bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Er:YAG Laser on Mineral Content of Sound Dentin in Primary Teeth
Authors: Guler Cigdem , Malkoc Meral Arslan , Gorgen Veli Alper , Dilber Erhan , Bulbul Mehmet
Published: August 2014

The aim of the present study was to evaluate the mineral content of sound dentin in primary teeth prepared using an Er:YAG laser at two different power settings. Thirty-six primary second molars were used in this study. Three dentin slabs were obtained from each tooth, and the slabs were randomly divided into three groups: Group A, control; Group B, Er:YAG laser at 3.5 W, 175 mJ, and 20 Hz, short pulse mode; and Group C, Er:YAG laser at 4 W, 200 mJ, and 20 Hz, medium-short pulse mode. One dentin slab per group was used to evaluate the dentinal morphology and surface roughness values using SEM and profilometer, respectively. Mineral content in the dentin slabs were calculated by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The data were analyzed by one-way analysis of variance and Tukey’s HSD tests. No significant differences in Ca, K, Mg, Na, and P levels or Ca/P ratio were found among the groups(P>0.05). SEM micrographs showed that surface irregularities increased with a higher power setting. The surface roughness after laser treatment in Group B and Group C was found to be similar, unlike Group A.
Challenges in Endobronchial Tuberculosis: From Diagnosis to Management
Authors: Kashyap Surender , Solanki Anjali
Published: August 2014

Despite the rapid advancement in diagnostic and therapeutic modalities, endobronchial tuberculosis (EBTB), defined as tuberculous infection of the tracheobronchial tree, continues to remain challenging for clinicians. Nonspecific respiratory symptoms along with normal chest radiograph in 10–20% of cases may be alleged for the diagnostic delay. Variable diagnostic yield with sputum microscopy might further compound the problem. In such cases, high resolution computed tomography (HRCT) works as a more sensitive tool and demonstrates involvement of tracheobronchial tree described classically as “tree-in-bud” appearance. Bronchoscopic biopsy is considered the most reliable method for confirmation of the diagnosis with 30% to 84% positivity in different series. Evolution of the disease is also unpredictable with frequent progression to bronchostenosis, therefore requiring regular follow-up and early intervention to halt the natural course. This review article elaborates various aspects of the disease with specific focus on diagnostic dilemma and recent advances in interventional bronchoscopy. In addition, this discussion evokes optimism for further research and introduction of innovative therapeutic modalities.
A Novel Adaptive Cuckoo Search for Optimal Query Plan Generation
Authors: Gomathi Ramalingam , Sharmila Dhandapani
Published: August 2014

The emergence of multiple web pages day by day leads to the development of the semantic web technology. A World Wide Web Consortium (W3C) standard for storing semantic web data is the resource description framework (RDF). To enhance the efficiency in the execution time for querying large RDF graphs, the evolving metaheuristic algorithms become an alternate to the traditional query optimization methods. This paper focuses on the problem of query optimization of semantic web data. An efficient algorithm called adaptive Cuckoo search (ACS) for querying and generating optimal query plan for large RDF graphs is designed in this research. Experiments were conducted on different datasets with varying number of predicates. The experimental results have exposed that the proposed approach has provided significant results in terms of query execution time. The extent to which the algorithm is efficient is tested and the results are documented.
Successful Nonoperative Treatment of Isolated Popliteus Tendon Avulsion Fractures in Two Adolescents
Authors: McKay Scott D. , Holt Andrew , Stout Thomas , Hysa Viola Qafalijaj
Published: August 2014

Isolated popliteal tendon avulsion fractures are relatively uncommon in the pediatric population as other posterolateral lateral structures are often involved. This report describes two skeletally immature male patients who presented with knee injuries without ligamentous instability and were subsequently diagnosed with isolated popliteus tendon avulsion fractures. Both of these patients were managed nonoperatively and had subjectively full recoveries. As the treatment for isolated popliteal tendon avulsion fractures is still unclear, the report here may contribute to strategies regarding conservative treatment of these injuries.
Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström’s Macroglobulinemia: Correlation withMYD88L265P Somatic Mutation Status, Clinical Features, and Outcome
Authors: Petrikkos Loizos , Kyrtsonis Marie-Christine , Roumelioti Maria , Georgiou George , Efthymiou Anna , Tzenou Tatiana , Panayiotidis Panayiotis
Published: August 2014

We performedIGHclonotypic sequence analysis in WM in order to determine whether a preferentialIGHgene rearrangement was observed and to assessIGHVmutational status in blood and/or bone marrow samples from 36 WM patients. In addition we investigated the presence ofMYD88L265P somatic mutation. AfterIGHVDJ locus amplification, monoclonal VDJ rearranged fragments were sequenced and analyzed.MYD88L265P mutation was detected by AS-PCR. The most frequent family usage wasIGHV3(74%);IGHV3-23andIGHV3-74segments were used in 26% and 17%, respectively. Somatic hypermutation was seen in 91% of cases.MYD88L265P mutation was found in 65,5% of patients and absent in the 3 unmutated. These findings did not correlate with clinical findings and outcome. Conclusion.IGH genes’repertoire differed in WM from those observed in other B-cell disorders with a recurrentIGHV3-23andIGHV3-74usage; monoclonalIGHVwas mutated in most cases, and a high but not omnipresent prevalence ofMYD88L265P mutation was observed. In addition, the identification of 3 patients with unmutatedIGHVgene segments, negative for theMYD88L265P mutation, could support the hypothesis that an extra-germinal B-cell may represent the originating malignant cell in this minority of WM patients.
Spectrums of Solvable Pantograph Differential-Operators for First Order
Authors: Ismailov Z. I. , Ipek P.
Published: August 2014

