survival and cure rates of cancers often reflect better surveillance, which facilitates detection of cancers at earlier stages. The development of a multianalyte chip-based RNA sensor platform would be valuable for enabling detection of circulating tumor cells (CTCs) from multiple types of cancers, potentially leading to earlier diagnosis and improved prognosis. CTCs may appear in blood before metastases develop, and PCR-based assays in patients with breast cancer have shown utility as a prognostic tool. Unfortunately, PCR-based analyses are not easily adapted to screening approaches for detection of multiple cancers. Direct methods for detecting CTCs are being investigated, but are limited in availability and applicability. Our multianalyte chip-based RNA biosensor platform enables point-of-contact detection of CTCs from multiple cancers simultaneously, using selected marker RNAs, and assists in determining the site of origin of the CTCs. The nanosensor is based on mass-sensitive detection of biospecific RNA sandwich-hybridization binding events using arrays of silicon nanowire cantilevers that are derivatized with cancer-specific optimized antisense oligonucleotides (ASOs) prior to on-chip assembly. To enhance its mass, a gold nanoprobe is first hybridized to the target RNA. In prostate cancer, for example, a non-coding RNA called DD3 appears to be a valuable molecular marker; exon 4 is prostate-specific, and DD3 levels are upregulated more than 100X in cancer, with average expression of about 6000 copies per cell. This approach provides excellent detection selectivity as well as sensitivity to single binding events, enabling detection of CTCs, earlier diagnosis, and, potentially, improvement in cancer survival.
[12] Molecular Classifications of Breast Carcinomas with Similar Terminology and Different Definitions: Are They the Same? Ping Tang and Jianmin Wang, University of Rochester Medical Center, Rochester, NY, and RTI Health Solution, Research Triangle Park, NC.
In the attempt to better predict prognosis, several molecular classifications, which divide breast carcinoma into basal and luminal/non-basal subtypes, exist in the literature based on expression patterns of molecular markers. Worse prognosis is associated basal subtype in these classifications (CK, ER/HER-2, and ER/PR/HER-2). Cytokeratin (CK) classification is based on expression profiles of CK markers: luminal subtype being CK8 and/or CK18 positive; basal subtype being CK5/6, CK14, and/or CK17 positive. ER/HER-2 classification is based on expression profiles of ER and HER2: luminal being ER positve/HER-2 negative or positive; basal being ER negative/HER-2 negative or positive. ER/PR/HER-2 (triple) classification is based on expression of ER, PR, and HER-2: basal being all three markers negative; non-basal: being at least one of the markers positive. Since all three classifications use similar terminology (basal and luminal/non-basal) but different definitions, it is critical to understand the precise relationship between these classifications. Here for the first time we directly compared these classifications in 195 cases of breast carcinoma, including 46 of high-grade (HG) and 53 of non-high grade (NHG) pure ductal carcinomas in situ (DCIS) and 44 of HG and 52 of NHG DCIS with coexisting invasive ductal carcinoma (IDC) by immunohistochemical (IHC) analysis of the expression patterns of CK5/6, 8, 18, 14,17, along with ER, PR, and HER-2. We found that (1) Different distributions of basal subtypes in HG carcinoma exist among these classifications, there are 17%, 70%, and 30% for HG DCIS and 48%, 82%, and 34% for HG DCIS/IDC for triple subtype, ER/HER-2 subtype, and CK subtype, respectively. (2) The distribution of basal subtype between HG DCIS and HG DCIS/IDC in triple classification is significantly different, 17% and 48%, respectively. (3) The distribution of both subtypes among three classifications is similar for NHG carcinoma (2%–8% for basal subtype, and 92%–98% for non-basal subtype. (4) The highest degree of correlation among these three classifications is observed for non-basal subtypes of NHG carcinoma (ranging 87%–98%); followed by non-basal (18–59%) and basal subtypes (7–34%) of HG carcinoma. Almost no correlation exists for NHG basal subtypes (0%–2%). In conclusion, the three classifications differ significantly, especially for HG carcinoma. More studies are needed to explore their relationships and evaluate their utility in predicting prognosis and guiding individualized therapy.
[13] Avian Influenza from a Virologist’s Perspective. Wallace Greene, Department of Pathology, Penn State University Medical School, Hershey, PA.
Avian influenza is caused by numerous influenza viruses that commonly occur every year and occasionally cause severe outbreaks in poultry flocks, but rarely produce infections in humans. Over the past few years, the avian influenza virus H5N1 has not only produced economically devastating outbreaks in poultry in many parts of the world, but also caused severe and deadly infections in humans. This presentation will explain the unique characteristics of the H5N1 virus that have elevated our level of concern and preparation for a pending pandemic to unprecedented levels. Comparisons will be made with this virus and those causing the pandemics of 1918, 1957, and 1968. Finally, current recommendations from the CDC, NIH, HHS, and WHO will be reviewed.
[14] Pathology of Post-Primary Tuberculosis in Humans and Mice: Contradiction of Long-Held Beliefs. Robert L. Hunter, Chinnaswamy Jagannath, and Jeffrey K. Actor. Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, Houston, TX.
Tuberculosis remains one of the world’s leading infectious causes of death. About 80% of all disease is due to post-primary (secondary) tuberculosis in the lung. Unfortunately, tissues of developing lesions are seldom available and there are no recognized models of post-primary tuberculosis. In the pre-antibiotic era when tissues were more abundant, several investigators described early post-primary tuberculosis as a lipid pneumonia quite different from the caseating granulomas commonly described today. We used histopathologic, immunohistochemical, and acid fast stains to examine tissues from 35 people with untreated primary and post-primary tuberculosis and compared the findings with those of mice with reactivation tuberculosis. The results confirmed that developing post-primary tuberculosis begins as a lipid pneumonia accompanied by bronchial obstruction in which infection is restricted to foamy alveolar macrophages. Cavities result from a combination of caseation of tuberculous pneumonia and microvascular occlusion characteristic of delayed-type hypersensitivity (DTH). Reactivation tuberculosis in the mouse begins as a similar tuberculous lipid pneumonia with bronchial obstruction and evidence for participation of DTH. Developing necrosis in both species is associated with localization of organisms within lipid droplets. These results suggest that reactivation tuberculosis in mice is a valuable model of developing human post-primary tuberculosis.
[15] Human Papilloma Virus (HPV) Vaccine: Old/New Approach to Cancer Prevention. Armand B. Glassman, Medical University of South Carolina, Charleston, SC.
HPV vaccination is projected to lower the incidence of cervical carcinoma (CC). Ten to twelve thousand cases of CC are diagnosed each year in the USA. There are approximately 4000 deaths annually from the disease. Cervical cytology screening (30–95% sensitivity and 78–100% specificity) has brought this number down over the years. In June 2006 the FDA approved the clinical use of a quadrivalent HPV vaccine. This study reviews HPV types, associated oncogenic potential, the types chosen for protection in the FDA approved vaccine, and vaccine efficacy. There are approximately 100 types of HPV, of which about 30 are sexually transmitted. High risk for malignant oncogenesis is a characteristic of types 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 82. HPV 16 is the most common cancer associated type resulting in ~50% of CC. HPV 18, another fairly common strain, has been associated with glandular as well as squamous carcinomas. The approved vaccine is made from non-infectious HPV-like particles representing the antigenicity of types 6, 11, 16, and 18. These four types cause ~70% of CC and ~90% of genital warts. Vaccination (3 doses at 0, 2, and 6 months) is recommended for women between the ages of 16 and 26 although clinical trials showed benefits in girls as young as 9. Efficacy studies of the vaccine were done on a group (~11,000) of women age 9 to 26 years. For HPV naive women 16–26 years of age the vaccine was 100% successful in preventing CC and >95% effective in preventing genital warts. There were no therapeutic effects of the vaccine on existing HPV disease and tests in men are ongoing. Antibodies were demonstrable for at least 5 years. An FDA approved HPV vaccine representing 4 types (two of which are the most common malignant oncogenic types) is available and should be given to women before the onset of sexual activity. Successful HPV vaccination is expected to reduce the incidence of CC by ~70%.
