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Abstracts of Presentations at the Association of Clinical Scientists’ 128th Meeting at Los Angeles, California, on 14 to 18 May 2008

This talk will review the progress that has been made in kidney transplantation over the past few decades and that has resulted in quite exceptional outcomes. It will also review the more recently introduced immunosuppressive agents, the way in which the allocation system has changed, and the new proposals to modify it yet further. Strategies to increase the number of living-donor transplants for those who are immunologically incompatible, or ABO blood type incompatible are altering this area as well. Some troubling complications of renal transplantation will also be discussed.

[2] Advances in Liver Transplantation. R. Mark Ghobrial, Liver Center, Methodist Hospital, Houston, TX.

Orthotopic liver transplantation (OLT) has become the treatment of choice for a wide variety of irreversible liver diseases. Refinements in organ preservation, more effective and safer immunosuppressive therapy, advances in patient care, and surgical techniques have contributed to the dramatic growth of liver transplantation over the past decade. At present, more than 6000 transplants in over 100 centers are performed every year in the United States, while more than 18,000 candidates await transplantation. The exponential increase of liver transplant candidates has vastly outgrown the number of livers available for transplantation from deceased donors after brain death (DBD). Disparity between availability and demand of organs has resulted in longer waiting periods prior to transplantation. Moreover, an increasing number of patients markedly deteriorate while waiting, require multiple hospital admissions, and life support in intensive care units. Even more critical is the progressive increase in the number of deaths of transplant candidates on the waiting lists. Such rising demand for OLT has fueled the development of innovative surgical techniques that utilize partial donor grafts that include split liver transplantation, adult-to-pediatric, and adult-to-adult living-donor transplants. However, living-donor transplantation is not without risks to the donor. Concern for donor safety coupled with recent donor deaths have been the impetus for utilization of older donors, donors with extended criteria, and donors after cardiac death (DCD). The adoption of the model for end stage liver stage (MELD) scores for organ allocation has provided a unique opportunity for objective stratification of transplant candidates based on severity of illness. Once considered experimental, transplantation for hepatitis B virus (HBV) is associated with survival rates that exceed 90%. This is largely due to the development of effective anti-viral therapy. In contrast, OLT for hepatitis C virus (HCV) faces multiple challenges. HCV recurrence has been universally documented in allograft recipients with allograft failure in a small number of patients in the short term and allograft destruction in a larger number of patients in the long term. A pressing need has therefore developed to identify antiviral regimens to treat or prevent recurrent disease. Current approaches utilize interferon alone or in conjunction with ribavirin. Low efficacy and poor tolerability of current antiviral agents have provided the impetus for development of other therapeutic agents. Anti-hepatitis C immunoglobulin has stimulated a lot of interest. Advancess in molecular research have yielded promising new agents that target HCV RNA synthesis and protein translation. Such future agents under development include anti-sense oligo-nucleotides, helicase, and replicase inhibitors. The multi-disciplinary approach for treatment of hepatocellular carcinoma has vastly improved survival rates. Transplantation for early stages is currently accompanied with superior survival outcomes. Further, pretransplant downstaging with ablative therapy has allowed successful transplantation of patients with advanced local disease. In summary, the last decade has witnessed major advances in surgical techniques, expansion of the donor pool, and development of successful therapies for HBV and HCC. Future challenges include successful therapy to treat HCV, to combat ischemia-reperfusion injuries, and to expand the donor pool to meet the needs of transplant candidates.

[3] Pathology of Lung Allograft Rejection. Michael N. Koss, Keck School of Medicine of USC, Los Angeles, CA.

A variety of forms of pulmonary rejection can occur in the setting of lung transplantation, including acute and chronic rejection and antibody-mediated rejection. Allograft biopsies have an overall sensitivity for diagnosis of acute cellular rejection (ACR) of 68% and a specificity of 97%. When the biopsy has 5 fragments of alveolated tissue containing at least 100 alveoli and one bronchiole, the sensitivity for diagnosis of ACR climbs to 92%. The initial targets of acute rejection are vessels and airways, but emphasis in diagnosis is on the vasocentric pattern of lymphocytic infiltrates. The ISHLT system grades biopsies based on the extent of perivascular lymphoid infiltrates, from none (A0) to rare (A1), mild (A2), moderate with extension into the interstitium (A3), and severe (A4) with diffuse interstitial lymphoid infiltrates and acute alveolar damage. Similarly, so-called lymphocytic bronchitis may be an airway manifestation of rejection; it is graded on severity and destructiveness of the lymphoid infiltrate (B1–B4). Reproducibility for A grades among pathologists is better (kappa statistic = 0.65) than for B grades. Chronic rejection usually manifests as obliterative bronchiolitis (OB) and occurs in 60–80% of transplants after 5–10 yr. There is chronic inflammation and fibrous tissue obliterating the airway lumens. Transbronchial biopsy has a specificity of 56% and a sensitivity of only 20% in this diagnosis. Proposed risk factors for OB include numbers and severity of acute rejection episodes, history of cytomegalovirus pneumonitis, GERD, reperfusion injury, and lymphocytic bronchitis. These etiologies may share damage to bronchiolar epithelium with uncovering of hidden epitopes, leading to auto-immunity, but the pathogenesis of OB is still debated. Antibody-mediated rejection is manifested by capillaritis, hemorrhage, and diffuse alveolar damage. Such rejection is rare. There is scant experience with C4d staining in these cases.

[4] You’re Not My Type: Histocompatibility Barriers to Organ Transplantation. J. Michael Cecka, David Geffen School of Medicine at UCLA, Los Angeles, CA.

The highly polymorphic HLA antigens play key roles in controlling the immune response and are major barriers to the transplantation of tissues and organs between individuals. While improvements in immunosuppression permit effective control of most cellular rejection, patients who have become sensitized to allogeneic HLA antigens from pregnancies, prior transplants, blood transfusions, and other sources still represent an important challenge in transplantation. Sensitized patients represent 33% of the waiting list for kidney transplants. These patients wait significantly longer to receive a transplant and, when transplanted, they have significantly higher rates of delayed graft function, primary nonfunction, and chronic graft failure than nonsensitized patients. Strategies to avoid or overcome donor-specific HLA antibodies in sensitized patients by desensitization, broader organ sharing, or paired organ exchanges between incompatible living donor pairs have had limited but promising success. Still the presence of anti-HLA antibodies carries risks ranging from catastrophic hyperacute rejection to accelerated antibody mediated rejection to chronic graft dysfunction and failure. Recently introduced solid-phase technologies for identifying and measuring anti-HLA antibodies are rapidly changing the way transplant candidates are evaluated and will facilitate new approaches to this important remaining barrier to organ transplantation.

[5] Current and Future Immune Monitoring Tools for Improving Diagnosis of Transplant Rejection. Elaine F. Reed, UCLA School of Medicine, Los Angeles, CA.

The development of robust immune monitoring assays to quantitate and characterize anti-donor alloimmune responses may permit the early identification of patients at risk of rejection and graft loss, optimization of drug regimens, monitoring response to therapy following intervention, and may guide the development of new therapeutic immunosuppressive therapies. Immunologic monitoring could also differentiate rejection from other forms of graft dysfunction such as primary non-function and drug toxicity. The potential of this approach to reduce the costs associated with graft rejection while maximizing patient and graft survival is tremendous. With recent development in our understanding of transplantation biology and therapeutics, there is an expectation that these tests may be used to identify tolerance as much as to predict rejection. Developments in flow cytometry and ELISPOT have brought about new approaches to measure alloreactive T cells via the direct and indirect recognition pathways. The recently developed solid phase flow cytometry and luminex techniques using purified HLA antigens have become useful for post-transplant monitoring because they are more sensitive and can detect small amounts of antibody. With the advent of high throughput genomic and proteomic technologies, new biomarkers of transplant outcome are being rapidly discovered and will be ripe for translation to diagnostics in the near future. The assays that have been informative to date will be discussed. This information leads to a better understanding of the mechanisms underlying allograft rejection and tolerance. Although this presentation reviews in vitro assays in the context of transplantation, these techniques have widespread application to research and diagnostics in other areas of clinical immunology.

[6] Pathology of Late Graft Failure in Small Bowel Transplantation. Bita V. Naini, S. B. Gordon, and G. Cortina, David Geffen School of Medicine at UCLA, Los Angeles, CA.

Small-bowel-transplant patients experience high rates of graft failure. The histologic features of late graft failure are not well-defined; this study aims to characterize these features. We performed a retrospective study of small bowel recipients at UCLA (1994–2007) who had graft failure and underwent enterectomy. Twelve cases were identified; 4 lost the graft after 100 days (late graft failure) and 8 before 100 days. We performed a comprehensive histological examination evaluating all standard and identifiable pathological changes. The following findings were present in 50% of cases: mucosal erosion/ulceration, epithelial injury/repair, undulating/wavi-form architecture, architectural distortion, crypt hyperplasia, villus blunting, apoptosis in >6/10 crypts, mucosal ischemic necrosis, organizing serositis, and serosal fibrosis. The following findings were present in <50% of cases: mucosal and submucosal fibrosis, granulomatous inflammation, muscularis propria inflammation, fibrosis and myocyte dropout, suppurative serositis, obliterative arteriopathy, and intravenous thrombi. Mucosal ulceration and undulating architecture were present respectively in 75% and 50% of late-graft-failure cases compared to total cases. Epithelial injury/repair was seen in 50% of late-graft failures and 67% of all cases. Submucosal fibrosis was seen in 25% of late-graft failures and 17% of all cases. Muscularis propria fibrosis and myocyte dropout were seen in none of the late-graft failures and 33% of all cases. Organizing serositis was seen in 75% of both groups. Obliterative arteriopathy was seen in 25% of late-graft failures and 8% of total cases. The majority of histologic features of late-graft failure are non-specific. However, the features that are frequently seen include discrete mucosal ulceration with mucosa showing undulating architecture and evidence of injury/repair. An infrequent but characteristic feature of late-graft failure is obliterative arteriopathy of large arteries within the serosa.

[7] Development of Severe Aplastic Anemia Following Orthotopic Liver Transplantation. John Lazarchick, Ginell Post, Gil Cortes, and R. Pollack, Medical University of South Carolina, Charleston, SC.

Aplastic anemia is a rare complication following orthotopic liver transplantation. We present a case of a 61-yr-old woman with a diagnosis of autoimmune hepatitis with progression to cirrhosis who underwent two orthotopic liver transplants. Her CBC following the second procedure included a hemoglobin of 9.8 g/dl, WBC of 8.6x10⁹/L, and a platelet count of 105x10⁹/L. She was readmitted to the hospital 5 weeks later with a skin rash diagnosed as either GVHD or drug reaction. Her CBC at that time included a hemoglobin of 8g/dl, WBC of 0.14x10⁹/L, and a platelet count of 17x10⁹/L. A bone marrow study done at this time showed cellularity of <5% consistent with a diagnosis of aplastic anemia. Immunohistochemical staining of the skin and marrow biopsies showed a predominance of CD3/CD8 positive lymphocytes suggestive of GVHD. A liver biopsy done at the same time showed no evidence of GVHD. FISH analysis showed 2% of her cells to be of male (donor) origin. She had no evidence of HLA antibodies. The differential diagnosis of aplastic anemia after solid organ transplant will be discussed, including orthotopic liver-related GVHD, transfusion-associated GVHD, and drug-induced marrow aplasia.

