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The purpose of this study was to identify correlates of HER2 status for patients with bilateral breast cancer.Data were collected prospectively in our institutional review board approved patient registry for all patients with asynchronous (ABBC) and synchronous (SBBC) bilateral infiltrating breast cancer whose HER2 assays were performed at our laboratory using FISH. Data were analyzed using the Wilcoxon rank sum and Chi-square test.Data were available for 98 tumors in 49 patients. Patients diagnosed with SBBC were more likely to be white (P = 0.023); to have bilateral HER2 non-amplified tumors (P = 0.022) and to be older at diagnosis (P = 0.025) compared to those with ABBC. Patients with one HER2 amplified tumor were likely to have at least one tumor be hormone receptor negative and to have ABBC.Only 16% of patients in this study had one tumor that was HER2 amplified; no patient had bilateral HER2 amplified tumors. more...

Background: GP88 (progranulin) is a growth and survival factor implicated in tumorigenesis and drug resistance for various cancers. GP88 tissue expression measured by IHC is inversely correlated with survival. We now evaluate GP88 tissue lung cancer small cell carcinoma (SCLC), non-small cell lung carcinoma (NSCLC) and mesothelioma. Methods: GP88 expression was determined by immunohistochemistry on tissue sections from normal lung, pleura, benign mesothelial proliferation, SCLC, NSCLC and mesothelioma. In the case of NSCLC, GP88 expression in tumor tissues from patients with localized (stage I-II) and locally advanced disease (IIIa-IIIb) was determined and correlated with clinical outcome disease-free survival (DFS) and overall survival (OS). Serum GP88 levels from patients with metastatic NSCLC was quantified by EIA and compared to GP88 levels from patients with COPD and from healthy individuals. Results: GP88 was significantly expressed in NSCLC (adenocarcinoma and squamous cell) and mesothelioma (epitheloid, sarcomatoid and biphasic subtypes) tumors. In contrast, normal lung tissue, benign lung disease, pleura, benign mesothelial proliferations and SCLC were negative. There was a significant increased risk of recurrence and death for patients with resected NSCLC (stage I,II) with high expression (GP88 IHC score 3+) compared to patients with GP88IHC score Conclusions: This is the first study investigating GP88 expression in various thoracic malignancies. Differences of GP88 expression were observed in different lung tumor types with NSCLC and mesothelioma being positive. Correlation with clinical outcomes in NSCLC demonstrates that measuring the GP88/Progranulin IHC tumor tissue expression using the anti-GP88/PGRN 6B3 antibody provides information on the risk of recurrence and death of patients with stages I-III NSCLC and that serum GP88 levels are elevated in advanced disease. Future research will focus on investigating the prognostic potential of GP88 tissue expression and serum levels for mesothelioma patients. Citation Format: Ginette Serrero, Martin J. Edelman, Pablo Bejarano, Douglas M. Hawkins, David Hicks, David N. Reisman, Olga Ioffe, Josephine Feliciano, Binbin Yue. Progranulin (GP88) expression in thoracic maligancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4339. doi:10.1158/1538-7445.AM2015-4339 more...

Background: The transcription factor SOX10 mediates the differentiation of neural crest-derived cells, and expression of SOX10 detected by IHC analysis is primarily used to support the diagnosis of melanoma. Expression of SOX10 has been recently reported in benign breast myoepithelial cells and triple negative breast cancer. The aim of the current study is to investigate the expression pattern of SOX10 in a cohort of invasive ductal carcinoma (IDC) tumors, and analyze its relationship with different clinicopathological features and clinical outcome. Methods: Four hundred-twenty eight cases of IDC of the breast diagnosed in our institution between 1997 and 2008 and having follow-up information were included in this study. Immunohistochemical studies for SOX10 (≥1% of tumor cells having nuclear staining designated as positive expression), ER, PR, HER2 and Ki-67 were performed on 25 pre-constructed tissue microassay blocks. The relationship between SOX10 expression and the clinicopathologic features, expression of ER, PR, HER2 and Ki-67, and clinical outcome were evaluated. Results: Among this cohort of patients, the majority of them were greater than 50 years old, with grade 1 or 2 tumors that were less than 2 cm and node negative. 81% of the tumors were ER positive, 75% were PR positive and 8% HER2 positive. The overall expression rate of SOX10 was 18%; however, its expression rates were significantly higher in high grade tumors (31%), and tumors that were ER (53%) and PR (41%) negative, and tumors with high Ki-67 expression (designed as >15% nuclear labeling, 47%). 51% of ER-/PR- tumors, 64% of triple negative tumors and 68% of Basal-like tumors expressed SOX10. SOX10 expression was not associated with patient age, tumor size, nodal status and over-expression of HER2. When we looked at the 4 different levels of SOX10 expression (1-25%, 26-50%. 51-75% and 76-100% of cells with positive nuclear staining) in different subgroups of breast cancer, we noted that the majority of all cases had high levels of SOX10 expression (76-100% cells with nuclear staining) regardless of their ER, PR, HER2 and Ki-67 status. No significant difference in overall survival (p value=0.8553) and disease-free survival (0.1810) between SOX10 positive and negative tumors was noted. Conclusion: Our data demonstrates a significant association between SOX10 expression and high grade, ER/PR negative, triple negative or basal-like IDC, suggesting their myoepithelial differentiation. Given that SOX10 has been recently described as a principle driver for melanoma, better understanding of SOX10 expression and signalling would provide important insight in development of novel diagnostic and therapeutic tools for SOX10 expressing tumors. Citation Format: Laura L Hoang, Jianmin Wang, David Tacha, Huijiao Chan, Bing Wei, Zhang Zhang, Hong Bu, David G Hicks, Ping Tang. Expression of SOX10 in invasive ductal carcinoma of the breast [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-14. more...