[16] Tau with Protein 14-3-3 Improves the Diagnosis of Creutfeldt-Jakob Disease. C R Hamlin and P Gambetti, Case Western Reserve School of Medicine, Cleveland, OH.
Creutfeldt-Jakob (C-J) disease can only be diagnosed with certainty by the trilogy of positive immunohistochemistry, characteristic morphology, and the confirmed presence of scrapie prion protein (PrPsc) in post-mortem brain tissue. Protein 14-3-3 is included, by international agreement, as part of the pre-mortem diagnostic work-up. However, its diagnostic accuracy is only moderately good, and several European laboratories have proposed using alternative brain-derived proteins, in particular Tau. This report describes the evaluation of Tau and its comparison to protein 14-3-3 in 112 individuals with subsequent post-mortem, autopsy-proven diagnoses, 61 with C-J disease and 51 with alternative neuropathology. Tau was quantified by the Biosource kit (KHB0042, Camarillo, CA) and protein 14-3-3 was classified as positive, ambiguous, or negative by immunoblot (primary antibody: 14-3-3ß (K-19) (Santa Cruz Biotechnology, Santa Cruz, CA). A cut-off value of 800 pg/ml for Tau is used: specimens with lower values are considered negative. Tau is measured on specimens ambiguous or positive for protein 14-3-3. If Tau is negative, then the specimen is considered negative for C-J disease, otherwise the specimen is considered positive. Tau need not be measured on negative protein 14-3-3 specimens. The diagnostic accuracy of protein 14-3-3 alone is 72%, with 29 ambiguous specimens excluded; with Tau added it is 83%, with no specimen excluded. The sensitivity and specificity for protein 14-3-3 are 94% and 41%, and with Tau added 89% and 77%. It is intended to introduce this combination of tests into routine use, provided the overall conclusions are maintained with 100 autopsy-proven C-J disease cases.
[17] Proton Pump Inhibitors but Not Histamine Type 2 Receptor Antagonists Are Associated with Clostridium difficile Infection for Patients in an Urban Medical Center. R. Shakov, S. Jayatilaka, R. Eddi, W. J. Baddoura, and V. A. DeBari, St. Joseph’s Regional Medical Center, Paterson, NJ, and School of Graduate Medical Education, Seton Hall University, South Orange, NJ.
C. difficile associated diarrhea (CDAD) has emerged as a major factor resulting in morbidity in hospitalized patients. In a study of five-year (2001–2005, inclusive) trends of incidence of CDAD among adults in an inner city medical center, it was determined that the overall annual incidence increased from 5.08 to 8.42 cases/103 admissions (p = 0.0005). Age distribution remained fairly constant for 2001–2004 but decreased significantly in 2005 (p=0.005); no significant change was observed for gender. During that period, we observed a decline in the use of histamine type 2 receptor antagonists (H2A) with a concomitant increase in the use of proton pump inhibitors (PPI) as a prophylactic measure to prevent stress ulcers. The usage of PPI correlated exactly (rs = 1.0; p = 0.017) with the increase in CDAD incidence. A case (n = 122)-control (n = 244) study for the final year was conducted, examining the association of PPI and H2A with CDAD. Use of PPI either pre- or during admission was associated with CDAD (odds ratio, OR = 2.61, 95% CI = 1.65 to 4.12; p = 0.0001); the association with H2A was not significant (OR = 1.06, 95% CI = 0.46 to 2.46; p = 1.00). If only first-time use during hospital stay is considered, PPI were also strongly associated with CDAD (OR = 2.57, 95% CI: 1.45 to 4.55; p = 0.0016) and H2A, again, were not associated with CDAD (OR = 0.86, 95%CI: 0.32 to 2.30;p = 1.00).These data suggest that the widespread prescription of PPI for stress ulcer prophylaxis in acute care facilities may be contributing to the increased incidence of CDAD.
[18] Deployed Medical Surveillance using Probabilistic and Statistical Modeling. Linda V. Hardie, Linda Steel-Goodwin, and Gregory W. Johnson, Office of the Surgeon General, Air Combat Command, Langley AFB, Hampton, VA; EqualNox Consulting, Newport News, VA.
Since 2000, the United States Air Force has been aggressively analyzing electronic medical information, collected at deployed medical facilities, to facilitate both retrospective and near-real time identification of medical concerns and capability gaps. For seven years, deployed medical treatment has been captured with the Global Expeditionary Medical System (GEMS) using a combination of structured and free-text data fields. Structured data fields allow the user to select from a predefined set of values. Data mining for specific values is performed on the structured data to build statistical predictive models. While structured data provides uniformity of data, it is too rigid to accurately capture all relevant medical information. To address this limitation, GEMS allows the user to enter free-text notes using natural language throughout the data entry process. Due to the unpredictability of free-text data, such data cannot be included in building statistical models. Instead, text mining and fuzzy logic are utilized to derive knowledge from the text and to build probabilistic models. An example of such a study was conducted on leishmaniasis, a public health reportable event, which is endemic at the current deployed locations. Using a structured diagnosis field that captures ICD-9 codes, 28 specific leishmaniasis cases were identified. Another 438 cases were discovered using text mining for non-healing insect bites on the free-text fields. Using both statistical and probabilistic models to identify medical encounters for non-healing insect bites, 466 cases have been identified. By combining knowledge obtained from both probabilistic and statistical models, the United States Air Force is better able to identify reportable events, disease outbreaks, disease trends, and potential operational improvements.
[19] Can We Better Define Roles for Clinical Laboratory Professionals in Radiation Event Medical Management? Jack W. Snyder, National Library of Medicine, Bethesda, MD.
Reports from Britain of possible human exposure to polonium-210 have reinvigorated concerns of public health and disaster preparedness officials regarding potential adverse health effects of radiation. To aid emergency personnel who respond to documented or perceived exposure or contamination of humans by radiation, the National Library of Medicine, CDC, and Office of the Assistant Secretary for Preparedness and Response have launched a multi-media, web-based electronic information tool known as REMM (Radiological Event Medical Management). To stimulate investigation, NIH also has published a strategic plan and research agenda for medical countermeasures against radiological and nuclear threats. To ensure a continuing and meaningful role in modern management of radiation events, including a role in specimen processing, worker safety, cytogenetic biodosimetry, provision of blood products, detection of sources of radiation, and deployment of other diagnostic and therapeutic modalities, laboratory scientists should periodically update not only their understanding of biological and environmental impacts of radiation, but also their knowledge of the U.S. National Response Plan and the response assets of federal (eg, DOE, EPA, DOD), state, and local agencies that may be authorized to engage clinical laboratories for various public health purposes. Serious efforts are needed to identify novel radiation biomarkers and to develop applied biological dosimetry assays monitored with clinical, deployable, and hand-held analytical systems. Reports (eg, those of the International Commission on Radiological Protection) emphasize that clinical laboratories, as integral parts of public health systems, should proactively define their specific capabilities and roles in furtherance of the goals of national and international radioprotection programs.
[20] Document Control: A College of American Pathologists’ Requirement. Sidney M Hopfer and Richard R Lindquist, University of Connecticut School of Medicine, Farmington, CT.