[8] Role for Killer Immunoglobulin-like Receptors and/or their Ligands in Hematopoietic Stem Cell Transplantation (HCT). David Senitzer, J. Y. Sun, A. Dagis, L. Gaidulis, M. M. Miller, and S. J. Forman, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA.

Killer Ig-like receptors (KIRs) are expressed on human NK and T cell subsets. KIRs transmit either inhibitory or activating signals regulating cell activities. Some inhibitory KIRs specifically recognize HLA-A, B, or Cw allotypes on target cells as inhibitory KIR ligands (iKIRLs). Most activating KIR ligands have not been well defined. We have analyzed the known iKIRLs and KIR genotypes in 544 primary allogeneic HCTs with T-replete grafts from unrelated or HLA mismatched related donors. There were 159 transplants with class I antigen level mismatched donors; 79 cases were without also being iKIRL mismatched; 80 cases were mismatched for the iKIRLs. The latter group had inferior overall survival (HR: 1.79; p = 0.003), and event-free survival (HR: 1.86; p = 0.001). Both relapse (HR: 1.90; p = 0.03) and non-relapse mortality (HR: 1.83; p = 0.01) contributed to the inferior survival of the group that were mismatched for both Class I and iKIRLs. The detrimental effect was also evident when 8/8 antigen-matched transplants (385) were analyzed for patients lacking iKIRLs. Overall survival in patients (218) lacking the HLA-Cw Group-1 or 2 iKIRL was significantly lower than that in patients (167) having the iKIRLs (HR: 1.39; p = 0.02). Patients lacking iKIRLs had higher non-relapse mortality (HR: 1.50; p = 0.03). KIR genotype data are available for 360 cases. Our algorithm calculates a score based on the KIR genotype of the patient compared to the KIR genotype of the donor. In 360 cases the score was found to be predictive of the incidence of aGVHD. Multivariate analyses confirmed that both iKIRLs and KIR genotype scores were independent factors affecting HCT outcomes. In T-replete, unrelated HCT, iKIRL mismatches, as well as the absence of iKIRLs, confer higher risk of a poor outcome. The algorithm for KIR genotype scoring produces results that are predictive of the incidence of aGVHD.

[9] Morphoproteomic Characterization of Signal Transduction Pathways in Adult CD133+ Stem Cells Derived from Human Bone Marrow. Jitakshi De and Robert E. Brown, University of Texas Health Science Center-Medical School at Houston, Houston, TX.

Bone-marrow-derived stem cells have the potential for regenerative medicine with respect to liver and myocardial injury. However, there are also data to implicate bone-marrow-derived stem cells in the origin of epithelial cancers. This study was designed to characterize the signal transduction pathways in bone-marrow-derived stem cells in order to gain a better understanding of their biology. CD133+ cells, the putative primitive stem cells, were isolated from the peripheral blood of normal donors and from a culture of commercially obtained bone marrow-derived stem cells. These were isolated and enriched using magnetic bead purification methodology. CD133+ expression was confirmed by flow cytometric analysis of the enriched populations. Immunohistochemical procedures using regular antibody and phosphospecific probes were applied to cytospin preparations to detect the following protein analytes: CD133; phosphorylated (p)-Akt (Ser 2448); p-mammalian target of rapamycin (mTOR [Ser 2448]); p-p70S6K (Thr 389); p-extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204); and p-nuclear factor (NF)-kappaBp65 (Ser 536). Peripheral blood mononuclear cells (PBMCs) depleted of CD133+ cells were concurrently evaluated and served as a control. The chromogenic signal (staining intensity) and the cellular compartmentalization of the signal (plasmalemmal, cytoplasmic, and/or nuclear) were evaluated by bright-field microscopy and scored on a scale of 0–3+. CD133+ cells from bone marrow and peripheral blood showed moderate to strong nuclear and plasmalemmal reactivity for p-Akt, p-mTOR, p-p70S6K, p-ERK 1/2, and p-NF-kappaBp65, and evidence of intracellular trafficking (ie, cytoplasmic reactivity in some cells and combined with nuclear reactivity in others). By contrast, the overall staining pattern in PBMCs was either less in the case of p-Akt, p-mTOR, and p-p70S6K or confined to a specific cellular subtype such as lymphocytoid and monocytoid cells. In summary, morphoproteomic analysis identifies constitutive activation of the pathways of convergence in adult CD133+stem cells derived from human bone marrow to include the PI3'-K/Akt/mTOR, ras/Raf kinase/ERK, and NF-kappaB pathways. This will serve as a framework to design therapies that preserve the pathways in such cells being used in regenerative medicine and opportunities for therapeutic intervention against their role in tumorigenesis.

[10] Opportunities and Controversies in Molecular Genetic Testing. Wayne W. Grody, David Geffen School of Medicine at UCLA, Los Angeles, CA.

Under the impetus of the Human Genome Project, a rapidly growing number of genes and mutations are becoming targets for molecular testing in patients and whole populations affected with, or at risk for, genetic disorders. The rapid pace of gene discovery, and the ease with which modern molecular biologic technology enables translation of these findings into clinical tests, has placed unprecedented demands on both the clinical laboratory and the clinicians who must decide if and when a particular test is appropriate and informative. Given the relatively small size of the specialty of medical genetics and the limited number of trained genetic counselors in the country, much of this test ordering and interpretive burden must fall on primary care physicians, with clinical laboratory scientists serving as gatekeepers and resident experts at many institutions. The situation becomes more acute as we move into the era of mass population molecular genetic screening, as is now mandatory for cystic fibrosis carrier detection. The development and implementation of that program took 12 years, and numerous issues surrounding genotype-phenotype correlation and psychosocial acceptance continue to be debated. Similar challenges face the acceptance of other potential targets of population genetic screening, as well as pharmacogenetic testing, whole-genome scanning, high-throughput DNA sequencing, predictive genetic testing, early embryo diagnosis, and other revolutionary applications made possible by new technologies. This presentation will present a contemporary overview of these challenges and opportunities, highlighting key diseases and tests along with the thorny ethical dilemmas they raise.

[11] Engineering Nanoparticles to Deliver Therapeutics; Research in the California NANOSystems Institute. Leonard H. Rome, David Geffen School of Medicine at UCLA and California NANOSystems Institute, Los Angeles, CA.

The California NanoSystems Institute (CNSI) is a research center at UCLA whose mission is to encourage university collaboration with industry and to enable the rapid commercialization of discoveries in nanosystems. Established in 2000 through a State of California initiative to create 4 Institutes of Science and Innovation, the CNSI is forging partnerships with industry as a way to accelerate technological advances and fuel California industry. CNSI members represent an interdisciplinary collaboration among UCLA and UCSB faculty from the life and physical sciences, engineering, and medicine. One area of research in the CNSI is directed toward engineering of nanoparticles to deliver therapeutics. Vaults are novel nano-scale particles first described in 1986 and found to exist in thousands of copies in most eukaryotic cells. They have an intricate shape composed of multiple arches reminiscent of cathedral vaults, hence their name. Vault size (~74 x 42 x 42 nm), shape, molecular composition, and facile assembly suggest that they have the potential to be engineered to deliver a wide variety of therapeutics. The baculovirus expression system is currently being used to produce recombinant vaults in order to test the concept that vaults can have a broad nanosystems application as malleable nanocapsules. Toward this aim, the particles are currently being designed with encapsulated fluorescent probes and enzymatically active protein domains. In addition, a number of strategies are under development to encapsulate chemically active small molecules, drugs, and nucleic acids into the vault particle. If successful, these vault nanocapsules can be bioengineered to allow their use in a wide variety of biological applications including drug delivery, biological sensors, enzyme delivery, controlled release, and nano-electrical machine (NEMS) applications.

[12] Microbial and Molecular Biomarkers for Inflammatory Bowel Disease Biology and Management. Jonathan Braun, David Geffen School of Medicine at UCLA, Los Angeles, CA.

The intestinal microbial community is enormously abundant (100 trillion cells in the colon alone) and diverse (500–1000 species). Physiologically, the microbiota capture nutrients and defend against alien pathogenic microbiota. However, in inflammatory bowel disease (IBD), they are the target of an inappropriate immune response resulting in chronic intestinal damage. IBD involves deficits in mucosal barrier and innate immune effector functions that control local activity of the resident microbiota and immunoregulation that controls the enthusiasm of the immune response to these microbial residents. Highlighted among these host traits are: (a) the level of epithelial mucin production and the structural isoforms of its abundant o-glycans; (b) mucosal epithelial and dendritic cells, and their products for microbial sensing and local anti-microbial responses; and (c) the formation and activity of regulatory CD4 and CD8 T cells in the mucosal immune system, which control the formation of colitigenic TH1, TH2, or TH17 CD4 T cells. The clinical challenge is to create tools to identify host susceptibility traits and specific resident microbiota, so that definitive personalized therapies can be selected for each patient. Several analytic approaches are emerging. Genome-wide association studies have already identified >10 genetic loci affecting one or more of these host traits, yielding disease-specific SNPs to identify pertinent traits in at-risk individuals. Serologies for a panel of index microbiota are useful in diagnosis, predicting disease course, and uncovering linked genetic susceptibility loci. The mosaic of susceptibility traits is confirmed by the clinical benefit of recent therapies targeting TNF-alpha, alpha-4 integrin, IL12/23, and CD3. Inflammatory bowel disease is thus a powerful example that bridges combinatorial genetics, immune monitoring, and microbiology to understand and treat chronic inflammatory disease.

[13] Mitochondrial Biology and Disease. Joseph C. Parker Jr., University of Louisville School of Medicine, Louisville, KY.

Not only do mitochondria provide energy (ATP) for all eukaryotes and multi-cellular organisms, but they produce most intracellular oxidants as radical oxygen species (ROS) creating intrinsic damage with subsequent energy loss. Mitochondria are derived from an endosymbiotic relationship between a eukaryote and prokaryotes leading to flexible, mobile micro-organelles (mitochondria, mt) that easily move through the cytoplasm to high energy sites within the cell. In humans, aging is associated with declining oxidative phosphorylation within these mitochondria associated with accumations of ROS, producing deletions, point-mutations, and oxidation in mtDNA. Mitochondrial cytopathies can affect many organs including the brain, eyes, heart, liver, pancreas, gastrointestinal tract, and bone marrow. Laboratory recognition of these disorders can be achieved with biochemical evaluations and muscle biopsies that demonstrate these abnormal mitochondria by histochemical and enzyme stains and electron micropscopy. Through binary fission, mitochondria reproduce in a haphazard fashion not controlled by nuclear DNA (nDNA) and typical cellular reproduction.The mitochondrial DNA (mtDNA) codes for 13-proteins, 22-tRNA’s, and 2-rRNA’s dealing with respiratory chain enzymes excluding succinic dehydrogenase, which is encoded by nuclear DNA. Mitochondrial cytopathies can be inherited through nuclear DNA defects as autosomal recessive and autosomal dominant diseases as well as through mitochondrial DNA defects with maternal or sporadic patterns. Using clinical history and physical examination, mitochondrial diseases can be suspected in individuals with maternal inheritance and/or associated multi-organ deficiencies. A muscle biopsy can support this impression when abnormal mitochondria are recognized with histochemical and oxidizing enzyme stains and confirmed with electron microscopy. Abnormal blood and CSF lactate and pyruvate levels may develop. Treatment for mitochondrial disorders is mainly supportive, but potential mitochondrial gene therapies are being considered.

[14] Cancer Prevention: Role of Preservation of Mitochondrial Function, Egil Fosslien, University of Illinois College of Medicine, Chicago, IL.

