Jamie Weydert

Low-Level Endotoxin Induces Potent Inflammatory Activation of Human Blood Vessels: Inhibition by Statins

Background—Low-level endotoxemia (ie, ≥50 pg/mL) in apparently healthy subjects was recently identified as a powerful, independent risk factor for atherosclerosis. Methods and Results—We treated human saphenous veins (HSVs) with low levels of endotoxin. Release of the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) was measured by ELISA. Superoxide was determined by using the fluorescent probe dihydroethidium (HE), and monocyte binding was assessed with calcein-labeled U-937 cells. Three- to 4-fold increases in MCP-1 and IL-8 release were observed at endotoxin concentrations of 100 pg/mL; these increases were inhibited by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin. Studies in cultured endothelial cells suggest that the mechanism is related to inhibition of isoprenylation (ie, geranylgeranylation) rather than cholesterol formation. Endotoxin produced dose-dependent increases in HE fluorescence that were inhibited by the superoxide dismutase mimics Tiron and MnTBAP. Endotoxin potently induced U-937 cell binding to HSV; binding was inhibited by both Tiron and atorvastatin. Toll-like receptor-4 expression was detected in cultured HSV endothelial and smooth muscle cells and in intact HSV. Conclusions—Clinically relevant levels of endotoxin, as reported in ambulatory populations, have profound inflammatory effects on intact HSV. Inhibition of endotoxin-induced vascular inflammation might contribute to the beneficial effects of statins in treating atherosclerosis.

Mandatory Second Opinion in Surgical Pathology Referral Material : Clinical Consequences of Major Disagreements

Second opinion in pathology is intended to expose clinically significant errors that have a direct impact on patient care. Before definitive treatment of referred patients, our institution requires a second opinion of outside surgical pathology slides. We sought to determine if this local standard of practice has a measurable impact on patient care via clinical and pathologic follow-up. 5629 second opinion surgical pathology cases seen at the University of Iowa Hospitals and Clinics were studied. Each case was classified as: no diagnostic disagreement, minor diagnostic disagreement, or major diagnostic disagreement by the second opinion pathologist at the time of referral. A major diagnostic disagreement was defined as a change in pathologic diagnosis with potential for significant change in treatment or prognosis. Major diagnostic disagreements were categorized by organ system and according to the clinical significance of the changed diagnosis based on clinical and pathologic follow-up. Second opinion surgical pathology resulted in 132 (2.3% of total cases) major diagnostic disagreements and 507 (9.0%) cases with minor disagreements. The organ systems involved in the majority of the major disagreements were the female reproductive tract (32), gastrointestinal tract (27), and skin (24). Of the 132 major diagnostic disagreements, 68 (1.2% of total cases reviewed) prompted changes in the clinical management as a result of the second opinion interpretation. These findings support the idea that mandatory second opinion is an important part of patient care in the referral setting.

Subinvolution of the Placental Site as an Anatomic Cause of Postpartum Uterine Bleeding: A Review

CONTEXT Subinvolution of the placental site is an anatomic cause of delayed postpartum uterine bleeding that may be underrecognized by general surgical pathologists. OBJECTIVE To review the physiology of uteroplacental arterial development and normal postpartum involution, and to present the characteristic clinical and histopathologic features of subinvolution. DATA SOURCES Literature review (MEDLINE via PubMed and Ovid) regarding the pathology and pathophysiology of placental site subinvolution. Review of the clinical and pathologic characteristics of our own institutions previously diagnosed cases of subinvolution from hysterectomy and endomyometrial curettage specimens. CONCLUSIONS Surgical pathologists must be aware of the cardinal histopathologic findings of subinvolution, and this diagnosis must be considered in every postpartum curettage or hysterectomy specimen presented to the surgical pathologist. Subinvolution of the placental site is an important diagnosis, as this process implies an idiopathic cause, rather than an iatrogenic cause, of postpartum uterine bleeding. The etiology of placental site subinvolution remains poorly characterized.

Comparison of fungal culture versus surgical pathology examination in the detection of Histoplasma in surgically excised pulmonary granulomas.