The College of American Pathologists (CAP) instructs all clinical laboratories to have a system in place that assures all copies of policies and procedures (P&P) are current, personnel understand these P&P, all P&P have been authorized for use by the laboratory director, all P&P are reviewed annually, and discontinued P&P are quarantined and retained for 2 or 5 years. The CAP also recommends a control log be available listing current P&P with locations of derivative documents (including dates P&P were activated or retired), identity of reviewer, and reason for changes. One of the authors (RRL) has developed a home-brew, data-base management system specifically for clinical laboratory use that addresses these CAP criteria. The system is available through intranet access and thereby accessible to all laboratory-associated locations, including satellite laboratories and phlebotomy stations on the hospital internal network. Access can be customized by location, individual password, or IP address. Policies and procedures that require review (P&P review by new technologists or annual review by director) are displayed at the time the user logs on to the system. Users are able to search each P&P by title or key word, enhancing their ability to find topics of interest. Changes to documents (including reasons for changes), identity of reviewers, and dates of review are recorded electronically by the system and are available to the system administrator. This document verification system (DVS) is available at the majority of laboratory workstations, negating the need for confusing hard-to-control derivative documents. The DVS enhances workflow, alleviates the need to have bulky P&P manuals near each workstation, and is a step closer to achieving the elusive paperless environment.
[21] Assessment of Physicians’ Ability to Interpret Urine Drug Screening Tesults. Roger L. Bertholf, and Gary M. Reisfield, Departments of Pathology and Community Health & Family Medicine, University of Florida Health Science Center, Jacksonville, FL.
Immunochemical screening methods designed to detect common drugs of abuse have been available for decades, and are usually included among the tests offered by reference and hospital-based clinical laboratories. Whereas test results generated by forensic toxicology laboratories typically undergo scientific and medical review by appropriately qualified individuals, results of urine drug testing (UDT) in clinical settings are often interpreted by physicians with no specialized training in clinical or analytical toxicology. The proper interpretation of UDT–upon which vital clinical decisions are made–requires an understanding of several factors, including the analytical sensitivity and specificity of the particular test for individual drug(s) and/or metabolite(s), the predictive value of positive and negative results, the pharmacokinetic variables that affect the timing and assortment of compounds that appear in the urine, and the recognition of potential positive and negative interferants. We designed a seven-question instrument to assess basic knowledge of UDT interpretation and administered the survey to physicians attending three regional opioid education conferences. One hundred and fourteen respondents (80%) completed the survey. Sixty-eight percent of these respondents indicated that they utilize UDT to monitor adherence in their patients on chronic opioid therapy. Among those using UDT, none answered all seven questions correctly, and only 24 (31%) answered at least four questions correctly. There were no significant differences in the percent of correct responses to any question between physicians who order UDT and those who do not. We conclude that misinterpretation of UDT results by clinicians is common.
[22] Atypical Cells in Body Cavity Fluids: an Algorithmic Approach to Diagnosis. Myra Wilkerson, Geisinger Medical Center, Danville, PA.
The peritoneal, pleural, and pericardial cavities each provide a large surface area that is frequently involved in reactive/reparative processes or invaded by malignant cells, both of which may lead to a serous effusion. Cytologic examination is a very reliable test for detection of malignant cells in effusions, with a reported sensitivity of 70–90%. In our laboratory, we process these fluids as cytospins and cell blocks. Cell block preparations offer a distinct advantage in that the cells are processed similarly to traditional tissues and the specimens are fixed in formalin and embedded in paraffin so that they can be sectioned multiple times for immunostaining. This presentation will review the morphologic features of a variety of atypical cells identified in effusions and explore an algorithmic approach using immunoperoxidase staining panels to arrive at a diagnosis. The discussion will focus on differentiating between reactive mesothelial proliferations, malignant mesothelioma, and a variety of metastatic malignancies.
[23] Utility of p16/ProEXC for Detection of Squamous Intraepithelial Lesions in Liquid-Based Cytology Specimens on Cell Block Sections. Haiyan Liu, Jianhui Shi, Myra L Wilkerson, and Fan Lin, Geisinger Medical Center, Danville, PA, and Lehigh Valley Hospital. Allentown, PA.
Our previous study showed p16 to be a highly sensitive marker in confirming the diagnosis of high-grade squamous intraepithelial lesion (HGSIL), but it exhibited low sensitivity for low-grade squamous intraepithelial lesion (LGSIL) in liquid-based cytology specimens on cell block sections (LBCBS). We recently demonstrated that a cocktail of p16 and ProEXC (a mixture of MCM2 and DNA topoisomerase IIA) could detect 100% of LGSIL in cervical biopsy specimens. To improve diagnostic sensitivity for LGSIL, we evaluated the same cocktail in LBCBS from 48 specimens. Three groups of cases were included: Group #1 (G1), 17 cases of LGSIL; Group #2 (G2), 15 cases of HGSIL; and Group #3 (G3), 16 negative/reactive cases. The diagnoses in all cases in G1 and G2 were confirmed by cervical biopsy. Immunostaining with monoclonal antibodies against p16 (Dako) and a cocktail of p16 and ProEXC (TriPath) were performed on the LBCBS. The results were recorded as negative (no staining) or positive (>3 atypical squamous cells, with both cytoplasmic and nuclear staining or nuclear staining). The results are summarized in the following table. All cases in G1 were positive for the p16/ProEXC cocktail, including the 4 p16-negative cases. In G3, 2 of 16 cases (10.5%) showed false immunoreactivity for both p16 and the p16/ProEXC cocktail with staining of metaplastic squamous cells or endocervical cells.
| Marker | G 1 (%) | G 2 (%) | G 3 (%) |
| p16 | 13/17 (76%) | 15/15 (100%) | 2/16 (10.5%) |
| p16 + ProEXC | 17/17 (100%) | 15/15 (100%) | 2/16 (10.5%) |
Our preliminary data indicate that a cocktail of p16 and ProEXC provides higher diagnostic sensitivity (100%) for both LGSIL and HGSIL than p16 alone and can potentially serve as a marker for screening of SIL in LBCBS.
[24] Utility of S100P in Diagnosis of Pancreatic Adenocarcinoma in Fine Needle Aspiration Biopsies and Surgical Specimens. Fan Lin, Hongbing Deng, Jianhui Shi, and Myra L Wilkerson, Geisinger Medical Center, Danville, PA.
Fine needle aspiration biopsy (FNAB) of the pancreas is used to establish a diagnosis of pancreatic carcinoma. Attempts have been made to identify specific markers for pancreatic ductal adenocarcinoma (PDA), but most are expressed in normal/reactive pancreatic ductal epithelium as well. Recently, S100P was identified as a marker for PDA, but its utility in FNAB specimens has not been reported. We immunohistochemically evaluated S100P on 26 cell block sections (CBS) and 36 alcohol-fixed direct smears in 44 cases of FNAB of the pancreas. The 44 cases were divided into 4 groups: Group 1 (G1), 24 cases (18 CBS and 23 smears) of PDA; Group 2 (G2), 6 cases (6 smears) with an atypical or suspicious diagnosis; Group (G3), 9 cases (5 CBS and 5 smears) of benign/reactive pancreatic ductal epithelium; and Group 4 (G4), 5 cases (3 CBS and 2 smears) of pancreatic endocrine tumor (PET). Clinical followup or surgery confirmed the diagnoses. Immunostain for S100P was also performed on 56 cases of PDA on surgical specimens. Only nuclear or nuclear and cytoplasmic staining were regarded as positive. Positive immunoreactivity for S100P was observed in 18 of 18 PDA cases in CBS and 23 of 23 smears, with 3+ and 4+ staining in 17 cases and 22 cases, respectively. All cases in G3 and G4 were negative for S100P, with the exception of 1 case of benign glandular epithelium on the smear that showed positive staining and strong background staining. Importantly, all cases in G2 were positive for S100P with >2+ positivity. All 56 cases from surgical specimens were positive for S100P. Our data confirm the utility of S100P for detection of PDA in surgical specimens and as a sensitive and specific marker for detection of PDA on FNAB specimens on both CBS and smears, but caution should be exercised in interpreting positive results on smears because background staining may be present.