The objective of this paper is to examine evidence that shows that reduction in exposure to mitochondrial toxins and support of mitochondrial function by nutritional supplements or therapeutic drugs can prevent cancer. Epidemiological, clinical, and in vitro studies showing detrimental effects of environmental toxins on mitochondrial function and reports that demonstrate beneficial effects of specific diets and dietary supplements were analyzed and evaluated. Findings: The rate of cancer cell glycolysis correlates inversely with lower mitochondrial bioenergetics, and primary dysfunction of the latter is associated with increased risk of neoplastic growth. Environmental toxins can degrade mitochondrial β-oxidation and oxidative phosphorylation (OXPHOS). As examples, phthalates–plasticizers widely used in food packaging, plastic bottles, cosmetics, floor tiles and other everyday products–can inhibit mitochondrial beta-oxidation, succinate dehydrogenase, cytochrome c reductase, and adenine nucleotide translocase, and decrease the rate of ATP synthesis. The carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (Dioxin, TCDD) promotes tumor growth: In mice, it lowers co-enzyme Q (CoQ) levels, reduces the mitochondrial inner membrane potential (m), reduces the synthesis of F₀F₁-ATP synthase, and lowers production of adenosine triphosphate (ATP). Sodium benzoate (E211), used to prevent mold growth in various soft drinks, can severely damage the mitochondrial genome. Gossypol in cottonseed oil, genistein in soybean oil, and erucic acid in rape seed oil have been shown to interfere with in vitro and in vivo mitochondrial energetics. Shift-work increases cancer risk; experimentally, melatonin has a concentration-dependent effect on the risk. In conclusion, many environmental toxins are associated with degradation of mitochondrial energetics and increased cancer rates. The risk of cancer may be reduced by avoiding or lowering exposure to environmental mitochondrial toxins and by supplementation with nutritional and medicinal agents that support mitochondrial energy metabolism.

[15] Morphoproteomic-Guided Modeling Incorporates Layers of Heterogeneous Knowledge and Converges on the NF-kappaB Pathway in Glioblastoma Multiforme. Robert E. Brown, Mary F. McGuire, Amy Law, and M. Sriram Iyengar, University of Texas Health Science Center at Houston, TX, and Geisinger Medical Center, Danville, PA.

Signaling pathways involved in tumorigenesis are influenced and defined by complex processes based on genomics, proteomics, and therapeutics that rely, at least in part, on morphologic structure and intracellular biochemical reactions. Understanding these complex interactions is facilitated by viewing them from the perspective of layers of heterogeneous knowledge. In this study, we applied such a layered modeling approach to identify a common mechanism for the tumorigenicity and chemo/radioresistance in glioblastoma multiforme (GBM) and to uncover novel therapeutic opportunities. This model is guided by morphoproteomics, which serves as a primary layer and incorporates genomic, preclinical, and therapeutic data that are comprised of the following: morphoproteomic data; genomic correlates; chemotherapeutic agents with or without cell cycle dependency and variable therapeutic efficacy in GBM; and drug-resistant molecules and mechanisms in GBM. Morphoproteomic data were obtained from immunohistochemical analysis on tissue microarrays in our laboratories and from a computer-assisted search of the US National Library of Medicine MEDLINE database. The layers of genomic, therapeutic and drug-resistant correlates were also assembled from MEDLINE, Ingenuity Pathway Analysis, and Gen Way Glioma Pathway.The morphoproteomics revealed constitutive activation of the PI3'-K/Akt/mammalian target of rapamycin and ras/Raf kinase/extracellular signal-regulated kinase (ERK) pathways with convergent and constitutive activation of the nuclear factor (NF)-kappaBp65 pathway in GBM. Knowledge contained in 134 related citations from the literature was classified into genomic, preclinical, and therapeutic layers. This analysis also demonstrated convergence on the NF-kappaB pathway. In conclusion, morphoproteomic-guided modeling together with layering of heterogeneous knowledge expedited the identification of the role of the NF-kappaB pathway in chemo/radioresistance in GBM and discloses novel opportunities for therapeutic intervention.

[16] Morphoproteomic Analysis on mTOR Pathway in Papillary Carcinoma of the Thyroid. Jing Liu and Robert E. Brown, University of Texas Health Science Center at Houston Medical School, Houston, TX.

Papillary carcinoma of the thyroid (PTC) is the most common malignant neoplasm of the thyroid. Tumorigenesis of this neoplasm is not quite understood. Morphoproteomics is a recently emerging method, which uses immunohistochemistry to identify the expression of signal transduction pathways and effectors and to determine their compartmentalization. We used this method to investigate the pathogenesis of PTC. In 20 cases of the conventional type of PTC, formalin-fixed, paraffin-embedded tissues were examined for the expression and localization of p-Akt(Ser 473), p-mTOR(Ser 2448), p-p70S6K(Thr 389), Skp2, and Ki-67. Both staining intensity (0 to 3+) and extensiveness (0 to 100%) were evaluated for the expression of these markers. The staining intensity was graded as weak(1+), moderate(2+), or strong(3+). Activation of mTOR was observed in all 20 cases with predominantly cytoplasmic and plasmalemmal staining (1+ to 3+) in 50% to 100% of tumor cells. p-Akt positivity was mainly cytoplasmic and nuclear, with an intensity between 1+ and 2+ and an extensiveness of 70 to 100%. p-p70S6K was positive (2+ to 3+) in >95% of tumor nuclei. Skp-2 and Ki-67 were expressed in <3% and <7% of tumor nuclei, respectively. The results indicate that there is constitutive activation of the mTOR pathway in PTC by evidence of moderate to strong expression of p-mTOR and p-p70S6K and some level of expression of p-Akt. Moreover, since the p-Akt expression intensity was in the weak to moderate range, other upstream pathways that activate mTOR may be operative. Paradoxically, this pathway is not resulting in cell cycle progression in PTC, as evidenced by the low Skp2 and Ki-67 expression rates in our study. Additional studies are required to clarify the role of the mTOR pathway in PTC.

[17] Morphoproteomic Evidence of Constitutively Activated Proinflammatory and Profibrogenic mTOR and NF-kappaB Pathways in Non-Alcoholic Steatohepatitis. Wei Feng, Wei Li, Zhenhong Qu, and Robert E. Brown, University of Texas Medical School, Houston, TX, and University of Rochester, Rochester, NY.

Nonalcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) with known complications of cirrhosis and hepatocellular carcinoma. The increasing incidence of NASH and the lack of effective treatment have prompted intensive studies on disease pathogenesis. Activation of both the mammalian target of rapamycin (mTOR) and the nuclear factor (NF)-B pathways is implicated in fibrogenic and inflammatory processes. However, the roles of mTOR and NF-B pathways in the development and progression of NASH are not well defined. Formalin-fixed, paraffin-embedded, tissue sections of 13 NASH cases (8 adult, 5 pediatric) were evaluated by H&E and trichrome stains for disease activity and fibrosis stage according to criteria set by NASH Clinical Research Network. Expression levels of -smooth muscle actin (SMA), mTOR pathway markers, phosphorylated (p)-mTOR(Ser 2448) and p-p70S6K(Thr 389), NF-B pathway markers, p-NF-Bp65(Ser 536), and interleukin (IL)-8 were evaluated by immunohistochemistry. SMA was used as a positive control stain for myofibroblastic cells. Positive staining was defined as distinct and appropriate pattern of cytoplasmic or nuclear staining. Intensity was graded as 1+ (weak), 2+ (moderate), or 3+ (strong). In all study cases, the expressions of cytoplasmic p-mTOR, nuclear p-p70S6K, nuclear p-NF-Bp65, and cytoplasmic IL-8 in inflammatory cells and myofibroblasts were significantly increased compared to other parenchymal cells. The levels of expression of p-mTOR, p-p70S6K, p-NF-Bp65, and IL-8 correlated positively with the degrees of inflammation and fibrosis (p <0.05). In conclusion, morphoproteomic analysis reveals constitutive activation of mTOR and NF-B pathways in the inflammatory cells and myofibroblasts as evidenced by increased cytoplasmic p-mTOR; nuclear translocation of p-p70S6K and p-NF-Bp65; and correlative expression of NF-B activation associated cytokine IL-8. These results suggest that the mTOR and NF-B pathways are involved in the pathogenesis of inflammatory and fibrogenic processes in NASH and provide a rationale for individualized targeted therapies to thwart disease progression.

[18] Aortic Intimal Sarcoma: A Case Study with Morphoproteomic Characterization and Therapeutic Implications. Bihong Zhao, Brandy Shattuck, Adel D. Irani, Anthony L. Estrera, L. Maximilian Buja, and Robert E. Brown, University of Texas Medical School at Houston, Houston, TX.

Aortic intimal sarcoma is defined as a malignant tumor arising in the intima of large blood vessels. It is a rare and highly malignant tumor with poor survival rate and most cases are resistant to chemotherapy. In this study, morphoproteomic analysis that combines the application of phosphospecific probes directed against putative sites of activation on protein analytes and cellular compartmentalization was carried out on a case of intimal sarcoma of the descending thoracic aorta. This was done in an attempt to define the chemoresistance of such tumors and uncover potential therapeutic applications for the patient. The tissue had been fixed in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. Immunohistochemistry utilized using phosphospecific probes and followed routine protocols described elsewhere. In 65% of tumor cells, there is nuclear staining of the mammalian target of rapamycin (mTOR phosphorylated on Ser 2448) and correlative expression of down-stream effectors, p-p70S6K (Thr389, 66%) and vascular endothelial growth factor (VEGF [92% of tumor cells]). The collaborative-parallel pathway of ras/Raf kinase/extracellular signal-regulated kinase (ERK) pathway that includes p-ERK 1/2 (Thr 202/Tyr 204) is also activated. Most importantly, there is constitutive activation of the nuclear factor (NF)-kappaB pathway, a putative chemoresistant pathway. That is to say, there is expression of p-NF-kappaBp65 (Ser 536) with nuclear translocation in the tumor cells. Finally, the impact of convergent signaling by these pathways on cell cycle progression in the tumor is underscored by a high proliferation index (Ki-67 at 75%) and associated high expression of Skp2, an S-phase kinase-associated protein, and cyclin D1 (Bcl-1).These findings support a role for the mTOR and NF-kappaB pathways in the development of high grade sarcoma of the aorta and suggest potential therapeutic targets.

[19] Molecular Classification of Breast Carcinoma by Immunohistochemical Analysis and its Clinical Significance. Ping Tang, Jianmin Wang, and Patria Bourne, University of Rochester Medical Center, Rochester, NY, and RTI Health Solution, Research Triangle Park, NC.

Breast cancer includes a group of heterogeneous diseases with distinct biological entities that have specific pathological features and biological behaviors. Although we have made significant progress in breast cancer detection and management, we have made little progress in determining which patients are most likely to benefit from which therapies and identifying patients at highest risk for recurrence. Using IHC-based molecular classification derived from gene expression profiling, we studied the distribution of molecular subtypes in two different aspects of breast carcinoma, mammary Paget’s disease and bone metastasis. For Paget’s disease, IHC analysis was performed on one representative section of 28 cases of MPD and 81 carcinomas without Paget’s disease (non-MPD, 38 DCIS and 43 IDC), as well as 12 cases of non-MPD nipple involvement, For bone metastasis, IHC analysis was performed on representative sections of 21 breast carcinomas with bone metastasis and 94 cases without bone metastasis. We used antibodies against ER, PR, AR, HER2, EGFR, CK5/6, CK14, CK17, CK8, and CK18. We found that (a) MPD is strongly associated with the ER and PR negative, HER2 positive, and high-grade non-basal HER2-over-expression subtype, which may be useful in guiding clinical management; and (b) bone metastasis is strongly associated with the ER positive/PR negative luminal B subtype of breast cancer. Expression rates of HER2 and EGFR may not be useful in predicting bone metastasis. In summary, Paget’s disease is strongly associated with the HER2 subtype and breast cancer with bone metastasis is strongly associated with the luminal B subtype.