CONTEXT Granulomatous pulmonary nodules are common in areas endemic for Histoplasma infection, and may require surgical excision to exclude neoplasia. Surgeons may elect to routinely send material directly to the clinical microbiology laboratory for fungal and mycobacterial cultures. OBJECTIVE To determine if tissue from surgically excised pulmonary granulomatous nodules removed from patients in a geographic area endemic for Histoplasma infection should be routinely submitted for fungal culture. DESIGN Retrospective review and comparison of surgical pathology histochemical findings and clinical microbiology results of 30 surgical (wedge) lung excisions that demonstrated granulomatous nodule at the time of frozen section. RESULTS Twenty cases demonstrated fungal organisms consistent with Histoplasma species via histochemical fungal stains. Of these 20 cases, 17 were tested in the microbiology laboratory using direct smear examination and fungal culture; Histoplasma was detected in 1 case (1/17). Eight cases revealed no organisms by surgical pathology. Of these, 6 were tested in the microbiology laboratory, and all 6 were negative by culture and direct smear (0/6). The remaining 2 cases demonstrated organisms other than Histoplasma by surgical pathology examination. CONCLUSIONS Surgical pathology examination of granulomatous pulmonary nodules detected Histoplasma organisms with greater sensitivity than culture and direct smear. There were no false-negative surgical pathology diagnoses when compared with microbiological results. These findings suggest that it is not necessary to routinely send material from solitary pulmonary granulomas for fungal culture when the material is removed from immunocompetent patients in a geographic area endemic for histoplasmosis.

A preliminary diagnosis service provides prospective blinded dual-review of all general surgical pathology cases in an academic practice.

Quality assurance of diagnostic accuracy in surgical pathology is an important part of a pathologists total quality management program. At our academic institution, the quality of diagnostic accuracy is monitored via dual-review of every general surgical pathology case, which accounts for nearly 20,000 cases per year. This comprehensive dual-review is achieved by operating a preliminary diagnosis service, staffed by a senior or board eligible resident. Analysis of a portion of our dual-review data (6300 cases) demonstrates an overall diagnostic concordance rate of 95.4% and a clinical major discrepancy rate of 0.29% between the preliminary diagnosis and staff pathologist diagnosis, comparable to other published rates. The incorporation of a preliminary diagnosis service into our academic surgical pathology practice has proven to be beneficial with regard to quality assurance and resident education. Other academic institutions may similarly benefit from the addition of such a service.

Regulation of 14-3-3σ expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation

Increased 14-3-3sigma expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3sigma mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpGs in the genes CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2-7, which did not express 14-3-3sigma. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3sigma expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3sigma expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3sigma expression in thyroid carcinomas is regulated by CpG island hypermethylation.

Telomerase Reverse Transcriptase Subunit Expression Is Associated with Chondrosarcoma Malignancy

Expression of the telomerase reverse transcriptase subunit telomerase reverse transcriptase gene is associated with most human malignancies. Because telomerase reverse transcriptase is rarely expressed in normal tissue, its presence in pathologic specimens is considered a marker of transformed cells. Moreover, high levels of expression have been correlated with poor prognosis in many cancers. Although telomerase activity has been found in chondrosarcomas, its prognostic significance in these malignant cartilage tumors is unknown. Malignancy in cartilage-derived tumors is assessed routinely by histomorphologic grading, but even well differentiated, low-grade lesions can metastasize. This unpredictable behavior greatly complicates the clinical treatment of cartilage tumors, making better prognostic indicators desirable. To address this issue we used immunohistochemistry to compare telomerase reverse transcriptase expression in a collection of 61 tumors consisting of malignant chondrosarcomas of varying grade and benign enchondromas. Associated case histories were reviewed to test the hypothesis that telomerase reverse transcriptase expression levels correlated with subsequent tumor recurrence. We found that the relative abundance of telomerase reverse transcriptase-expressing cells correlated significantly with grade and recurrence. These findings indicate that telomerase reverse transcriptase immunostaining may be a useful adjunct to the conventional three-level grading system.

RABL6A Promotes Oxaliplatin Resistance in Tumor Cells and Is a New Marker of Survival for Resected Pancreatic Ductal Adenocarcinoma Patients.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early recurrence following pancreatectomy, rapid progression, and chemoresistance. Novel prognostic and predictive biomarkers are urgently needed to both stratify patients for clinical trials and select patients for adjuvant therapy regimens. This study sought to determine the biological significance of RABL6A (RAB, member RAS oncogene family-like protein 6 isoform A), a novel pancreatic protein, in PDAC. Analyses of RABL6A protein expression in PDAC specimens from 73 patients who underwent pancreatic resection showed that RABL6A levels are altered in 74% of tumors relative to adjacent benign ductal epithelium. Undetectable RABL6A expression, found in 7% (5/73) of patients, correlated with improved overall survival (range 41 to 118 months with 3/5 patients still living), while patients with RABL6A expression had a worse outcome (range 3.3 to 100 months, median survival 20.3 months) (P = 0.0134). In agreement with those findings, RABL6A expression was increased in pancreatic cancer cell lines compared to normal pancreatic epithelial cells, and its knockdown inhibited pancreatic cancer cell proliferation and induced apoptosis. Moreover, RABL6A depletion selectively sensitized cells to oxaliplatin-induced arrest and death. This work reveals that RABL6A promotes the proliferation, survival, and oxaliplatin resistance of PDAC cells, whereas its loss is associated with extended survival in patients with resected PDAC. Such data suggest RABL6A is a novel biomarker of PDAC and potential target for anticancer therapy.