[25] Fine Needle Aspiration Biopsy of Soft Tissue Tumors. Shaobo Zhu, Penn State Milton Hershey Medical Center, Hershey, PA
Fine needle aspiration (FNA) biopsy plays an important role in the diagnosis of soft tissue tumors. This presentation will consider the advantages and limitations of FNA for soft tissue tumors, cytologic features of major soft tissue tumors (fibrous and fibrohistiocytic tumor, adipose tissue tumors, smooth muscle and skeletal muscle tumors, peripheral nerve tumors, and miscellaneous tumors), and ancillary diagnostic methods for soft tissue tumors (immunohistochemistry, cytogenetics, and molecular analysis).
[26] Evaluation of Oral Lesions Using Liquid-Based Cytology. Philip R. Foulis, Leah Strickland-Marmol, and Marion Ridley, James A. Haley Veterans Hospital and University of South Florida, Tampa, FL.
Oral cavity lesions detected during routine clinical surveillance can either be biopsied or re-evaluated. The squamous mucosa of the oral cavity can be sampled easily by brush cytology. We independently examined cytologic brushings of oral cavity lesions via the monolayer technique, using the ThinPrep technique (Cytyc Corp., Marlborough, MA). We compared these results to concurrently performed biopsies to determine if the cytologic evaluation of oral lesions provides useful information that could aid in the decision between ‘watchful waiting’ or surgical biopsy. We performed 23 brushings of oral lesions in a group of Veterans attending an Otolaryngology Clinic. We had excellent correlation for benign epithelial lesions (10/10), squamous cell carcinoma (6/6), and HPV change (1/1). Five benign submucosal lesions were not detected, since the overlying epithelium was either benign or reactive. One biopsy of mild dysplasia was interpreted as suspicious for malignancy. Leukoplakia, a precursor of oral squamous cell carcinoma, affects 3% of the adult male Caucasian population in the United States, where progression of the lesion is estimated to be 10%. In 2006, squamous cell carcinoma of the oral cavity comprised 2.21% of new cancer cases and 1.31% of cancer deaths. The incidence of carcinoma increases in patients with a history of tobacco, ethanol abuse, or chronic irritation. When present in combination, these risk factors increase the risk exponentially. All of these risk factors are present in our population, and they are often present together. Cytologic brushing by the monolayer technique is a useful, non-invasive, and easy to perform screening tool for evaluation of epithelial lesions in the oral cavity. Visualization and brushing of oral lesions may avoid the necessity for surgical biopsy.
[27] Chondroblastoma of Bone: The Hershey Medical Center Experience. Elizabeth E. Frauenhoffer, Penn State University M.S. Hershey Medical Center, Hershey, PA.
Chondroblastoma (Chbl) is an uncommonly encountered bone tumor. The purpose of this study was to characterize the clinical, pathological, and radiographic features of chondroblastomas diagnosed at M.S. Hershey Medical Center (MSHMC), employing multidisciplinary expertise. Methods: The pathology database was searched for patients given a tissue diagnosis of Chbl. Pathology of available tumors and patient records were reviewed. Results: 25 patients diagnosed with Chbl from years 1979 to 2007 were identified (1.5% of patients with primary bone tumors at MSHMC). Patients (12 male, 13 female) ranged in age from 3 months to 58 years (average 17 years). Common sites of involvement were femur (12), humerus (7); other sites (8). Preoperative imaging studies were available in 21 patients; 14 of these studies suggested Chbl. Three studies suggested lytic lesions or other non-specific diagnoses. Diagnosis of Chbl was made by fine needle aspiration in 4 cases; frozen section diagnosis of Chbl was rendered in 13 cases prior to curettage or biopsy. With rare exceptions, the histologic features were characteristic and pathognomic of Chbl. All patients were initially treated by curettage. Followup was available in 19 patients, ranging from 1 month to 25 years (average 50 months). All were alive with either stable localized disease or with no evidence of disease. Five patients had local recurrences, 2 of these had lung metastases; both are alive without disease after 10 years. In conclusion, the frequency of presentation, radiographic features, pathologic features, and clinical followup of patients with Chbl seen at MSHMC is similar to that reported in the literature. Chbl is readily diagnosed using a multidisciplinary approach. The behavior of these tumors is typically indolent, even in patients with atypical locations, multiple recurrences, and metastases.
[28] Bizarre Parosteal Osteochondomatous Proliferation with Translocation t(1;17)(q32;q21). Supriya Kuruvilla, Rex Marco, A. Kevin Raymond, and Nina Tatevian, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX, and University of Texas MD Anderson Cancer Research Center, Houston, TX.
Parosteal reactive lesions can be diagnostically challenging in all age groups, but especially in the children, where the implications of an erroneous diagnosis of malignancy can be potentially devastating. These lesions are often clinically ambiguous, with rapid but circumscribed, non-infiltrative growth patterns, and histological atypia; without overt features of malignancy. There is a history of trauma in a significant subset of cases. Histologically, this ambiguity often remains unresolved, not least due to the pathologist’s inexperience with this rare lesion. These lesions are composed of a disorganized mass of bone, cartilage, and fibrous tissue which may have significant histological atypia. The differential diagnosis includes benign and malignant (ie, osteoasarcoma) entities, and therefore has profound clinical impact. The infrequency of these lesions in the general surgical pathology practice makes ancillary studies such as molecular and cytogenetic aids particularly valuable. To illustrate the utility of these tools, we present a case of bizarre parosteal osteochondromatous proliferation, (ie, so-called Nora’s Lesion) arising in the soft tissues overlying the right distal ulna of an 8-year-old female. The current tumor has a balanced translocation that has been previously described in this lesion [ie, t(1;17)(q32;q21)]. This finding of a reproducible cytogenetic abnormality provides confirmatory diagnostic evidence. It also brings to question the appropriate classification of this group of lesions. They are currently viewed as a "tumor-like lesion," but the cytogenetic data suggest that Nora’s lesion might better be viewed as a benign neoplasm. Thus, cytogenetics can provide an invaluable element to the diagnosis and treatment of such lesions.
[29] Goblet Cell Carcinoid of the Appendix. Sonal Kamat, Peter Farmer, and Jela Bandovic, North Shore University Hospital, Manhasset, NY.
Goblet cell carcinoid (GCC) of the appendix is a rare neoplasm that shares histological features of both adenocarcinoma and carcinoid tumor. While its malignant potential remains unclear, GCC’s are more aggressive than conventional carcinoids. We present a case of a 68-year-old female who presented with acute appendicitis and the goblet cell carcinoid was an incidental finding on histopathological examination of the appendix. A 7.0 x 1.5 cm tissue specimen from the appendectomy was received. The appendix was perforated. The serosa was covered by a grey white fibrinous exudate. No grossly identifiable solid lesion was noted. Histopathological examination showed acute perforative appendicitis. Also seen were small uniform nests and clusters of signet ring cells, predominantly submucosal in location and infiltrating the muscularis propria with focal extension to the subserosal layer. These were located close to the site of perforation of the appendix. The tumor diameter was less than 2 cm. Proximal and serosal resection margins were free of tumor. Immunohistochemical studies showed the neoplastic cells positive for synaptophysin and focally positive for cytokeratin 20, cytokeratin 7, carcinoembryonic antigen, and mucicarmine. These findings were supportive for goblet cell carcinoid. In conclusion, the behavior of the GCC is more aggressive than that of the classic carcinoid, particularly if the tumor has transmural involvement, extends into the cecum, is larger than 2 cm, and shows the presence of signet ring cells. Metastasis has been documented particularly to the ovary. Because of this, a right hemicolectomy is recommended for this tumor type especially if it has spread beyond the appendix and if the tumor diameter exceeds 2 cm.