[20] Significance of Overexpression of Alpha-Methylacyl-coenzyme A Racemase in Hepatocellular Carcinoma. Wei Li, Robert E. Brown, Zhaoping Zhang, and D. Tan, University of Texas Medical School at Houston, and University of Texas MD Anderson Cancer Canter, Houston, TX.

Alpha-methylacyl-coenzyme A racemase (AMACR), an immunomarker for prostatic adenocarcinoma, has been shown to be expressed in a variety of other neoplasms. This study aims to evaluate immunohistochemical expression of the AMACR in neoplastic and non-neoplastic liver lesions and to assess its value in the diagnosis of hepatocellular carcinoma (HCC). Formalin-fixed, paraffin-embedded, tissue sections of 51 HCC (14 well, 22 moderately, and 15 poorly differentiated), 9 hepatocellular adenoma (HCA), 48 cirrhotic nodules (CN), and 16 normal liver tissues (NLT) were immunostained for AMACR. Expression of AMACR is significantly enhanced in HCC tissue compared with non-HCC tissue. High expression of AMACR was found in 82 % of HCC including 86 % of well-differentiated HCC. In contrast, only 11 % of HCA, 13 % of CN, and 6 % of NLT showed high expression for AMACR. Clinico-pathological evaluation showed a significant correlation between AMACR expression and venous invasion and capsular invasion by HCC. Our results suggest that AMACR staining may serve as a useful marker for the differential diagnosis of well-differentiated HCC from HCA. Increased AMACR expression and its association with tumor venous invasion suggest that AMACR plays a role in HCC development and progression.

[21] Dual Functions of Bid in Etoposide-induced DNA Damage in Human Hepatocellular Carcinoma Cells. George G. Chen, Gang Song, Davor Kin-Fan Chau, Ji Miao, and Paul B. S. Lai, Chinese University of Hong Kong, Hong Kong, China.

Overexpression of Bid in hepatocellular carcinoma (HCC) cells can increase the sensitivity of tumor cells to chemotherapeutic agents. Growing evidence shows that Bid plays a dual role in DNA damage response, but the role of Bid in etoposide-induced-DNA damage has not been investigated. Our experiments were performed in HCC cells containing wide-type or mutant p53. The cells were treated with low (10 µM) or high (100 µM) concentrations of etoposide, causing reparable and irreparable DNA damage respectively. With a high dose of etoposide, Bid sensitized cells to apoptosis. However, at a low dose of etoposide, Bid activated the S phase checkpoint through the up-regulation of p21 and p27. While the irreparable damage was being carried out, Bid was translocated to the mitochondria to release cytochrome c into the cytosol, which activated caspases 9 and 3 and led to cell death. The level of Bid protein was up-regulated after the p53 gene was introduced into the p53-negative HCC cells. However, the overexpression of Bid led to the notable down-regulation of the cellular level of p53, indicating that Bid may act upstream of p53. The negative effect of Bid on p53 may be activated as a form of feedback regulation of cell growth in response to DNA damage. In addition, the etoposide-induced phosphorylation of Akt and c-Jun was inhibited by Bid but the phosphorylation of ERK1/2 was enhanced, which suggests that the phosphorylation of Akt/c-Jun and that of ERK1/2 may have different roles in DNA damage and apoptosis. In conclusion, Bid can either exhibit S phase checkpoint activation or play a pro-apoptotic role in HCC cells, depending on different degrees of etoposide-induced DNA damage. The elucidation of these intricate mechanisms of Bid points to the development of a possible therapeutic option that combines cytotoxic therapies for HCC. (This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region Project No: CUHK 4534/06M.)

[22] Mucosal Vaccines: The Promise and The Challenge. Kathleen A. Kelly, Cheryl I. Champion, Valerie Kickhoefer, and Leonard H. Rome, David Geffen School of Medicine, UCLA, Los Angeles, CA.

The majority of pathogens gain entry through mucosal surfaces and mucosal immune responses are important for protecting these surfaces against infection. However, the majority of vaccines are injected and poor inducers of mucosal immune responses. This is primarily due to the difficulty in measuring antibody and mucosal T cell responses following the mucosal application of vaccines. As a result, there are only a handful of FDA-approved mucosal vaccines. An ideal mucosal vaccine would target mucosal inductive sites and stimulate the production of appropriate innate and adaptive immune responses to clear the target pathogen. We have evaluated novel mucosal nano-delivery systems as vaccines for producing protective T cell responses in the genital mucosa. We reasoned that encasing an immunogenic chlamydial protein within a nanoparticle would protect the protein at mucosal surfaces and allow delivery of intact immunogenic proteins to mucosal surfaces. Further, nanoparticles can be appropriately sized to mimic microbes and stimulate phagocytosis by dendritic cells. Dendritic cell incorporation of immunogenic protein is necessary for the production of immune responses. In pre-clinical studies using these mucosal nano-delivery systems containing a chlamydial peptide as a vaccine, we found significant protection following vaginal challenge and the production of Chlamydia trachomatis-responsive T cell and antibody responses. Thus, unique mucosal nano-delivery systems have the promise of protecting mucosal surfaces.

[23] Where the Textbooks Went Wrong: Differences Between the Life Styles of M. tuberculosis and M. bovis. Robert L. Hunter, University of Texas–Houston Medical School, Houston, TX.

The introduction of antibiotics caused a decline in human tuberculous tissues for study. While this was an important advance in medicine, it forced researchers to look for other sources of tissue. The disease in the rabbit produced by M. bovis seemed to be the best model of human pulmonary tuberculosis because rabbits develop caseating granulomas and pulmonary cavities. The cavities arise from expansion of caseating granulomas that erode into bronchi and discharge their contents. This pathogenic mechanism dominates current textbooks on human tuberculosis and is wrong. Cavities in humans develop by slow progression of tuberculous lipid pneumonia that rapidly undergoes necrosis. In the formative stages, it is a pneumonic, not a granulomatous process. At least 80% of cases undergo spontaneous regression without forming cavities. Since the research community has failed to recognize this process, there has been little research and the mechanisms of spontaneous regression of tuberculosis remain unexplored. The fundamental difference between diseases produced by M. bovis and M. tuberculosis is that M. bovis must complete its life cycle and escape to new hosts in the short life of an animal. Consequently, it grows progressively in many organs, producing ulcerated lesions. Severely affected cattle shed organisms into the environment from the pharynx, lung, GI track, kidneys, and udders. M. tuberculosis, in contrast, infects a person and becomes dormant (hides) for 10–30 years before inducing a cavity in the lung from which it can escape for decades more to infect new generations of people. The process of becoming dormant, inducing lipid pneumonia that undergoes necrosis to produce a cavity in an otherwise immune individual, is unique to M. tuberculosis.

[24] Human T Lymphotropic Virus Type I: A Reluctant and Elusive Pathogen. Armand Glassman, Medical University of South Carolina, Charleston, SC.

Human T lymphotropic virus type 1 (HTLV-1) is a type-C retrovirus that has been associated with adult T cell leukemia/lymphoma (ATLL), tropical spastic paraparesis (TSP), HTLV associated myelopathy (HAM), and Sjorgen syndrome. HTLV I/II is transmitted by white blood cells, sexual contact, contaminated needles/syringes, breast milk, and other perinatal exposures. An HTLV viral oncoprotein transactivator (Tax) enhances viral transcription and blocks cellular regulatory mechanisms, permitting cell growth and division. As ATLL progresses, Tax expression is lost. Testing for HTLV I/II has been required for blood and blood products in the USA since 1988. Tests available are based on enzyme immunoassay (EIA) and Western blot (WB) techniques. There is some antibody cross-reactivity for types I/II. Lifetime risk for ATLL in patients with HTLV-I antibodies is 1–2%. Risks for HAM and/or TSP are unknown. The rate of infection ranges from approximately 20/100,000 for blacks of Caribbean background, 3/100,000 for Africans, and 0.1/1000,000 for whites of European descent. Greater than 95% of people infected remain asymptomatic. Various reports have stated that the specificity (true negative/true negative + true positive) is >99%. There are false positives and true positives, which present clinical and counseling problems. Algorithms for repeated EIA tests followed by WB testing have been proposed. The epidemiologic role of HTLV-I in disease causality, the need for universal testing of blood donors, and the guidance for transfusion medicine personnel in counseling/treatment continue to evolve.

[25] Anti-tumor Necrosis Factor-alpha Therapy in a Patient with Rheumatoid Arthritis Provokes Latent Leishmaniasis Infection: A Case Report. Gillian Franklin, Joel Greenspan, and Sheng Chen, North Shore-Long Island Jewish Medical Center, New York, NY.

Anti-tumor necrosis factor-alpha (anti-TNF-) therapy has been reported to increase the risk of opportunistic infections including rare case reports of leishmaniasis. We report here a case of cutaneous leishmaniasis that occurred in a patient with rheumatoid arthritis on anti-TNF therapy. The patient is a 42-yr-old Brazilian woman who immigrated to the USA in 1999. The patient had spent several months on the Mediterranean coasts of Spain and southern Italy at 11 and 4 months respectively prior to the initial appearance of her lesion. In December 2004 she apparently plucked a nasal hair and developed a boil that evolved into a 4 cm ulcer around the right nostril over a period of three years. During that 3-yr time frame the patient was seen by multiple clinicians, several biopsies were performed, and numerous diagnostic impressions were suggested. A correct diagnosis of cutaneous leishmaniasis was finally made in June 2007 following another biopsy, which revealed the presence of numerous leishmaniasis amastigotes within macrophages. Subsequent culture studies with species typing at the CDC identified the organisms to be Leishmania donovani complex (Leishmania chagasi), which is endemic only in the New World. There was marked clinical improvement of the nasal lesion following treatment with liposomal amphotericin B. Since this case of cutaneous leishmaniasis occurred in a patient who had been undergoing anti-TNF- therapy for rheumatoid arthritis, we believe the case is an example of cutaneous leishmaniasis that was incited from latency by anti-TNF- therapy.

[26] The Impact of Organ Transplantation on Heart and Lung Pathology. Michael C. Fishbein, David Geffen School of Medicine, UCLA, Los Angeles, CA.

The successful application of solid organ transplantation has had a major impact on the survival of patients with end-stage heart and lung disease; 5-yr survivals are now over 70% and 50%, respectively. Transplantation has had a major impact on diagnostic surgical pathology and clinical laboratories, and has stimulated research in transplant-related laboratory sciences, such as immunology and microbiology. Entire hearts and lungs, once only encountered at autopsy, are now surgical pathology specimens, requiring labor-intensive, time-consuming dissection and the evaluation of complicated and rare entities. Native hearts with bypass grafts, artificial valves, pacemakers, defibrillators, assist devices, conduits, stents, and repaired congenital lesions are common explants. Relatively rare and diagnostically difficult pulmonary lesions such as interstitial lung diseases are also often encountered. After transplantation, endomyocardial and transbronchial biopsies are common specimens as numerous surveillance biopsies are performed whether or not there is clinical evidence of organ dysfunction. These medical biopsies are among the most challenging and demanding occurrences in surgical pathology, requiring rapid and accurate diagnosis on small specimens in life-threatening situations. A misdiagnosis in these immunosuppressed patients may prove to be a fatal mistake. In patients with evidence of graft dysfunction, there is a broad differential diagnosis that includes cellular and/or antibody mediated rejection, infection, neoplasm, drug reaction, and recurrence of the original disease of the native organ. Proper evaluation requires, and is assisted by, special histochemical and immunohistochemical staining. The pathologist practicing at a transplant center is faced with considerable new challenges, but there is also the extremely rewarding opportunity to play an integral role in the management of the post-transplant patient and to contribute to advancing knowledge in transplant-related sciences.

