Daniza Mandich

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)

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.

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.

S-100A4 Protein and Mesothelin Expression in Dysplasia and Carcinoma of the Extrahepatic Bile Duct

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.

Immunohistochemical detection of immunoglobulin light chain expression in B-cell non-Hodgkin lymphomas using formalin-fixed, paraffin-embedded tissues and a heat-induced epitope retrieval technique.

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 &kgr; or &lgr; light chain restriction (Cohen &kgr; 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.

IFNγ Enhances CD64-Potentiated Phagocytosis of Treponema pallidum Opsonized with Human Syphilitic Serum by Human Macrophages

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.

Esophageal polypoid dysplasia of gastric foveolar phenotype with focal intramucosal carcinoma associated with Barrett's esophagus.

We describe a rare case of esophageal polypoid dysplasia with gastric phenotype and focal intramucosal carcinoma associated with Barretts 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 Barretts 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 Barretts intestinal metaplasia.

Implementation of a high-quality biospecimen program to support molecular medicine.

200 Background: The successful implementation of a tumor genomics program relies heavily upon the collection of high quality tumor tissue samples. Although there has been an evolution towards utilizing formalin-fixed paraffin embedded (FFPE) tissue, many research centers continue to rely upon frozen fresh tissue for these types of analyses. A comprehensive effort is required to supply high-volume and high-quality tissue for research. Most community hospitals, even with superb pathology departments, are not well suited to deliver consistent tissue samples without a concerted programmatic effort. As part of the NCI Community Cancer Centers Program (NCCCP), we undertook the development of such capability at our hospital. In addition, as a member of H. Lee Moffitt Cancer Centers Total Cancer Care program, we received grant funding to help support this comprehensive effort. Our patients and clinicians expressed a strong desire to participate in this type of translational research. This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. METHODS We developed a comprehensive staffing model to implement this program, including a program coordinator, consenters, pathology assistant, lab aide, and data manager. We developed superb relationships with surgeons, interventional radiologists, pathologists and staffs to assure appropriate referrals and processes, and implemented quality checks as a standard. We developed relationships with Moffitt, The Cancer Genome Atlas, and other research efforts, which help provide funding. RESULTS We successfully implemented a program which resulted in high levels of patient and provider satisfaction, high numbers of fresh frozen and FFPE tissues (nearly 3,000 over 3 years), high quality pass rates, low ischemia time, and high satisfaction on the part of our research partners. We have incorporated best practices in our tissue handling protocols. CONCLUSIONS We successfully implemented a comprehensive cancer genomics bio-specimen program utilizing dedicated staff, working with patients and clinicians closely, and assuring careful coordination of all efforts.

Immunohistochemical Identification of Estrogen and Progesterone Receptor Proteins in Paraffin-Embedded Tissue: A Protocol Used in a Busy Immunohistochemistry Laboratory

Abstract The identification of estrogen and progesterone receptor proteins in breast cancer cells by immunohistochemistry is used to identify patients who may benefit from hormonal therapy. An immunoperoxidase staining procedure is described for the demonstration of these proteins in routinely fixed, paraffin-embedded tissue sections using overnight primary antibody incubations (clones 6Fll and PgR 636) and the sensitive Envision+detection system. In our hands, this protocol has produced quality results that are reproducible, cost-effective, and easy to interpret using a visual scoring system. The protocol is easily incorporated into laboratories already performing immunohistochemical staining. (The J Histotechnol 25:000, 2002)