Search

Background: Obesity and physical activity have been hypothesized to affect breast cancer risk partly via the androgen signaling pathway. We conducted the first study to evaluate these associations by tumor androgen receptor (AR) status., Methods: Height, weight, and physical activity were assessed using questionnaires in the Nurses' Health Study. AR, estrogen receptor (ER), and progesterone receptor (PR) status were determined using immunohistochemistry on tumor tissue and medical/pathology reports., Results: A total of 1,701 AR(+) and 497 AR(-) cases were documented during 26 years of follow-up of 103,577 women. After adjusting for ER/PR status and other risk factors, the relative risks (RR) and 95% confidence intervals (95% CI) for every 5 kg/m(2) increase in body mass index (BMI) were 1.07 (1.01-1.13) for AR(+) and 1.16 (1.05-1.29) for AR(-) tumors (P-heterogeneity = 0.17). The RRs (95% CIs) per 5 hours of brisk walking/week were 0.87 (0.73-1.04) for AR(+) and 0.67 (0.45-0.99) for AR(-) tumors (P-heterogeneity = 0.22). Further, BMI, but not physical activity, associations differed significantly across ER/PR/AR subtypes (P-heterogeneity = 0.04 and 0.63, respectively). The RRs (95% CIs) for 5 kg/m(2) increase in BMI were 1.23 (1.04-1.45) for ER(+)PR(+)AR(-), 1.19 (1.01-1.39) for ER(-)PR(-)AR(-), 1.15 (1.08-1.23) for ER(+)PR(+)AR(+), and 0.88 (0.75-1.03) for ER(+)PR(-)AR(+) tumors., Conclusions: Higher BMI was associated with an increased risk of both AR(+) and AR(-) breast tumors in postmenopausal women, whereas physical activity, including brisk walking, was associated with a reduced risk of both subtypes. In addition, a significant positive association was observed between higher BMI and ER(-)PR(-)AR(-) tumors., Impact: The similar associations observed by AR status suggest that mechanisms other than androgen signaling underlie these two breast cancer risk factors., (©2015 American Association for Cancer Research.)

Background: Mammographic density (MD) is a strong breast cancer risk factor. We previously reported associations of percent mammographic density (PMD) with larger and node-positive tumors across all ages, and estrogen receptor (ER)-negative status among women ages <55 years. To provide insight into these associations, we examined the components of PMD [dense area (DA) and nondense area (NDA)] with breast cancer subtypes., Methods: Data were pooled from six studies including 4,095 breast cancers and 8,558 controls. DA and NDA were assessed from digitized film-screen mammograms and standardized across studies. Breast cancer odds by density phenotypes and age according to histopathologic characteristics and receptor status were calculated using polytomous logistic regression., Results: DA was associated with increased breast cancer risk [OR for quartiles: 0.65, 1.00 (Ref), 1.22, 1.55; P(trend) <0.001] and NDA was associated with decreased risk [ORs for quartiles: 1.39, 1.00 (Ref), 0.88, 0.72; P(trend) <0.001] across all ages and invasive tumor characteristics. There were significant trends in the magnitude of associations of both DA and NDA with breast cancer by increasing tumor size (P(trend) < 0.001) but no differences by nodal status. Among women <55 years, DA was more strongly associated with increased risk of ER(+) versus ER(-) tumors (P(het) = 0.02), while NDA was more strongly associated with decreased risk of ER(-) versus ER(+) tumors (P(het) = 0.03)., Conclusions: DA and NDA have differential associations with ER(+) versus ER(-) tumors that vary by age., Impact: DA and NDA are important to consider when developing age- and subtype-specific risk models., (©2015 American Association for Cancer Research.)

Imaging plays a central role in the diagnosis of cancer and the evaluation of therapeutic efficacy in patients with cancer. Because macroscopic imaging is noninvasive and quantitative, the development of specialized instruments for small animals has spurred increasing utilization in preclinical cancer studies. Some small-animal imaging devices are miniaturized derivatives of clinical imaging modalities, including computed tomography, magnetic resonance imaging, positron-emission tomography, single-photon emission computed tomography, and ultrasonography. Optical imaging, including bioluminescence imaging and fluorescence imaging, has evolved from microscopic cellular imaging technologies. Here, we review how current imaging modalities are enabling high-resolution structural imaging with micrometer-scale spatial resolution, thus allowing for the quantification of tumor burden in genetically engineered and orthotopic models of cancer, where tumors develop within organs not typically accessible to measurements with calipers. Beyond measuring tumor size, imaging is increasingly being used to assess the activity of molecular pathways within tumors and to reveal the pharmacodynamic efficacy of targeted therapies. Each imaging technology has particular strengths and limitations, and we discuss how studies should be carefully designed to match the imaging approach to the primary experimental question., (© 2015 Cold Spring Harbor Laboratory Press.)

The development of tools in computational pathology to assist physicians and biomedical scientists in the diagnosis of disease requires access to high-quality annotated images for algorithm learning and evaluation. Generating high-quality expert-derived annotations is time-consuming and expensive. We explore the use of crowdsourcing for rapidly obtaining annotations for two core tasks in com- putational pathology: nucleus detection and nucleus segmentation. We designed and implemented crowdsourcing experiments using the CrowdFlower platform, which provides access to a large set of labor channel partners that accesses and manages millions of contributors worldwide. We obtained annotations from four types of annotators and compared concordance across these groups. We obtained: crowdsourced annotations for nucleus detection and segmentation on a total of 810 images; annotations using automated methods on 810 images; annotations from research fellows for detection and segmentation on 477 and 455 images, respectively; and expert pathologist-derived annotations for detection and segmentation on 80 and 63 images, respectively. For the crowdsourced annotations, we evaluated performance across a range of contributor skill levels (1, 2, or 3). The crowdsourced annotations (4,860 images in total) were completed in only a fraction of the time and cost required for obtaining annotations using traditional methods. For the nucleus detection task, the research fellow-derived annotations showed the strongest concordance with the expert pathologist- derived annotations (F-M =93.68%), followed by the crowd-sourced contributor levels 1,2, and 3 and the automated method, which showed relatively similar performance (F-M = 87.84%, 88.49%, 87.26%, and 86.99%, respectively). For the nucleus segmentation task, the crowdsourced contributor level 3-derived annotations, research fellow-derived annotations, and automated method showed the strongest concordance with the expert pathologist-derived annotations (F-M = 66.41%, 65.93%, and 65.36%, respectively), followed by the contributor levels 2 and 1 (60.89% and 60.87%, respectively). When the research fellows were used as a gold-standard for the segmentation task, all three con- tributor levels of the crowdsourced annotations significantly outperformed the automated method (F-M = 62.21%, 62.47%, and 65.15% vs. 51.92%). Aggregating multiple annotations from the crowd to obtain a consensus annotation resulted in the strongest performance for the crowd-sourced segmentation. For both detection and segmentation, crowd-sourced performance is strongest with small images (400 × 400 pixels) and degrades significantly with the use of larger images (600 × 600 and 800 × 800 pixels). We conclude that crowdsourcing to non-experts can be used for large-scale labeling microtasks in computational pathology and offers a new approach for the rapid generation of labeled images for algorithm development and evaluation.