Determination of HER2 status by fluorescence in situ hybridization (FISH) in breast carcinoma correlates well with response to targeted therapy and prognosis. However, manual time consuming methods and quantification aspects of the procedure may be challenging for some laboratories. We examined the feasibility of automating these components of the FISH assay using a tissue microarray (TMA-118 clinically annotated cases) and a series of 41 whole sections. An in situ hybridization automated staining workstation was used to automate a programmed overnight start, on line baking, deparaffinization, cell conditioning, protease digestion, and prehybridization buffer washing. Dual label probe/target codenaturation/hybridization and stringency washing were done off line. The HER2 and CEP17 spot counts were quantified, and the HER2/CEP17 ratio calculated, via an imaging workstation. Results were benchmarked against manual counts for whole sections, and bright field in situ hybridization [silver in situ hybridization (SISH)] for the TMA. Automated FISH results using whole sections correlated well with manual results: HER2/CEP17 ratio correlation coefficient r = 0.9154, r = 0.8380, P < 0.0001. Correlation between automated and manual TMA FISH results was also excellent, and disease-free survival was significantly shorter (P < 0.001) for the HER2 amplified cases. Automation of the laborious manual prehybridization and image quantification components of FISH using directly labeled probes is feasible. Operational gains and enhanced consistency are inherent in this automated approach to HER2 clinical FISH testing. more...

Objectives: To understand the effect of adjuvant cryotherapy on glottic wound healing after endoscopic CO2 laser cordectomy. Study Design: Canine acute injury model with videolaryngostroboscopic and histopathologic outcomes analysis. Methods: Twelve adult male dogs underwent bilateral endoscopic CO2 laser transmuscular cordectomy followed by randomized unilateral endoscopic glottic cryotherapy. The animals were randomly divided into four groups and underwent videolaryngostroboscopy followed by sacrifice at 0, 2, 6, and 12 weeks postoperatively. Three untreated male dogs served as controls. Histopathologic sections were prepared with Alcian blue, Giemsa, hematoxylin-eosin, movat's, Masson's trichrome, and picrosirius stains. Results: Videostroboscopy demonstrated an earlier restoration of glottic volume and a return of mucosal waves among vocal folds treated with combined therapy by 6 weeks posttreatment. The mean depth of inflammatory reaction in the vocal cords treated with combined therapy was 1.07 mm versus 1.15 mm in vocal cords treated with CO2 laser therapy alone. At 2 and 6 weeks postoperatively, combined treatment was associated with a decreased volume of collagen. At 12 weeks postoperatively, combined treatment was associated with greater collagen organization, normalized collagen histoarchitecture, and decreased keratinization. Conclusions: Adjuvant cryotherapy appears to alter glottis-specific wound healing, leading to decreased and more organized collagen formation and decreased keratinization with a resultant improvement in glottic function, when compared with CO2 laser surgery alone, in an acute canine injury model. Studies in humans are ongoing to further evaluate the clinical potential of cryotherapy on glottic wound healing. more...

PurposePatients with breast cancer experience progression and respond to treatment in diverse ways, but prognostic and predictive tools for the oncologist are limited. We have used gene expression data to guide the production of hundreds of novel antibody reagents to discover novel diagnostic tools for stratifying carcinoma patients.Patients and MethodsOne hundred forty novel and 23 commercial antisera, selected on their ability to differentially stain tumor samples, were used to stain paraffin blocks from a retrospective breast cancer cohort. Cox proportional hazards and regression tree analysis identified minimal panels of reagents able to predict risk of recurrence. We tested the prognostic association of these prospectively defined algorithms in two independent cohorts.ResultsIn both validation cohorts, the Kaplan-Meier estimates of recurrence confirmed that both the Cox model using five reagents (p53, NDRG1, CEACAM5, SLC7A5, and HTF9C) and the regression tree model using six reagents (p53, PR, Ki67, NAT1, SLC7A5, and HTF9C) distinguished estrogen receptor (ER) –positive patients with poor outcomes. The Cox model was superior and distinguished patients with poor outcomes from patients with good or moderate outcomes with a hazard ratio of 2.21 (P = .0008) in validation cohort 1 and 1.88 (P = .004) in cohort 2. In multivariable analysis, the calculated risk of recurrence was independent of stage, grade, and lymph node status. A model proposed for ER-negative patients failed validation in the independent cohorts.ConclusionA panel of five antibodies can significantly improve on traditional prognosticators in predicting outcome for ER-positive breast cancer patients. more...

Summary Recently published clinical trial data have produced compelling evidence for increased survival when Herceptin is administered to patients whose tumors are HER2 amplified. Therefore, the accuracy of HER2 status is essential to determine which patients should or should not receive Herceptin. Although HER2 results obtained by FISH and IHC are often in agreement, there is a persistent group of cases in which results are discordant, particularly among tumors with intermediate results. A multivariable analysis was undertaken to determine relative significance of various clinical and pathologic findings for patients diagnosed with infiltrating ductal carcinoma, and a data model was produced that predicts which patients are most likely to have HER2 amplified tumors. Correlates of HER2 amplification were higher Scarff–Bloom–Richardson grade, younger age at diagnosis, and a comedo ductal carcinoma in situ component. more...

