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Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Considered broadly, syphilis can be conceptualized as a dualistic process in which spirochete-driven inflammation, the cause of clinical manifestations, coexists to varying extents with bacterial persistence. Inflammation is elicited in the tissues, along with the persistence of spirochetes to keep driving a robust immune response while evading host defenses; this duality is best exemplified during the florid, disseminated stage called secondary syphilis (SS). SS lesions typically contain copious amounts of spirochetes along with a mixed cellular infiltrate consisting of CD4+ T cells, CD8+ T cells, NK cells, plasma cells, and macrophages. In the rabbit model, Tp are cleared by macrophages via antibody-mediated opsonophagocytosis. Previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. Herein, we used monocyte-derived macrophages to study Tp-macrophage interactions ex vivo. In the absence of HSS, monocyte-derived macrophages internalized low numbers of Tp and secreted little cytokine (e.g., TNF). By contrast, these same macrophages internalized large numbers of unopsonized Borrelia burgdorferi and secreted robust levels of cytokines. Maturation of macrophages with M-CSF and IFNγ resulted in a macrophage phenotype with increased expression of HLA-DR, CD14, inducible nitric oxide synthase, TLR2, TLR8, and the Fcγ receptors (FcγR) CD64 and CD16, even in the absence of LPS. Importantly, IFNγ-polarized macrophages resulted in a statistically significant increase in opsonophagocytosis of Tp accompanied by enhanced production of cytokines, macrophage activation markers (CD40, CD80), TLRs (TLR2, TLR7, TLR8), chemokines (CCL19, CXCL10, CXCL11), and TH1-promoting cytokines (IL-12, IL-15). Finally, the blockade of FcγRs, primarily CD64, significantly diminished spirochetal uptake and proinflammatory cytokine secretion by IFNγ-stimulated macrophages. Our ex vivo studies demonstrate the importance of CD64-potentiated uptake of opsonized Tp and suggest that IFNγ-activated macrophages have an important role in the context of early syphilis. Our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.

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Biliary brush cytology is an important diagnostic tool in the evaluation of biliary strictures. Here, we evaluated 64 patients with biliary strictures who underwent endoscopic retrograde cholangiopancreatography with bile duct brushings. We assessed the utility of combining routine Papanicolaou-stained cytologic evaluation with immunocytochemical expression of insulin-like growth factor mRNA-binding protein-3 (IMP3). Definitive diagnoses were obtained via tissue resection/autopsy, biopsy, fine needle aspiration, or clinical progression of disease. Thirty-nine of the 64 patients were ultimately diagnosed with malignancy. The sensitivity of routine cytology for the detection of malignancy was 33.3%, immunocytochemical-IMP3 expression was 64.1%, and the combined sensitivity was 71.8%. The specificity of each method was 100%. The sensitivity of IMP3 immunocytochemical staining in the detection of malignancy in biliary brushings was superior to routine PAP-stained cytologic evaluation. Moreover, the combined use of biliary brushing cytology and IMP3 immunohistochemistry proved superior to the use of either method alone.

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We describe a rare case of esophageal polypoid dysplasia with gastric phenotype and focal intramucosal carcinoma associated with Barrett's esophagus. A 69-year-old man with a long history of gastroesophageal reflux disease was initially seen at an outside institution for evaluation of significant dysphagia. Screening upper gastrointestinal endoscopic evaluation revealed a large intraluminal polypoid lesion occluding the distal portion of the esophagus. Surgery was performed with resection of the distal esophagus and proximal stomach. The histopathologic examination of this lesion revealed an exuberant polypoid gastric epithelium with areas of low-grade dysplasia, high-grade dysplasia, and focal intramucosal carcinoma. A few residual foci of specialized intestinal metaplasia consistent with Barrett's esophagus without dysplasia were identified at the proximal and distal ends of the lesion. Immunohistochemically, this lesion revealed a pattern of expression of apomucins (MUC5AC diffusely positive, MUC1 and MUC6 focally positive, and MUC2 negative) consistent with a gastric foveolar phenotype. In addition, in the dysplastic areas, there was high Ki-67 labeling index and no overexpression of p53 protein. In our opinion, this case represents a precursor lesion of an extremely well-differentiated adenocarcinoma of gastric foveolar phenotype that has been previously documented in the stomach and in the duodenum and that now for the first time we report in the esophagus in association with Barrett's intestinal metaplasia.