[27] An Unusual Case with Population Consequences. M. Kent Froberg, Duluth Clinic and University of Minnesota Duluth; Virginia Regional Medical Center, Virginia, MN.

The pathological features of an unusual tumor are presented as an unknown case exercise. The tumor occurred as multiple, cutaneous, exophytic, firm masses on the face, head and neck of the affected individuals. Gross findings included central necrosis and hemorrhage. Histological sections showed the tumor masses to be confined to the dermis and comprised of fairly homogeneous cells with round nuclei and scant cytoplasm. Immunophenotyping demonstrated the tumor cells to be of primitive neuroendocrine phenotype. Eventually the tumor was observed in relatives of the proband and these tumors were found to have the identical genetic makeup as the original lesions. Hosts appeared to have no immune or inflammatory response to these tumors. Subsequent molecular studies indicated that the affected population of individuals lacked sufficient genetic diversity within the peptide binding regions of their MHC genes to recognize tumor proteins as exogenous antigens. The mode of transmission and the role of the founder effect in the epidemiology of these tumors are discussed, as are the population consequences of this disease.

[28] Immunohistochemical Characterization of Cytokine Expression in Eosinophilic Esophagitis and Gastroesophageal Reflux Disease. Usa Tantib-haedhyangkul, Carla Davis, Mark Gilger, Angela Major, and Nina Tatevian, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX.

Rapid increase in the number of patients with severe esophageal eosinophilia has been noted in recent years. Patients who were thought to have gastroesophageal reflux (GERD) but did not respond to medical GERD management were diagnosed as eosinophilic esophagitis (EE). Histologic characteristics of EE include severe squamous epithelial hyperplasia, eosinophilic microabscesses, distribution of eosinophils in the upper epithelial layer, and >15 eosinophils per high power field (HPF); in general, GERD shows lesser intensity of eosinophilic inflammation. Chemokine eotaxin-3 and cytokine IL-5 are associated with eosinophilic infiltration of the tissue. CD4 T-cells are important for cytokine IL-5 production in allergic diseases and CD8 T-cells are found to be prominent suppressors in inflammatory diseases. In this study we assessed the immunohistochemical expression of eotaxin-3, IL-5, CD4, and CD8 in formalin-fixed, paraffin embedded esophageal tissue in both diseases in comparison to normal controls (NC). Ten EE, 8 GERD, and 10 NC cases were chosen upon review of pathology and clinical records for the past 3 years at Texas Children’s Hospital. Surprisingly, eotaxin-3 is present in mature superficial squamous epithelial cells but absent in basal proliferative layer in all three groups. IL-5 is located in the intravascular spaces in all 3 groups, with focal extravasation in EE. No statistically significant difference is found in the number of CD4+ T-cells among all groups; however, CD4+ cells are qualitatively increased in EE (medians = 10.4, 3.6, and 1.6/HPF in EE, GERD, and NC, respectively). The number of CD8+ T-cells is increased in both EE and GERD groups compared with NC (p<0.01). No statistically significant difference of CD8+ cells is detected between EE and GERD groups (medians = 7.5, 4.7, and 0.4/ HPF in EE, GERD, and NC, respectively). The number of eosinophils correlates inversely with CD4/CD8 ratio in EE (p = 0.042). These results strongly suggest a role of IL-5 and CD8 T-cells in the immunopathogenesis of EE and GERD.

[29] Adrenocortical Neoplasms of Unknown Malignant Potential in Young Children. Wanghai Zhang and Atif Ahmed. Howard University Hospital and Children’s National Medical Center, Washington DC.

Adrenal cortical neoplasms in pediatric patients are rare. About 14 new cases per year in patients younger than age 20 years are diagnosed in the United States. The clinical manifestations and biologic behavior of these neoplasms are distinct from their histologically similar counterparts in adults. We report 3 cases of adrenocortical neoplasms in young children and review the clinical presentation, pathology and follow-up data. Pathologic evaluations included histology and immunostains with p53 and Ki-67. The patients (2 girls and 1 boy) presented at 21–28 months of age with signs of virilization. Serum testosterone levels were elevated in two cases (5 and 23 ng/dl). Imaging studies revealed neoplasms confined to the left adrenal gland in all 3 cases. No evidence of disease was identified elsewhere. At pathology, the adrenalectomy specimens, weighing less than 59 g, revealed tumors ranging in diameter from 3 to 6 cm. The tumors were grossly confined to the adrenal glands and had histologic and cytologic characteristics of adrenocortical neoplasms. Furthermore, features associated with an increased probability of a malignant clinical behavior in adult patients were identified in all cases and included broad fibrous bands, capsular and vascular invasion, nuclear pleomorphism, mitotic rate >15/20 HPF, and presence of atypical mitoses. Immunohistochemical reactivities of p53 and Ki-67 were 30–75% and 10–30%, respectively. One patient is alive without evidence of disease for 9.7 yr; the other two patients for 5 and 8 mo, respectively. Despite histological and immunopheno-typical evidence of malignancy, adrenocortical neoplasms in patients <3 years of age have a benign clinical course with no metastasis or recurrence. Such tumors should be classified regardless of histology as of unknown malignant potential.

[30] Can Molecular Studies Improve Survival in Patients with Cancers from Unknown Primary Sites? Guy diSibio, Harbor-UCLA Medical Center, Torrance, CA.

Cancers presenting as metastases from unknown primary site (CUPS) are common in clinical practice and generally have a poor prognosis, with median survival rates as low as 4 months after diagnosis. The uncertain origin of these malignancies often causes frustration on the part of both clinician and patient, who may undertake an aggressive, expensive, and often fruitless diagnostic workup to identify the primary site. Even when successful, such efforts do not necessarily increase patient survival. A more limited, cost-effective workup for CUPS is therefore usually advocated, with the goal of targeting appropriate treatments against specific histologic patterns. Sundry molecular diagnostic tests for various malignancies, some now commercially available, have spurred clinical interest in their potential for expanding our understanding of cancer and improving patient survival. Are such tests, however, sufficiently justified for inclusion into a cost-effective CUPS workup? A literature review was undertaken to address this question and to identify current and emerging molecular studies that might be considered as appropriate adjunct tests during the workup of CUPS. While no large-scale studies were found to indicate a clear improvement over non-molecular approaches, several small-scale studies suggest a potential role for molecular analyses to resolve specific problems in CUPS diagnosis and treatment.

[31] Diagnostic Accuracy of Fine Needle Aspiration in Breast Lesions: Implications for Future Use. Christina Day, Amber Fimbres, Naghmeh Salami, Neda Moatamed, and Sophia Apple, Geffen UCLA Medical Center, Los Angeles, CA.

The use of fine needle aspiration (FNA) in diagnosing breast lesions has recently declined. Core needle biopsies have increased, partially because FNA cannot distinguish between in-situ and invasive carcinoma. Also, cytology samples are not optimal for assessing prognostic markers. We sought to evaluate the role of FNA for breast lesions at our institution. A retrospective review of breast FNA samples from 2002–2006 was performed, followed by identification of cases with corresponding surgical tissue. FNAs were mostly from palpable breast lesions. Pathology residents and fellows collected samples using 23- or 25-gauge needles. A total of 829 FNAs were performed; 256 (30.8%) had histologic follow-up. FNAs numbered 103 in 2002, 176 in 2003, 198 in 2004, 200 in 2005, and 152 in 2006. Cases were placed into 1 of 4 categories: non-diagnostic (9%), benign (77.5%), atypical/suspicious (5.5%), or malignant (8%). Tissue was available for 28 non-diagnostic, 144 benign, 37 of A/S, and 47 malignant cases. Overall sensitivity/specificity (S/S) for FNA was 83/92% respectively. Positive and negative predictive values were 83 and 92%, respectively. There were no false-positive cases, indicating a PPV of 100% for diagnosis of malignancy. The false-negative rate was 5.5%. In diagnosing breast lesions by FNA, the literature reports a range of 65–98% and 82–100% for S/S. At UCLA the overall S/S for FNA was 83 and 92%, respectively. Although the national trend is away from FNAs of breast lesions, this has not been our experience. While FNA may not be ideal for evaluating suspicious lesions, we believe that FNA for clinically benign palpable lesions and recurrent carcinoma has significant value.

[32] PTEN Soft Tissue Hamartoma. Kyle Kurek, Emily Howard, Lucile Tenant, Joseph Upton, Steven Fishman, John Mulliken, Antonio Perez-Atayde, and Harry Kozakewich, Children’s Hospital, Harvard Medical School, Boston, MA.

The PTEN hamartoma tumor syndrome includes a spectrum of disorders associated with germline mutations in the PTEN tumor suppressor gene on 10q23.3, most notably Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Both are associated with the development of multiple cutaneous, stromal, and visceral hamartomas, usually by late adolescence, as well as a predisposition to certain malignancies. We have recently recognized and herein expand our prior observations upon a unique soft tissue vascular lesion that is an early clinical finding in many PTEN patients. Cases were identified from PTEN patients registered in the Vascular Anomalies Center as well as from a retrospective review of unusual intramuscular arteriovenous malformations and vascular hamartomas in our database. Chart reviews were completed for each patient. Thirty-five patients were identified (13 M, 22 F), 23 with a clinical diagnosis of a PTEN syndrome (19 confirmed genetically). The lesions presented with pain and swelling, commonly in the extremities, by 15 yr of age in most patients (n = 29; range 0–15 yr; mean 7 yr). Lesions were most often solitary, intramuscular, and diameter 3–25 cm; when excised, they frequently recurred. Histologically, the lesions were hamartomatous masses of adipose, fibrous, and myxoid tissue with abundant blood vessels. The vascular component consisted of tortuous, thick-walled arteries with marked concentric smooth muscle hyperplasia, clusters of thin and thick-walled dysplastic veins, numerous small vessels of indeterminate type, and occasional arteriovenous communications. Lymphoid aggregates were common. Bone occurred in several lesions and Schwannian hyperplasia in one. This distinctive hamartomatous lesion is highly associated with PTEN mutations and its identification should prompt a thorough clinical investigation.

[33] Molecular Testing and the Autopsy: Quo Vadis? Jack W. Snyder, Potomac, MD.