The term 'field effect' (also known as field defect, field cancerization, or field carcinogenesis) has been used to describe a field of cellular and molecular alteration, which predisposes to the development of neoplasms within that territory. We explore an expanded, integrative concept, 'etiologic field effect', which asserts that various etiologic factors (the exposome including dietary, lifestyle, environmental, microbial, hormonal, and genetic factors) and their interactions (the interactome) contribute to a tissue microenvironmental milieu that constitutes a 'field of susceptibility' to neoplasia initiation, evolution, and progression. Importantly, etiological fields predate the acquisition of molecular aberrations commonly considered to indicate presence of filed effect. Inspired by molecular pathological epidemiology (MPE) research, which examines the influence of etiologic factors on cellular and molecular alterations during disease course, an etiologically focused approach to field effect can: (1) broaden the horizons of our inquiry into cancer susceptibility and progression at molecular, cellular, and environmental levels, during all stages of tumor evolution; (2) embrace host-environment-tumor interactions (including gene-environment interactions) occurring in the tumor microenvironment; and, (3) help explain intriguing observations, such as shared molecular features between bilateral primary breast carcinomas, and between synchronous colorectal cancers, where similar molecular changes are absent from intervening normal colon. MPE research has identified a number of endogenous and environmental exposures which can influence not only molecular signatures in the genome, epigenome, transcriptome, proteome, metabolome and interactome, but also host immunity and tumor behavior. We anticipate that future technological advances will allow the development of in vivo biosensors capable of detecting and quantifying 'etiologic field effect' as abnormal network pathology patterns of cellular and microenvironmental responses to endogenous and exogenous exposures. Through an 'etiologic field effect' paradigm, and holistic systems pathology (systems biology) approaches to cancer biology, we can improve personalized prevention and treatment strategies for precision medicine.

The categorization of intraductal proliferative lesions of the breast based on routine light microscopic examination of histopathologic sections is in many cases challenging, even for experienced pathologists. The development of computational tools to aid pathologists in the characterization of these lesions would have great diagnostic and clinical value. As a first step to address this issue, we evaluated the ability of computational image analysis to accurately classify DCIS and UDH and to stratify nuclear grade within DCIS. Using 116 breast biopsies diagnosed as DCIS or UDH from the Massachusetts General Hospital (MGH), we developed a computational method to extract 392 features corresponding to the mean and standard deviation in nuclear size and shape, intensity, and texture across 8 color channels. We used L1-regularized logistic regression to build classification models to discriminate DCIS from UDH. The top-performing model contained 22 active features and achieved an AUC of 0.95 in cross-validation on the MGH data-set. We applied this model to an external validation set of 51 breast biopsies diagnosed as DCIS or UDH from the Beth Israel Deaconess Medical Center, and the model achieved an AUC of 0.86. The top-performing model contained active features from all color-spaces and from the three classes of features (morphology, intensity, and texture), suggesting the value of each for prediction. We built models to stratify grade within DCIS and obtained strong performance for stratifying low nuclear grade vs. high nuclear grade DCIS (AUC = 0.98 in cross-validation) with only moderate performance for discriminating low nuclear grade vs. intermediate nuclear grade and intermediate nuclear grade vs. high nuclear grade DCIS (AUC = 0.83 and 0.69, respectively). These data show that computational pathology models can robustly discriminate benign from malignant intraductal proliferative lesions of the breast and may aid pathologists in the diagnosis and classification of these lesions.

Mammographic density reflects the amount of stromal and epithelial tissues in relation to adipose tissue in the breast and is a strong risk factor for breast cancer. Here we report the results from meta-analysis of genome-wide association studies (GWAS) of three mammographic density phenotypes: dense area, non-dense area and percent density in up to 7,916 women in stage 1 and an additional 10,379 women in stage 2. We identify genome-wide significant (P<5 × 10(-8)) loci for dense area (AREG, ESR1, ZNF365, LSP1/TNNT3, IGF1, TMEM184B and SGSM3/MKL1), non-dense area (8p11.23) and percent density (PRDM6, 8p11.23 and TMEM184B). Four of these regions are known breast cancer susceptibility loci, and four additional regions were found to be associated with breast cancer (P<0.05) in a large meta-analysis. These results provide further evidence of a shared genetic basis between mammographic density and breast cancer and illustrate the power of studying intermediate quantitative phenotypes to identify putative disease-susceptibility loci.

Repressive histone tail modifications have been associated with molecular breast cancer subtypes. We investigated whether histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) were associated with tumor features and subtypes while adjusting for prospectively collected reproductive and lifestyle breast cancer risk factors. We have tissue microarray data with immunohistochemical marker information on 804 incident cases of invasive breast cancer diagnosed from 1976-2000 in the Nurses' Health Study. Tissue microarray sections were stained for global H3K9me3 and H3K27me3, and scored into four categories. Multivariate odds ratios (OR) and 95 % confidence intervals (CI) were calculated using logistic regression models for tumor features and subtypes, adjusting for breast cancer risk factors. While there were no significant associations between H3K9me3 and tumor features, H3K27me3 was significantly associated with lower grade tumors compared to high grade tumors in the multivariate model (OR = 1.95, 95 % CI 1.35-2.81, p = 0.0004). H3K27me3 was suggestively associated with estrogen receptor-positive (ER+) tumors (OR = 1.47, 95 % CI 0.97-2.23, p = 0.07). In subtype analyses, H3K27me3 was positively associated with the luminal A subtype compared to all other subtypes (OR = 1.42, 95 % CI 1.14-1.77, p = 0.002), and was inversely associated with HER2-type (OR = 0.58, 95 % CI 0.37-0.91, p = 0.02) and basal-like breast cancer (OR = 0.52, 95 % CI 0.36-0.76, p = 0.0006). In the largest immunohistochemical examination of H3K9me3 and H3K27me3 in breast cancer, we found that H3K27me3 positivity, but not H3K9me3, was associated with lower grade tumors and the luminal A subtype after adjusting for reproductive and lifestyle breast cancer risk factors.

Large-scale pharmacogenomic high-throughput screening (HTS) studies hold great potential for generating robust genomic predictors of drug response. Two recent large-scale HTS studies have reported results of such screens, revealing several known and novel drug sensitivities and biomarkers. Subsequent evaluation, however, found only moderate interlaboratory concordance in the drug response phenotypes, possibly due to differences in the experimental protocols used in the two studies. This highlights the need for community-wide implementation of standardized assays for measuring drug response phenotypes so that the full potential of HTS is realized. We suggest that the path forward is to establish best practices and standardization of the critical steps in these assays through a collective effort to ensure that the data produced from large-scale screens would not only be of high intrastudy consistency, so that they could be replicated and compared successfully across multiple laboratories., (©2014 American Association for Cancer Research.)

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-β and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Genes encoding components of the PI3K-AKT-mTOR signaling axis are frequently mutated in cancer, but few mutations have been characterized in MTOR, the gene encoding the mTOR kinase. Using publicly available tumor genome sequencing data, we generated a comprehensive catalog of mTOR pathway mutations in cancer, identifying 33 MTOR mutations that confer pathway hyperactivation. The mutations cluster in six distinct regions in the C-terminal half of mTOR and occur in multiple cancer types, with one cluster particularly prominent in kidney cancer. The activating mutations do not affect mTOR complex assembly, but a subset reduces binding to the mTOR inhibitor DEPTOR. mTOR complex 1 (mTORC1) signaling in cells expressing various activating mutations remains sensitive to pharmacologic mTOR inhibition, but is partially resistant to nutrient deprivation. Finally, cancer cell lines with hyperactivating MTOR mutations display heightened sensitivity to rapamycin both in culture and in vivo xenografts, suggesting that such mutations confer mTOR pathway dependency.