Ultrasensitive bright field in situ hybridization assays using enzyme metallography (EnzMet) have been developed and validated, but little is known regarding the applicability of EnzMet for immunophenotypic detection of protein via IHC. Superior resolution via discrete metallographic deposits offers the potential for enhancing high-resolution immunophenotyping. Using high-complexity tissue microarrays (TMAs), 88 common solid tumors were evaluated by automated EnzMet (Nanoprobes and Ventana). Targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. Results were compared with conventional IHC diaminobenzidine (DAB) immunostaining. There was full concordance between the EnzMet and conventional IHC results. Furthermore, the EnzMet reaction products did not appreciably diffuse, were dense and sharply defined, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, allowing more tissue to be screened in an efficient manner. This preliminary study shows the utility of using enzyme metallography for high-resolution immunophenotyping in TMAs. more...

Tissue microarrays (TMA) are valuable tools for validating results of array-based comparative genomic hybridization (ACGH) and other translational research applications requiring independent verification of genomic gains and losses by fluorescence in situ hybridization (FISH). However, spatial orientation and accurate manual tracking of the TMA cores is challenging and prone to error. Image analysis combined with core tracking software, implemented via an automated FISH scanning workstation, represents a new approach to FISH and TMA-based validation of novel genomic changes discovered by ACGH in breast and other cancers. Automated large-scale tissue microarray validation FISH studies of genomic gains and losses identified by ACGH for breast cancer are feasible using an automated imaging scanner and tracking/classifying software. Furthermore, by leveraging the bifunctional fluorescent and chromogenic properties of the alkaline phosphatase chromogen fast red K and combining the technology with FISH, correlative and simultaneous phenotype/genotype studies may be enabled. more...

We performed this study to determine the prognostic significance of clinical tumor size, pathologic measurement of residual tumor, and number of positive axillary nodes in the surgical specimen relative to overall survival for patients who underwent primary induction chemotherapy for advanced breast cancer.Data, collected prospectively between 1997 and 2002, included clinical tumor-node-metastasis stage, age at diagnosis, hormone receptor status, type of preoperative chemotherapy, histological type, surgical procedure, pathologic measurement in centimeters of residual breast tumor, and the number of positive axillary nodes in the surgical specimen. Univariable correlates of residual breast disease were assessed by using the chi2 test. Recursive partitioning analysis was used to determine the prognostic significance of clinical tumor size, residual tumor size, and pathologic node involvement relative to overall survival. Survival was estimated by using the method of Kaplan and Meier and compared by using the log-rank test. A P value of.05 was considered significant.Data were available for 85 patients with advanced breast cancer. Although univariable analysis identified increasing age, clinically involved axillary nodes, and a higher clinical tumor-node-metastasis stage as predictors of an increased risk of residual disease, recursive partitioning analysis identified more than three involved axillary nodes in the surgical specimen, with or without any measurable residual breast disease, as the most significant predictor of decreased survival (P.001).Pathologic axillary node involvement was the most significant predictor of decreased survival for patients who had undergone primary induction chemotherapy for advanced breast cancer. more...

We report the development of techniques and instrumentation for clinical application of NIR Raman spectroscopy for breast cancer diagnosis. Potential clinical applications include optical biopsy at the time of mammography and intra-operative margin assessment. more...

Summary Clinical and in vitro evidence supports the concept that human epidermal growth factor receptor-2 ( HER2 ) gene amplification prediction of response to anthracycline-based chemotherapy in breast cancer is not a direct effect of HER2 overexpression, but the result of coamplification of topoisomerase II-alpha ( TOP2A ). We investigated the relationship of TOP2A to HER2 genomic alterations by fluorescence in situ hybridization (FISH) and the correlations with polysomic states for chromosome 17 (CEP17). One hundred thirty-eight cases of breast cancer HER2 gene amplified by 2-color FISH ( HER2 /CEP17) were reevaluated with a 3-color probe set ( HER2 /CEP17/ TOP2A ) to investigate the frequency of coamplification and deletion of TOP2A . TOP2A was never amplified in the absence of HER2 amplification and was coamplified with HER2 in 68 (50%) of 137 cases; HER2 gene copy number was higher than the TOP2A copy number ( P HER2 amplification, 23 (16%) showed a monoallelic deletion of TOP2A . Of the 43 cases not amplified for HER2 , 27 (63%) were CEP17 eusomic, 13 (30%) polysomic, and 3 (7%) monosomic. Of the HER2 nonamplified cases, 2 (5%) showed monoallelic deletion of both the HER2 and TOP2A . The current study demonstrates the complex interrelationship between the HER2 and TOP2A genes in breast cancer. The clinical implications of TOP2A amplification and deletion in breast cancer need to be further defined. If TOP2A gene dosage can be confirmed to correlate with tumor responsiveness to anthracycline-based therapy in the clinical setting, FISH testing for TOP2A status may be warranted to aid in the selection of the most appropriate therapy. more...

Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2 -driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control. The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing. more...

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nuclear hormone receptor that is involved in a wide range of cellular processes. Although it is known that PPAR-gamma plays an important role in cell cycle control, inflammation, apoptotic cell death, and other cellular processes, the role of PPAR-gamma in the normal and pathological function of growth plate chondrocytes has not been investigated. The purpose of this study was to determine if PPARs are expressed in growth plate chondrocytes and to describe the biological effect of PPAR activation in these cells. The results demonstrate the presence of three PPAR isoforms (alpha, delta, and gamma) in growth plate cartilage. Activation of PPAR-gamma by ciglitazone in growth plate chondrocytes inhibits T(3) induced terminal differentiation and promotes apoptosis through increased levels of caspase 3/7 activity and decreased expression of the anti-apoptotic protein Bcl-2. more...