[30] Immune Complex Glomerulonephritis in Wilms Tumor. Ewa Elenberg, Baylor College of Medicine, Houston, TX.
The risk of end stage renal disease (ESRD) is very low in patients (Pts) with unilateral Wilms tumor, although Pts with WAGR or associated genitourinary anomalies are at higher risk of late occurring ESRD. Nephropathy is attributed to hyperfiltration in the remnant kidney. Immune complex glomerulonephritis (ICG) has not been previously reported in Pts with Wilms tumor. Objective: To report 2 cases of ICG in Pts with unilateral Wilms tumor. Methods: Retrospective chart review. Pt #1: a boy with cryptorchidism, diagnosed with unilateral Wilms tumor at age 3 yr. Pt #2: a girl with WAGR and unilateral Wilms tumor diagnosed at age 2 yr. Both were treated by tumor resection and chemotherapy. Results: Pt #1: At 2 mo post-surgery, proteinuria (3.8 g/24 hr) and elevated serum creatinine level were noted. Renal biopsy was revealed ICG with subendothelial deposits with infrequent mesangial deposits and full-house immunofluorescence. At 5 mo post-surgery, the Pt developed ESRD requiring hemodialysis. Pt #2: At 4 yr post-surgery, the Pt developed proteinuria (2 g/24 hr). The renal biopsy revealed changes typical of ICG. Therapy with enalapril and prednisone was started. Due to no response, prednisone was replaced with mycophenolate mofetil (MMF). After 4 mo, proteinuria improved to 0.5 g/24 hr; renal function continued to be normal. Conclusions: This is the first report of ICG in Pts with unilateral Wilms tumor with rapid progression to ESRD in the first Pt, but successful response to MMF in the second Pt. Despite low risk for progressive proteinuria in Wilms tumor Pts, it is prudent to monitor for proteinuria and to perform a renal biopsy if proteinuria is progressive. MMF therapy may be attempted to decrease proteinuria and to delay the onset of ESRD.
[31] Clonality of Tumor-Infiltrating Lymphocytes in Testicular Seminoma. Kyle Kurek, Evgeny Yakirevich, Alvaro Laga, and Murray Resnick. Rhode Island Hospital and Brown University Medical School, Providence, RI.
Testicular seminoma is characterized by a prominent lymphoid infiltrate. The goal of this study was to evaluate whether a tumor-specific response is detectable in seminoma by analysis of T-cell clonality and to assess the impact of FoxP3+ regulatory T-cells on this response. Clonality was assessed in 12 seminoma cases, including 2 tumors with corresponding lymph node metastases. Laser capture microdissection (LCM) was performed on CD3 stained tumor sections. CD3+ lymphocytes were captured separately from tumor cell nests and from surrounding peritumoral stromal aggregates. T-cell clonality was assessed by PCR analysis of TCR 
gene rearrangements followed by microcapillary electrophoresis. FoxP3 was analyzed by immunohistochemistry on paraffinized sections. Analyses of whole tissue sections did not demonstrate clonal lymphocytic populations. However, LCM revealed oligoclonal (2–4 clones) TCR gene rearrangements in the majority (92%) of tumors and lymph nodes, both within tumor nests and in peritumoral areas. The majority (75%) of cases contained intratumoral clones that were distinct from those in peritumoral areas, indicative of clonal heterogeneity. However, at least one T-cell clone was identical between intratumoral and peritumoral lymphocytes. Two lymph nodes contained clones identical to those in the primary tumor. The number of FoxP3+ cells in the tumor cell nests was significantly higher than in the peritumoral areas. However, these cells represented only a minor proportion (14%) of the tumor-infiltrating lymphocytes in the majority of cases. This study demonstrates an oligoclonal expansion of T-lymphocytes despite the presence of regulatory T-cells. Heterogeneity of lymphocytic clones may reflect a variety of tumor antigens. Characterization and expansion of these clones may lead to the development of novel immunotherapies in the treatment of seminoma patients.
[32] PSVI, PSVII, and PSH Are Apoptosis Inducers in SKOV 3 Cells. Xue Xiao, Peng Bai, Hong-Xiang Ying, Jianguo Xiao, and He Wang, Sichuan University, Chengdu, China, and University of Texas Medical School–Houston, Houston, TX.
Rhizoma paridis is a traditional Chinese medicine that has been used to treat cancer in China for hundreds of years. It has been shown that Paris polyphylla Smith var stenophylla Franch has significant antitumor activity in vitro. In this study, three compounds of the herb were isolated, characterized, and their activities on ovarian SKOV3 carcinoma cell line and apoptosis were investigated. The compounds of the rhizome plant were isolated and identified with chromatographic and spectroscopic techniques. Flow cytometry, immunohistochemical staining and Western blotting were applied to characterize cell apoptosis and protein expression. Three active compounds, paris saponin VI (PSVI), paris saponin VII (PSVII), and paris saponin H (PSH) were isolated and characterized. All three compounds were disaccharide, trisaccharide, and tetrasaccharide glucosides of pennogenin. The LC50 of PSVI, PSVII, and PSH to SKOV3 were 12.38, 44.94, and 9.14 µmol/L respectively. The apoptosis rates of the SKOV3 were 2.0% (0 hr), 7.9% (24 hr), 72.9% (48 hr), and 97.9 (72 hr) with concentration of 10 µmol/L of compounds, while at the 24 hr point the rates were 7.9% (1 µmol/L ), 23.3% (5 µmol/L), 80.0% (10 µmol/L), and 94.5% (20 µmol/L). Expression of erk1/2, bcl-2, bad, and bax was decreased while cytochrome c, caspase-3 and caspase-9 in SKOV3 was increased statistically after 12 hr of application of the compounds. PSVI, PSVII, and PSH elicited release of cytochrome c and apoptosis-inducing factors in a dose- and time-dependent manner. In conclusion, isolated compounds of Rhizoma paridis, PSVI, PSVII, and PSH, elicit programmed cell death of SKOV3. The mitochondrial apoptotic pathway may be involved in the apoptosis induced by PSVI, PSVII, and PSH.
[33] Tobacco-Specific Carcinogen NNK Inhibits Cell Death and Promotes Cell Growth of Lung Epithelium. George G Chen, Ming-Yue Li, Johnson Yip, Michael Hsin, Tony SK Mok, Anthony PC Yim. Chinese University of Hong Kong, Shatin, Hong Kong, China.
4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the tobacco-specific nitrosamine, is a known carcinogen for the development of lung cancer. However, the molecular mechanism responsible for its action is still not clear. Extracellular signal-regulated kinase (ERK) and nuclear factor kappaB (NF-
B) have a role in the promotion of cell proliferation and growth. We thus explored whether and how NNK influenced ERK and NF-B and stimulated the growth of lung cancer cells. Results showed that NNK up-regulated NF-B expression and induced ERK activation. NNK translocated p65 into the nucleus from the cytoplasm and enhanced the NF-kB DNA binding activity. Both U0126 (a specific inhibitor of ERK) and SN50 (a specific inhibitor of NF-B) blocked most of NF-B transcriptional activity in the cells treated by NNK. Unlike U0126, NNK-mediated increased ERK activation was not affected by SN50. ERK activation appeared to have a role upstream of NF-B in the cells treated by NNK. The levels of HO-1, p21, and c-IAP-2 as well as Bcl-2 were up-regulated by NNK in a time-dependent manner. In contrast, the expression of Bad was down-regulated. Accompanied by the above molecular changes, the proliferation of the lung cells was significantly increased by NNK. Inhibition of ERK and NF-kB activation led to the suppression of NNK-mediated expression of these proteins, indicating that NNK may regulate cell proliferation through an ERK- and NF-B-dependent pathway. In conclusion, the activation of ERK and NF-B is involved in NNK-mediated changes in HO-1, p21, c-IAP-2, Bcl-2, and Bad, which favor proliferation and growth of lung cancer cells.