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Biliary tract brush cytology is one of the favored methods of evaluating lesions of the pancreatobiliary tract. However, although its specificity has been reported to be high (91-100%), the sensitivity is lower (30-88%). In this study we applied KOC and S100A4 protein immunocytochemistry to assess their potential use as adjunct markers in differentiating benign from malignant cells, and improve the diagnostic sensitivity of this method for pancreatobiliary malignancies. The authors examined KOC and S100A4 protein expression in 44 alcohol-fixed cytology specimens obtained by biliary brushings. Diagnoses included: (1) benign/atypical favor reactive (20 cases), (2) atypical/not diagnostic of malignancy (3 cases), and (3) suspicious for malignancy/malignant (21 cases). Alcohol-fixed Papanicolaou-stained slides (PAP) were stained with monoclonal antibody to KOC/L523S and polyclonal antibody to S100A4 protein. Results were recorded as negative or positive. Twenty-four cases were confirmed positive for adenocarcinoma and 20 cases were negative. The sensitivity and specificity of cytology was 83 and 95%, KOC showed a sensitivity of 92% and specificity of 95%. S100A4 protein showed a sensitivity of 79% and a specificity of 95%. The combined use of KOC and S100A4 protein showed a sensitivity of 100% and a specificity of 95%, respectively. The concurrent use of KOC and S100A4 protein improves the diagnostic sensitivity of biliary brushings cytology and demonstrates similar specificity as cytology alone in the diagnosis of pancreatobiliary malignancy.

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We evaluated the immunocytochemical expression of GPC3 in archival material obtained from fine needle aspiration of hepatic lesions to assess the sensitivity and specificity of this marker in cytological material and its potential diagnostic utility in differentiating hepatocellular carcinoma (HCC) from other primary benign or malignant hepatic tumors and from metastatic lesions in the liver. Forty-nine FNAs of the liver obtained between January 2000 and June 2006 were identified from our cytology files. Cytological diagnoses (confirmed by tissue diagnosis and/or clinical follow-up) included: 7 adenomas, 1 focal nodular hyperplasia (FNH), 24 HCCs, and 17 metastatic tumors. On the basis of the histological, clinical and/or radiological follow-up, 20 of 24 (83.3%) FNAs confirmed positive for HCC-expressed GPC3. All the seven adenomas and the only FNH were negative for GPC3. Sixteen out of seventeen metastatic malignancies were negative for GPC3. The only case expressing GPC3 was an anaplastic carcinoma with neuroendocrine features of unknown origin. In this study, the sensitivity of GPC3 in the diagnosis of HCC in the cytological material was 83.3%, the specificity 96%, the positive predictive value (PPV) 95% and negative predictive value (NPV) was 85.7%. Immunocytochemical staining for GPC3 in alcohol-fixed FNA material is a highly sensitive and specific method capable of distinguishing HCC from other benign and malignant hepatic lesions and from the great majority of metastatic lesions.

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We evaluated the immunocytochemical (ICC) expression of K homology domain containing protein overexpressed in cancer (KOC) in pancreatic endoscopic ultrasound-guided fine needle aspirates (EUS-FNAs) to assess its potential use as an adjunct in differentiating nonneoplastic (GI epithelium) and benign neoplastic epithelia (benign epithelial pancreatic neoplasms) from pancreatic adenocarcinoma cells. Forty-eight cases of EUS-FNAs with histological and/or clinical follow-up data were selected for this study. Alcohol-fixed and PAP-stained slides were stained with monoclonal antibody to KOC/L523S (clone 69.1). Results were recorded as negative or positive. KOC expression was present in 35/40 (88%) of adenocarcinomas (Ac) and was negative in all eight benign cases. The sensitivity and specificity were as follows: cytology 85 and 100%, KOC 88 and 100%; combination of cytology and KOC 95 and 100%. We conclude that KOC ICC expression on alcohol-fixed smears along with cytology improves the sensitivity of EUS-FNAs in the diagnosis of pancreatic Ac, and KOC reactivity is especially useful in differentiating Ac from nonneoplastic gastrointestinal epithelium and benign neoplastic epithelia.