Site‐directed mutations of human hemoglobin at residue 35β: A residue at the intersection of the α1β1, α1β2, and α1α2 interfaces

Because Tyr35β is located at the convergence of the α1β1, α1β2, and α1α2 interfaces in deoxyhemoglobin, it can be argued that mutations at this position may result in large changes in the functional properties of hemoglobin. However, only small mutation‐induced changes in functional and structural properties are found for the recombinant hemoglobins βY35F and βY35A. Oxygen equilibrium‐binding studies in solution, which measure the overall oxygen affinity (the p50) and the overall cooperativity (the Hill coefficient) of a hemoglobin solution, show that removing the phenolic hydroxyl group of Tyr35β results in small decreases in oxygen affinity and cooperativity. In contrast, removing the entire phenolic ring results in a fourfold increase in oxygen affinity and no significant change in cooperativity. The kinetics of carbon monoxide (CO) combination in solution and the oxygen‐binding properties of these variants in deoxy crystals, which measure the oxygen affinity and cooperativity of just the T quaternary structure, show that the ligand affinity of the T quaternary structure decreases in βY35F and increases in βY35A. The kinetics of CO rebinding following flash photolysis, which provides a measure of the dissociation of the liganded hemoglobin tetramer, indicates that the stability of the liganded hemoglobin tetramer is not altered in βY35F or βY35A. X‐ray crystal structures of deoxy βY35F and βY35A are highly isomorphous with the structure of wild‐type deoxyhemoglobin. The βY35F mutation repositions the carboxyl group of Asp126α1 so that it may form a more favorable interaction with the guanidinium group of Arg141α2. The βY35A mutation results in increased mobility of the Arg141α side chain, implying that the interactions between Asp126α1 and Arg141α2 are weakened. Therefore, the changes in the functional properties of these 35β mutants appear to correlate with subtle structural differences at the C terminus of the α‐subunit.

Neoplastic pulmonary cytology: why all the fuss over "NSCLC"?

The recent accumulation of epidemiologic and molecular research focused on nonsmall cell lung cancer (NSCLC) in combination with the development of novel/targeted therapies has caused pathologists to critically evaluate the issue of the histologic subclassification of such tumors. The use of the generic term NSCLC, once fully endorsed and accepted by our oncology colleagues, has recently been taken to task, and we are being asked, ‘‘Can you tell if it is a squame or an adeno?’’ by our clinical partners on a routine basis. This shift in framework in the diagnostic reporting of lung cancer poses practical challenges in the arena of diagnostic pulmonary cytology. Although cytology is an excellent discriminator of small cell carcinoma versus nonsmall cell carcinoma, it is less reliable in the subclassification of NSCLC. Furthermore, we are being asked to provide molecular-level information on small biopsy specimens (epidermal growth factor receptor [EGFR] gene mutations, KRAS gene mutation, ALK-EML4 gene fusion, etc), which is even more problematic for many cytologic specimens (aspirates, brushes, washes) that may have very limited residual specimen for testing. How does all this impact the day to day sign out of pulmonary cytology specimens? A few points for consideration are presented below. The reader can draw his or her own conclusions, and we will welcome a spirited and critical debate in this journal. 1. What are the most important goals of sampling a pulmonary mass via aspiration or exfoliative cytology techniques? The primary goal is to establish, with certainty, the biologic nature of the mass; benign (eg, inflammatory) versus malignant. If a lesion can be placed into the ‘‘malignant’’ category, then the second objective is to determine whether it is a primary or metastatic lesion. Ancillary studies, such as immunocytochemistry, can be very helpful in this regard, particularly when the clinical picture is unclear, and are worthwhile uses of residual material. When the lesion is a primary lung tumor, the distinction between small cell and nonsmall cell histology is important for proper classification. These diagnostic objectives have not changed despite the molecular-driven changes in pulmonary oncologic practice. 2. What is the clinical significance of distinguishing NSCLC subtypes on cytologic specimens? The principal (albeit not sole) driver of the interest in specifying subtypes of NSCLC in pathology reports is the advent of bevacizumab (Avastin), a vascular endothelial growth factor (VEGF) receptor inhibitor, as a treatment of NSCLC. Briefly, bevacizumab was found to have an unacceptable risk of life-threatening pulmonary hemorrhage in patients who have squamous cell (compared with nonsquamous cell) morphology. Data on