The invasion and metastasis of tumor cells is a major cause of mortality in cancer patients. In the current study, we investigated the expression of fascin, an actin-bundling motility-associated protein, in 210 invasive breast carcinomas with corresponding 5-year clinical follow-up. Fascin expression was compared with hormone receptor (ER/PR) status, HER2 status, cancer grade, cancer stage, metastasis pattern, disease-free survival, and overall survival. Fascin expression was seen in 16% (33/210) of the cases and correlated with ER negativity (22/33, P0.001), PR negativity (21/33, P0.001), Bloom-Richardson grade 3 (19/29, P0.001), and advanced stage (stage 3 or 4, P = 0.04). There was no correlation between fascin expression and HER2 status or pattern of metastases. Patients whose tumors were positive for fascin showed both a decreased mean disease-free survival (74.44 versus 100.52 months, P = 0.002) and mean overall survival (77.58 versus 98.98 months, P = 0.002), independent of tumor stage and HER2 status, but not independent of ER/PR status or cancer grade. Given fascin's role in altering cell motility, overexpression may contribute to a more aggressive clinical course in ER/PR-negative breast cancers. If so, then fascin may represent a new molecular target for therapeutic intervention in patients with ER-negative breast cancer. more...

Epithelioid sarcoma is a distinctive, aggressive soft tissue tumor typically presenting as a subcutaneous or deep dermal mass in the distal extremities of young adults. Molecular genetic data of well-characterized cases of epithelioid sarcoma are sparse. A recent cytogenetic study of epithelioid sarcoma by conventional metaphase comparative genomic hybridization reported recurrent gains at chromosome 11q13, a region containing many genes, including the cyclin D1 gene. Cyclin D1 is a positive cell cycle regulator that is overexpressed in a variety of neoplasms, including mantle cell lymphoma and breast carcinoma. The objective of this study was to examine cyclin D1 expression in epithelioid sarcoma. Of 24 cases evaluated, 23 (96%) displayed cyclin D1 nuclear expression using immunohistochemical evaluation. Eight cases, which expressed cyclin D1 by immunohistochemistry, were evaluated by fluorescence in situ hybridization (FISH) and RNA in situ hybridization (RISH) for amplification of the cyclin D1 gene and messenger RNA (mRNA) expression, respectively. Seven of eight cases showed a typical eusomic state. One case showed pseudoamplification due to aneusomy/polysomy. There was no evidence of cyclin D1 gene amplification or messenger RNA overexpression detected by FISH or RNA in situ hybridization analyses, respectively. Our data clearly demonstrate that cyclin D1 protein is upregulated in epithelioid sarcoma, suggesting a role for this cell cycle regulator in the pathogenesis of epithelioid sarcoma. The high level of cyclin D1 protein expression in epithelioid sarcoma appears to be regulated by translational and/or post-translational mechanisms. more...

Diagnostic anatomic pathologists play an important role in the care of patients through their careful evaluation of morphological features in routinely prepared histological sections stained with Hematoxylin and Eosin. Morphological assessment of tissue sections, backed by over one hundred years of experience is powerful and allows for the accurate classification and diagnosis of the majority of disease states within pathologically altered tissues. However, the appearance of cells and their architectural arrangement within a morphologically complex tissue represents only a fraction of the information, which is contained within a histological section. These tissues also contain all of the cellular proteins and expressed genes, which help to ultimately determine the biological behavior of cells, as well as provide clues to the origins and pathogenesis of disease states. Technical and theoretical advances in our understanding of cellular biology have provided pathologists with powerful tools to probe beyond pure morphology into the abnormalities in both protein and gene expression that underlie human disease. These tools, which include immunohistochemistry and in situ hybridization, are playing an increasingly important role in diagnostic pathology, as well as in translational research. This review will focus on the emerging role of in situ hybridization within clinical and research laboratories, and will highlight a number of technical advances that have expanded the application of this technology. more...

Psoriatic arthritis (PsA) is an inflammatory joint disease characterized by extensive bone resorption. The mechanisms underlying this matrix loss have not been elucidated. We report here that blood samples from PsA patients, particularly those with bone erosions visible on plain radiographs, exhibit a marked increase in osteoclast precursors (OCPs) compared with those from healthy controls. Moreover, PsA PBMCs readily formed osteoclasts in vitro without exogenous receptor activator of NF-kappaB ligand (RANKL) or MCSF. Both osteoprotegerin (OPG) and anti-TNF antibodies inhibited osteoclast formation. Additionally, cultured PsA PBMCs spontaneously secreted higher levels of TNF-alpha than did healthy controls. In vivo, OCP frequency declined substantially in PsA patients following treatment with anti-TNF agents. Immunohistochemical analysis of subchondral bone and synovium revealed RANK-positive perivascular mononuclear cells and osteoclasts in PsA specimens. RANKL expression was dramatically upregulated in the synovial lining layer, while OPG immunostaining was restricted to the endothelium. These results suggest a model for understanding the pathogenesis of aggressive bone erosions in PsA. OCPs arise from TNF-alpha-activated PBMCs that migrate to the inflamed synovium and subchondral bone, where they are exposed to unopposed RANKL and TNF-alpha. This leads to osteoclastogenesis at the erosion front and in subchondral bone, resulting in a bidirectional assault on psoriatic bone. more...