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The objective of this study was to evaluate the coexpression patterns of hormonal markers in breast cancer tissue and their relationship with pathologic characteristics and epidemiologic risk factors. We evaluated the expression of 17 markers by immunohistochemistry in 842 invasive breast carcinomas collected in a population-based case-control study conducted in Poland. Based on marker correlations, factor analysis identified four major coexpression patterns (factors): "nuclear receptor factor" [estrogen receptor (ER)-alpha, progesterone receptor, androgen receptor, cyclin D1, and aromatase], "estrogen metabolism/ER-beta factor" (ER-beta, peroxisome proliferator-activated receptor-gamma, steroid sulfatase, estrogen sulfonotransferase, and cytochrome P450 1B1), "HER2 factor" (human epidermal growth factor receptor 2, E-cadherin, cyclooxygenase-2, aromatase, steroid sulfatase), and "proliferation factor" (cytokeratin 5, cytokeratin 5/6, epidermal growth factor receptor, P53). Three of these factors corresponded to molecular subtypes previously defined by expression profiling; however, the estrogen metabolism/ER-beta factor seemed to be distinctive. High scores for this factor were associated with high tumor grade (P heterogeneity = 0.02), younger age at menarche (P heterogeneity = 0.04), lower current body mass index among premenopausal women (P heterogeneity = 0.01), and older age at menopause (P heterogeneity = 0.04). High scores for the proliferation factor were also associated with early menarche (P heterogeneity < 0.0001), and in contrast to the estrogen metabolism/ER-beta factor, higher current body mass index among premenopausal women (P heterogeneity = 0.03). Our analysis of hormonal pathway markers independently confirmed several previously defined molecular subtypes identified by gene expression profiling and augmented these findings by suggesting the existence of additional relationships related to ER-beta and enzymes involved in hormone metabolism.

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The distinction between malignant mesothelioma and adenocarcinoma is a diagnostic challenge in cytologic specimens of effusion fluids. As for today, no single antibody has demonstrated absolute sensitivity or specificity for Mesothelioma. D2-40 and podoplanin have recently been recognized to stain mesothelial cells. Our aim for this study was to evaluate the utility of these two markers as indicators of mesothelial cells using cell blocks by comparison with two other established mesothelial markers. A total of 40 cell blocks of effusion fluids including cases of epithelioid mesotheliomas, metastatic carcinomas and benign cases with reactive mesothelial cells were selected. A panel of immunostains including D2-40, podoplanin, CK5, and calretinin was performed. D2-40 and podoplanin were positive in 100% of mesothelioma cases in comparison to metastatic adenocarcinoma cases where the positivity was 0%. It is concluded that D2-40 and podoplanin are very useful markers for mesotheliomas. Since these markers are extremely helpful in differentiating epithelioid mesothelioma from metastatic adenocarcinoma, they shall be a valuable addition to the battery of markers used to differentiate the two entities.

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Evidence suggests that breast cancer hormone receptor status varies by etiologic factors, but studies have been inconsistent. In a population-based case-control study in Poland that included 2,386 cases and 2,502 controls, we assessed ER-alpha and PR status of tumors based on clinical records according to etiologic exposure data collected via interview. For 842 cancers, we evaluated ER-alpha, ER-beta, PR and HER2 levels by semiquantitative microscopic scoring of immunostained tissue microarrays and a quantitative immunofluorescence method, automated quantitative analysis (AQUAtrade mark). We related marker levels in tumors to etiologic factors, using standard regression models and novel statistical methods, permitting adjustment for both correlated tumor features and exposures. Results obtained with different assays were generally consistent. Receptor levels varied most significantly with body mass index (BMI), a factor that was inversely related to risk among premenopausal women and directly related to risk among postmenopausal women with larger tumors. After adjustment for correlated markers, exposures and pathologic characteristics, PR and HER2 AQUA levels were inversely related to BMI among premenopausal women (p-trend = 0.01, both comparisons), whereas among postmenopausal women, PR levels were associated directly with BMI (p-trend = 0.002). Among postmenopausal women, analyses demonstrated that BMI was related to an interaction of PR and HER2: odds ratio (OR) = 0.86 (95% CI = 0.69-1.07) for low PR and HER2 expression vs. OR = 1.78 (95% CI = 1.25-2.55) for high expression (p-heterogeneity = 0.001). PR and HER2 levels in breast cancer vary by BMI, suggesting a heterogeneous etiology for tumors related to these markers.