Investigations of fetal, infant, and adult sudden death can be stymied by lack of histological markers. Postmortem genetic testing (PMGT), however, can facilitate identification of morphologically invisible disease. Pathologists who understand and employ DNA-friendly techniques for procurement of postmortem tissue (eg, blood, liver, spleen, muscle, skin) can enhance the value of the autopsy, especially when the cause of death may involve drug use, pulmonary embolism, or abnormalities of cardiac conduction. For examples, analysis of drug-related deaths can benefit from identification of genes that code for variant alleles of the cytochromes p450 (eg, 2D6, 3A4), analysis of fatal pulmonary embolism can benefit from identification of 3 common mutations (factor V-Leiden G1691A, prothrombin G20210A, and MTHFR C677T), and analysis of sudden infant and childhood deaths can benefit from identification of mutations in genes that code for cardiac ion channels. The results of PMGT may provide a rational basis for pharmacogenomic, coagulational, or cardiological screening of family members to uncover toxicological risk, thrombotic risk, familial cardiomyopathies, or genetic arrhythmias and to adopt appropriate therapeutic and preventive strategies. By contrast, PMGT of stillbirths may be based on need to address parental desire for diagnostic closure, need to identify infectious agents as a sporadic cause of stillbirth to reassure parents and clinicians regarding risk for future pregnancies, and need to reduce unnecessary testing. Viral agents including rubella, human cytomegalovirus (CMV), parvovirus B19, herpes simplex virus (HSV), lymphocytic choriomeningitis virus (LCMV), and varicella zoster virus (VZV) are known to cause intrauterine deaths, but the roles of other viruses and difficult-to-culture organisms in stillbirth are uncertain, and need more research. Nevertheless, testing stillborns for viral agents can be a useful adjunct to histopathological and other examinations at autopsy. Finally, introduction of large-scale genetic screening may have significant, unforeseen, desirable or undesirable effects on the practice of pathology.

[34] The Anti-Doping Movement in Sports: Are We Winning? Anthony W. Butch, Geffen School of Medicine, UCLA, Los Angeles, CA.

Doping refers to the use by individuals of prohibited substances that have the potential to enhance athletic performance in professional and amateur competitive sporting events. The practice of doping has been part of competitive events throughout the history of sports. Substances such as strychnine, caffeine, cocaine, and alcohol were used by athletes for stamina in the nineteenth century. In 1928, the International Amateur Athletic Federation was the first organization to ban doping agents (stimulants) and other sports federations quickly followed its lead. However, the doping problem continued to grow with the availability of synthetic hormones and their popularity in strength/ endurance sports. Drug testing was first conducted at the 1968 Olympic Games in Mexico and was subsequently implemented by the majority of international federations. Unfortunately anabolic steroids were difficult to detect until the introduction of reliable and accurate test methods that utilize gas chromatographic separation techniques with mass spectrometric detection. In 1999, the International Olympic Committee established the World Anti-Doping Agency (WADA), which is an independent international agency with support from intergovernmental organizations, governments, sports organizations, and public authorities. WADA has developed international standards for anti-doping work and operates under the principle of a level playing field in the spirit of sport. WADA laboratories routinely monitor anabolic androgenic steroids (both exogenous and naturally produced steroids), beta-2-agonists, hormone antagonists, diuretics/ masking agents, stimulants, street drugs, glucocorticosteriods, and beta-blockers. The challenge to stay ahead of cheaters is a daunting task, as illustrated by the designer steroid tetrahydro-gestrinone-BALCO scandal, and the widespread use of growth hormone in USA professional sports. To win the doping battle, additional approaches besides drug testing need to be identified and implemented.

[35] Drugs of Abuse Testing: So Many Limitations. Frederick L. Kiechle and Deanna D. H. Franke, Memorial Regional Hospital, Hollywood, FL.

Testing for drugs of abuse (DOA) has evolved from chromatographic screening technologies followed by positive confirmations to screening with immunoassays and confirmation. Immunoassays have been promoted from a confirmatory test to a first-line screening technique. NACB guidelines encourage laboratories to report detection thresholds, parent compounds, and metabolites that are detected and not detected, as well as drugs that can cause false-positives. We evaluated the performance of the Roche Integra device and the MedTox point-of-care (POC) device in routine clinical urine specimens. Both systems failed to detect more than 50% of the top 7 (Integra 5, MedTox 4) DOA in Broward County, FL. There were 13 discrepancies in 21 urine specimens tested over 7 days. Following chart review, single drug discrepancies were classified as MedTox device or automated reader failures (6), specificity-related (6), sensitivity-related (2), and unknown causes (4). In conclusion, the heterogeneous limitations that DOA screening with immunoassays introduce suggest that great caution should be exercised before their use is implemented in a healthcare system.

[36] Cocaine Metabolite Assay Results in Patients Taking Amoxicillin: Is There Interference? Roger L. Bertholf and Gary M. Reisfield, University of Florida Health Science Center, Jacksonville, FL.

Urine drug testing for drugs of abuse (UDT) in SAMHSA-regulated laboratories is a 2-stage process involving screening immunoassays and confirmation by gas chromatography-mass spectrometry (GC/MS). The GC/MS analysis is highly specific, virtually eliminating the possibility of a false positive result. Screening immunoassays, on the other hand, are susceptible to interferences by virtue of their antibody-based selectivity. Confirmation of positive immunoassay results by GC/MS is necessary when drug testing results are potentially susceptible to legal challenge, as in workplace or sports UDT programs. However, UDT has become increasingly common in clinical medicine, most notably in the treatment of chronic pain with opioid analgesics. UDT in the clinical setting is not regulated, and often includes only the screening immunoassay. Hence, there is a potential for misinterpretation if the screening results are falsely positive or negative. Numerous sources on the World Wide Web, and some accounts in the published medical literature, cite amoxicillin as a potential cause of false positive cocaine metabolite (benzoylecgonine) screening results, although we were unable to locate any published studies demonstrating that such interference exists. Thirty-three subjects, all of whom had been prescribed amoxicillin, were recruited from an urban primary care clinic population and asked to return after 2 or 3 days to provide a urine specimen. The specimens were screened by 4 UDT immunoassays for cocaine metabolite. The results of all 4 immunoassays were in agreement, with 28 negative, 3 sub-threshold positive, and 2 positive results. GC/MS analysis of all 5 positive specimens confirmed the presence of benzoylecgonine. The study failed to demonstrate any interference by amoxicillin, although the statistical power of the limited sample size does not exclude the possibility of amoxicillin-induced false positive results.

[37] Quantification of the Major Lipoprotein Fractions Using an Electrophoresis System. Clive R. Hamlin, Case Western Reserve School of Medicine, Cleveland, OH.

The National Cholesterol Education Program (NCEP), through a series of guideline reports, centers the treatment goals on lowering LDL cholesterol. Recommended classification for lipid levels are listed for LDL, HDL, and total cholesterol and a similar classification is given for triglycerides. The Friedewald formula is still widely used to calculate LDL, despite its well-known limitations. Direct LDL immunoassays are a possible improvement, but issues of incomplete analytic specificity, cost, and insurance concerns exist. I have evaluated an electrophoresis system for the quantification of the major lipid sub-groups (Helena QuickGel Cholesterol). This provides an acceptable, though not perfect, alternative for measuring LDL cholesterol in small laboratories. Once the manufacturer’s claims have been verified, there is nothing more to be done, as no known reference materials exist for the lipoprotein fractions. My situation is illustrative of the difficulty that all laboratory directors face in providing LDL cholesterol values in which one can have confidence, and with which many clinical decisions are made.

[38] Glycated Albumin Analysis on an Automated Analyzer. Greg D. Horton, Barry I. Freedman, and Zak K. Shihabi, Wake Forest University Medical School, Winston-Salem, NC.

Tests such as HbA1c, fructosamine, and glycated collagen are used to assess the degree of glycemic control in diabetes. These tests reflect the degree and the duration of hyperglycemia. The fructosamine assay, also known as glycated albumin, reflects the last 3 weeks of control; however, it is not often utilized in clinical practice for reasons that include being subject to interference from reducing substances. An enzymatic reaction was recently introduced for glycated albumin (Lucica GA-L, Asahi Kasei Pharma Corporation, Chiyoda-ku, Tokyo, Japan). It is based on hydrolyzing enzymatically the glycated albumin into glycated amino acids, which in turn are oxidized to amino acids and hydrogen peroxide. We adapted this test for automation on the ADVIA 1650 (Siemens Diagnostic Solutions, Tarrytown, NY). Test results were linear between 0 and 1.8 (g/dl; r = 0.998) for the glycated albumin. The within-run CV at 0.47 g/dl was 0.6 % (n = 12). The correlation coefficient with % HbA1c in patients without nephropathy was r = 0.88 based on percentage of glycated albumin (%); and r = 0.92 based on the absolute glycated albumin (g/dl). However, in a large group (n=254) of diabetics including those with nephropathy the correlation for the glycated albumin as g/dl with the % of HbA1c was 0.73. Because of this high correlation, the assay for patients with stable albumin can be simplified by eliminating the need to index it as percentage of albumin. The reference range was 9–14% for the percentage of the total (n = 22), and 0.34–0.62 g/dl for the absolute glycated albumin (n = 22). Lipemia, ascorbate, and elevated bilirubin did not interfere, but high hemolysis (>200 mg/dl) did interfere with the reading since it absorbed light at the same wavelength as the test. This test can be used to assess the degree of control of hyperglycemia in different settings: directly as an absolute value for patients with stable albumin; as a ratio to total albumin in patients with hypoalbuminemia; and as a better marker than HbA1c in diabetics with a reduced red blood cell half life (eg, patients with nephropathy or on dialysis). In conclusion, this glycated albumin test correlates well with HbA1c, has good precision, and is simple to automate.

[39] The New Theory of Vitamin D Function May Greatly Surge the Test Volume. Pai C. Kao and Ta-Jen Wu, Mayo Clinic, Rochester, MN.

Vitamin D testing has been used very conservatively and has mainly focused on 1,25-dihydroxyvatmin D, which is an active hormone that regulates intestinal calcium absorption and reflects kidney and parathyroid functions. Currently the focus is shifting from 1,25-dihydroxyvitamin D to 25-hydroxyvitamin D, the functionless precursor, and its serum level (ng/ml), which is thousands of times greater than the active hormone, 1,25-dihydroxyvitamin D (pg/ml) level, and is very difficult to determine. The test for 25-hydroxyvitamin D can be automated; in a large reference laboratory the volume has increased from a few hundred assays per month to almost a thousand samples per day. The new theory suggests that vitamin D or sunlight exposure prevent a variety of diseases from type 1 diabetes to cancers and may even increase longevity. Most of the evidence is based on epidemiological studies, such as a report that children living at the high latitude of Finland have 35 times greater chance to develop type 1 diabetes than children in South China, which is close to the equator. Again, patients living in the cloudy Seattle area have a greater chance of getting colon cancer than those living in sunny Texas. Sunshine is only one factor; various other factors have not been considered. In addition to epidemiologic data, biochemical studies have broadened the function of vitamin D. For example, among 1200 women, those with higher serum 25-hydroxyvitamin D levels had longer telomeres in their leukocytes, suggesting longer cell life. Such data are the basis for the new theory of vitamin D function and suggest that physicians test patients to prevent vitamin D insufficiency. However, the true benefits of this approach to disease prevention remain to be determined.

[40] The Medical Algorithms Project: An Online Clinical Informatics Resource to Support Medical Decision-Making. M. Sriram Iyengar and John R. Svirbely, University of Texas Health Science Center at Houston, TX, and TriHealth Hospitals, Cincinnati, OH.