[34] Contributions of Laboratory Automation to Food Safety. W. Jeffrey Hurst Sr., The Hershey Company Technical Center; Hershey, PA, and Department of Comparative Medicine, M. S. Hershey Medical Center, Hershey, PA.
Recently the general news and scientific press has focused on the topic of food safety. In January 2007, the New York Times published an article entitled "When Bad Things Come from Good Food" highlighting incidents with fresh produce. While time does not permit a discussion of overall food safety, this contribution will describe selected topic areas including but not limited to the detection of food allergens, pesticides, and pathogens, and the uses of automation-based technology to help solve analytical issues. Furthermore, it will describe how technology, ranging from biosensors and spectroscopy to direct mass spectrometry and microarrays, can be used to address current and potential future analytical challenges in the food safety arena.
[35a] Acetaminophen Treatment Confers Cardioprotection and Increased Longevity in Aging Male F344XBN Rats. Ernest M Walker Jr., Devashish Desai, Kevin Rice, Lucy Dornon, Sylvestre Cansino, Mark Studeny, John McGinty IV, and Eric Blough, Marshall University, Huntington WV.
Fischer 344 X Brown Norway rats demonstrate progressive age-associated changes in cardiac structure and function. Mechanisms may involve damage to cardiomyocytes by reactive oxygen species (ROS, oxidative stress) and age-associated cardiac remodeling. Acetaminophen reduces ROS content of various tissues, which may help explain its cardioprotective effects. Acetaminophen was administered orally in drinking water (30 mg/kg/day) to male Fischer 344/Brown Norway rats starting at age 27 months and continued for the lifetimes of the rats. Cardiac structural and functional changes were monitored by periodic echocardiographic (ECHO) and electrocardiographic (ECG) testing. Slide-mounted control and treated heart samples were stained and evaluated microscopically. A number of untreated and treated rats were utilized for survival observations. Untreated rats exhibited average survival times of 34–35 months compared to 40+ months for acetaminophen treated. Treatment significantly (p <0.05) (a) decreased the incidence of cardiac arrhythmias (PACs and PVCs), (b) reduced the incidences of mitral, tricuspid, and aortic valve regurgitations, and (c) prevented or greatly reduced cardiac hypertrophy. Sections of control rat hearts revealed changes indicative of ischemic damage including changes simulating myocardial infarctions in humans; these changes were not seen in treated hearts. In conclusion, oral acetaminophen treatment of male Fischer 344/Brown Norway F1 rats significantly (a) increased longevity, (b) provided appreciable cardioprotection as judged from significant decreases in the incidence of cardiac arrhythmias, valvular regurgitation, or incompetence, and decreased hypertrophy, (c) decreased ROS in aging rat hearts, and (d) provided cardioprotection against age-associated ischemic changes and changes characteristic for myocardial infarctions. (Supported by McNeil Pharmaceuticals Grant 991-546-518 to EMW and EB.)
[35b] Chronic Acetaminophen Treatment Decreases Cardiac Oxidative Stress in Aging F344XBN Rats. Devashish Desai, Sarath Meduru, Ernest M Walker Jr., Deborah Preston, Kevin Rice, Paulette Wehner, and Eric Blough, Marshall University, Huntington WV.
Cardiovascular disease remains the foremost cause of death and disability in the rapidly increasing aged population. Age-associated elevation of reactive oxygen species (ROS) levels is strongly correlated with cardiovascular disease. Recent studies have suggested that acetaminophen possesses antioxidant properties and may serve as a cardioprotective agent. We examined whether chronic treatment with a therapeutic dose of acetaminophen decreases age-associated myocardial oxidative stress. Aging male Fischer 344/NNiaHSd X Brown Norway/BiNia (FBN) rats (27month old; n = 8) were treated with acetaminophen (30 mg/kg/day; orally in drinking water) for 6 months. Age-matched control rats (n = 8) did not receive any drug treatment. Immunohistochemical and immunoblot analyses for markers of oxidative stress were employed to assess effects of chronic acetaminophen treatment on myocardial ROS accumulation and signaling. TUNEL assay was employed to examine the extent of myocardial apoptosis. OxyBlot analysis indicated that protein oxidation in acetaminophen treated hearts was 79.7 ± 4.65% of controls (p <0.001). Immunohistochemical analyses showed that protein tyrosine nitration and hydroethidium staining were lower in the treated hearts, compared to controls. The percentage of TUNEL-positive nuclei in treated hearts was 81 ± 1.4 % of control (p <0.05) in 33 mo treated hearts. Immunoblot analyses showed that expression of phospho-AMPK and phospho-JNK was significantly lower in treated hearts, compared to controls. These findings suggest that chronic acetaminophen treatment may decrease aging associated myocardial oxidative stress and apoptosis. Decrease in ROS accumulation in the myocardium may be the mechanism of this protective effect. (Supported by McNeil Pharmaceuticals Grant 991-546-518.)
[35c] Chronic Acetaminophen Treatment Influences Indices of Oxidative Stress in Aorta of Aging F344XBN Rats. Sarath Meduru, Devashish Desai, Kevin Rice, Sunil Kakarla, Ernest M Walker Jr, and Eric Blough, Marshall University, Huntington WV.
Cardiovascular disease (CVD) is the leading cause of mortality in aged individuals. Age associated CVD is thought to be caused in part by gradual oxidative damage to biomolecules. We previously reported that aging in the Fisher 344 X Brown Norway (FBN) rat aorta is characterized by increased levels of ROS and alterations in cell signaling. Acetaminophen was found to scavenge free radicals in ischemia-reperfusion studies. It remains unknown if chronic acetaminophen administration influences ROS accumulation and signaling in the aging aorta. We examined if chronic treatment with a therapeutic dose of acetaminophen attenuates age-associated increase in aortic ROS accumulation and signaling. FBN rats (27 month old; n = 8) were subjected to 6 months of treatment with a therapeutic dose of acetaminophen (30 mg/kg/day); age-matched untreated FBN rats served as controls (n = 8). Immunoblotting and immunohistochemical analysis were used to examine protein oxidation, protein nitration, and various indices of oxidative stress in the aorta. Protein oxidation and hydroethidine staining levels were lower in treated aortas, compared to controls. Immunoblotting analysis revealed that activated levels of cJun-N-terminal kinase (JNK) were 74.73 ± 3.15% and 87.39 ± 3.14%, respectively, in control and treated aortae. Phospho-Erk1/2 levels were 78.7 ± 0.49%, 99.45 ± 1.85%, activated p38-MAPK levels were 65.03 ± 1.65%, 67.65 ± 1.96%, and activated AMPK levels were 88.62 ± 2.26%, 106.86 ± 3.89%, respectively, in aortas from control and treated animals. These data suggest that chronic acetaminophen treatment alters age associated ROS signaling in FBN rat aorta. (Supported by McNeil Pharmaceuticals Grant 991-546-518.)
[36] Mycophenolic Acid Assay by Tandem Mass Spectrometry: Analytical and Clinical Experience for Immunosuppression in Organ Transplantation. Christine N. Papadea, Nicole Weimert, and Walt Uber, Medical University of South Carolina, Charleston, SC.