On January 1st, 2012, the Center for Medicare and Medicaid Services (CMS) implemented a policy in conjunction with the National Correct Coding Initiative (NCCI) to pay pathologists and laboratories for immunohistochemistry (IHC) cocktail stains as a single unit of Current Procedural Terminology (CPT) code 88342, regardless of how many individually interpretable antibodies are included in the cocktail. Medicare has, in the past, paid for IHC cocktail stains based on the American Medical Association’s prescription: 1 unit of CPT code 88342 was payable for each separately interpreted and reported antibody (eg, via unique color expression compared with other markers in the cocktail). Knowledgeable sources indicate that this new policy is a reaction by CMS to a dramatic increase in the number of IHC charges on physician and laboratory claims during the past 2 or 3 years. This period roughly corresponds to the observed proliferation of in-office and other histology laboratory arrangements entered into, particularly but not exclusively, by urologists and gastrointestinal practitioners. Overutilization of IHC stains—the application of diagnostic markers in medically suspect situations (eg, staining all of a patient’s biopsy specimens instead of just 1 or 2 tumor-rich samples, applying a stain to rule out a condition not suggested by the H&E slides)—by these physicians, and perhaps by a minority of overly aggressive pathologists as well, is a natural cause for alarm among government payers and private medical insurers alike. As taxpayers and as ethical practitioners of the medical arts, we are highly sympathetic to the goal of CMS and other parties to eliminate waste and abuse in the laboratory industry. Notwithstanding, it is the strong opinion of leaders in the field of IHC testing that the … more...

Hyperostosis cranii ex vacuo is a condition that may develop after successful ventricular shunting for hydrocephalus. It is characterized by diffuse thickening of the skull (particularly the inner table) involving the calvarium and the base. It is a diagnosis that distinguishes marked calvarial thickening from thickening of the skull because of other causes. It is an entity that needs to be considered in patients who have diffuse thickening of the skull, have premature closure of sutures, and who have been treated for hydrocephalus in the past. more...

Clinical laboratory testing for HER-2/neu gene amplification by fluorescence in situ hybridization is not widely used in diagnostic pathology laboratories. A bright field alternative permitting direct visualization of gene amplification using conventional microscopy may be more readily incorporated into routine diagnostic pathology practice. Interobserver reproducibility represents an important component of the validation of such an assay. We tested the hypothesis that a first-generation bright field alternative to fluorescence in situ hybridization, a Nanogold (Nanoprobes, Inc, Yaphank, NY, USA) (or gold-label)/autometallographic assay for HER-2/neu gene amplification in breast carcinoma, can be reproducibly interpreted by pathologists. Reference standard was direct fluorescence in situ hybridization supplemented by RNA/RNA in situ hybridization. Reproducibility of selected conventional histologic parameters was captured based on a hematoxylin and eosin slide accompanying the GOLDFISH preparation (gold-facilitated autometallographic in situ hybridization) as an indication of comparative reproducibility. The average kappa among GOLDFISH observers was 0.84, which was at least or concordant of observers scoring nuclear grade (kappa = 0.50) and the presence of in situ carcinoma (kappa = 0.57) by conventional histopathology. The GOLDFISH assay was specifically designed for qualitative interpretation, thus obviating the need for oil immersion microscopy and signal enumeration, and its interpretation was highly reproducible among five pathologists. more...

Context.—Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. Objectives.—To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. Design.—We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. Results.—Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. Conclusion.—Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer. more...

A patient with remittent multiple sclerosis presented subacutely macular visual dysfunction and superior altitudinal hemianopia documented with static and kinetic perimetry. MRI showed extensive demyelination in both temporo-occipital optic radiations. This is the first reported case of altitudinal hemianopia due to multiple sclerosis. more...

Purpose: The aims of this study were to investigate whether osteonecrosis affects the mandibular condyle and to determine whether this condition could be diagnosed with magnetic resonance imaging (MRI). Materials and Methods: The study was based on 50 temporomandibular joints in 44 patients in whom MRI and surgery were performed for painful internal derangements. At the time of surgery, a core biopsy specimen was obtained from the marrow of the mandibular condyles, and the histological observations were correlated to T1- (proton density) and T2-weighted MR images. Results: Histological evidence of osteonecrosis was found in nine joints (18%). Bone marrow edema, which may be a precursor of osteonecrosis, was found in nine other joints. The MRI was 78% sensitive and 84% specific for the diagnosis of osteonecrosis of the mandibular condyle. However, the positive predictive value was only 54% because of a number of false-positive MRI diagnoses. Conclusion: Osteonecrosis can affect the mandibular condyle, and this condition can be diagnosed with MRI. A combination of edema arid sclerosis of the bone marrow was the most reliable MRI sign of osteonecrosis. The cause, its clinical significance, and the need for treatment are unknown. more...

Rationale and Objectives. The authors' purpose was to develop a magnetic resonance (MR) imaging technique for examining the hip joint of a rabbit with a clinical MR imager. Materials and Methods. Fourteen hips of 10 male New Zealand white rabbits were examined on a 1.5-T MR imager with a specially designed surface coil. Field of view was 3–8 cm, matrix was 256–512 × 192–256, section thickness was 0.9–2.0 mm, and spacing was 0.3–0.5 mm. The hips were sectioned and evaluated by means of light microscopy. Measurements of cartilage thickness from MR images were correlated with those from histologic specimens. Results. The resolution obtainable with a 1.5-T imager was adequate for imaging articular cartilage when a debrided rabbit hip specimen was placed in the center of a 3.5-cm single-loop coil. Rabbit hip cartilage had a trilaminar appearance on MR images. The coefficient of correlation between cartilage-thickness measurements was .81. Conclusion. MR imaging of rabbit hip cartilage can be performed on a 1.5-T clinical MR imaging unit. Limitations due to the field strength can be overcome with efficiently designed surface coils. more...