PTEN dysfunction plays a crucial role in the pathogenesis of hereditary and sporadic cancers. Here, we show that PTEN homodimerizes and, in this active conformation, exerts lipid phosphatase activity on PtdIns(3,4,5)P3. We demonstrate that catalytically inactive cancer-associated PTEN mutants heterodimerize with wild-type PTEN and constrain its phosphatase activity in a dominant-negative manner. To study the consequences of homo- and heterodimerization of wild-type and mutant PTEN in vivo, we generated Pten knockin mice harboring two cancer-associated PTEN mutations (PtenC124S and PtenG129E). Heterozygous Pten(C124S/+) and Pten(G129E/+) cells and tissues exhibit increased sensitivity to PI3-K/Akt activation compared to wild-type and Pten(+/-) counterparts, whereas this difference is no longer apparent between Pten(C124S/-) and Pten(-/-) cells. Notably, Pten KI mice are more tumor prone and display features reminiscent of complete Pten loss. Our findings reveal that PTEN loss and PTEN mutations are not synonymous and define a working model for the function and regulation of PTEN., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: The PI3K-AKT signaling pathway regulates all phenotypes that contribute to progression of human cancers, including breast cancer. AKT mediates signal relay by phosphorylating numerous substrates, which are causally implicated in biologic responses such as cell growth, survival, metabolic reprogramming, migration, and invasion. Here a new AKT substrate is identified, the adherens junction protein Afadin, which is phosphorylated by AKT at Ser1718. Importantly, under conditions of physiologic IGF-1 signaling and oncogenic PI3K and AKT, Afadin is phosphorylated by all AKT isoforms, and this phosphorylation elicits a relocalization of Afadin from adherens junctions to the nucleus. Also, phosphorylation of Afadin increased breast cancer cell migration that was dependent on Ser1718 phosphorylation. Finally, nuclear localization of Afadin was observed in clinical breast cancer specimens, indicating that regulation of Afadin by the PI3K-AKT pathway has pathophysiologic significance., Implications: Phosphorylation of the adhesion protein Afadin by AKT downstream of the PI3K pathway, leads to redistribution of Afadin and controls cancer cell migration.

Introduction: The ThinPrep Imaging System ([Imager], Cytyc Corporation, Hologic, Inc.) for cervical cytology is commonly employed in laboratories. It uses a proprietary stain that allows measurement of cellular DNA content. We evaluated the initial effect of implementing the Imager stain on atypical squamous cells of undetermined significance (ASCUS) and squamous intraepithelial lesion (SIL) diagnoses, and high-risk human papillomavirus (hrHPV) rates., Materials and Methods: This study included 264 Imager-stained ASCUS cases from the first 5 months of Imager use. All cases were analyzed for hrHPV using Hybrid Capture II assay (Digene Corporation, Qiagen). ASCUS/SIL ratios and hrHPV-positive rates were calculated for the laboratory overall and for 3 cytopathologists. ASCUS/SIL ratios were also calculated for 5 cytotechnologists. These metrics were compared with those from the equivalent 5-month period 1 year prior. Slide review of all Imager-stained ASCUS cases was performed to assess for potential factors involved in ASCUS misinterpretation., Results: The proportion of ASCUS diagnoses increased after Imager stain introduction, from 1.79% to 3.14% (P < 0.001). The proportion of SIL diagnoses also increased, from 0.69% to 1.1% (P < 0.001). However, the hrHPV-positive rate declined from 61.3% to 53.6% (P = 0.104). Review of Imager-stained ASCUS cases showed marked nuclear hyperchromasia, particularly in benign squamous metaplastic cells., Conclusions: Imager stain implementation, required for the ThinPrep Imaging System, led to significant increase in ASCUS rates while the hrHPV-positive rate declined. Cytopathologists will need to recalibrate thresholds for an ASCUS diagnosis when interpreting Imager-stained slides. In particular, nuclear hyperchromasia in squamous metaplastic cells should not be overdiagnosed as ASCUS., (Copyright © 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Gene set enrichment analysis (GSEA) associates gene sets and phenotypes, its use is predicated on the choice of a pre-defined collection of sets. The defacto standard implementation of GSEA provides seven collections yet there are no guidelines for the choice of collections and the impact of such choice, if any, is unknown. Here we compare each of the standard gene set collections in the context of a large dataset of drug response in human cancer cell lines. We define and test a new collection based on gene co-expression in cancer cell lines to compare the performance of the standard collections to an externally derived cell line based collection. The results show that GSEA findings vary significantly depending on the collection chosen for analysis. Henceforth, collections should be carefully selected and reported in studies that leverage GSEA.

Triple-negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective targeted therapy and, as a consequence, in general has a poor outcome. To identify new therapeutic targets in TNBC, we performed a short hairpin RNA (shRNA) screen for protein kinases commonly amplified and overexpressed in breast cancer. Using this approach, we identified AKT3 as a gene preferentially required for the growth of TNBCs. Downregulation of Akt3 significantly inhibits the growth of TNBC lines in three-dimensional (3D) spheroid cultures and in mouse xenograft models, whereas loss of Akt1 or Akt2 have more modest effects. Akt3 silencing markedly upregulates the p27 cell-cycle inhibitor and this is critical for the ability of Akt3 to inhibit spheroid growth. In contrast with Akt1, Akt3 silencing results in only a minor enhancement of migration and does not promote invasion. Depletion of Akt3 in TNBC sensitizes cells to the pan-Akt inhibitor GSK690693. These results imply that Akt3 has a specific function in TNBCs; thus, its therapeutic targeting may provide a new treatment option for this tumor subtype.

Sarcomas of soft tissue and bone are rare neoplasms that can be separated into a large number of different diagnostic entities. Over the years, a number of diagnostic markers have been developed that aid pathologists in reaching the appropriate diagnoses. Many of these markers are sarcoma-specific proteins that can be detected by immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) sections. In addition, a wide range of molecular studies have been developed that can detect gene mutations, gene amplifications or chromosomal translocations in FFPE material. Until recently, most sequencing-based approaches relied on the availability of fresh frozen tissue. However, with the advent of next-generation sequencing technologies, FFPE material is increasingly being used as a tool to identify novel immunohistochemistry markers, gene mutations, and chromosomal translocations, and to develop diagnostic tests., (© 2013 John Wiley & Sons Ltd.)

Two large-scale pharmacogenomic studies were published recently in this journal. Genomic data are well correlated between studies; however, the measured drug response data are highly discordant. Although the source of inconsistencies remains uncertain, it has potential implications for using these outcome measures to assess gene-drug associations or select potential anticancer drugs on the basis of their reported results.