By using the methods of operator theory, all solvable extensions of minimal operator generated by first order pantograph-type delay differential-operator expression in the Hilbert space of vector-functions on finite interval have been considered. As a result, the exact formula for the spectrums of these extensions is presented. Applications of obtained results to the concrete models are illustrated.
The Optimization Model of Target Recognition Based on Wireless Sensor Network
Authors: Dou Zheng , Sun Yu , Lin Yun
Published: August 2014

In the application of opportunistic networking in wireless sensor network, the technology of target recognition is very important. However, since the sensor reports are typically inconsistent, incomplete, or fuzzy, the technology of target recognition whereby sensor reports is a major challenge. In this paper, based on the minimization of inconsistencies among the sensor reports, a new optimization model of target recognition is presented by using a convex quadratic programming (QP) formulation. Firstly, the description method of sensor report is introduced and then we talk about how to set up this new optimization model of target recognition by using the wireless sensor network reports and how to calculate the solution of this new optimization model. Finally, theory analysis and numeric simulation indicate that this optimization model can generate reasonable fusion results, which is similar to the Dempster-Shafer (D-S) evidence inference model. Furthermore, in contrast to D-S evidence inference model, this optimization model can fuse sensor reports of the form more general than that allowed by the D-S evidence inference model without additional processes. Meantime, it can deal with the high conflict sensor reports.
A Miracle That Accelerates Operating Room Functionality: Sugammadex
Authors: Dogan Erdal , Akdemir Mehmet Salim , Guzel Abdulmenap , Yildirim Mehmet Besir , Baysal Yildirim Zeynep , Kuyumcu Mahir , Gümüş Abdurrahman , Akelma Hakan
Published: August 2014

Background.Sugammadex offers a good alternative to the conventional decurarisation process currently performed with cholinesterase inhibitors. Sugammadex, which was developed specifically for the aminosteroid-structured rocuronium and vecuronium neuromuscular blockers, is a modified cyclodextrin made up of 8 glucose monomers arranged in a cylindrical shape.Methods.In this study, the goal was to investigate the efficacy of sugammadex. Sugammadex was used when there was insufficient decurarisation following neostigmine. This study was performed on 14 patients who experienced insufficient decurarisation (TOF<0.9) with neostigmine after general anaesthesia in the operating rooms of a university and a state hospital between June, 2012, and January, 2014. A dose of 2 mg/kg of sugammadex was administered.Results.Time elapsed until sugammadex administration following neostigmine37 ± 6min, following sugammadex it took2.1 ± 0.9min to reach TOF≥ 0.9, and the extubation time was3.2 ± 1.4min. No statistically significant differences were detected in the hemodynamic parameters before and after sugammadex application. From the time of administration of sugammadex to the second postoperative hour, no side effects or complications occurred. None of the patients experienced acute respiratory failure or residual block during this time period.Conclusion.Sugammadex was successfully used to reverse rocuronium-induced neuromuscular block in patients where neostigmine was insufficient.
Hippocampal Proliferation Is Increased in Presymptomatic Parkinson’s Disease and due to Microglia
Authors: Doorn Karlijn J. , Drukarch Benjamin , van Dam Anne-Marie , Lucassen Paul J.
Published: August 2014

Besides dopamine-deficiency related motor symptoms, nonmotor symptoms, including cognitive changes occur in Parkinson's disease (PD) patients, that may relate to accumulation ofα-synuclein in the hippocampus (HC). This brain region also contains stem cells that can proliferate. This is a well-regulated process that can, for example, be altered by neurodegenerative conditions. In contrast to proliferation in the substantia nigra and subventricular zone, little is known about the HC in PD. In addition, glial cells contribute to neurodegenerative processes and may proliferate in response to PD pathology. In the present study, we questioned whether microglial cells proliferate in the HC of established PD patients versus control subjects or incidental Lewy body disease (iLBD) cases as a prodromal state of PD. To this end, proliferation was assessed using the immunocytochemical marker minichromosome maintenance protein 2 (MCM2). Colocalization with Iba1 was performed to determine microglial proliferation. MCM2-positive cells were present in the HC of controls and were significantly increased in the presymptomatic iLBD cases, but not in established PD patients. Microglia represented the majority of the proliferating cells in the HC. This suggests an early microglial response to developing PD pathology in the HC and further indicates that neuroinflammatory processes play an important role in the development of PD pathology.
Abstract P1-05-07: Prognostic relevance of Claudin-2 expression in metastatic breast cancer
Authors: Hedenfalk I , Kimbung S , Kovacs A , Skoog L , Einbeigi Z , Walz T , Malmberg M , Loman N , Fernö M , Hatschek T ,   TEX Study Group
Published: August 2014