A 32-year-old, previously healthy woman with a diffuse, complex tumoral process is described. While she was being evaluated for primary infertility, a laparoscopic procedure revealed a disseminated peritoneal process associated with bilateral ovarian lesions. A clinical diagnosis of stage III ovarian cancer was made and total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy were performed. Pathologic studies showed an admixture of leiomyomatosis peritonealis disseminata (LPD) endometriosis, and an extensive multicystic mesothelial proliferation. Although endometriosis is known to occur in conjunction with either LPD or multicystic mesothelioma, to our knowledge this is the first report of all three lesions occurring simultaneously. more...

Parathyroid hormone-related protein is a critical autocrine regulator of endochondral ossification in the growth plate, as demonstrated by the severe disruption of growth-plate structure and function in parathyroid hormone-related protein-deficient transgenic mice. In the present study, the effects of parathyroid hormone-related protein on the synthesis of collagen mRNA and protein were studied in short-term cultures of isolated chick growth-plate chondrocytes. Parathyroid hormone-related protein selectively inhibits type-X collagen protein synthesis with no significant effect on type-II collagen protein synthesis. These effects were present in all maturationally distinct populations of chondrocytes separated by countercurrent centrifugal elutriation. In cultures of resting chondrocytes, the onset of type-X collagen expression was inhibited, while the synthesis of type-X collagen was decreased in cultures of hypertrophic chondrocytes. Synthesis of type-II and type-X collagen mRNA was examined by nonradioactive in situ hybridization with synthetic oligonucleotide cDNA probes, and the level of expression was quantified using digital image analysis. Dose-dependent suppression of type-X collagen gene expression by parathyroid hormone-related protein was observed, with no significant effect on type-II collagen mRNA detected. The results were confirmed by analysis of Northern blots of total chondrocyte mRNA. These experiments demonstrated differential transcriptional regulation of type-II and type-X collagen, with selective suppression of type-X collagen expression, by parathyroid hormone-related protein in growth-plate chondrocytes. In addition, excellent agreement was found between traditional protein and mRNA analyses and microscopic digital image analysis techniques, supporting the use of this convenient and sensitive assay method. Parathyroid hormone-related protein inhibits chondrocyte maturation and is known to stimulate proliferation, suggesting that this autocrine factor may function to regulate premature hypertrophy in the growth plate. more...

Lead toxicity is a major public health problem in the United States. The skeleton serves as the major reservoir for ingested lead, where it is incorporated into bone matrix during calcification. While lead in bone has been considered inactive, mounting clinical and epidemiological data has shown a strong correlation between lead exposure and adverse effects on stature in children. These epidemiologic data suggest a direct effect of lead on skeletal development, but whether it reflects a systemic effect, a specific effect on osteoblasts, or an effect on the epiphyseal growth plate is as yet unclear. This study examined the effects of lead on parameters of cartilage biology in isolated chondrocytes. Changes in growth plate chondrocyte phenotype were assessed utilizing an established avian growth plate chondrocyte model. Low, sublethal doses of lead caused specific and significant effects on a number of important markers of growth plate chondrocyte phenotype, including suppression of alkaline phosphatase and both type II and type X collagen expression at the protein and mRNA levels, and a decrease in thymidine incorporation. In contrast, proteoglycan synthesis was stimulated relative to controls in lead-treated cultures, suggesting that the alterations in collagen and DNA synthesis and alkaline phosphatase activity are not due to cytotoxity. The data demonstrate important regulatory effects of lead on growth plate chondrocytes in cell culture and suggest an inhibitory effect on the process of endochondral bone formation. The growth plate may be one of the key target tissues accounting for the adverse effects of chronic lead exposure on skeletal development. more...

Giant cell tumor (GCT) of bone is a locally aggressive neoplasm with a high incidence of recurrence, usually at the site of previous osseous involvement. Primary and recurrent intraosseous lesions typically are lytic and do not show evidence of tumor-associated osteogenesis. Rarely, GCT recurs or is primary within soft tissue, and not infrequently, these extraosseous lesions show metaplastic bone formation that is visible radiographically. The authors report two recurrent and one primary case of extraosseous GCT, all of which exhibited significant deposits of metaplastic bone localized to the periphery of the lesions. In situ hybridization showed messenger RNA (mRNA) for transforming growth factor beta1 (TGF-beta1) and transforming growth factor beta2 (TGF-beta2) in neoplastic stromal cells and osteoclast-like giant cells within the recurrent and primary extraosseous tumors as well as in active osteoblasts on the surfaces of recently formed spicules of metaplastic bone. In situ hybridization also revealed mRNA for TGF-beta1 and TGF-beta2 in primary intraosseous tumors from these cases and from four cases in which neither extraosseous recurrence nor osseous metaplasia was identified. In the microenvironment of the extraosseous soft tissue, production of these osteoinductive growth factors by GCT may have a paracrine effect on mesenchymal progenitor cells, thereby stimulating the osteoblastic differentiation and metaplastic bone formation associated with these lesions. more...