Introduction: Understanding whether mammographic density (MD) is associated with all breast tumor subtypes and whether the strength of association varies by age is important for utilizing MD in risk models., Methods: Data were pooled from six studies including 3414 women with breast cancer and 7199 without who underwent screening mammography. Percent MD was assessed from digitized film-screen mammograms using a computer-assisted threshold technique. We used polytomous logistic regression to calculate breast cancer odds according to tumor type, histopathological characteristics, and receptor (estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER2)) status by age (<55, 55-64, and ≥ 65 years)., Results: MD was positively associated with risk of invasive tumors across all ages, with a two-fold increased risk for high (>51%) versus average density (11-25%). Women ages <55 years with high MD had stronger increased risk of ductal carcinoma in situ (DCIS) compared to women ages 55-64 and ≥ 65 years (P(age-interaction) = 0.02). Among all ages, MD had a stronger association with large (>2.1 cm) versus small tumors and positive versus negative lymph node status (P's < 0.01). For women ages <55 years, there was a stronger association of MD with ER-negative breast cancer than ER-positive tumors compared to women ages 55-64 and ≥ 65 years (P(age-interaction) = 0.04). MD was positively associated with both HER2-negative and HER2-positive tumors within each age group., Conclusion: MD is strongly associated with all breast cancer subtypes, but particularly tumors of large size and positive lymph nodes across all ages, and ER-negative status among women ages <55 years, suggesting high MD may play an important role in tumor aggressiveness, especially in younger women.

Increasing evidence points to an important role for the ribosome in the regulation of biological processes and as a target for deregulation in disease. Here, we describe a SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry approach to probing mammalian riboproteomes. Using a panel of cell lines, as well as genetic and pharmacological perturbations, we obtained a comparative characterization of the cellular riboproteome. This analysis identified a set of riboproteome components, consisting of a diverse array of proteins with a strong enrichment for RNA-binding proteins. Importantly, this global analysis uncovers a high incidence of genetic alterations to riboproteome components in cancer, with a distinct bias toward genetic amplification. We further validated association with polyribosomes for several riboproteome components and demonstrate that enrichment at the riboproteome can depend on cell type, genetics, or cellular stimulus. Our results have important implications for the understanding of how ribosomes function and provide a platform for uncovering regulators of translation., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Advancements in molecular biology have unveiled multiple breast cancer promoting pathways and potential therapeutic targets. Large randomized clinical trials remain the ultimate means of validating therapeutic efficacy, but they require large cohorts of patients and are lengthy and costly. A useful approach is to conduct a window of opportunity study in which patients are exposed to a drug pre-surgically during the interval between the core needle biopsy and the definitive surgery. These are non-therapeutic studies and the end point is not clinical or pathological response but rather evaluation of molecular changes in the tumor specimens that can predict response. However, since the end points of the non-therapeutic studies are biologic, it is critical to first define the biologic changes that occur in the absence of treatment. In this study, we compared the molecular profiles of breast cancer tumors at the time of the diagnostic biopsy versus the definitive surgery in the absence of any intervention using the Nanostring nCounter platform. We found that while the majority of the transcripts did not vary between the two biopsies, there was evidence of activation of immune related genes in response to the first biopsy and further investigations of the immune changes after a biopsy in early breast cancer seem warranted.

Although the incidence of human papillomavirus (HPV)-associated anal neoplasia is increasing, interobserver and intraobserver reproducibility in the grading of biopsy specimens from this area remains unacceptably low. Attempts to produce a more reproducible grading scheme have led to the use of biomarkers for the detection of high-risk HPV (HR-HPV). We evaluated the performance of standard morphology and biomarkers p16, ProEx C, and Ki-67 in a set of 75 lesions [17 nondysplastic lesions, 23 low-grade squamous intraepithelial lesions (LSIL)/condyloma, 20 high-grade squamous intraepithelial lesions (HSIL), 15 invasive squamous cell carcinomas] from the anal and perianal region in 65 patients and correlated these findings with HPV subtype on the basis of a type-specific multiplex real-time polymerase chain reaction assay designed to detect HR-HPV. A subset of cases with amplifiable HPV DNA was also sequenced. HSIL was typically flat (15/20), and only a minority (4/20) had koilocytes. In contrast, only 1 LSIL was flat (1/23), and the remainder were exophytic. The majority of LSIL had areas of koilocytic change (20/23). HR-HPV DNA was detected in the majority (89%) of invasive carcinomas and HSIL biopsies, 86% and 97% of which were accurately labeled by strong and diffuse block-positive p16 and ProEx C, respectively. LSIL cases, however, only infrequently harbored HR-HPV (13%); most harbored low-risk HPV (LR-HPV) types 6 and 11. Within the LSIL group, p16 outperformed ProEx C, resulting in fewer false-positive cases (5% vs. 75%). Ki-67 was also increased in HR-HPV-positive lesions, although biopsies with increased inflammation and reactive changes also showed higher Ki-67 indices. These data suggest that strong and diffuse block-positive nuclear and cytoplasmic labeling with p16 is a highly specific biomarker for the presence of HR-HPV in anal biopsies and that this finding correlates with high-grade lesions.

Introduction: Estrogen receptor (ER) and progesterone receptor (PR) testing are performed in the evaluation of breast cancer. While the clinical utility of ER as a predictive biomarker to identify patients likely to benefit from hormonal therapy is well-established, the added value of PR is less well-defined. The primary goals of our study were to assess the distribution, inter-assay reproducibility, and prognostic significance of breast cancer subtypes defined by patterns of ER and PR expression., Methods: We integrated gene expression microarray (GEM) and clinico-pathologic data from 20 published studies to determine the frequency (n = 4,111) and inter-assay reproducibility (n = 1,752) of ER/PR subtypes (ER+/PR+, ER+/PR-, ER-/PR-, ER-/PR+). To extend our findings, we utilized a cohort of patients from the Nurses' Health Study (NHS) with ER/PR data recorded in the medical record and assessed on tissue microarrays (n = 2,011). In both datasets, we assessed the association of ER and PR expression with survival., Results: In a genome-wide analysis, progesterone receptor was among the least variable genes in ER- breast cancer. The ER-/PR+ subtype was rare (approximately 1 to 4%) and showed no significant reproducibility (Kappa = 0.02 and 0.06, in the GEM and NHS datasets, respectively). The vast majority of patients classified as ER-/PR+ in the medical record (97% and 94%, in the GEM and NHS datasets) were re-classified by a second method. In the GEM dataset (n = 2,731), progesterone receptor mRNA expression was associated with prognosis in ER+ breast cancer (adjusted P <0.001), but not in ER- breast cancer (adjusted P = 0.21). PR protein expression did not contribute significant prognostic information to multivariate models considering ER and other standard clinico-pathologic features in the GEM or NHS datasets., Conclusion: ER-/PR+ breast cancer is not a reproducible subtype. PR expression is not associated with prognosis in ER- breast cancer, and PR does not contribute significant independent prognostic information to multivariate models considering ER and other standard clinico-pathologic factors. Given that PR provides no clinically actionable information in ER+ breast cancer, these findings question the utility of routine PR testing in breast cancer.