The Medical Algorithms Project (Medal) is a clinical informatics resource that can be used to support decision-making and evidence-based medicine. Medal ( www.medal.org) is a free online resource with over 11,500 formulae, scales, scores, tables, and other computational techniques selected from the peer-reviewed biomedical literature. Content is divided into 45 chapters reflecting different medical specialties. Each algorithm has an information web-page that describes its purpose, provides brief details of the computation, and lists supporting references. From here the user can search medical journal databases (such as PubMed) and can access an Excel spreadsheet that encodes the algorithm’s content. About 400 algorithms have also been implemented as easy-to-use interactive web forms. Since its inception in 1999 the Medal resource has achieved several significant accomplishments. It is the first item returned by web searches for the terms ‘medical algorithms’ and ‘algorithmic medicine.’ In September 2004, free registration was initiated and as of January 2008 there are more than 92,000 registered users from around the World with about 50 new users registering every day. In addition, all Veterans administration facilities enjoy free non-login access. Forty percent of the registered users are physicians and another 40% are medical students or nurses. A series of books compiling algorithms related to various medical specialties (rheumatology, travel medicine, military medicine, etc) is being prepared. Medal provides clinicians with objective evidence-based criteria to support diagnostic and therapeutic decisions. When integrated into clinical systems, algorithms can provide documentation of clinical care and reduce medical errors. To achieve these goals it will be necessary to integrate medical algorithms with the electronic health record (HER) and to automate their execution.

[41] Machine Vision Systems for Automation of Clinical Laboratory Quality Inspections. Charles D. Hawker, ARUP Laboratories and University of Utah, Salt Lake City, UT.

For two decades, manufacturing and pharmaceutical plants, bottling companies, and virtually all other industries have used machine vision systems (cameras, lasers, infrared, X-ray, gamma-ray, etc) to inspect products for the quality and accuracy of sizes, fill levels, bottle seals, labeling, lot numbers, expiration dates, and much more. Clinical laboratory automation vendors are now incorporating similar systems to inspect specimens for correct specimen type, serum volumes above the packed red cells, and interferences such as fibrin, hemolysis, icterus, and lipemia. Such systems require extensive computer software for analysis and data interpretation. As cost reduction pressures on laboratories increase and the supply of medical technologists continues to diminish, automation becomes increasingly important. Automated systems are being developed that can replace human inspection of specimens to determine their correctness and suitability for the ordered tests. Working with two third-party automation integrators as well as engineers in our University’s College of Engineering, our laboratory has developed an automated system to determine if a specimen is within predefined minimum and maximum volumes for automated testing. We are also developing an automated system for camera inspection of the patient name on the client’s label to determine if the specimen has been mislabeled. Even if they don’t have accurate statistics, most laboratories are aware that they have occasional mislabeled specimens, and require employees to inspect all specimens. This project has been an extremely difficult challenge because of the variety of font sizes, label orientations on tubes, and the arrangement of data on the labels. A project proposal has been submitted to CLSI to develop a specimen labeling standard in order to reduce mislabeled specimens and improve patient care.

[42] Cost Effective Consultations by Telepathology. Robert L. Hunter, University of Texas–Houston Medical School, Houston, TX.

Pathologists are caught in a bind between an increasing demand for subspecialty expertise and the need to be dispersed in small groups near patients and surgeons. This is intensified by increasing demand for quality assurance and demonstration of competency. For several years, we have used Axis surveillance cameras to transmit live microscopic images over the internet. Our goal was to facilitate collaboration between pathologists located at different sites as effectively as with a two-headed microscope. Recent modifications enable use of any digital camera capable of producing live images on a computer. The images are viewed remotely using video conferencing software. We evaluated several cameras and video conferencing options. The cameras all worked equivalently. We found that most video conferencing programs could not support live microscopic images. The most effective and one of the least expensive programs was GoToMeeting. This system requires a microscope with a digital camera attached to a computer with a moderately fast bidirectional internet connection. Microscopic images can be viewed and discussed by up to 25 remote sites. Any one of the sites can transmit images as well. We now use this system for regularly scheduled conferences and ad hoc consultations. For example, pathologists at 2 hospitals 10 miles apart gather around multiheaded microscopes for daily QA conferences. They review problem cases by viewing and discussing each other’s slides. The results are recorded in a log book that satisfies all regulatory requirements for quality assurance and maintenance of competency. While not yet ideal, the system facilitates collaboration among pathologists at a distance, enhances learning, and provides subspecialty expertise that is not feasible for isolated individuals or small groups.

[43] Applying Statistical Quality Control to Her-2/neu Immunohistochemistry (IHC) through Digital Imaging. Jeffrey W. Prichard, Geisinger Medical Center, Danville, PA.

The FDA has approved trastuzumab (Herceptin; Genentech, San Francisco, CA) for use in treating breast cancer with the requirement that a quantitative IHC test be used to assess if the staining of the target oncoprotein reaches a quantitative threshold value of intensity associated with drug efficacy. We use the HercepTest kit (Dako; Glostrup, Denmark). The HercepTest provides breast cancer cell lines (MDA175, MDA-231, and SKBR3) with known HER2 oncoprotein expression to assess the staining process. This project applied image analysis to HER2 IHC controls in order (a) to provide quantitation of HercepTest kit controls, (b) to apply common laboratory control methods to the results, (c) to establish quantifiable acceptable limits for control results in order to monitor variability between runs and (d) to detect process errors. We performed 84 runs of HER2 stains over a 6-mo period using the ACIS II Automated Cellular Imaging System (Dako North America, Carpinteria, CA). We established quality control target values for the SKBR3 cell line control: mean 3.12 units; 95% confidence interval (CI) for mean 3.08–3.17; standard deviation (SD) 0.2; 95% CI for SD 0.18–0.24; 2 SD range 2.71–3.53; and coefficient of variation 6.50%. Measurements of the controls are now plotted on a Levy-Jennings chart and Westgard rules are applied to detect errors in the staining process. Control results exceeding 2 SD are repeated. If the repeated control is within 2 SD, the run is accepted. If a control result exceeds 3 SD or repeatedly exceeds 2 SD, an investigation is performed to identify the source of error. The most common reason for a control failure is a focus failure in the robotic microscope caused by dust or fingerprints on the cover slip.

[44] Development of a Specimen Tracking Tool for Surgical Pathology. Myra L Wilkerson, Geisinger Medical Center, Danville, PA.

Surgical pathology traditionally involves a series of processes through which specimens are tracked with manual logs. This makes it difficult to know where a specimen is at a specific point in time, how long the specimen spends at a given point, and how to identify bottlenecks. The goals for this project were to identify steps in the workflow at which a specimen can be tracked and to develop a real-time Web-based tracking monitor that can be viewed by all pathology personnel. We first identified steps in the workflow at which a specimen could be tracked electronically using data collected in the pathology information system (CoPath Plus version 2.7, Cerner Corp., Kansas City, MO). We then developed a tracking monitor to display tracking events on an interactive bar graph that is deployed via an intranet Web page and updated every 5 min. The Web page was developed using ColdFusion (Adobe Systems, San Jose, CA) and the database was created in Microsoft SQL Server 2000 (Microsoft Corp., Redmond, WA). Each 5-min snapshot contains data about every specimen that falls within the ‘accessioned’ tracking event through the ‘billing reviewed’ tracking event. Users can determine at a glance the status of a specific specimen, what steps it has already passed through, and where it needs to go. Our group is continuing to develop and refine rules for this tracking event monitor, including a more granular tracking monitor for histology events. We are developing a tracking ticker that can run on a user’s computer desktop, adding e-mail notification for cases that have entered an ‘overdue’ status, and placing large-screen monitors in surgical pathology, transcription, and histology areas so that the overall system status can be checked instantly by any worker in those areas.

[45] Patient Safety in the Clinical Laboratory: New Approaches to Managing Laboratory Errors, Elizabeth A. Wagar. Geffen School of Medicine, UCLA, Los Angeles, CA.

Patient safety is an increasingly prominent topic in healthcare. By some estimates, as many as 100,000 deaths related to healthcare errors may occur per year. The clinical laboratory is an important element of patient safety, given the volume and significance of the diagnostic and therapeutic information generated in the average clinical laboratory. This presentation describes the elements of a clinical laboratory patient-safety project, focusing on the importance of patient and specimen identification. Sequential implementation of 3 patient safety projects at UCLA will be described that resulted in a decrease in specimen identification errors for mislabeled specimens (p <0.01) and a trend analysis decline for mislabeled, unlabeled, and specimen/requisition mismatched specimens. A study of specimen labeling errors in a College of American Pathologists (CAP) Q-PROBES was performed by 147 participating laboratories and examined >3.3 million labels. The data defined the consensus specimen identification error rate at 0.92 errors/1000 labels. Laboratories that maintained a 24/7 phlebotomy service and employed a quality monitor for specimen identification had statistically significant fewer errors (p = 0.02 and p = 0.008, respectively).

[46] Systemic Iron Regulation in Health and Disease. Tomas Ganz, Geffen School of Medicine, UCLA, Los Angeles, CA.

In the face of varying dietary supply of iron and varying iron losses through bleeding, iron concentrations in plasma and extracellular fluid and body iron stores are homeostatically regulated. The principal regulator of iron concentrations and stores is hepcidin, a 25-amino acid peptide produced by hepatocytes. Hepcidin regulates iron flows by binding to the sole known cellular iron efflux channel, ferroportin, and inducing its internalization and degradation. By this mechanism, hepcidin inhibits the entry into plasma of dietary iron from duodenal enterocytes, of recycled and stored iron from macrophages, and of stored iron from hepatocytes. Production of hepcidin is homeostatically regulated by holotransferrin, and by erythropoietic activity, and pathologically regulated by inflammation. During inflammation, high concentrations of hepcidin cause iron sequestration in macrophages, hypoferremia, and anemia of inflammation. We developed and validated the first serum ELISA for hepcidin. In 24 healthy subjects (12 men, 12 women) serum hepcidin concentrations ranged from 18 to 237 ng/ml and appropriately correlated with estimated iron stores and with serum ferritin (r = 0.79, 0.89). Urinary hepcidin concentrations in healthy subjects ranged from 70 to 1762 ng/mg creatinine and correlated well with serum hepcidin (r = 0.82). In physiological validation studies in healthy volunteers, we observed the expected transient rise of serum hepcidin in response to iron ingestion, and a transient fall in response to blood donation. Expected alterations in hepcidin levels were also observed in a variety of clinical conditions associated with iron disturbances. Serum hepcidin concentrations were undetectable or low in patients with iron deficiency anemia (ferritin <10 ng/ml), iron-depleted HFE hemochromatosis, and juvenile hemochromatosis. Serum hepcidin concentrations were high in patients with inflammation (CRP >10 mg/dl), multiple myeloma, or chronic kidney disease.

[47] Rapid Quantification of HbS in Sickle Cell Patients Using the Variant II Turbo. Henry D. Edwards and Zak K. Shihabi, Wake Forest University School of Medicine, Winston-Salem, NC.

The objective of this work is to describe and validate a rapid (90 sec) analysis of HbS using the Variant II Turbo apparatus (Bio-Rad Laboratories, Hercules, CA). Monitoring the level of HbS in pre-operative transfusion therapy is important for improving the outcome in patients with sickle cell disease. Ion exchange chromatography by HPLC as performed on the Variant II Turbo is a common procedure for accurate and rapid quantification of HbA1c. Here we show that the rapid gradient used for the HBA1c quantification can also be used directly to quantify HbS in those patients already confirmed to have HbS without other abnormal Hb variant and who also have HbF <15%. Fresh EDTA blood samples were loaded onto the instrument without any treatment such as those for HbA1c. The HbS percentage was read directly from the instrument display. It was linear between 5–95%. HbS measurements correlated well with those obtained by agarose electrophoresis (Sebia Inc., Norcross, GA) (r = 0.98; y = 0.99x + 0, n = 60). HbF >25 % can elute in several peaks similar to HbA, rendering its quantification a challenge. On the other hand, when the HbF level is <15%, as in majority of patients, it elutes as one major fraction that is identifiable by the instrument software. In these patients, the correlation for HbF (<15%) with the electrophoresis was high, r = 0.96 with a regression of y = 0.67x + 0.65, n = 18. In this case, HbA can also be calculated by subtraction. The precision, calculated as CV, of a sample containing 42.3% HbS was 0.9% (n = 16). In conclusion, the Variant II turbo can accurately quantify HbS in sickle cell patients in 90 sec with full automation using the same gradient as the HbA1c assay; in contrast, 2 hr are needed to quantify HbS by agarose electrophoresis.