Infiltration of regional lymph nodes by macrophages has been demonstrated after total joint arthroplasty. Although lymph nodes regulate the immune response, neither cytokine production nor the degree of immunological activation of cells within these nodes after total joint arthroplasty has been investigated. Pelvic lymph nodes were obtained from five patients who had had a total of eleven arthroplasties in seven hips three to twenty years before a pelvic staging procedure for adenocarcinoma (of the prostate in four patients and of the endometrium in one). All lymph nodes had polyethylene or metal debris as well as effacement of the normal nodal architecture by a histiocytic infiltrate. These changes were bilateral in the patients who had had an arthroplasty of one hip. Analysis of specimens from pelvic lymph nodes on the side of the involved hip demonstrated intense immunohistochemical staining of histiocytes for the major histocompatibility complex class-II antigen HLA-DR, a marker of histiocyte immune activation. In contrast, staining was absent in specimens from the contralateral lymph nodes as well as in those from seven patients who had had a prostatectomy but not a hip arthroplasty. Immunohistochemical staining for interleukin-1beta, tumor necrosis factor-alpha, and interleukin-6 demonstrated a much greater expression of these cytokines in the involved lymph nodes.Additions improvements in total joint replacement will be facilitated by a more thorough understanding of the biological response to the components and materials of implants. While local biological factors leading to failure of prostheses are currently under intense investigation, the mechanisms and importance of regional and systemic immune responses to wear debris require further study. more...

Endochondral ossification in fracture healing of rats at 4, 8, 11, 14, and 21 days was analyzed using immunological and molecular probes for markers of the chondrocyte and osteoblast phenotype. These markers were osteocalcin, type I and type II collagen, including the probes homologous to the alternatively spliced forms of alpha 1 type II collagen, type IIA and type IIB. Histologic examination was performed on serial sections of the same tissue blocks to correlate cellular morphology with the immunohistochemical and in situ hybridization findings. At the junction of the cartilaginous and osseous tissue, an overlap of phenotype and morphology was noted. At the 8-day time point, the cells with chondrocyte morphology expressed intracellular message for osteocalcin and type I collagen. Immunohistochemical analysis of these cells also demonstrated intracellular osteocalcin. However, high levels of the type IIA collagen mRNA, which has previously been associated with less differentiated mesenchymal precursor cells, were expressed in both chondrocytes and osteoblasts. At the later time point (21 days) there was a substantial decrease in the number of cells displaying shared phenotypic characteristics. In situ hybridization and immunohistochemistry have permitted identification of an overlapping or shared phenotype in osteoblasts and chondroblasts in fracture callus. The findings raise important questions regarding the possible plasticity of mesenchymal cell phenotypes within the dynamic environment of fracture healing. Additional examination of these issues will further define factors involved in origin, differentiation, and maturation of bone and cartilage cells. more...

153 Background: Long ischemic times prior to tissue fixation can lower the accuracy of immunohistochemical staining (IHC). The purpose of this study was to identify factors that impact the time to fixation (TTF), and to determine if TTF impacts prognostic factor (PF) patterns and if TTF can be changed with simple interventions. Methods: Surgeries performed between 2008 and June 2011 for which TTF was noted and PF testing was done were reviewed. TTF, patient age and race, surgeon, date and type of surgery, tumor size, nodal status, grade, and status of ER/PR and HER2 by IHC were noted. TTF was dichotomized into > or more...

180 Background: AR after negative SNB is a rare event. The purpose of this study was to identify factors associated with AR. Methods: A breast cancer (BC) database was reviewed identifying 4 cases of AR among 424 (0.9%) patients with a negative SNB over a 5 year period. Demographic, pathologic and treatment data were collected. Patients with AR were compared those without using standard methods for contingency tables to identify factors associated with AR. Results: Tumor and treatment factors are shown in the table. There was no difference in age, race, menopausal status, tumor size or palpability, or the average number of SLNs harvested between the groups. Recurrences were only seen in patients with triple negative (TN), high grade tumors. AR occurred in 12.5% of pN0(i+) BCs compared to 6% of pN0 BCs (p more...

e21144 Background: Studies show that women with low vitamin D levels have an increased risk of breast cancer incidence and mortality, but there is a lack of research examining vitamin D levels and ... more...

Objective: Epidemiologic studies show that women with low 25-OH vitamin D levels have an increased risk of breast cancer incidence and mortality. However, there is a lack of research examining vitamin D levels and prognostic variables in breast cancer patients. The aim of this study is to identify the associations between 25-OH vitamin D levels, demographic variables, and prognostic pathological and genetic characteristics of breast cancers. Method: This study cohort consists of 155 women who underwent breast cancer surgery at the University of Rochester between 1/2009 and 9/2010. Vitamin D levels were obtained in the 1-year period before and after surgery (74% of vitamin D levels within 6 months). Prognostic variables included age, race, menopausal status, Oncotype DX score, TNM staging, ER/PR status, and HER2 expression. ANCOVA, linear regression, and logistic regression were used to determine the association between prognostic variables and 25-OH vitamin D levels, while controlling for relevant covariates (age, race, and month of blood draw). Results: Non-Caucasian (OR = 3.8; P < 0.01) and premenopausal (OR = 3.5; P < 0.01) breast cancer patients were significantly more likely to have suboptimal 25-OH vitamin D levels than Caucasian and postmenopausal patients, respectively. Women with invasive breast tumors were more likely to have suboptimal vitamin D levels (invasive OR = 2.4; P = 0.10) and lower mean 25-OH vitamin D levels (invasive: 30.5 ng/mL vs. in situ : 36.9 ng/mL; P = 0.04). A significant correlation ( r = −0.42; P = 0.04) between decreasing vitamin D levels and increasing Oncotype score was noted. Breast cancer patients who had ER- and triple-negative breast tumors were more likely to have suboptimal levels of 25-OH vitamin D (ER-OR = 2.4; P = 0.07; triple-negative OR = 2.6; P = 0.09). Conclusions: Breast cancer patients with suboptimal vitamin D levels were more likely to have tumors with more aggressive tumor profiles, worse prognostic markers (ER- and triple-negative tumors), and higher recurrence risk (Oncotype scores), lending support to previous research that found decreased breast cancer survival among vitamin D deficient individuals. Further research is needed to elucidate the biological relationship between vitamin D and prognostic breast cancer markers. This abstract is one of the 14 highest scoring abstracts of those submitted for presentation at the 35th Annual Meeting of the American Society of Preventive Oncology, held March 5–8, 2011 in Las Vegas, NV. more...