The site of relapse is known to be a strong prognostic factor of survival from metastatic breast cancer. The liver is the third most frequent site affected and patients with hepatic metastases have an inferior prognosis relative to patients with bone and lung metastases. Predicting the likelihood of liver metastasis after adjuvant therapy of primary breast cancer is challenging. The aim of this study was to identify and characterize genes associated with breast cancer liver metastases. We thus performed global transcriptional profiling of 120 metastatic lesions from different anatomical sites from breast cancer patients enrolled in a randomized clinical trial1 and found that while the expression of other matrix remodelling and tight-junction molecules was down-regulated in liver metastases, Claudin-2 (CLDN2) was significantly up-regulated (p = 0.001). Furthermore, high expression of CLDN2 was also observed amongst patients diagnosed with a liver metastasis, irrespective of the specific site of the metastasis that was profiled, compared to patients without hepatic metastases (p = 0.001). We further investigated the prognostic significance of CLDN2 protein expression by immunohistochemical staining of primary tumours and matched lymph node metastases from a larger cohort of patients (n∼200) with metastatic breast cancer. We found CLDN2 to be significantly more frequently expressed in lymph node metastases compared to primary tumours (p = 0.013). Also, consistent with mRNA expression levels, more frequent expression of CLDN2 protein was observed in the primary tumours of patients who eventually developed a liver metastasis (p = 0.027). Interestingly, high expression of CLDN2 in the primary tumour was associated with a shorter time to recurrence (p = 0.032). Specifically, high expression of CLDN2 in the primary tumour was associated with a shorter progression to liver metastasis (p = 0.002). These data highlight a potential role of CLDN2 in the development and prognosis of breast cancer liver metastases.

1. Hatschek, T., et al. Individually tailored treatment with epirubicin and paclitaxel with or without capecitabine as first-line chemotherapy in metastatic breast cancer: a randomized multicenter trial. Breast Cancer Res Treat. 2012, 131(3):939–47.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-07.
Abstract P1-07-03: Quantification of HER2 expression at the single cell level and HER2 intratumoral heterogeneity of breast cancer tissue samples using automated image analysis
Authors: Geretti E , Paragas V , Onsum M , Kudla A , Moulis S , Luus L , Wickham T , McDonagh C , MacBeath G , Hendriks B
Published: August 2014

HER2 is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or FISH testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to accurate HER2 assessment and patient stratification may include intratumoral heterogeneity, lack of assays that are quantitative and/or objective, and differences between measuring HER2 at the protein vs. DNA level. We have developed a novel immunofluorescence method for absolute quantitation of HER2 protein expression at the single cell level on formalin-fixed, paraffin-embedded (FFPE) patient samples. Our assay utilizes automated image analysis software to identify cells, classify them as tumor or non-tumor cells, and quantitate the level of HER2 staining for each tumor cell. The HER2 staining level is then converted to absolute HER2 receptor numbers per cell using an array composed of cell lines that span a range of HER2 levels. This cell pellet array standard is stained in parallel with each tissue sample. With this approach we quantitate HER2 expression at the single cell level and describe the heterogeneity of HER2 expression within breast cancer tissue samples. Our assay provides additional insight into the absolute level of HER2 expression and heterogeneity of HER2 expression within tumors, and allows for direct comparison with the currently existing HER2 assessments by standard IHC and/or FISH. This new assay could be used to increase understanding of the relationship between HER2 expression and patient response to HER2-targeted therapies.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-03.
Abstract P1-15-12: Single Nucleotide Polymorphism (SNP) Bayesian networks (BNs) Predict Risk of Chemotherapy-Induced Side Effects in Patients with Breast Cancer Receiving Dose Dense (DD) Doxorubicin/Cyclophosphamide Plus Paclitaxel (AC+T)
Authors: Schwartzberg LS , Sonis ST , Walker MS , Weidner SM , Alterovitz G
Published: August 2014

Background: Despite advances in supportive care, patients undergoing standard chemotherapy for breast cancer are at significant, but unequal, risk for the development of treatment-induced side effects. Current methods for predicting risk are inconsistent and unreliable. Because the pathobiology of many side effects are regulated by the synergistic interaction of simultaneously expressed genes, we reasoned that a novel approach to risk prediction could be achieved by the development of clusters of SNPs defined by BNs. The present study was designed to assess the feasibility of identifying BNs that were associated with regimen-related diarrhea, oral mucositis, nausea and vomiting, fatigue, cognitive dysfunction and neuropathy in patients with breast cancer treated with dose-dense AC+T. The primary endpoint was an area under the receiver operating characteristic curve (aROC) >0.80.