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Analysis of gene expression data suggests that breast cancers are divisible into molecular subtypes which have distinct clinical features. This study evaluates whether pathologic features and etiologic associations differ among molecular subtypes. We evaluated 804 women with invasive breast cancers and 2,502 controls participating in a Polish Breast Cancer Study. Immunohistochemical stains for estrogen receptor alpha, progesterone receptor, human epidermal growth factor receptors (HER2 and HER1), and cytokeratin 5 were used to classify cases into five molecular subtypes: luminal A, luminal B, HER2-expresing, basal-like, and unclassified. Relative risks were estimated using adjusted odds ratios and 95% confidence intervals. We observed that compared with the predominant luminal A tumors (69%), other subtypes were associated with unfavorable clinical features at diagnosis, especially HER2-expressing (8%) and basal-like (12%) tumors. Increasing body mass index significantly reduced the risk of luminal A tumors among premenopausal women (odds ratios, 0.71; 95% confidence intervals, 0.57-0.88 per five-unit increase), whereas it did not reduce risk for basal-like tumors (1.18; 0.86-1.64; P(heterogeneity) = 0.003). On the other hand, reduced risk associated with increasing age at menarche was stronger for basal-like (0.78; 0.68-0.89 per 2-year increase) than luminal A tumors (0.90; 0.95-1.08; P(heterogeneity) = 0.0009). Although family history increased risk for all subtypes (except for unclassified tumors), the magnitude of the relative risk was highest for basal-like tumors. Results from this study have shown that breast cancer risk factors may vary by molecular subtypes identified in expression studies, suggesting etiologic, in addition to clinical, heterogeneity of breast cancer.

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We evaluated the expression of S100A4 protein and mesothelin in dysplasia and carcinoma of the extrahepatic bile duct (EBD) and their potential use as adjuncts for differentiating carcinomatous and significant high-grade dysplastic epithelium from reactive or inflammatory glandular atypia of the EBD. We used immunohistochemical analysis on formalin-fixed tissue sections from 10 cases of carcinoma, 6 cases of high-grade dysphasia (HGD), 4 cases of low-grade dysplasia (LGD), and 10 cases of benign or reactive or inflammatory epithelium from the EBD. Expression of S100A4 protein was observed in 8 invasive carcinomas (80%), 5 HGD/carcinoma in situ cases (83%), and 0 LGDs. Mesothelin was expressed in 5 (50%) of 10 adenocarcinomas, 1 (17%) of 6 HGD/adenocarcinoma in situ cases, and 0 LGDs. No case of normal or reactive epithelium was positive for S100A4 protein or mesothelin. Mesothelin has moderate sensitivity and high specificity, whereas S100A4 protein is sensitive and specific for the identification of carcinoma and HGD of the EBD. S100A4 protein alone or combined with mesothelin can be used as an adjunct in differentiating carcinomatous and significant high-grade dysplastic epithelium from LGD and reactive or inflammatory glandular atypia of the EBD.

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Definitive diagnosis of B-cell non-Hodgkin lymphomas often requires demonstration of B-cell monoclonality. Immunohistochemical detection of monotypic immunoglobulin light chain expression, and thereby B-cell monoclonality, may be accomplished readily using fresh cell suspensions or frozen tissue sections. However, immunohistochemical detection of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues is more difficult; with few exceptions, techniques suitable for formalin-fixed, paraffin-embedded tissues are not widely available. This report describes and validates a method for detecting immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues using a heat-induced epitope retrieval technique. This method was evaluated in a series of 113 cases of B-cell non-Hodgkin lymphoma, including 73 cases with correlative flow cytometric immunophenotyping data. Monotypic light chain expression was demonstrated in 91 (81%) of 113 cases, including several small core biopsy specimens with extremely limited tissue. Compared with the reference method (flow cytometric immunophenotyping), the specificity of the assay was 100%. Interobserver reproducibility was excellent, with 87% concordance between two independent observers categorizing cases as indeterminate, suggestive or diagnostic of kappa or lambda light chain restriction (Cohen kappa statistic: 0.81). In summary, the described method permits demonstration of immunoglobulin light chain expression in formalin-fixed, paraffin-embedded tissues in approximately 80% of cases of B-cell non-Hodgkin lymphoma with a high degree of specificity and excellent interobserver reproducibility. The assay is sufficiently robust for diagnostic use in small biopsies in which fresh tissue is unavailable.

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