The aim of therapeutic monitoring of mycophenolic acid (MPA) is to maintain adequate immunosuppression and to minimize the risk of toxicity and graft rejection. Most clinical assays for MPA in the USA are performed by high performance liquid chromatography (LC) with UV detection. Combining LC with tandem mass spectrometric detection (LCMSMS) confers high sensitivity and specificity to the analysis. Our MPA assay by LCMSMS and its application in clinical practice at our institution are described. Method: MPA and its inactive glucuronide metabolite (MPAG) are extracted from plasma (75 µl) after the addition of ZnSO4 and the internal standard carboxybutoxy ether (MPAC) in acetonitrile. After an 8-min elution from a dC18 reversed phase column using an LC gradient constructed with two solvents (methanolic or aqueous, each containing 2 mM ammonium acetate and 0.1% formic acid), the three well-separated compounds are detected by triple quadrupole mass spectrometry operated with electrospray positive ionization in multiple reaction mode. Precursor ions MPA m/z 321, MPAG m/z 514, and MPAC m/z 421 are monitored. Seven MPA standards (0 to 25 mcg/mL) in plasma are used to calibrate each run, which includes three controls. Quantification by integrated peak area ratio (MPA/MPAC) is calculated with software. Results: Between-day (n = 94) reproducibility: CV <5% across the linear range, 0.7–25 µg/ml. Accuracy evaluation (38 samples) of our assay (y) agreed well with results obtained by a reference laboratory (x) using LCMSMS as shown by regression analysis: y =1.05 –0.032x, r2 = 0.97. Discussion: The assay is reproducible and accurate. In 11 months, ~400 samples have been tested for MPA to assist clinical management of patients. Therapeutic trough levels (1.3–4 µg/ml) depend on concomitant immunosuppression. A case report illustrating the clinical value of MPA analysis will be presented. Our next goal is to quantify MPAG along with MPA.
[37] Role of Ascorbic Acid in the Toxicity of Metals. Kazimierz S. Kasprzak, Konstantin Salnikow, and Monika Kaczmarek, National Cancer Institute at Frederick, Frederick, MD.
The aim of this investigation is to test the hypothesis that destruction of cellular ascorbate is the critical step in the mechanisms of toxicity and carcinogenesis induced by heavy metals such as nickel, chromium, cobalt, and possibly others. It has been known for centuries that exposure of humans to heavy metals may result in a variety of acute or chronic toxic effects, including cancer. The molecular mechanisms of these effects include structural and functional damage inflicted on target biomolecules directly by metal ions, due to non-specific binding, and/or indirectly by reactive intermediates, eg, oxygen radicals, of metal-mediated redox reactions. Our recent observations indicate that owing to the latter, ascorbate, the abundant natural antioxidant, is quickly depleted in cultured cells and animal tissues exposed to Ni(II) or Cr(VI) ions. This leads to inhibition of cellular proline hydroxylases, enzymes whose function depends on non-heme Fe(II), 2-oxoglutarate, and ascorbate. Among other functions, proline hydroxylation is crucial for activation of hypoxia-inducible factor HIF-1a, and proper assembly of collagen, extracellular matrix, surfactants, and complement. Thus, exposure to Ni(II), Cr(VI), or Co(II) results in activation of HIF-dependent expression of genes typical for hypoxia (eg, erythropoietin) in Ni(II)-treated animals, and in causing lung diseases like asthma and acute respiratory distress syndrome in nickel refinery workers. Most importantly, our experiments also indicate that the adverse effects of metals on proline hydroxylation and the resulting pathologies can be prevented by ascorbate supplementation. This has been observed in both cultured cells and gulonolactone oxidase knock-out mice, which, like humans, do not synthesize ascorbate. These results suggest a possibility of using vitamin C in prevention of occupational lung diseases, including cancer, in metallurgy workers.
[38] Diagnostic Laboratory Tests for Paroxysmal Nocturnal Hemoglobinuria (PNH) Revisited. Jonathan S Krauss, Medical College of Georgia, Augusta, GA.
The diagnostic laboratory approach to PNH was recently reviewed (
Ann Clin Lab Sci 2003;33:401–406
[39] Therapy-Related Acute Myeloid Leukemia with t(15:17) Translocation. John Lazarchick, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC.
The occurrence of therapy-related acute myelogenous leukemia with t(15:17)(q22;q12) translocation following cytotoxic chemotherapy is a rare event. We present the case of a 63-year-old male with a history of fibrohistiocytoma diagnosed initially in 1975 with metastatic recurrence in 2000. He received both radiation and chemotherapy over the course and was disease-free when seen in 2006 complaining of weakness and easy bruising. A CBC then revealed a hemoglobin of 11 g/dl, WBC of 1 k/µl, and a platelet count of 19 k/µl. His marrow aspirate showed an increase in promyelocytes with Auer rods present; his biopsy was only 20% cellular and markedly left-shifted. The cells were CD13, CD33, and CD117 positive. Both FISH and cytogenetic studies revealed a t(15:17) translocation consistent with a diagnosis of acute promelocytic leukemia. He responded to all trans-retinoic acid/idarubicin therapy and he is in complete remission. Although the efficacy of current cytotoxic agents to treat malignancies has improved, the incidence of therapy-related myelodysplastic syndrome/acute leukemia is increasing. The overall incidence, pathophysiology, and prognosis of this complication will be reviewed.
[40] CD52 Antigen May Be a Therapeutic Target for Eosinophilic Rhinosinusitis. Ping L. Zhang, Jared R. Pennington, Jeffery W. Prichard, Thomas M. Blasick, Adam M. Brown, and Santosh Potdar, Geisinger Medical Center, Danville, PA.
Allergic rhinosinusitis involves several types of inflammatory cells. The dominant inflammatory cells include mast cells, eosinophils, lymphocytes, and monocytes/macrophages. Since eosinophils are one type of inflammatory cells related to allergy, we investigated whether eosinophils present in rhinosinusitis were potential targets for CD52 antibody treatment in this study. First, we found that circulating eosinophils in renal recipients were almost completely depleted after an intravenous bolus of treatment with Campath-1H, a humanized antibody against CD52 antigen. Second, we constructed a tissue microarray block using archival tissue blocks containing sinus contents for immunohistochemical staining. Morphologically, eosinophils, lymphocytes, and monocytes were stained positively for CD52. Analysis with the ChromaVision system showed that rhinosinusitis with prominent eosinophils (eosinophilic rhinosinusitis, n = 15) had significantly higher myeloperoxidase staining scores (475.2 ± 109.7 arbitrary units) than rhinosinusitis with lymphocytes as a dominant component (lymphocytic rhinosinusitis, n = 15) 141.7 ± 38.3* arbitrary units; (*p<0.05, statistically significant by unpaired t-test to compare the two groups). The finding supports that there were more eosinophils in eosinophilic rhinosinusitis than in lymphocytic rhinosinusitis. In addition, eosinophilic rhinosinusitis had significant higher scores of CD52 staining (3639.1 ± 565.2 arbitrary units) vs lymphocytic rhinosinusitis (1806.3 ± 364.0* arbitrary units), whereas marker staining scores for master cells (CD117), B lymphocytes (CD20), T lymphocytes (CD4 and CD8), and monocytes (CD68) were similar between the two groups. The different levels of CD52 expression between the two groups further support that eosinophils expressed CD52 antigen. This study indirectly indicates that CD52 might be used as a target for treating rhinosinusitis, especially the eosinophilic type.
[41] Heparin-Induced Thrombocytopenia (HIT); a Devastating Problem and Diagnostic Dilemma. Atheer Ahmed and Hamid Al-Mondhiry, Penn State University, Hershey Medical Center, Hershey, PA.