Methods: After informed consent, 78 patients with breast cancer who had received at least 3 cycles of (DD) AC+T were enrolled and saliva samples were collected. Salivary DNA was isolated and analyzed on Illumina Omni microarrays (2.5 × 106 SNPs). Standard supportive therapy for prevention and management of side effects was provided. The frequency and severity of diarrhea, oral mucositis, fatigue, cognitive dysfunction, neuropathy, and nausea and vomiting were determined using Patient Care Monitor©, a validated patient-reported symptom assessment instrument. BNs were developed for each of the six side effects using Bayesian methods including ten-fold internal cross-validation.

Results: The incidence of clinically significant side effects was higher than anticipated by the literature and are reported in Table 1 along with the accuracy and aROC.

Conclusions: We identified SNP-BNs associated with moderate to severe common side effects of AC+T regimens used to treated breast cancer. These BNs, sourced from salivary SNPs, should provide  clinicians with accurate actionable information which predicts side effect risk for patients who are scheduled to receive dose-dense doxorubicin/cyclophosphamide and paclitaxel for the treatment of breast cancer.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-15-12.
Abstract P2-01-05: Parallel DNA and RNA profiling of EpCAM-positive cells in blood of metastatic breast cancer (MBC) patients confirm their malignant nature
Authors: Magbanua MJM , Hauranieh L , Sosa EV , Pendyala P , Scott JH , Rugo HS , Park JW
Published: August 2014

Background: Molecular studies of EpCAM-positive cells found in the blood of cancer patients have been limited to immuno-enriched samples containing a high background of leukocytes. We developed a process to isolate rare EpCAM-positive cells and subjected them to DNA and RNA profiling.

Methods: Blood was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). Duplicate samples of 20 cells were isolated from the same enriched blood from 53 MBC patients and then subjected to DNA and RNA profiling in parallel. For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays. For non-tumor controls, RNA profiling was performed on matched leukocytes (CD45+) isolated from the same enriched blood samples from 44 of the 53 patients.

Results: Differential gene expression analysis between EpCAM-positive cells and matched leukocytes confirmed the up-regulation of EPCAM and other genes including MUC1 and KRT19 (adjusted p <0.05). In addition, EpCAM-positive cells showed a significant down-regulation of the leukocyte-specific marker PTPRC (encodes CD45) as well as CD44 and VIM, markers associated with stem cellness and epithelial to mesenchymal transition, respectively. Unsupervised hierarchical clustering analysis of RNA profiles showed that EpCAM-positive cells clustered away from the leukocytes. Genome-wide copy number analysis of EpCAM-expressing cells revealed gains (e.g. 1q and 8q), losses (e.g. 8p and 16q), and focal amplifications (e.g. on 8q and 11q including CCND1) frequently seen in primary breast cancers.

Discussion: We demonstrate the feasibility of isolation and molecular analysis of highly pure EpCAM-positive cells in the blood. Our results strongly suggest that epithelial cells captured from blood of MBC patients are malignant in nature and are therefore circulating tumor cells. This approach can serve as a non-invasive source of highly pure metastatic tumor tissue for further molecular characterization. This study was supported by CALGB and BCRF.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-05.
Abstract P2-02-02: Prognostic significance of the ratio of absolute neutrophil to lymphocyte counts for breast cancer patients in neoadjuvant setting
Authors: Koh YW , Lee HJ , Ahn J-H , Lee JW , Gong G
Published: August 2014

Background: The baseline absolute neutrophil count/absolute lymphocyte count ratio (NLR) has potential prognostic importance in solid tumors, including breast cancer. However, NLR as a predictive factor of the pathologic complete response (pCR) or as a prognostic factor has not been fully studied in breast cancer patients who have received neoadjuvant chemotherapy.

Materials and Methods: Using receiver operating characteristic curve analyses, we retrospectively examined the predictive and prognostic impact of the NLR at the diagnosis in 401 patients with primary breast cancer who were treated with neoadjuvant chemotherapy, followed by definitive surgical resection. Of the 401 included patients, 72 (18%) received anthracycline-based, 285 (71%) received anthracycline- and taxane-based, 37 (9.2%) received herceptin-based, and 7 (1.7%) received fluorouracil-based regimens. The significance of NLR was analyzed according to clinicopathologic variables and clinical outcomes.

Results: Forty-three (10.7%) patients achieved pCR. On univariate analysis, high NLR (> 2.2) correlated with poor recurrence-free survival (RFS; P = 0.001) and overall survival (OS; P = 0.003). Subgroup analysis of the non-pCR group showed that NLR was significant for RFS and OS (P = 0.002 and P = 0.007, respectively). Multivariate analysis showed NLR to be an independent prognostic factor for RFS and OS (P = 0.002 and P = 0.007, respectively).

Subgroup analysis of the pCR group showed that patients with high NLR have a higher recurrent rate than low NLR (30.8% vs. 13.3%). The NLR was not related to pCR (P = 0.098).