A major goal in translational cancer research is to identify biological signatures driving cancer progression and metastasis. A common technique applied in genomics research is to cluster patients using gene expression data from a candidate prognostic gene set, and if the resulting clusters show statistically significant outcome stratification, to associate the gene set with prognosis, suggesting its biological and clinical importance. Recent work has questioned the validity of this approach by showing in several breast cancer data sets that "random" gene sets tend to cluster patients into prognostically variable subgroups. This work suggests that new rigorous statistical methods are needed to identify biologically informative prognostic gene sets. To address this problem, we developed Significance Analysis of Prognostic Signatures (SAPS) which integrates standard prognostic tests with a new prognostic significance test based on stratifying patients into prognostic subtypes with random gene sets. SAPS ensures that a significant gene set is not only able to stratify patients into prognostically variable groups, but is also enriched for genes showing strong univariate associations with patient prognosis, and performs significantly better than random gene sets. We use SAPS to perform a large meta-analysis (the largest completed to date) of prognostic pathways in breast and ovarian cancer and their molecular subtypes. Our analyses show that only a small subset of the gene sets found statistically significant using standard measures achieve significance by SAPS. We identify new prognostic signatures in breast and ovarian cancer and their corresponding molecular subtypes, and we show that prognostic signatures in ER negative breast cancer are more similar to prognostic signatures in ovarian cancer than to prognostic signatures in ER positive breast cancer. SAPS is a powerful new method for deriving robust prognostic biological signatures from clinically annotated genomic datasets.

In recent decades, epidemiology, public health, and medical sciences have been increasingly compartmentalized into narrower disciplines. The authors recognize the value of integration of divergent scientific fields in order to create new methods, concepts, paradigms, and knowledge. Herein they describe the recent emergence of molecular pathological epidemiology (MPE), which represents an integration of population and molecular biologic science to gain insights into the etiologies, pathogenesis, evolution, and outcomes of complex multifactorial diseases. Most human diseases, including common cancers (such as breast, lung, prostate, and colorectal cancers, leukemia, and lymphoma) and other chronic diseases (such as diabetes mellitus, cardiovascular diseases, hypertension, autoimmune diseases, psychiatric diseases, and some infectious diseases), are caused by alterations in the genome, epigenome, transcriptome, proteome, metabolome, microbiome, and interactome of all of the above components. In this era of personalized medicine and personalized prevention, we need integrated science (such as MPE) which can decipher diseases at the molecular, genetic, cellular, and population levels simultaneously. The authors believe that convergence and integration of multiple disciplines should be commonplace in research and education. We need to be open-minded and flexible in designing integrated education curricula and training programs for future students, clinicians, practitioners, and investigators.

Background: Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported., Results: We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker., Conclusions: This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research.

ProExC expression has been shown to perform similarly to p16 as an aid in the diagnosis of cervical dysplasia but has not been well characterized in head and neck squamous cell carcinomas (SCC). The purpose of this study is to determine whether ProExC performs similarly to p16 as a prognostic marker in oropharyngeal SCC and to evaluate the threshold of ProExC and p16 staining that correlates with survival. ProExC, p16, and human papillomavirus DNA in situ hybridization were performed on tissue microarray (TMA) cores and whole sections from 62 patients with oropharyngeal SCC. Sensitivity and specificity for high-risk HPV and correlation with overall survival (OS), cancer-specific survival (CSS), and time to distant metastasis (TDM) were calculated for ProExC and p16 at different thresholds. ProExC did not prove to be a robust marker. It showed strong correlation with OS at a 66% threshold on TMA cores, but correlation with OS was lost on whole sections. It also exhibited low sensitivity (53.7%) on TMA cores and low specificity on whole sections (65%). ProExC at a 33% threshold exhibited unacceptably low specificity and did not correlate with OS, CSS, or TDM. Sensitivity and specificity of p16 varied predictably with threshold: higher sensitivity and lower specificity with lower thresholds and vice versa for higher thresholds. p16 at a 50% threshold offers a balance between sensitivity and specificity, and correlates with OS, CSS, and TDM on whole sections; correlation with TDM is lost on TMA cores. These findings indicate that ProExC does not perform well enough to be used as a prognostic marker in oropharyngeal SCC. p16 should be used and scored as positive when at least half the tumor is strongly stained.

Soft-tissue sarcomas are a group of malignant tumours whose clinical management is complicated by morphological heterogeneity, inadequate molecular markers and limited therapeutic options. Receptor tyrosine kinases (RTKs) have been shown to play important roles in cancer, both as therapeutic targets and as prognostic biomarkers. An initial screen of gene expression data for 48 RTKs in 148 sarcomas showed that ROR2 was expressed in a subset of leiomyosarcoma (LMS), gastrointestinal stromal tumour (GIST) and desmoid-type fibromatosis (DTF). This was further confirmed by immunohistochemistry (IHC) on 573 tissue samples from 59 sarcoma tumour types. Here we provide evidence that ROR2 expression plays a role in the invasive abilities of LMS and GIST cells in vitro. We also show that knockdown of ROR2 significantly reduces tumour mass in vivo using a xenotransplantation model of LMS. Lastly, we show that ROR2 expression, as measured by IHC, predicts poor clinical outcome in patients with LMS and GIST, although it was not independent of other clinico-pathological features in a multivariate analysis, and that ROR2 expression is maintained between primary tumours and their metastases. Together, these results show that ROR2 is a useful prognostic indicator in the clinical management of these soft-tissue sarcomas and may represent a novel therapeutic target., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.

Leiomyosarcoma (LMS) is a malignant, soft-tissue tumor for which few effective therapies exist. Previously, we showed that there are three molecular subtypes of LMS. Here, we analyzed genes differentially expressed in each of the three LMS subtypes as compared to benign leiomyomas and then used the Connectivity Map (cmap) to calculate enrichment scores for the 1309 cmap drugs in order to identify candidate molecules with the potential to induce a benign, leiomyoma-like phenotype in LMS cells. 11 drugs were selected and tested for their ability to inhibit the growth of three human LMS cell lines. We identified two drugs with in vitro efficacy against LMS, one of which had a strongly negative enrichment score (Cantharidin) and the other of which had a strongly positive enrichment score (MG-132). Given MG-132's strong inhibitory effect on LMS cell viability, we hypothesized that LMS cells may be sensitive to treatment with other proteasome inhibitors and demonstrated that bortezomib, a clinically-approved proteasome inhibitor not included in the original cmap screen, potently inhibited the viability of the LMS cell lines. These findings suggest that systematically linking LMS subtype-specific expression signatures with drug-associated expression profiles represents a promising approach for the identification of new drugs for LMS.

The morphological interpretation of histologic sections forms the basis of diagnosis and prognostication for cancer. In the diagnosis of carcinomas, pathologists perform a semiquantitative analysis of a small set of morphological features to determine the cancer's histologic grade. Physicians use histologic grade to inform their assessment of a carcinoma's aggressiveness and a patient's prognosis. Nevertheless, the determination of grade in breast cancer examines only a small set of morphological features of breast cancer epithelial cells, which has been largely unchanged since the 1920s. A comprehensive analysis of automatically quantitated morphological features could identify characteristics of prognostic relevance and provide an accurate and reproducible means for assessing prognosis from microscopic image data. We developed the C-Path (Computational Pathologist) system to measure a rich quantitative feature set from the breast cancer epithelium and stroma (6642 features), including both standard morphometric descriptors of image objects and higher-level contextual, relational, and global image features. These measurements were used to construct a prognostic model. We applied the C-Path system to microscopic images from two independent cohorts of breast cancer patients [from the Netherlands Cancer Institute (NKI) cohort, n = 248, and the Vancouver General Hospital (VGH) cohort, n = 328]. The prognostic model score generated by our system was strongly associated with overall survival in both the NKI and the VGH cohorts (both log-rank P ≤ 0.001). This association was independent of clinical, pathological, and molecular factors. Three stromal features were significantly associated with survival, and this association was stronger than the association of survival with epithelial characteristics in the model. These findings implicate stromal morphologic structure as a previously unrecognized prognostic determinant for breast cancer.