CMF is a rare neoplasm of bone, comprising less than 1% of primary bone tumors. The neoplasm was first described in 1948 by Jaffe and Lichtenstein as a separate entity from chondrosarcomas, having slightly different plain film characteristics and a benign clinical course. As of 1985, Kreicbergs, Lonnquist, and Willems estimated that approximately 400 cases have been published. Recently, we encountered a CMF with an unusual clinical presentation. The efficacy of MRI studies is shown. more...

The molecular classification for breast carcinomas has been used in clinical studies with a simple surrogate panel of immunohistochemistry (IHC) markers. The objective of this current project was to study the molecular classification of commonly used breast cancer cell lines by IHC analysis. Seventeen breast cancer cell lines were harvested, fixed in formalin and made into cell blocks. IHC analyses were performed on each cell block with antibodies to estrogen receptor (ER), progesterone receptor (PR), HER2, EGFR, CK5/6, Ki-67 and androgen receptor (AR). Among the 17 cell lines, MCF-7 and ZR-75-1 fell to Luminal A subtype; BT-474 to Luminal B subtype; SKBR-3, MDA-MD-435 and AU 565 to HER2 over-expression subtype; MDA-MB-231, MCF-12A, HBL 101, HS 598 T, MCF-10A, MCF-10F, BT-20, 468 and BT-483 to basal subtype. MDA-MB-453 belonged to Unclassified subtype. Since each subtype defined by this IHC-based molecular classification does show a distinct clinical outcome, attention should be paid when choosing a cell line for any study. more...

The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that plays an important role in tumor growth, invasion and metastasis. EGFR Positive immunoreactivity ranged from focal to diffuse in overexpression has been shown in many different tumors and multiple EGFR-targeted therapies are currently being investigated. Renal epithelioid angiomyolipoma (epiAML) is an aggressive and potentially malignant variant of conventional angiomyolipoma (cAML). Currently, the status of EGFR expression in epiAML is unknown. We compared EGFR protein expression, as well as expression levels of several other tumor regulatory proteins including c-Kit, p53, alpha-catenin, beta-catenin and osteopontin in 6 cases of cAML and 10 cases of epiAML by semiquantitative immunohistochemistry (IHC). EGFR gene amplification was analyzed by fluorescent in situ hybridization (FISH). Within epiAML, EGFR immunostaining was significantly higher when compared to cAML (90% vs. 16.7%, p more...

Insulin-dependent diabetics may manifest evidence of autoimmune diseases involving endocrine or other organs. Rare cases of a peculiar fibrous and inflammatory lesion of the breast in diabetic patients have been previously described; however, the pathologic and clinical features that uniquely characterize these cases have not been defined or distinguished from other chronic inflammatory and fibrosing conditions in the breast. We studied eight patients with breast masses and longstanding insulin-dependent diabetes and compared them with 36 nondiabetic or short-duration diabetic patients with fibrosis and chronic mastitis. The longstanding diabetic patients presented with clinical breast masses ranging in size from 2 to 6 cm. Six of the eight patients had documented diabetic nephropathy, retinopathy, or neuropathy. Pathologically, these lesions showed lymphocytic lobulitis and ductitis, lymphocytic vasculitis (predominantly B cell), and dense keloid-like fibrosis that in many cases (six of eight) contained peculiar epithelioid cells embedded in dense fibrous stroma. We have provisionally labeled these cells "epithelioid fibroblasts" (EFBs). Although the features of lymphocytic lobulitis, ductitis, and/or vasculitis may occasionally be encountered in nondiabetic breast biopsies, EFBs appear to be unique to the diabetic condition. Control cases of chronic mastitis in nondiabetic or short-duration diabetes patients failed to show the complete constellation of lymphocytic lobulitis and ductitis, vasculitis, keloidal fibrosis, and EFBs. Diabetic mastopathy may represent an immune reaction to abnormal matrix accumulation. A hypothesis is presented. more...

A 24 year old male with a history of eczema, recurrent mild infections, and thrombocytopenia consistent with the Wiskott-Aldrich syndrome (WAS) presented with a mediastinal mass, generalized lymphadenopathy, splenomegaly, and severe thrombocytopenia. Studies of immune function including immunoglobulin levels and T-cell subsets were normal. Furthermore, his T lymphocytes proliferated normally in response to phytohemagglutinin, concanavalin A, and the combination of neuraminidase/galactose oxidase. However, their proliferative responses to anti-CD43 antibody and periodate were diminished, consistent with the clinical diagnosis of WAS. An initial inguinal lymph node biopsy surprisingly revealed Kaposi sarcoma. However, following splenectomy to increase the platelet count, biopsy of the mediastinal mass revealed T-cell large cell lymphoma. Studies of biopsied tissue for the presence of Epstein-Barr virus and cytomegalovirus were negative, as were studies of blood, including the polymerase chain reaction, for the presence of the human immunodeficiency virus (HIV). This is the first report of Kaposi sarcoma arising in a patient with a congenital immunodeficiency syndrome. Although Kaposi sarcoma can arise in the face of the severe immunosuppression that follows allograft transplantation and in patients infected with HIV, we postulate that longevity in the face of mild immunosuppression was the major factor in the development of Kaposi sarcoma in this patient. more...