Conclusion: Our results suggest that the NLR is an independent prognostic factor for RFS and OS in breast cancer patients receiving neoadjuvant chemotherapy and may provide additional prognostic information for patients not achieving a pCR to determine who might profit from further therapy.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-02-02.
Abstract P2-04-05: Prolonged targeted overexpression of Aurora-A in mammary epithelium promotes mammary adenocarcinoma with genomic instability
Authors: Treekitkarnmongkol W , Katayama H , Sen S
Published: August 2014

Aurora kinase-A (referred to as Aurora-A) identified as a sereine/threonine kinase induces mitotic progression process including centrosome amplification, bipolar spindle formation and chromosome segregation through modulation of activity and localization of its interacting proteins during cell division. Beside its essential roles in mitosis, cancer profiling studies have revealed that Aurora-A function is implicated in tumor relevant signaling pathways and overexpressed in many human tumors. The findings showed highly frequent overexpression of Aurora-A in human breast ductal carcinoma in situ and primary invasive ductal breast carcinoma. Moreover, the Aurora-A overexpression correlates with poor prognosis in breast carcinoma, deregulated overexpression of Aurora-A associates with centrosome anomalies and chromosome instability in rodent models of mammary caricinogenesis.

However, published studies in several transgenic mouse models attempting to address the role of Aurora-A in tumorigenesis and genomic instability have raised conflicting results. In this report we targeted expression of Aurora-A transgene in an inducible mouse model in which expression of Aurora-A was restricted to mammary epithelium during multiple pregnancy and lactation cycles under the activity of β-lactoglobulin promotor. The results demonstrate that overexpression of Aurora-A could induce tumorigenesis in mouse mammary epithelium. The tumor incidence was 70% of Aurora-A transgenic mice at 16 months of age. Of note, overexpression of Aurora-A led to genomic instability. Comparative genomic hybridization (CGH) array analyses revealed loss of Trim12a, Trim12c, Trim30b, Trim30d, Trim30e, Cdkn2d, Pten, Cep55 and gain of Adam6. Notably, Aurora-A overexpression caused nuclear accumulation of cyclin D1, activation of AKT, overexpression of TPX2 and PLK1and loss of ERα expression in tumors. Our findings indicated that prolonged Aurora-A expression in mammary epithelium leads to mammary adenocarcinomas with genomic instability. Aurora-A driven adenocarcinoma reveals deregulation of critical tumor relevant genetic pathways involving both oncogenes and tumor suppressor genes. These transgenic mouse model findings indicate that Aurora-A may be an important therapeutic target for mammary cancer.

Keywords: Aurora-A, carcinogenesis.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-05.
Abstract P2-04-07: Modeling orthotopic and metastatic progression of mammary tumors to evaluate the efficacy of TGF-β inhibitors in a pre-clinical setting
Authors: Biswas T , Yang J , Zhao L , Sun L
Published: August 2014

Preclinical models are elemental in understanding drug efficacy and safety in vivo. Many existing models of breast cancer metastasis use immune compromised animals and do not effectively characterize the differential stages of metastasis. Transforming growth factor-β (TGF-β) has been shown to be a major contributor in epithelial-mesenchymal transition and enhances various steps during metastasis.

The aim of this study is to develop mouse models of tumor metastasis using murine mammary cells in a syngeneic background. This will help illustrate the effect of TGF-β inhibitors on metastasis in an immune competent environment, and also allow determining the optimal time of drug administration. We demonstrate that TGF-β signaling has differential effects on a pair of murine mammary cell lines derived from PyMT tumors in C57Bl/6 mice. These cell lines represent luminal (Py 230) and basal (Py 8119) epithelial cell populations in the tumor. In vitro characterization revealed differences in viability, motility, and transcriptional and translational activation of Smad. Py 230 (clonal luminal) cells are growth inhibited and exhibit epithelial-mesenchymal transition on treatment with TGF-β, whereas Py 8119 (basal cells) are non-responsive to both effects. Furthermore, intra cardiac injections were performed using luciferase-GFP labeled PyMT cells in syngeneic background and animals were treated with either placebo or TβRI-KI (kinase inhibitor for TGF-β receptor I). Results from Py 8119 cell injections showed that TβRI-KI treatment enhanced bone and brain metastases. Further results will shed light on the effect of TβRI-KI during specific stages of metastasis progression.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-04-07.
Abstract P2-06-05: Single-cell RNA sequencing of paclitaxol-treated breast cancer cell lines to find individual cell response
Authors: Vaske CJ , Lee W , Benz SC , Sanborn JZ , Emerson BM , Pourmand N , Lopez Diaz F
Published: August 2014

Cancer treatments act on a population of cells, each of which may experience different individual responses to treatment. Such differential response will result in resistance to treatment even if a majority of cancerous cells are eliminated. To examine differential cell response, we simultaneously profiled the gene expression and mutation spectrum of individual cells from the MDAMB231 cell line using next generation sequencing of isolated RNA. A total of 23 transcriptomes were characterized from paclitaxel-treated and paclitaxel-surviving cells. We found significant different changes in mutation rates between paclitaxel treated cells, with a dose-dependent increase in single nucleotide changes in RNA in paclitaxel-treated cells. Cells undergoing exposure to paclitaxel also showed higher pathway activity in SRC, as well as an integrin switch from ITGB1 to ITGB3. In contrast, cells that survived a high dose of paclitaxel showed an insignificant number of single nucleotide changes, suggesting that these cells either evaded initial paclitaxel exposure or were better able to repair the effects of paclitaxel exposure. Despite the RNA sequence similarity between surviving and untreated cells, there were changes in gene expression and pathway activities including higher PI3K activity. Paclitaxel-surviving cells also showed activation of pathways associated with higher proliferation.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-05.
Abstract P2-06-07: Exome sequencing identifies somatic mutations in basal-like breast cancer before and after neoadjuvant chemotherapy
Authors: Jiang Y-Z , Yu K-D , Shao Z-M
Published: August 2014