[48] The PFA-100 and the Bleeding Time: Twentieth Century Hemostatic Screening Tests. Jonathan S. Krauss, Medical College of Georgia, Augusta GA.

The bleeding time test (BT) is a time-honored ex vivo measure of platelet function that has been employed as a hemostatic screening method for nearly a century. Blood is blotted on a filter paper every 30 sec until bleeding stops; BT data may be improved by integration of the area occupied by the blotted blood. There have been several BT modifications: (a) the Duke (1910) method used lancet-puncture of the ear lobe, whereas (b) the Ivy (1935) method employed the forearm and a sphygmomanometer. In 1969 (c) Mielke standardized the bleeding time assay by using an incisional template. A modification (d) of this technique later became commercially available (Simplate, General Diagnostics). By 1990, it had become apparent, however, that the BT was neither an accurate, nor a precise technology (Rodgers and Levin). The PFA-100 (Dade-Behring), an in vivo platelet function test, evolved from procedures developed in the 1980s. This test employs a high shear (5000–6000/sec) environment for citrated whole blood and measures the closure time for platelets to form a plug in a 150 µm aperture in a membrane coated with collagen and either ADP or epinephrine. The PFA-100 method is von Willebrand factor-dependent and is more accurate and precise than the BT method, although the PFA-100 has some drawbacks as a screening test. According to the College of American Pathologists, currently 1218 labs perform the PFA-100 test and 2939 labs perform the BT test.The author’s institution has dropped the template BT and replaced it with the PFA-100. Although the PFA-100 is superior to the BT for von Willebrand disease screening, its role for therapeutic monitoring of various platelet-related disorders remains to be established. Apparently, the PFA-100 has not yet replaced the BT.

[49] Use of Normal B-cells as a Negative Control Significantly Impacts the Results of ZAP-70 Expression. S. Alexanian, F. Chaves, Q. Kong, and S. Song, Geffen School of Medicine, UCLA, Los Angeles, CA.

ZAP-70 serves as a surrogate marker for immunoglobulin VH mutation status (iVHms) in CLL patients and is widely used in molecular risk stratification of CLL. ZAP-70 expression as detected by flow cytometry was originally defined as being positive if >20%; such results can be seen in about 50% of CLL cases with unmutated VH genes. Although the technology of ZAP-70 detection by flow cytometry has evolved significantly since 2003, it remains challenging due to lack of proper controls, meaningful thresholds, and standardized analysis and reporting of results. Most labs use T/NK-cells, where ZAP-70 is constitutively expressed, as the positive and sole control for ZAP-70 levels in neoplastic B-cells. Consequently, far more CLL cases are positive for ZAP-70 than the 50% predicted by iVHms. In order to establish consistent quality control standards and provide insight in standardizing the detection of ZAP-70 by multiparametric flow cytometry, a new gating strategy utilizing simultaneous positive and negative controls has been developed. Data were retrospectively collected from 95 CLL patients who had ZAP-70 studies performed between 01/2005 and 09/2007. The ZAP-70 expression for each case was determined by both the more common approach where T/NK-cells are used as a sole positive control (Strategy 1), and our new gating strategy where normal B-cells are added as negative controls (Strategy 2). The results were compared using the t test. ZAP-70 expression averaged 41.36% when Strategy 1 was used for data analysis, and 13.86% when Strategy 2 was employed (p < 0.001). In conclusion, use of normal B-cells as negative controls, in conjunction with T/NK-cell positive controls, yields dramatically different ZAP-70 results when compared to a traditional gating strategy. This difference is clinically significant as it changes the interpretation of numerous cases from positive to negative. This finding demonstrates the significant impact our new gating strategy has on ZAP-70 results and highlights the critical need for further standardization. In this retrospective study, gene sequencing for iVHms could not be assessed; further studies incorporating such gene sequencing are vital in elucidating the marked differences and standardization of ZAP-70 detection, as well as in determining the most accurate gating strategy.

[50] Platelet Inventory Management through Collaborative Efforts with Engineers Expert in Supply Chain Management. W. Rogers, B. DeMare, F. Khan, Y. Ling, I. Patino, T. Chung, H. Sussmann, F. Erhun, and MJ Fontaine, Stanford University, Stanford, CA.

Stanford Hospital Transfusion Service (SHTS) transfuses 10,406 apheresis platelets per year, supplied primarily by Stanford Blood Center (SBC), but had a platelet outdate rate of 17.3% between November 2005 and April 2006. In order to reduce platelet wastage, collaboration was established between SHTS, SBC, and the Stanford University Department of Management Science and Engineering (MSE). The team from MSE analyzed raw data provided by SBC regarding orders, shipments, usage, and expiration dates of platelets from January 2006 to April 2007 using Microsoft Excel and multiple custom-written macros. Analysis was focused on (1) appropriate usage of platelets in relation to expiration date, (2) forecasting platelet usage, and (3) inventory organization. This initial analysis, performed using a first-in, first-out mathematical model, showed 34.8% unexplained platelet allocations. Information regarding ABO and Rh type, CMV status, and HLA antigen requirements was relayed to the MSE team to help explain the perceived misallocation and to guide future analysis. The distribution of either ABO/Rh type or CMV positive/negative status was equivalent for both outdated vs transfused platelet categories, reflective of an imbalanced donor base/customer demand. SHTS investigation into platelet ordering practices revealed a standing order of 32-"current inventory" without considering the daily transfusion rate which was 31.2 per day vs 20.7 on the weekend. The first corrective action was to decrease platelet collections on Thursdays by 10%. Subsequently, the rate of outdated platelets dropped from 17.3% to 10.3%. Furthermore this collaborative, multidisciplinary effort between hospital, blood bank, and engineers with expertise in supply chain management resulted in implementation of process improvement based on an electronic system that monitors platelet donations based on platelet transfusion rates and inventories.

[51] Implementation of Pharmacogenomic Biomarkers into Clinical Practice. Alan H. B. Wu, University of California, San Francisco, CA

Medical errors contribute to significant morbidity and mortality in the United States each year. The majority of these errors are caused by unanticipated adverse reactions (ADRs) to therapeutic drugs. Pharmacogenomics has the promise of reducing medication errors. While personalized medicine has been discussed as a new frontier for laboratory medicine for >10 yr, it has only been through the recent Food and Drug Administration (FDA) product relabeling initiative that pharmacogenomics has evolved from an idealized concept into real clinical practice today. Warning labels recommending genotyping prior to initial drug use introduces a medicolegal concern to selected therapeutic drugs that have significant inter-individual pharmacokinetic and pharmacodynamic variability. Properly implemented clinical pharmacogenomics will reduce the incidence of adverse events, enable the selection of the best drug for treatment of a disease, and/or determine the best dosage to achieve the desired therapeutic goal. The requirements for pharmacogenomics include the availability of FDA-cleared testing platforms and reagents, a clinical need for testing, adequate test reimbursement, and a mechanism to report and interpret results. It is important to show that pharmacogenomic testing leads to "actionable" medical decisions that can either improve drug efficacy or avoid adverse events, while demonstrating cost effectiveness (eg, reduced hospital length-of-stay, unnecessary outpatient visits or procedures). Pharmacogenomic tests of the highest interest include polymorphisms in thiopurine methyltransferase for azothioprine and 6-mercaptopurine, cytochrome P450 2C9 and vitamin K reductase complex for warfarin, UDP-glucuronosyltransferase 1A1 for irinotecan, 2D6 for tamoxefin, and HLA-B*5701, *5801, and *1502 for abacavir, allopurinol, and carbamazepine, respectively. A successful pharmacogenomic program is a multidisciplinary effort of basic pharmaceutical sciences, clinical pharmacy, medical genetics, and laboratory medicine. Each stakeholder must be consulted prior to initiating a clinical testing service.

[52] ESR Spin-Labeling in Biomedicine and Pharmacology. Henry M. Zeidan and Iman Zeidan, University of Southern Nevada School of Pharmacy, Henderson, NV

Applications of the spin-label and spin probe techniques in the applied fields of clinical and biomedical research are emerging. Specifically, these techniques are being applied to areas of pharmacological assay in drug treatment, detection of drug metabolites, and the mechanism of drug interaction with cellular constituents. We illustrate by means of specific examples the directions that electron paramagnetic resonance spin labeling and the spin probe are taking in biomedicine. The main objectives of this presentation are: (1) to address the basic principles for the application of spin-labeling in biomedicine and pharmacology, (2) to provide examples about the application of spin-labeling and magnetic resonance spectroscopy in exploring the active site of enzymes and proteins of clinical interest, (3) to address the application of the dual spin-label methodology at the molecular level in medical research, and (4) to assess the mode of action of drugs as explored by spin-labeling methods.

[53] Alveolar-arterial Oxygen Gradient ( A-a), Pneumonia Severity Index (PSI), and Outcomes in Patients Hospitalized with Community-Acquired Pneumonia (CAP). M. Q. Moammar, H. M. Azam, A. I. Blamoun, A. O. Rashid, M. Ismail, M. A. Khan, and V. A. DeBari, School of Graduate Medical Education, Seton Hall University, South Orange, NJ.

The A-a provides a useful physiological assessment of ventilation-perfusion abnormalities but has not been studied as a potential index for decisions about patients with CAP. The objectives of this study were: (a) to examine the association between the A-a and PSI and (b) to determine how well each of these indices predicted clinical outcomes, length of stay (LOS) and survival, in a cohort of patients hospitalized with CAP. This study was conducted at a 750-bed urban teaching hospital. It examined a retrospective cohort of 255 CAP patients (125 male, 130 female; age: 21 to 99 yr; median: 71 yr; IQR: 53–82 yr) developed over a 2-yr period. Association between the CAP and A-a gradient was examined by linear and non-linear regression and non-parametric correlation, and for subjects bifurcated by low-risk/moderate-to-high risk, 2 logistic models, receiver-operator characteristic curve analysis and binary logistic regression. The decision levels (DL) for both PSI and A-a were compared as predictors of LOS and survival. Correlation between PSI and A-a was strong ( = 0.76; p <0.0001) and was best modeled by a curvilinear relationship (2nd order polynomial; r² = 0.51). Both logistic models indicated an excellent association (p <0.001) of A-a and PSI and yielded an optimal DL for the A-a of <89 mm Hg. Inter-test agreement of A-a with PSI was 76.9% ( = 0.60; 95% CI: 0.47 to 0.72; p < 0.0001). At <89 mmHg, the odds ratios for LOS were similar to those at PSI 90 in predicting LOS in the range 3–7 days, inclusive. There was no significant difference in the ability of A-a and PSI to predict survival for either the low-risk (p = 0.363) or high-risk group (p = 0.951). The A-a correlates well with PSI and performs comparably in predicting 2 major outcomes (length of stay and survival) in subjects with CAP.

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