The purpose of this case report is to recognize heparin therapy complications and to interpret laboratory testing in the context of clinical findings of venous thromboembolism. A 60-year-old man presented with septic arthritis of the knee. He was managed with antibiotics and unfractionated heparin for venous thrombosis prophylaxis. Later, he developed lower extremity deep venous thrombosis and was treated with IV heparin and inferior vena cava (IVC) filter placement. One day later, the patient developed sudden onset dyspnea with pre-syncope. A V/Q scan indicated high probability of bilateral pulmonary emboli and he was transferred to our medical center for management. Upon arrival, his clinical condition was stable with the exception of mild dyspnea. Echocardiogram showed thrombi in the heart chambers with a pressure-formed opening. Review of the medical record revealed a significant decrease in platelet count following heparin administration. At this point, the diagnosis of HIT was suspected. The patient underwent emergency embolectomy; the IVC filter was found in his right ventricle. After the patient received tirofiban, cardiopulmonary bypass (CPB) was performed with heparin used in the CPB circuit. Post-operatively, argatroban was started. HIT antibody testing was performed and was negative. This made HIT diagnosis unlikely and dalteparin was started. Later, as the platelet count declined again, HIT antibody testing was repeated and the results were positive. Argatroban was restarted and the platelet count became normalized. HIT is a condition characterized by a decrease in platelet count and high thrombogenic state following heparin administration, with a frequency of 1–5%. The diagnosis of HIT relies on clinical suspicion, confirmed by lab testing. The latter includes immunoassay and functional assays. Immunoassays have high sensitivity with low specificity, while functional assays carry high specificity, but low sensitivity.
[42] Utilization of Therapeutic Plasma Exchange (TPE) in the Treatment of Two Unusual Neurologic Diseases. Fouad Boctor, Ion Bucaloiu, Adel Makary, Scott Friedenberg, Daniel Leberfinger, Myra L Wilkerson, and William Jeffreys, Geisinger Medical Center, Danville, PA.
Therapeutic plasma exchange (TPE) is used for treatment of a wide range of antibody-mediated diseases. In the present report we describe two unusual neurological cases that were treated with (TPE). Case 1: A 46-year-old male who has had intermittent problems with muscle twitching, muscle spasm, and pain that occurred all over his body for the last 18 years. The patient had several EMG’s and was diagnosed as having Isaac’s syndrome. His family history was positive for neurological disorders including multiple sclerosis, myasthenia gravis, and tremors. The patient received 58 TPE procedures, averaging one every 9 weeks. The patient’s pain and muscle cramps usually improve after TPE. Isaac’s syndrome is a rare acquired neurological disorder characterized by intermittent or continuous muscle contraction. Autoimmune mechanisms, eg, production of an autoantibody against potassium channels, have been described. Case 2: A 66-year-old female complained of abdominal, neck, back, and shoulder pain for 3 weeks. Neurophysiological studies were consistent with an acute sensorimotor polyneuropathy having both axonal and demyelinating disease consistent with Guillain-Barré syndrome (GBS). The patient’s CBC showed Hb 12.8 gm/dl, WBC 8.6 x 103/µl with 48% lymphocytes. The lymphocytes were small to medium size, with round nuclei characterized by mature nuclear chromatin. The phenotyping showed mature monotypic B lymphocytes, which were brightly positive for CD 19, CD20, surface immunoglobin lambda light chain, FMC-7, CD45, CD25, CD11c, and CD103, and negative for T cell markers. This phenotype was consistent with hairy cell leukemia (HCL). The patient was treated with daily TPE for seven days. By the third TPE, her symptoms improved and she was discharged on the seventh day. Neurological complications have been reported in less than 5% of patients with HCL.
[43] The Chemistry of Chocolate. Donald J. Cannon, University of Tampa, Tampa, FL.
Chocolate has been consumed for centuries. It was part of Mayan culture, revered by early Mexicans, and has been cultivated throughout the planet Today this product of the cacao bean is touted as beneficial for one’s health. From benefits that reduce oxidative stress, elevate HDL cholesterol, and reduce blood pressure, to mood elevation and stimulation of sex drive, the consumption of chocolate is on the rise. No longer solely a contributor to weight gain, generator of pimples, or source of tooth decay, research on chocolate consumption is receiving a thumbs up. Considering that chocolate sales in the USA exceed $16 billion a year and it is estimated that Americans consume 12 pounds a year of the light, dark, and white products of this bean, recent beneficial claims should positively affect chocolate consumption. The story behind the claims lies with the amazing blend of over 300 chemicals in chocolate. Led by caffeine, theobromine, phenylethylamine, anandamide, and many more flavonoids, xanthine alkaloids, and endogenous neurotransmitters, the pleasure and addiction that this product generates is chemical. In addition further insight into the chemistry of pleasure and addiction can be explained to the benefit of all. From three distinct types af cocoa beans, a multibillion dollar industry has been created employing thousands and seducing grateful consumers. Chemistry joins the history, economics, and politics of chocolate cultivation, preparation, trade, and consumption.
[44] Biochemical Markers of Bone Turnover in Metastatic Bone Disease. Laurence M. Demers, Penn State University, Milton S. Hershey Medical Center, Hershey, PA.
Pathologic bone resorption is associated with a broad range of human disease conditions including osteoporosis, rheumatoid arthritis, hypercalcemia of malignancy, and tumor metastasis to bone. In malignancy, many common solid tumors like breast and prostate cancer have a high rate of metastases to bone and are the cause of considerable morbidity, vertebral fractures, and bone pain in the cancer patient. The process of bone resorption is mediated by osteoclasts in the microenvironment of bone that elaborate enzymes and factors that promote the resorptive processes on the bone surface. The process of osteoclastogenesis that is regulated by the RANKL/RANK/OPG axis controls the number of osteoclasts available for bone resorption. RANKL is a member of the TNF superfamily that binds to its cognate receptor RANK expressed on osteoclast progenitor cells as well as mature osteoclasts. In the presence of tumor cells in the bone microenvironment, cellular cross talk and the exchange of growth and other stimulating factors between the osteoclast and tumor cell occurs, leading to enhanced bone resorption and an increase in the osteolytic process that leads to demineralised bone and an increase in bone pain and vertebral fractures. Over the past several years, potent anti-resorptive therapies such as the bisphosphonates have been used to suppress bone resorption in cancer patients with bone metastases, reducing bone pain and the incidence of vertebral fractures. Biochemical markers of bone turnover such as the type 1 collagen telopeptides and bone specific alkaline phosphatase have been used to guide anti-resorptive therapy, improve therapy compliance, and select the proper dose of therapy in patients with osteolytic cancer metastases.
[45] Development of Monitoring and Treatment Strategies in a Model of Glutaric Acidemia. William J. Zinnanti, Jelena Lazovic, Russell E. Jocobs, and Keith C. Cheng, Pennsylvania State University College of Medicine, Hershey, PA, and California Institute of Technology, Pasadena, CA.
Using a mouse model, we have developed new monitoring and treatment strategies for the neurometabolic disorder known as glutaric acidemia type I (GA-1). GA-1 is an inherited metabolic disorder of lysine and tryptophan breakdown that presents with striatal lesions anatomically and symptomatically similar to Huntington’s disease. Affected children commonly suffer acute brain injury during a catabolic state associated with non-specific illness. The mechanisms underlying brain injury and age-dependent susceptibility have been unknown and the lack of a diagnostic marker heralding brain injury has impeded intervention efforts. Here we show in a mouse model that pathologic events begin in the neuronal compartment, as enhanced lysine accumulation in the immature brain allows increased glutaric acid production resulting in age-dependent
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