Purpose: Among the five breast cancer subtypes, basal-like breast cancers (BLBCs) have drawn much attention, because they are associated with poor clinicopathological features, lack of effective treatment targets, and have poor prognosis. We designed the experiment to test the hypothesis that significant mutational evolution of BLBCs might occur during chemotherapy.

Methods: We performed exome sequencing (100-fold coverage) to examine the exomes of 11 paired BLBC samples (pretreatment tumor biopsies and posttreatment tumors). The molecular subtype of BLBC was proven by gene-expression profile as well as by immunohistochemistry assay. Firstly, we sequenced the tumor genomic DNA obtained before and after neoadjuvant chemotherapy and their matched normal breast tissues. After analyzing and comparing potential somatic single nucleotide variations (SNVs) and insertions & deletions (indels), we focused on the functional mutations in certain genes or pathways. Furthermore, after validation of the mutations by Sanger sequencing, we investigated their biological functions (e.g. drug-resistance, cell proliferation, etc.).

Results: Compared with pretreatment biopsies, analysis of whole-exome sequence data identified and validated TEKT4 (tektin 4) — a constitutive protein of microtubules in cilia, flagella, basal bodies and centrioles — as a significantly mutated gene in posttreatment tumors with a mutation frequency of approximately 17.6% in an independent extension cohort of 84 paired BLBC samples. TEKT4 mutations are biologically relevant, as ectopic expression of mutant TEKT4 increased paclitaxel resistance of BLBC cell lines both in vitro and in vivo. Additionally, compared with the normal breast tissues, 16 significantly mutated genes were identified and validated in the paired cancer samples, including TP53, ARID1A, BEND5, LCT, COL4A4, GAP43, COL6A6, LOC728369, STAP1, KCNK17, LZTS2, TST, IL9R, HDLBP, RASL11A and FAM22D. These genes have functions that could be broadly grouped into five biological classes: (i) MAPK signaling, (ii) PI3K/AKT signaling, (iii) cell cycle, (iv) metabolism and (v) loss of genome stability.

Conclusion: Our data reveal the novel concept that significant evolution might occur during chemotherapy and mutational heterogeneity could be a property of BLBCs. In addition, the identification of somatic mutations as biomarkers for pCR (pathologic complete remission) prediction and prognosis might help us optimize choice for sequential therapy and improve patients' survival. Prospective clinical trials based on these findings might illustrate the drug-resistant mechanism and identify resistance pathways that can be exploited for therapeutic selection.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-07.
Abstract P2-09-04: DNA methylation landscapes in ER-negative breast cancer
Authors: Worsham MJ , Chen KM , Chitale D , Divine G
Published: August 2014

The biological significance of DNA methylation in the regulation of gene expression and its role in cancer is increasingly recognized. The underlying hypothesis of this study is that strategic global approaches will identify aberrantly methylated genes that underlie the pathogenesis of ER-negative (ER−) breast cancer (BC). We used the Infinium HumanMethylation450 BeadChip to profile the methylome of ER− breast cancers. The 450K array includes 485,577 cytosine positions of the human genome. From these cytosine sites, 99.3% are CpG dinucleotides.

Whole genomic DNA from 20 primary ER− and 8 normal breast tissue samples were assayed for genome-wide methylation using the Illumina 450K array. We had 8634 hypermethylated CpGs or 1.8% of the 485,577 sites on the 450K array. The proportion with hypermethylation was higher for promoter regions (2.1% vs 1.5%), and highest for the “FirstExon” promoter subregion. Of the 8634 CpGs, 2980 (adjusted p = 0.05) were differentially methylated between tumor and normal samples. This resulted in 206 genes with significant hypermethylation (all mean breast cancer betas >= −0.2, and ratio of tumor to normal mean beta >= 2.0).

Estrogen receptor-negative BC is a more aggressive form than ER positive BC with approximately double the incidence in African Americans than in Caucasian Americans. The emerging differential methylation pattern within hormone receptor negative breast cancers would further help stratify them into distinct subgroups. Promotor methylation being potentially reversible, methylated genes may serve as future molecular targets for demethylating therapies.