Leiomyosarcoma (LMS) is a malignant tumor of smooth muscle cells for which few effective therapies exist. A subset of LMS cases express macrophage colony-stimulating factor (CSF1) and the resultant tumor-associated macrophage (TAM) infiltration predicts poor clinical outcome. Further, TAMs have been shown to increase tumor angiogenesis. Here, we analyzed 149 LMS cases by immunohistochemistry for vascular marker CD34 and show that high microvessel density (MVD) in nongynecological LMS cases significantly predicts poor patient outcome. The majority of high MVD cases were also CSF1-positive, and when combining high MVD with CSF1 expression, an even stronger prognostic correlation with patient outcome was obtained. Gene expression profiling revealed that MVD has a stronger correlation with CSF1 expression than with expression of vascular endothelial growth factor isoforms, which have traditionally been used as markers of angiogenesis and as anti-angiogenic therapeutic targets. Finally, patterns of CSF1 expression and TAM recruitment remained consistent between primary tumors and their metastases, and between primary tumors and those grown as xenografts in mice, highlighting the stability of these features to the biology of LMS tumors. Together, these findings suggest an important role for CSF1 and the resulting TAM infiltration in the pathological neovascularization of LMS tumors and provide a rationale for CSF1-targeted therapies in LMS., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The Tissue of Origin Frozen (TOO-FRZ) assay from Pathwork Diagnostics has been cleared by the Food and Drug Administration as a diagnostic study for malignancies of unknown primary. The goal of this study was to evaluate the performance of TOO-FRZ on a diverse collection of malignancies. We collected a diverse set of 49 malignancies. We classified each case into 1 of 4 groups: common morphology from a tissue type included in the TOO-FRZ assay (n=29), uncommon morphology from a tissue type included in the TOO-FRZ assay (n=10), tumor from a tissue type not included in the TOO-FRZ assay (n=3), and malignancies of unknown primary (n=7). We found strong diagnostic performance for common morphologies from tissue types on the TOO-FRZ [overall accuracy=26 of 29 (90%, 95% CI, 73% to 97%)], with perfect performance in all tissue types except gastric (0 of 2) and pancreatic (1 of 2) tissues. There was a significant decline in performance for uncommon morphologies from tissue types included in the TOO-FRZ assay [6 of 10 (60%) cases with an indeterminate result, 1 of 10 (10%) cases with an incorrect prediction, and 3 of 10 (30%) with a correct prediction] and for tumors from tissue types not included in the assay (incorrect prediction in 2 of 3 cases). For the 7 malignancies of unknown primary in our study set, the TOO-FRZ provided a likely clinically useful result in only 2 of 7 cases. These results provide an insight into the strengths and limitations of this molecular assay for the surgical pathologist, and our findings suggest future directions for research in this area.

Objectives: To develop the diagnostic criteria for benign and neoplastic conditions of the urinary tract using probe-based confocal laser endomicroscopy (pCLE), a new technology for dynamic, in vivo imaging with micron-scale resolution. The suggested diagnostic criteria will formulate a guide for pCLE image interpretation in urology., Methods: Patients scheduled for transurethral resection of bladder tumor (TURBT) or nephrectomy were recruited. After white-light cystoscopy (WLC), fluorescein was administered as contrast. Different areas of the urinary tract were imaged with pCLE via direct contact between the confocal probe and the area of interest. Confocal images were subsequently compared with standard hematoxylin and eosin analysis., Results: pCLE images were collected from 66 participants, including 2 patients who underwent nephrectomy. We identified key features associated with different anatomic landmarks of the urinary tract, including the kidney, ureter, bladder, prostate, and urethra. In vivo pCLE of the bladder demonstrated distinct differences between normal mucosa and neoplastic tissue. Using mosaicing, a post hoc image-processing algorithm, individual image frames were juxtaposed to form wide-angle views to better evaluate tissue microarchitecture., Conclusions: In contrast to standard pathologic analysis of fixed tissue with hematoxylin and eosin, pCLE provides real time microscopy of the urinary tract to enable dynamic interrogation of benign and neoplastic tissues in vivo. The diagnostic criteria developed in this study will facilitate adaptation of pCLE for use in conjunction with WLC to expedite diagnosis of urinary tract pathology, particularly bladder cancer., (Published by Elsevier Inc.)

We studied the efficacy of 2 germinal center B-cell markers, HGAL and LMO2, in the separation of lymphomas derived from small B cells, particularly follicular lymphoma (FL) and marginal zone lymphoma occurring in nodal, extranodal, splenic, and bone marrow sites using immunohistochemical analysis for CD10, BCL6, BCL2, HGAL, and LMO2. Our results showed that HGAL and LMO2 are sensitive and specific markers for detecting FL in nodal and extranodal sites. In contrast, all markers were down-regulated in FL infiltrates in the bone marrow. CD10 and HGAL were expressed in a subset of FLs in the bone marrow and were highly correlated with each other and with CD21, a marker of follicular dendritic cells. We conclude that HGAL and LMO2 should be considered in immunohistochemical panels used for the routine workup of lymphomas derived from small B cells. In the bone marrow, staining for HGAL or CD10 can be helpful in making a diagnosis of FL, although they are absent in a subset of cases.

Leiomyosarcomas (LMSs) constitute approximately one quarter of all sarcomas and are usually defined by morphologic criteria and/or immunoreactivity for actin or desmin. Among high-grade lesions, the distinction from undifferentiated pleomorphic sarcoma (UPS) can be problematic, and previous studies have shown that a significant number of LMS cases may be hiding under the diagnosis of UPS. We recently described 3 novel molecular LMS subtypes that are distributed similarly over LMSs of gyneocologic and non-gyneocologic origins. The group 1 subtype shows an improved disease-specific survival compared with the other 2 groups that is independent of histologic grade. Group 1 comprises approximately 25% of all LMSs, and is defined by a shared pattern of gene expression, a distinct pattern of genomic changes, and reactivity for at least 3 of 5 immunohistochemistry (IHC) markers (smooth muscle gamma actin, calsequestrin 2, human muscle cofilin2, myosin light chain kinase, and sarcolemmal membrane associated protein), as tested on 271 cases of LMS in tissue microarrays. These IHC markers have not been well characterized in non-LMS sarcomas. Here we provide a characterization of these 5 markers across normal tissues, an additional 59 cases of LMS, and a wide range of 565 non-LMS soft tissue tumors from 44 diagnostic categories, with a focus on UPS. When analyzed individually, the 5 markers were found to be expressed in many sarcomas other than LMSs. However, when analyzed by the same criteria used for the recognition of group 1 LMSs, in which a case is scored positive when at least 3 of 5 markers reacted, coordinate expression was seen in significant numbers of cases from only 3 diagnostic groups that included 22% of leiomyomas (n=22), 16% of gastrointestinal stromal tumors (n=43), and 18% of endometrial stromal sarcomas (n=11). In addition, 5% (n=57) of UPSs showed a staining pattern similar to that seen in group 1 LMSs. To further examine the possibility that group 1 LMS constitutes a small part of cases diagnosed as UPS, we examined the expression of the top 500 genes from the group 1 LMS expression signature in 29 UPSs by complementary DNA microarray. Unsupervised hierarchical clustering of 29 UPS expression showed that 2 (7%) had coordinated high levels of expression of genes from the group 1 LMS signature, a rate similar to that seen by IHC analysis. These findings show that group 1 LMS IHC markers smooth muscle gamma actin, calsequestrin 2, human muscle cofilin2, myosin light chain kinase, and sarcolemmal membrane associated protein when coordinately expressed have specificity for a subset of LMS when compared with other sarcomas, and may be useful for the recognition of group 1 LMS cases within cases diagnosed as UPS.

