Richard W. Cartun

Differences in Risk Factors for Breast Cancer Molecular Subtypes in a Population-Based Study

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 α, 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; Pheterogeneity = 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; Pheterogeneity = 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. (Cancer Epidemiol Biomarkers Prev 2007;16(3):439–43)

Immunocytochemical evidence of Listeria, Escherichia coil, and Streptococcus antigens in Crohn's disease

Abstract Background/Aims Infectious agents have long been suspected of playing a role in the initiation of Crohns disease. The objective of this study was to search for likely microbial agents in diseased tissues using immunocytochemical techniques. Methods Intestines and mesenteric lymph node specimens of 21 patients from two French families with a high frequency of Crohns disease and from Connecticut were studied. The microbial agents searched for included Bacteroides vulgatus, Borrelia burgdorferi, Escherichia coil, Listeria monocytogenes, Streptococcus spp., bovine viral diarrhea virus, influenza A virus, measles virus, parainfluenza virus, and respiratory syncytial virus. Results Seventy-five percent of the patients with Crohns disease (12 of 16) were positively labeled with the antibody to Listeria. Macrophages and giant cells immunolabeled for this antigen were distributed underneath ulcers, along fissures, around abscesses, within the lamina propria, in granulomas, and in the germinal centers of mesenteric lymph nodes. In addition, 57% (12 of 21) of the cases contained the E. coli antigen, and 44% (7 of 16) contained the streptococcal antigen. The immunolabeling for the latter two agents also occurred within macrophages and giant cells, distributed in a pattern similar to that of Listeria antigen. Conclusions The results suggest that Listeria spp., E. coli, and streptococci, but not measles virus, play a role in the pathogenesis of Crohns disease.

Bacterial Translocation to Mesenteric Lymph Nodes Is Increased in Cirrhotic Rats With Ascites

BACKGROUND/AIMS Cirrhotic patients are predisposed to develop spontaneous bacteremias and/or peritonitis, mainly caused by enteric bacteria. The aim of this study was to investigate if bacterial translocation, which is the passage of bacteria from the intestinal lumen to regional lymph nodes and/or the systemic circulation, is increased in a rat model of cirrhosis. METHODS Rats were studied after 12-16 weeks of CCl4 inhalation, when samples of mesenteric lymph nodes, blood, liver, and spleen for standard bacteriologic cultures and a fragment of colon and liver for histology were obtained. Immunostaining of the cecum was performed using a polyclonal anti-Escherichia coli antibody. RESULTS A significantly greater proportion of rats with cirrhosis and ascites (5 of 9; 56%) had positive mesenteric lymph node cultures compared with cirrhotics without ascites (0 of 9) and normal controls (0 of 12) (P < 0.01). In one cirrhotic rat, E. coli was isolated from both mesenteric lymph nodes and ascites. Rats with cirrhosis and ascites had significantly greater cecal submucosal edema and inflammation than rats with no ascites and controls. Immunoreactivity with E. coli was present in the cecal wall in 3 of 5 animals with E. coli translocation to mesenteric lymph nodes. CONCLUSIONS In cirrhotic rats, bacterial translocation is increased after the development of ascites and may be a major factor in the development of spontaneous infections in cirrhosis.

Immunocytochemical analysis of estrogen and progesterone receptors in benign stromal lesions of the breast. Evidence for hormonal etiology in pseudoangiomatous hyperplasia of mammary stroma.

Five cases of pseudoangiomatous hyperplasia of mammary stroma, together with seven examples of mammary hamartoma, were probed with monoclonal antibodies H222 and KD68 to investigate the possible role of estrogen and progesterone receptor expression in the pathogenesis of these benign stromal proliferations. All five cases of pseudoangiomatous hyperplasia showed patchy, intense labelling of the stromal cells with progesterone receptor antibodies, a pattern contrasting markedly with the absence of immunoreactivity in normal (nongestational) mammary stroma or the stromal component of common juvenile mammary hyperplasia. The stroma of the hamartoma group labeled inconsistently, with the notable exception of three myoid hamartomas. Stromal immunoreactivity was diffuse and intense in two of these, and patchy and distinct in the remaining case. These findings (a) support the contention that pseudoangiomatous hyperplasia represents a localized form of stromal overgrowth with a hormonal (primarily progestagenic) etiology and (b) further highlight the heterogeneity of socalled mammary hamartomas by demonstrating dramatically different progesterone receptor immunoreactivity patterns in myoid lesions as compared with other hamartoma variants.

Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry.

Estrogen receptor (ER) status in breast cancer is currently the most important predictive biomarker that determines breast cancer prognosis after treatment with endocrine therapy. Although immunohistochemistry has been widely viewed as the gold standard methodology for ER testing in breast cancer, lack of standardized procedures, and lack of regulatory adherence to testing guidelines has resulted in high rates of “false-negative” results worldwide. Standardized testing is only possible after all aspects of ER testing—preanalytical, analytical, and postanalytical, have been closely controlled. A meeting of the “ad-hoc committee” of expert pathologists, technologists, and scientists, representing academic centers, reference laboratories, and various agencies, issued standardization testing recommendations, aimed at optimization of clinical ER testing environment, as a step toward improved standardized testing.

Decreased platelet deposition and smooth muscle cell proliferation after intramural heparin delivery with hydrogel-coated balloons.

In vitro and in vivo studies have demonstrated both anticoagulant and antiproliferative effects of heparin. The purpose of this study was to assess the effect of local intramural delivery of heparin, using heparin-coated hydrogel balloons, on platelet deposition and early smooth muscle cell proliferation after in vivo balloon angioplasty. Methods and ResultsThe effects of local heparin delivery were assessed during balloon angioplasty of porcine peripheral arteries. All balloon dilatations were performed with oversized hydrogel balloons coated with a known quantity of heparin. Balloon dilatations in contralateral vessels with uncoated hydrogel balloons served as study controls. The pharmacokinetics of heparin delivery were assessed using 3H-heparin to quantitate heparin wash-off from the balloon surface, heparin delivery to the arterial wall, and intramural persistence of drug. Platelet deposition at 1 hour after balloon injury was quantified using 111In-labeled platelets. Smooth muscle cell proliferation was assessed 6 to 7 days after angioplasty with immunohistochemical staining for proliferating cell nuclear antigen. 3H-heparin wash-off from the hydrogel balloon surface occurred rapidly, with approximately 95% of the heparin coating disappearing within 10 seconds in the intact circulation. Approximately 2% of heparin on the balloon surface was delivered intramurally at the time of angioplasty. Intramural heparin dissipated rapidly, although small amounts of intramural heparin could still be detected for at least 48 hours. In comparison to control vessels, there was less 111In-platelet deposition (P = .002) and less medial smooth muscle cell proliferation (P = .03) in heparin-treated vessels. ConclusionsLocal intraluminal delivery of heparin at the time of balloon angioplasty with heparin-coated hydrogel balloons results in intramural deposition of drug that persists for at least 48 hours. This in vivo technique significantly decreases platelet deposition and early smooth muscle cell proliferation after angioplasty injury.

Utility of glypican-3 in differentiating hepatocellular carcinoma from other primary and metastatic lesions in FNA of the liver: an immunocytochemical study.

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.

Variation in breast cancer hormone receptor and HER2 levels by etiologic factors: A population-based analysis

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‐α and PR status of tumors based on clinical records according to etiologic exposure data collected via interview. For 842 cancers, we evaluated ER‐α, ER‐β, PR and HER2 levels by semiquantitative microscopic scoring of immunostained tissue microarrays and a quantitative immunofluorescence method, automated quantitative analysis (AQUA™). 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.

Immune Evasion and Recognition of the Syphilis Spirochete in Blood and Skin of Secondary Syphilis Patients: Two Immunologically Distinct Compartments

Background The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of Treponema pallidum (Tp) to escape immune recognition while simultaneously inducing inflammation. Methods To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. Ex vivo opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions in vivo. Results Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56−/CD16+ NK-cell subset in blood. Skin lesions, which had visible Tp by IHC and substantial amounts of Tp-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of Tp in conjunction with monocyte activation, most spirochetes were not internalized. Conclusions Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of Tp subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.

KOC (K homology domain containing protein overexpressed in cancer) and S100A4-protein immunoreactivity improves the diagnostic sensitivity of biliary brushing cytology for diagnosing pancreaticobiliary malignancies.

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.