Support: Komen Foundation: KG110218

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-09-04.
Abstract P2-10-02: Clinical validation of the PAM50 risk of recurrence (ROR) score for predicting residual risk of distant-recurrence (DR) after endocrine therapy in postmenopausal women with ER+ early breast cancer (EBC): An ABCSG study
Authors: Gnant M , Filipits M , Mlineritsch B , Dubsky P , Jakesz R , Kwasny W , Fitzal F , Rudas M , Knauer M , Singer C , Greil R , Ferree S , Storhoff J , Cowens JW , Schaper C , Liu S , Nielsen T ,   the ABCSG
Published: August 2014

Aim: To assess the performance of the ROR score in predicting DR for postmenopausal patients with ER+ EBC treated with tamoxifen or tamoxifen followed by anastrozole when the PAM50 test is performed in a routine hospital pathology laboratory.

Background: The multi-analyte gene-expression tests (Oncotype DX, MammaPrint) that are currently used in clinical practice to assess prognosis after endocrine therapy in primary ER+ breast cancer are performed in a central referral laboratory. PAM50 is a 50-gene test in development that is designed to be performed in local routine hospital pathology laboratories and has been optimized to separate intrinsic disease subtypes which are used to generate a ROR score. This ROR score has been clinically validated and demonstrated to contain more prognostic information than Oncotype Dx RS in the TransATAC patient population (Dowsett, SABCS, 2011).

Methods: 1,251 women from the prospective randomized ABCSG-8 trial were entered into this study: the entry criteria were the availability of their FPET block and informed consent. Three unstained 10 micron sections and 1 H&E slide for each patient were sent to an independent academic pathology laboratory at BCCA where tissue review, manual micro-dissection and RNA extraction were performed. PAM50 analysis was then conducted on 250 ng of the extracted RNA using the NanoString nCounter® technology: both intrinsic subtype and ROR score were calculated. The median follow up of the patients entered into this study is 10 years. 912 are node negative and 339 are node positive. The baseline characteristics of the patients entered showed them to be a representative sample of the overall ABCSG-8 trial population. The primary analysis is designed to test whether the continuous ROR score adds prognostic value over and above the standard clinical variables, using a likelihood ratio test.

Results: Results are currently being derived and will be presented in full at SABCS.

Discussion: This large clinical trial serves both as a clinical validation study of the PAM 50 test and as a demonstration that a complex multi-analyte gene-expression test can be performed in a routine hospital pathology laboratory while meeting the same quality metrics as a central referral laboratory. The results of the primary analysis, when combined with the results already reported from the Trans ATAC population, yield Level 1 evidence for clinical validity of the PAM50 test for predicting the risk of DR in postmenopausal women with ER+ EBC and form the basis of the design of the clinical utility studies of the PAM 50 test.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-02.
Abstract P2-10-09: The incidence of false negative of HER2/Neu status in primary breast cancer in the era of standardized testing: a Canadian prospective study.
Authors: Hanna W , Barnes PJ , Chang MC , Gilks B , Magliocco A , Rees H , Robertson S , SenGupta SK , Nofech-Mozes S
Published: August 2014

The efficacy of trastuzumab in the treatment of primary breast cancer has mandated accurate and timely testing of all patients with a new diagnosis of breast cancer. Testing is centralized in designated laboratories across Canada with adherence to guidelines and mandatory participation in quality assurance programs. The Canadian testing algorithm recommends starting with immunohistochemistry (IHC) followed by in situ hybridization (ISH) for equivocal cases. Early HER2 testing showed that approximately 25–30% of invasive breast cancer is HER2 positive. Recent data shows that the HER2/neu positive rate in breast cancer in Canada is 17.6%.

Design: The study was designed to assess the rate of false-negative HER2 tests based on the IHC-first algorithm used in 8 pathology centres across Canada. Surgical excisions with invasive carcinoma were tested using the standardized local methodology for both IHC and ISH. The cases were scored by the local breast pathologist and in 2 of 8 centers image analysis was used in the evaluation of ISH. We compared consecutive HER2-negative IHC results (score 0/1+) to the corresponding ISH (either silver or fluorescence) result. False negative cases were defined as a negative IHC with an ISH ratio of≥ 2, since these patients are eligible for trastuzumab therapy.

Results: 715 cases were analyzed by IHC using Ventana 4B5 (287), HercepTest (253), or SP3 (175), and by ISH kits: Vysis FISH (303), Ventana SISH (412). The HER2 and CEP17 counts were available in all cases.

There were 4 cases with an ISH score ≥2 (4B5: 2/4, HercepTest 1/4, SP3 1/4). In 3 additional cases the absolute HER2 copy number was ≥6 but the HER2/CEP17 amplification ratio was <2 due to an increased number of CEP 17 signal (“polysomy 17”) or amplification of the pericentromeric region. The overall rate of false negative cases was 0.98% (7/715). These cases had a low level of amplification (ratio 2 to 2.45) or an absolute HER2 count of 6–8.