PAX (paired box) genes encode a family of transcription factors that regulate organogenesis in a variety of organs. Very little is known about the role of PAX8 in endocrine cell development and the expression of PAX8 in neuroendocrine tumors. The purpose of this study was to analyze PAX8 immunohistochemical expression in gastroenteropancreatic and pulmonary well-differentiated neuroendocrine tumors to determine whether PAX8 can reliably distinguish pancreatic neuroendocrine tumors from neuroendocrine tumors of other anatomic sites and other pancreatic non-ductal neoplasms. In total, 221 well-differentiated neuroendocrine tumors were evaluated: 174 primary neuroendocrine tumors (66 pancreatic, 31 ileal, 21 pulmonary, 20 gastric, 17 rectal, 11 appendiceal, and 8 duodenal) and 47 neuroendocrine tumors metastatic to the liver (31 pancreatic, 11 ileal, 2 pulmonary, 2 duodenal, and 1 rectal). Fifteen solid-pseudopapillary neoplasms and six acinar cell carcinomas of the pancreas were also evaluated. PAX8 was positive in 49/66 (74%) primary pancreatic neuroendocrine tumors. PAX8 expression did not correlate with World Health Organization categorization, grade, size, functional status, or the presence of liver or lymph node metastasis. PAX8 expression was identified in 0/31 (0%) ileal, 0/21 (0%) pulmonary, 2/20 (10%) gastric, 5/17 (29%) rectal, 1/11 (9%) appendiceal, and 6/8 (75%) duodenal neuroendocrine tumors. PAX8 was positive in 4/15 (27%) solid-pseudopapillary neoplasms of the pancreas, whereas all acinar cell carcinomas (0/6) lacked immunoreactivity. Among liver metastases, only pancreatic neuroendocrine tumors (20/31, 65%) were PAX8 positive, whereas no cases of ileal (0/11), pulmonary (0/2), duodenal (0/2), and rectal (0/1) neuroendocrine tumor metastases were PAX8 positive. PAX8 is expressed in primary and metastatic pancreatic well-differentiated neuroendocrine tumors, and its expression can reliably distinguish pancreatic from ileal and pulmonary well-differentiated neuroendocrine tumors. Duodenal neuroendocrine tumors and a subset of rectal, gastric, and appendiceal neuroendocrine tumors may also express PAX8. PAX8 expression can distinguish pancreatic neuroendocrine tumors from acinar cell carcinomas, but its utility in distinguishing neuroendocrine tumors from solid-pseudopapillary neoplasms is limited.

Purpose: We recently described two types of stromal response in breast cancer derived from gene expression studies of tenosynovial giant cell tumors and fibromatosis. The purpose of this study is to elucidate the basis of this stromal response--whether they are elicited by individual tumors or whether they represent an endogenous host reaction produced by the patient., Experimental Design: Stromal signatures from patients with synchronous dual primaries were analyzed by immunohistochemistry on a tissue microarray (n = 26 pairs) to evaluate the similarity of stromal responses in different tumors within the same patient. We also characterized the extent to which the stromal signatures were conserved between stromal response to injury compared to the stromal response to carcinoma using gene expression profiling and tissue microarray immunohistochemistry., Results: The two stromal response signatures showed divergent associations in synchronous primaries: the DTF fibroblast response is more likely to be similar in a patient with multiple breast primaries (permutation analysis P = 0.0027), whereas CSF1 macrophage response shows no significant concordance in separate tumors within a given patient. The DTF fibroblast signature showed more concordance across normal, cancer, and biopsy site samples from within a patient, than across normal, cancer, and biopsy site samples from a random group of patients, whereas the CSF1 macrophage response did not., Conclusions: The results suggest that the DTF fibroblast response is host-specific, whereas the CSF1 response may be tumor-elicited. Our findings provide further insight into stromal response and may facilitate the development of therapeutic strategies to target particular stromal subtypes., (©2010 AACR.)

Lymph node status is one of the most important predictors of survival in pancreatic ductal adenocarcinoma. Surgically resected pancreatic adenocarcinoma is often locally invasive and may invade directly into peripancreatic lymph nodes. The significance of direct invasion into lymph nodes in the absence of true lymphatic metastases is unclear. The purpose of this study was to retrospectively compare clinical outcome in patients with pancreatic ductal adenocarcinoma with direct invasion into peripancreatic lymph nodes with patients with node-negative adenocarcinomas and patients with true lymphatic lymph node metastasis. A total of 380 patients with invasive pancreatic ductal adenocarcinoma classified as pT3, were evaluated: ductal adenocarcinoma with true lymphatic metastasis to regional lymph nodes (248 cases), ductal adenocarcinoma without lymph node involvement (97 cases), and ductal adenocarcinoma with regional lymph nodes involved only by direct invasion from the main tumor mass (35 cases). Isolated lymph node involvement by direct invasion occurred in 35 of 380 (9%) patients. Overall survival for patients with direct invasion of lymph nodes (median survival, 21 mo; 5-year overall survival, 36%) was not statistically different from patients with node-negative adenocarcinomas (median survival, 30 mo; 5-year overall survival, 31%) (P=0.609). Patients with node-negative adenocarcinomas had an improved survival compared with patients with lymph node involvement by true lymphatic metastasis (median survival, 15 mo; 5-year overall survival, 8%) (P<0.001) regardless of the number of lymph nodes involved by adenocarcinoma. There was a trend toward decreased overall survival for patients with 1 or 2 lymph nodes involved by true lymphatic metastasis compared with patients with direct invasion of tumor into lymph nodes (P=0.056). However, this did not reach statistical significance. Our results indicate that patients with isolated direct lymph node invasion have a comparable overall survival with patients with node-negative adenocarcinomas as opposed to true lymphatic lymph node metastasis.

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the Escherichia coli 30S subunit suggest that ribosome assembly occurs via multiple parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery single-particle profiling (DSP), an application of time-resolved electron microscopy, was used to obtain more than 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome-assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates.