Conclusion: Our observation confirms that IHC is an adequate test to predict negative HER2 status in primary breast cancer in surgical excision specimens, even when different antibodies and IHC platforms are used. The study supports and justifies the Canadian algorithm of IHC followed by ISH in equivocal cases in view of the extremely low percentage of false negative cases observed. This reflects the strict adherence to internal protocols and mandatory participation in quality assurance programs. These results provide further confirmation that the vast majority of patients eligible for trastuzumab are not deprived from an effective treatment by using this algorithm.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-09.
Abstract P2-10-13: CD4 positive tumor-infiltrating lymphocytes are associated with improved prognosis in node-negative breast cancer
Authors: Schmidt M , van de Sandt L , Boehm D , Sicking I , Battista M , Lebrecht A , Solbach C , Koelbl H , Gehrmann M , Rahnenführer J , Hengstler JG
Published: August 2014

Background: The prognostic significance of CD8 positive cytotoxic lymphocytes in breast cancer is well accepted. However, the role of CD4 positive lymphocytes is ambiguous. We examined the prognostic relevance of CD4 positive tumor-infiltrating lymphocytes in previously published node-negative breast cancer cohorts using RNA expression.

Methods: Microarray based gene-expression data for CD4 (203547_at) were analyzed in four previously published cohorts (Mainz, Rotterdam, Transbig, Yu) of node-negative breast cancer patients not treated with adjuvant therapy (n = 824). A meta-analysis of previously published cohorts was performed using a random effects model. Prognostic significance of CD4 on metastasis-free survival (MFS) was examined in the whole cohort and in different molecular subtypes (ER+/HER2−, ER−/HER2−, HER2+). Independent prognostic relevance was analyzed using multivariate Cox regression.

Results: Higher RNA expression of CD4 was related to better MFS in a meta-analysis of the whole cohort (HR 0.66, 95% CI 0.49–0.90, p = 0.0074). Prognostic significance was most pronounced in the HER2+ positive molecular subtype (HR 0.32, 95% CI 0.14–0.75, p = 0.0091) as compared to ER+/HER2− (HR 0.62, 95% CI 0.29–1.32, P>0.05) and ER−/HER2− (HR 0.61, 95% CI 0.28–1.35, P>0.05) carcinomas of the breast. CD4 showed independent prognostic significance (HR 0.60, 95% CI 0.37–0.96, p = 0.032) in multivariate analysis. In addition to CD4, only histological grade of differentiation (HR 2.43, 95% CI 1.50–3.94, P < 0.001) and tumor size (HR 1.87, 95% CI 1.19–2.94, p = 0.007), but not age, HER2 status or hormone receptor status retained an independent prognostic association with MFS.

Conclusion: CD4 positive tumor-infiltrating lymphocytes have independent prognostic significance in node-negative breast cancer. A higher expression is associated with improved outcome.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-13.
Abstract P2-10-15: Evaluation of Prognostic and Predictive Performance of Breast Cancer Index and Its Components in Hormonal Receptor-Positive Breast Cancer Patients: A TransATAC Study
Authors: Sgroi DC , Sestak I , Zhang Y , Erlander MG , Schnabel CA , Goss PE , Cuzick J , Dowsett M
Published: August 2014

Background: The Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial compared the efficacy and safety of 5 years of anastrozole with tamoxifen as adjuvant treatment for postmenopausal women with localized HR+ breast cancer. At a median follow-up of 10 years, a statistically significant improvement with anastrozole vs. tamoxifen for disease-free survival, time to recurrence and time to distant recurrence was observed. The HOXB13:IL17BR gene expression ratio (H/I) quantifies recurrence risk in ER positive (ER+) breast cancer patients and is predictive of benefit from endocrine therapy. Molecular Grade Index (MGI) is a five-gene index that provides quantitative and objective molecular assessment of tumor grade and proliferative status. Breast Cancer Index (BCI) combines H/I and MGI into a continuous risk model that provides a likelihood of distant recurrence in patients treated with endocrine therapy, and efficacy from neoadjuvant chemotherapy. In the current analysis, evaluation of the prognostic and predictive performance of BCI, H/I and MGI in the ATAC study cohort was conducted.

Methods: Under the TransATAC protocol, formalin-fixed, paraffin-embedded (FFPE) blocks of primary tumor were collected from HR+ patients from each monotherapy arm. The current study examined samples collected from the United Kingdom, which constituted 79% of the collection. RNA extracted from 1102 samples from the TransATAC study was amplified, converted to cDNA and subjected to RT-PCR with primers and probes to HOXB13, IL17BR, BUB1A, CENPA, NEK2, RACGAP1 and RRM2. H/I, MGI and BCI were calculated and risk groups were determined using pre-specified cutpoints.

Results: Of 1102 tumor specimens assayed, 29 failed QC criteria, leaving 1073 samples for analysis. Detailed results on the prognostic and predictive performance of BCI, H/I and MGI will be presented. Data on whether BCI and its components provided independent prognostic information in the presence of classical variables, their prognostic value for risk of late recurrence, interaction by treatment arms, and comparative performance vs other models will also be discussed.

Discussion: The ATAC trial has established the long-term efficacy and safety of anastrozole over tamoxifen as initial adjuvant treatment for post-menopausal early stage breast cancer patients. Continued efforts are needed to improve on quantification of residual risk in patients who were treated with endocrine therapy to guide decision-making in selecting additional adjuvant chemotherapy and/or administering extended endocrine treatment. This study will help to establish the strategy to more effectively select patients for adjuvant therapies.

Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-15.