The tumour microenvironment (TME) plays an important role in tumour survival and growth, but little is known about the degree of preservation between different stromal response patterns found in primary tumours and their metastases. We have previously identified gene expression profiles for two distinct stromal signatures in breast carcinoma of fibroblast (aka DTF) and macrophage (aka CSF1) response and found them to be correlated with clinicopathological features, including outcome. In this study, we compare the DTF fibroblast and CSF1 macrophage stromal response patterns in primary breast and colorectal cancers to their matched lymph node metastases. In both breast and colorectal cancer, there was a significant positive correlation between the CSF1 macrophage signature in the primary tumours and the matched lymph node metastases, as assessed by immunohistochemical markers. No such correlation was observed for the DTF fibroblast signature. A similar result was seen in independent analysis of two published gene expression microarray datasets. The variations of these stromal reaction patterns from the primary to the metastasis shed light on the relationship between the neoplastic cells and the non-neoplastic cells in the TME. The preservation of the CSF1 macrophage response pattern in metastases lends support to targeting the CSF1 pathway in cancer., (Copyright 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Recent advances in the study of the tumor microenvironment have revealed significant interaction between tumor cells and their surrounding stroma in model systems. We have previously shown that two distinct stromal signatures derived from a macrophage (CSF1) response and a fibroblastic (DTF-like) response are present in subsets of invasive breast cancers and show a correlation with clinical outcome. In the present study we explore whether these signatures also exist in the stroma of ductal carcinoma in situ (DCIS). We studied the signatures by both gene expression profile analysis of a publically available data set of DCIS and by immunohistochemistry (IHC) on a tissue microarray of DCIS and invasive breast cancer cases. Both the gene expression and immunohistochemical data show that the macrophage response and fibroblast expression signatures are present in the stroma of subsets of DCIS cases. The incidence of the stromal signatures in DCIS is similar to the incidence in invasive breast cancer that we have previously reported. We also find that the macrophage response signature is associated with higher grade DCIS and cases which are ER and PR negative, whereas the fibroblast signature was not associated with any clinicopathologic features in DCIS. A comparison of 115 matched cases of DCIS and invasive breast cancer found a correlation between the type of stromal response in DCIS and invasive ductal carcinoma (IDC) within the same patient for both the macrophage response and the fibroblast stromal signatures (P = 0.03 and 0.08, respectively). This study is a first characterization of these signatures in DCIS. These signatures have significant clinicopathologic associations and tend to be conserved as the tumor progresses from DCIS to invasive breast cancer.

Follicular lymphoma (FL) can exhibit variant histologic patterns that can lead to confusion with other B-cell lymphomas and reactive conditions. Diagnostic markers such as CD10 and BCL2 may be difficult to interpret in variant FL patterns, and are often diminished or absent in the interfollicular and diffuse components. We evaluated 2 recently characterized germinal center B-cell markers, human germinal center associated lymphoma (HGAL), and LIM-only transcription factor 2 (LMO2), in 127 FL patient biopsies (94 nodal, 33 extranodal), and correlated the findings with histologic pattern, cellular composition, grade, and additional immunostains (CD20, CD3, CD21, CD10, BCL2, and BCL6). Architectural patterns included predominantly follicular (75%) and follicular and diffuse components (25%); 10 cases showed marginal zone differentiation and 3 were floral variants. Eighty-nine cases were low grade (38 grade 1; 51 grade 2) and 38 were grade 3 (29 grade 3A and 9 grade 3B). HGAL had the highest overall sensitivity of detecting FL and was superior in detecting the interfollicular and diffuse components compared with BCL2, LMO2, CD10, and BCL6. All 28 cases that lacked CD10, expressed HGAL, and the majority also expressed LMO2. Our results show that HGAL and LMO2 are sensitive markers for FL diagnosis. The addition of HGAL and LMO2 to the immunohistologic panel is beneficial in the work-up of nodal and extranodal B-cell lymphomas and the efficacy of HGAL in detecting the follicular, interfollicular and diffuse components of FL is of particular value in the setting of variant immunoarchitectural patterns.

Invasive micropapillary carcinoma (IMPC) of the urinary tract is a well-described variant of the urothelial carcinoma with aggressive clinical behavior. Recent studies have proposed that patients with IMPC on transurethral resection should be treated with radical cystectomy regardless of the pathologic stage. Despite the potentially important therapeutic implications of this diagnosis, interobserver variation in the diagnosis of IMPC has not been studied. Sixty digital images, each from hematoxylin and eosin-stained slides, representing 30 invasive urothelial carcinomas (2 images per case), were distributed to 14 genitourinary subspecialists and each pathologist was requested to classify cases as IMPC or not. These cases included "classic" IMPC (n=10) and urothelial carcinoma with retraction and variably sized nests that might potentially be regarded as IMPC (n=20). The following 13 morphologic features were recorded as positive/negative for all cases independent of the reviewers' diagnoses: columnar cells, elongate nests or processes, extensive stromal retraction, lumen formation with internal epithelial tufting, epithelial ring forms, intracytoplasmic vacuolization, multiple nests within the same lacunar space, back-to-back lacunar spaces, epithelial nest anastomosis/confluence, marked nuclear pleomorphism, peripherally oriented nuclei, randomly distributed nuclei, and tumor nest size. In addition, a mean tumor nest size was calculated for each image based on the number of nuclei spanning the width of the nests. Interobserver reproducibility was assessed and the morphologic features were correlated with the classic IMPC and nonclassic/potential IMPC groups. In addition, the relationships between morphologic features, pathologists' interpretations, and case type (classic IMPC vs. nonclassic/potential IMPC) were evaluated using unsupervised hierarchical clustering analysis. Interobserver reproducibility for a diagnosis of IMPC in the 30 study cases was moderate (kappa: 0.54). Although classification as IMPC among the 10 "classic" IMPC cases was relatively uniform (93% agreement), the classification in the subset of 20 invasive urothelial carcinomas with extensive retraction and varying sized tumor nests was more variable. Multiple nests within the same lacunar space had the highest association with a diagnosis of classic IMPC. These findings suggest that more study of IMPC is needed to identify the individual pathologic features that might potentially correlate with an aggressive outcome and response to intravesical therapy.

Endometrial and endocervical adenocarcinomas may seem histologically identical and it can be difficult to determine primary site of origin based on morphology alone. As the distinction is significant and cannot always be made on the basis of clinical findings, various immunohistochemical panels have been proposed to aid in determining site of origin. Stains for vimentin, estrogen receptor (ER), progesterone receptor (PR), monoclonal carcinoembryonic antigen, p16 and ProExC, and HPV in situ hybridization (ISH), were performed on 283 tissue microarray (TMA) cores and 38 whole sections. The TMA consisted of 214 endometrial carcinomas, 33 endocervical adenocarcinomas, and 36 problematic cases. The endometrial and endocervical carcinomas represented usual endometrioid and mucinous types, and special variants (uterine serous carcinoma, uterine clear cell carcinoma, minimal deviation endocervical adenocarcinoma, cervical small cell carcinoma, adenoid basal cell carcinoma, mesonephric carcinoma). Univariate analysis showed that 6 markers (vimentin, ER, PR, p16, ProExC, and HPV ISH) performed well in distinguishing between endocervical and endometrial origin for the usual endometrioid and mucinous types. Multivariate analysis showed that vimentin, p16, and HPV ISH are the strongest predictors of site. Using a script written in R, the diagnostic accuracy of all possible combinations of markers was evaluated and it was shown that a 3 marker panel including vimentin, ER, or PR, and an HPV marker (p16, ProExC, or HPV ISH) is optimal for determining site of origin for usual endometrial and endocervical adenocarcinomas. However, these panels do not perform well with special variant carcinomas.

The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility.