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1.  OvMark: a user-friendly system for the identification of prognostic biomarkers in publically available ovarian cancer gene expression datasets 
Molecular Cancer  2014;13(1):241.
Background
Ovarian cancer has the lowest survival rate of all gynaecologic cancers and is characterised by a lack of early symptoms and frequent late stage diagnosis. There is a paucity of robust molecular markers that are independent of and complementary to clinical parameters such as disease stage and tumour grade.
Methods
We have developed a user-friendly, web-based system to evaluate the association of genes/miRNAs with outcome in ovarian cancer. The OvMark algorithm combines data from multiple microarray platforms (including probesets targeting miRNAs) and correlates them with clinical parameters (e.g. tumour grade, stage) and outcomes (disease free survival (DFS), overall survival). In total, OvMark combines 14 datasets from 7 different array platforms measuring the expression of ~17,000 genes and 341 miRNAs across 2,129 ovarian cancer samples.
Results
To demonstrate the utility of the system we confirmed the prognostic ability of 14 genes and 2 miRNAs known to play a role in ovarian cancer. Of these genes, CXCL12 was the most significant predictor of DFS (HR = 1.42, p-value = 2.42x10−6). Surprisingly, those genes found to have the greatest correlation with outcome have not been heavily studied in ovarian cancer, or in some cases in any cancer. For instance, the three genes with the greatest association with survival are SNAI3, VWA3A and DNAH12.
Conclusions/Impact
OvMark is a powerful tool for examining putative gene/miRNA prognostic biomarkers in ovarian cancer (available at http://glados.ucd.ie/OvMark/index.html). The impact of this tool will be in the preliminary assessment of putative biomarkers in ovarian cancer, particularly for research groups with limited bioinformatics facilities.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-241) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-241
PMCID: PMC4219121  PMID: 25344116
2.  A systems approach to analysis of molecular complexity in breast cancer 
In this issue of Clinical Cancer Research, Andre et al. apply high-resolution arrays to elucidate copy number anomalies in breast cancer. They identify a series of genomic regions that may contain oncogenes and tumor suppressor genes that contibute to breast carcinogenesis and that serve as potential therapy targets.
doi:10.1158/1078-0432.CCR-08-2620
PMCID: PMC4161015  PMID: 19147744
3.  Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth 
Nature medicine  2011;17(11):1498-1503.
Intra-abdominal tumors, such as ovarian cancer1,2, have a clear predilection for metastasis to the omentum, an organ primarily composed of adipocytes. Currently, it is unclear why tumor cells preferentially home to and proliferate in the omentum, yet omental metastases typically represent the largest tumor in the abdominal cavities of women with ovarian cancer. We show here that primary human omental adipocytes promote homing, migration and invasion of ovarian cancer cells, and that adipokines including interleukin-8 (IL-8) mediate these activities. Adipocyte–ovarian cancer cell coculture led to the direct transfer of lipids from adipocytes to ovarian cancer cells and promoted in vitro and in vivo tumor growth. Furthermore, coculture induced lipolysis in adipocytes and β-oxidation in cancer cells, suggesting adipocytes act as an energy source for the cancer cells. A protein array identified upregulation of fatty acid–binding protein 4 (FABP4, also known as aP2) in omental metastases as compared to primary ovarian tumors, and FABP4 expression was detected in ovarian cancer cells at the adipocyte-tumor cell interface. FABP4 deficiency substantially impaired metastatic tumor growth in mice, indicating that FABP4 has a key role in ovarian cancer metastasis. These data indicate adipocytes provide fatty acids for rapid tumor growth, identifying lipid metabolism and transport as new targets for the treatment of cancers where adipocytes are a major component of the microenvironment.
doi:10.1038/nm.2492
PMCID: PMC4157349  PMID: 22037646
4.  BRCA1/2 MUTATION ANALYSIS IN 41 OVARIAN CELL LINES REVEALS ONLY ONE FUNCTIONALLY DELETERIOUS BRCA1 MUTATION 
Molecular oncology  2013;7(3):567-579.
Mutations in BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors.
The BRCA1/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines.
The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G>A, and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311.
The incidence of BRCA1/2 deleterious mutations 1/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective pressure against BRCA1/2 in cell culture similar to the selective pressure seen in the clinic after treatment with chemotherapy. PARP inhibitors may be useful in patients with BRCA1 deleterious mutations or gene methylation.
doi:10.1016/j.molonc.2012.12.007
PMCID: PMC4106023  PMID: 23415752
BRCA1/2; ovarian; mutation; methylation; parp inhibitor; olaparib; veliparib
5.  Integrative analysis of proteomic signatures, mutations and drug responsiveness in the NCI 60 cancer cell line set 
Molecular cancer therapeutics  2010;9(2):257-267.
Aberrations in oncogenes and tumor suppressors frequently affect the activity of critical signal transduction pathways. To analyze systematically the relationship between the activation status of protein networks and other characteristics of cancer cells, we performed reverse phase protein array (RPPA) profiling of the NCI60 cell lines for total protein expression and activation-specific markers of critical signaling pathways. To extend the scope of the study, we merged those data with previously published RPPA results for the NCI60. Integrative analysis of the expanded RPPA data set revealed 5 major clusters of cell lines and 5 principal proteomic signatures. Comparison of mutations in the NCI60 cell lines with patterns of protein expression demonstrated significant associations for PTEN, PIK3CA, BRAF and APC mutations with proteomic clusters. PIK3CA and PTEN mutation enrichment were not cell lineage-specific but were associated with dominant yet distinct groups of proteins. The five RPPA-defined clusters were strongly associated with sensitivity to standard anti-cancer agents. RPPA analysis identified 27 protein features significantly associated with sensitivity to paclitaxel. The functional status of those proteins was interrogated in a paclitaxel whole genome siRNA library synthetic lethality screen, and confirmed the predicted associations with drug sensitivity. These studies expand our understanding of the activation status of protein networks in the NCI60 cancer cell lines, demonstrate the importance of the direct study of protein expression and activation, and provide a basis for further studies integrating the information with other molecular and pharmacological characteristics of cancer.
doi:10.1158/1535-7163.MCT-09-0743
PMCID: PMC4085051  PMID: 20124458
NCI60; reverse phase protein arrays; signal transduction
6.  A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation 
BMC Cancer  2014;14:120.
Background
Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC.
Methods
Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A.
Results
Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr308 or pAKT-S473 suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression.
Conclusions
Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.
doi:10.1186/1471-2407-14-120
PMCID: PMC3941949  PMID: 24559118
Ovarian cancer; Proteomics; ARID1A/BAF250a; PIK3CA mutation; AKT; Phosphorylation
7.  VATS lobectomy facilitates the delivery of adjuvant docetaxel-carboplatin chemotherapy in patients with non-small cell lung cancer 
Journal of Thoracic Disease  2013;5(5):578-584.
Background
To evaluate the safety and tolerability of docetaxel/carboplatin regimen in the post-operative setting of patients with non-small cell lung cancer (NSCLC).
Methods
Enrolment of 133 patients with stage Ib - IIIa NSCLC was undertaken in an open-label, single arm study to assess the safety and tolerability of docetaxel (75 mg/kg) and carboplatin (AUC 5.5) administered for 3 cycles after resection for curative intent. The primary endpoint of the study was safety, as reflected by a febrile neutropenia rate of <10%. Other endpoints assessed protocol compliance and the impact of minimally invasive surgical technique.
Results
Patient accrual was completed at 1 center in the US and 10 centers in China in <6 months. Febrile neutropenia complicated treatment in 12 patients (9.0%), below the predetermined safety threshold of 14 patients. Four VATS and 8 open thoracotomy patients experienced febrile neutropenia (P=0.26). Completion of the three-cycle adjuvant regimen was achieved in 86% (95% CI, 77-95%) of patients. Sixty-two of 66 VATS patients compared to 53 of 67 open thoracotomy patients received all three doses according to protocol (P<0.01). Thirteen serious adverse events (9.8%) and no deaths were attributed to the study regimen.
Conclusions
In this rapidly accrued study, docetaxel and carboplatin were well-tolerated in the adjuvant treatment of NSCLC. Adjuvant treatment compliance was higher among patients undergoing a minimally invasive surgical approach. (ClinicalTrials.gov number NCT00883675).
doi:10.3978/j.issn.2072-1439.2012.02.05
PMCID: PMC3815738  PMID: 24255769
Non-small cell lung cancer; adjuvant chemotherapy; docetaxel; carboplatin
8.  Standard chemotherapy with or without bevacizumab in advanced ovarian cancer: quality-of-life outcomes from the International Collaboration on Ovarian Neoplasms (ICON7) phase 3 randomised trial 
The Lancet Oncology  2013;14(3):236-243.
Summary
Background
In the Gynecologic Cancer Intergroup International Collaboration on Ovarian Neoplasms 7 (ICON7) trial, bevacizumab improved progression-free survival in patients with ovarian cancer when used in combination with first-line chemotherapy and as a single-drug continuation treatment for 18 cycles. In a preliminary analysis of a high-risk subset of patients, there was also an improvement in overall survival. This study aims to describe the health-related quality-of-life (QoL) outcomes from ICON7.
Methods
ICON7 is a randomised, multicentre, open-label phase 3 trial. Between Dec 18, 2006, and Feb 16, 2009, after a surgical procedure aiming to debulk the disease, women with International Federation of Gynecology and Obstetrics (FIGO) high-risk stage I–IV epithelial ovarian cancer were randomly allocated (1:1) by computer program and block randomisation to receive either six cycles of standard chemotherapy (total 18 weeks) with carboplatin (area under the curve 5 or 6) and paclitaxel (175 mg/m2) alone or with bevacizumab (7·5 mg/kg) given intravenously with chemotherapy and continued as a single drug thereafter (total 54 weeks). The primary QoL endpoint was global QoL from the European Organisation for Research and Treatment of Cancer quality-of-life questionnaire–core 30 at week 54, analysed by ANOVA and adjusted for baseline score. Analyses were by intention to treat. The ICON7 trial has completed recruitment and remains in follow-up. This study is registered, number ISRCTN91273375.
Findings
764 women were randomly assigned to the standard chemotherapy group and 764 to the bevacizumab group. At baseline, 684 (90%) of women in the standard chemotherapy group and 691 (90%) of those in the bevacizumab group had completed QoL questionnaires. At week 54, 502 (66%) women in the bevacizumab group and 388 (51%) women in the standard chemotherapy group provided QoL data. Overall, the mean global QoL score improved during chemotherapy by 7·2 points (SD 24·4) when analysed for all women with data at baseline and week 18. The mean global QoL score at 54 weeks was higher in the standard chemotherapy group than in the bevacizumab group (76·1 [SD 18·2] vs 69·7 [19·1] points; difference 6·4 points, 95% CI 3·7–9·0, p<0·0001).
Interpretation
Bevacizumab continuation treatment seems to be associated with a small but clinically significant decrement in QoL compared with standard treatment for women with ovarian cancer. The trade-off between the prolongation of progression-free survival and the quality of that period of time needs to be considered in clinical practice when making treatment decisions.
Funding
Roche and the National Institute for Health Research through the UK National Cancer Research Network.
doi:10.1016/S1470-2045(12)70567-3
PMCID: PMC3596061  PMID: 23333117
9.  A brief mindfulness-based cognitive behavioral intervention improves sexual functioning versus wait-list control in women treated for gynecologic cancer 
Gynecologic oncology  2012;125(2):320-325.
Goal
The goal of this study was to evaluate a mindfulness-based cognitive behavioral intervention for sexual dysfunction in gynecologic cancer survivors compared to a wait-list control group.
Methods
Thirty-one survivors of endometrial or cervical cancer (mean age 54.0, range 31–64) who self-reported significant and distressing sexual desire and/or sexual arousal concerns were assigned either to three, 90-minute mindfulness-based cognitive behavior therapy sessions or two months of wait-list control prior to entering the treatment arm. Validated measures of sexual response, sexual distress, and mood, as well as laboratory-evoked physiological and subjective sexual arousal were assessed at pre-, one month post-, and 6-months following treatment.
Results
There were no significant effects of the wait-list condition on any measure. Treatment led to significant improvements in all domains of sexual response, and a trend towards significance for reducing sexual distress. Perception of genital arousal during an erotic film was also significantly increased following the intervention despite no change in physiologically-measured sexual arousal.
Conclusions
A brief mindfulness-based intervention was effective for improving sexual functioning. Geographic restrictions permitted only a select sample of survivors to participate, thus, the generalizability of the findings is limited. Future studies should aim to develop online modalities for treatment administration to overcome this limitation.
doi:10.1016/j.ygyno.2012.01.035
PMCID: PMC3438201  PMID: 22293042 CAMSID: cams2293
Mindfulness; Psychoeducation; Sexual dysfunction; Gynaecologic cancer; Sexual desire; Sexual arousal
10.  Proteomic Profiling Identifies Pathways Dysregulated in Non-small Cell Lung Cancer and an Inverse Association of AMPK and Adhesion Pathways with Recurrence 
Journal of Thoracic Oncology  2010;5(12):1894-1904.
Introduction
The identification of key pathways dysregulated in non-small cell lung cancer (NSCLC) is an important step toward understanding lung pathogenesis and developing new therapeutic approaches.
Methods
Toward this goal, reverse-phase protein lysate arrays (RPPA) were used to compare signaling pathways between NSCLC tumors and paired normal lung tissue from 46 patients and assess their association with clinical outcome.
Results
After RPPA quantification of 63 proteins and phosphoproteins, tissue pairs were randomized to a training set (n = 25 pairs) and test set (n = 21 pairs). In the training set, 15 protein markers were differentially expressed between tumors and normal lung (p ≤ 0.01), including markers in the PI3K/AKT and p38 MAPK signaling pathways (e.g., p70S6K, S6, p38, and phospho-p38), as well as caveolin-1 and β-catenin. A four-protein signature (p70S6K, cyclin B1, pSrc(Y527), and caveolin-1) independent of histology classified specimens as tumor versus normal with a predicted accuracy of 83%, sensitivity of 67%, and specificity of 100%. The signature was validated in the test set, correctly classifying all normal tissues and 14 of 21 tumor tissues. RPPA results were confirmed by immunohistochemistry for caveolin-1 and p70S6K. In tumors from patients with resected NSCLC, expression of proteins in the energy-sensing AMPK pathway (pLKB1, AMPK, p-Acetyl-CoA, pTSC2), adhesion, EGFR, and Rb signaling pathways was inversely associated with NSCLC recurrence.
Conclusions
These data provide evidence for dysregulation of several pathways including those involving energy sensing and adhesion that are potentially associated with NSCLC pathogenesis and disease recurrence.
doi:10.1097/JTO.0b013e3181f2a266
PMCID: PMC3374718  PMID: 21124077
NSCLC; Proteomics; Recurrence; AMPK; Adhesion
11.  Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress 
EMBO Molecular Medicine  2012;4(2):125-141.
Cancer cells are metabolically stressed during tumour progression due to limited tumour vascularity and resultant nutrient, growth factor and oxygen deficiency that can induce cell death and inhibit tumour growth. We demonstrate that Rab25, a small GTPase involved in endosomal recycling, that is genomically amplified in multiple tumour lineages, is a key regulator of cellular bioenergetics and autophagy. RAB25 enhanced survival during nutrient stress by preventing apoptosis and autophagy via binding and activating AKT leading to increased glucose uptake and improved cellular bioenergetics. Unexpectedly, Rab25 induced the accumulation of glycogen in epithelial cancer cells, a process not previously identified. Strikingly, an increase in basal ATP levels combined with AKT-dependent increases in glucose uptake and glycogen storage allowed maintenance of ATP levels during bioenergetic stress. The clinical relevance of these findings was validated by the ability of a Rab25-dependent expression profile enriched for bioenergetics targets to identify patients with a poor prognosis. Thus, Rab25 is an unexpected regulator of cellular bioenergetics implicated as a useful biomarker and potential therapeutic target.
doi:10.1002/emmm.201100193
PMCID: PMC3306554  PMID: 22253197
AKT; bioenergetic; cell death; glycogen; Rab25
12.  A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers 
Clinical proteomics  2010;6(4):129-151.
Introduction
The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA).
Methods
Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection.
Results
Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic “fingerprint” was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling.
Conclusion
Thus, the robustness of RPPA and stability of the functional proteomic “fingerprint” facilitate the study of the functional proteome in non-microdissected breast tumors.
doi:10.1007/s12014-010-9055-y
PMCID: PMC3116520  PMID: 21691416
Functional proteome; RPPA; Breast cancer; Kinase signaling; Steroid signaling
13.  Somatic Mutations in BRCA1 and BRCA2 Could Expand the Number of Patients That Benefit From Poly (ADP Ribose) Polymerase Inhibitors in Ovarian Cancer 
Journal of Clinical Oncology  2010;28(22):3570-3576.
Purpose
The prevalence of BRCA½ mutations in germline DNA from unselected ovarian cancer patients is 11% to 15.3%. It is important to determine the frequency of somatic BRCA½ changes, given the sensitivity of BRCA-mutated cancers to poly (ADP ribose) polymerase-1 (PARP1) inhibitors and platinum analogs.
Patients and Methods
In 235 unselected ovarian cancers, BRCA½ was sequenced in 235, assessed by copy number analysis in 95, and tiling arrays in 65. 113 tumors were sequenced for TP53. BRCA½ transcript levels were assessed by quantitative polymerase chain reaction in 220. When available for tumors with BRCA½ mutations, germline DNA was sequenced.
Results
Forty-four mutations (19%) in BRCA1 (n = 31)/BRCA2 (n = 13) were detected, including one homozygous BRCA1 intragenic deletion. BRCA½ mutations were particularly common (23%) in high-grade serous cancers. In 28 patients with available germline DNA, nine (42.9%) of 21 and two (28.6%) of seven BRCA1 and BRCA2 mutations were demonstrated to be somatic, respectively. Five mutations not previously identified in germline DNA were more commonly somatic than germline (four of 11 v one of 17; P = .062). There was a positive association between BRCA1 and TP53 mutations (P = .012). BRCA½ mutations were associated with improved progression-free survival (PFS) after platinum-based chemotherapy in univariate (P = .032; hazard ratio [HR] = 0.65; 95% CI, 0.43 to 0.98) and multivariate (P = .019) analyses. BRCA½ deficiency, defined as BRCA½ mutations or expression loss (in 24 [13.3%] BRCA½–wild-type cancers), was present in 67 ovarian cancers (30%) and was also significantly associated with PFS in univariate (P = .026; HR = 0.67; 95% CI, 0.47 to 0.96) and multivariate (P = .008) analyses.
Conclusion
BRCA½ somatic and germline mutations and expression loss are sufficiently common in ovarian cancer to warrant assessment for prediction of benefit in clinical trials of PARP1 inhibitors.
doi:10.1200/JCO.2009.27.2997
PMCID: PMC2917312  PMID: 20606085
14.  Functional proteomics can define prognosis and predict pathologic complete response in patients with breast cancer 
Clinical proteomics  2011;8(1):11.
Purpose
To determine whether functional proteomics improves breast cancer classification and prognostication and can predict pathological complete response (pCR) in patients receiving neoadjuvant taxane and anthracycline-taxane-based systemic therapy (NST).
Methods
Reverse phase protein array (RPPA) using 146 antibodies to proteins relevant to breast cancer was applied to three independent tumor sets. Supervised clustering to identify subgroups and prognosis in surgical excision specimens from a training set (n = 712) was validated on a test set (n = 168) in two cohorts of patients with primary breast cancer. A score was constructed using ordinal logistic regression to quantify the probability of recurrence in the training set and tested in the test set. The score was then evaluated on 132 FNA biopsies of patients treated with NST to determine ability to predict pCR.
Results
Six breast cancer subgroups were identified by a 10-protein biomarker panel in the 712 tumor training set. They were associated with different recurrence-free survival (RFS) (log-rank p = 8.8 E-10). The structure and ability of the six subgroups to predict RFS was confirmed in the test set (log-rank p = 0.0013). A prognosis score constructed using the 10 proteins in the training set was associated with RFS in both training and test sets (p = 3.2E-13, for test set). There was a significant association between the prognostic score and likelihood of pCR to NST in the FNA set (p = 0.0021).
Conclusion
We developed a 10-protein biomarker panel that classifies breast cancer into prognostic groups that may have potential utility in the management of patients who receive anthracycline-taxane-based NST.
doi:10.1186/1559-0275-8-11
PMCID: PMC3170272  PMID: 21906370
Breast Cancer; Functional Proteomics; Prognosis; Prediction
15.  14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis 
PLoS ONE  2011;6(1):e15864.
Background
In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2.
Principal Findings
Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis.
Conclusions
Cancer cells show differential proliferative sensitivity to changes in O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression.
doi:10.1371/journal.pone.0015864
PMCID: PMC3018427  PMID: 21249227
16.  Functional Proteomic Analysis of Advanced Serous Ovarian Cancer using Reverse Phase Protein Array: TGFβ Pathway Signaling Indicates Response to Primary Chemotherapy 
Purpose:
Using Reverse Phase Protein Array (RPPA) we measured protein expression associated with response to primary chemotherapy in patients with advanced-stage high-grade serous ovarian cancer.
Experimental Design:
Tumor samples were obtained from forty-five patients with advanced high-grade serous cancers from the Gynecology Tumor Bank at the British Columbia Cancer Agency. Treatment consisted of platinum-based chemotherapy following debulking surgery. Protein lysates were prepared from fresh frozen tumor samples and 80 validated proteins from signaling pathways implicated in ovarian carcinogenesis were measured by RPPA. Normalization of Ca-125 by the 3rd cycle of chemotherapy was chosen as the primary outcome measure of chemotherapy response. Logistic regression was used for multivariate analysis to identify protein predictors of Ca-125 normalization, and Cox regression to test for the association between protein expression and PFS. A significance level of p ≤ 0.05 was used.
Results:
The mean age at diagnosis was 56.8 years. EGFR, YKL-40 and several TGFβ pathway proteins (c-Jun N-terminal kinase JNK, JNK phosphorylated at residues 183 and 185, PAI-1, Smad3, TAZ) showed significant associations with Ca-125 normalization on univariate testing. On multivariate analysis, EGFR (p < 0.02), JNK (p < 0.01), and Smad3 (p < 0.04) were significantly associated with normalization of Ca-125. Contingency table analysis of pathway-classified proteins revealed that the selection of TGFβ pathway proteins was unlikely due to false discovery (p < 0.007, Bonferroni-adjusted).
Conclusion:
TGFβ pathway signaling likely plays an important role as a marker or mediator of chemoresistance in advanced serous ovarian cancer. On this basis, future studies to develop and validate a useful predictor of treatment failure are warranted.
doi:10.1158/1078-0432.CCR-09-2502
PMCID: PMC2877659  PMID: 20460476
ovarian cancer; functional proteomics; chemotherapy response; reverse phase protein array; Ca-125
17.  Integrative Analysis of Cyclin Protein Levels Identifies Cyclin B1 as a Classifier and Predictor of Outcomes in Breast Cancer 
Purpose
We studied the expression levels of cyclins B1, D1, and E1 and the implications of cyclin overexpression for patient outcomes in distinct breast cancer subtypes defined by clinical variables and transcriptional profiling.
Experimental Design
The expression levels of cyclins B1, D1, and E1 were quantified in 779 breast tumors and 53 cell lines using reverse phase protein arrays and/or transcriptional profiling.
Results
Whereas cyclin E1 overexpression was a specific marker of triple-negative and basal-like tumors, cyclin B1 overexpression occurred in poor prognosis hormone receptor–positive, luminal B and basal-like breast cancers. Cyclin D1 overexpression occurred in luminal and normal-like cancers. Breast cancer subgroups defined by integrated expression of cyclins B1, D1, and E1 correlated significantly (P < 0.000001) with tumor subtypes defined by transcriptional profiling and clinical criteria. Across three hormone receptor–positive data sets, cyclin B1 was the dominant cyclin associated with poor prognosis in univariate and multivariate analyses. Although CCNE1 was present in significantly higher copy numbers in basal-like versus other subtypes (ANOVA P < 0.001), CCNB1 gene copy number did not show gain in breast cancer. Instead, cyclin B1 expression was increased in tumors with co-occurrence of TP53 mutations and MYC amplification, a combination that seems to characterize basal-like and luminal B tumors. CCNB1 gene expression was significantly correlated with PLK, CENPE, and AURKB gene expression.
Conclusion
Cyclins B1, D1, and E1 have distinct expressions in different breast cancer subtypes. Novel PLK, CENPE, and AURKB inhibitors should be assessed for therapeutic utility in poor prognosis cyclin B1–overexpressing breast cancers.
doi:10.1158/1078-0432.CCR-08-3293
PMCID: PMC2887710  PMID: 19470724
18.  Characterization of a Naturally Occurring Breast Cancer Subset Enriched in Epithelial-to-Mesenchymal Transition and Stem Cell Characteristics 
Cancer research  2009;69(10):4116-4124.
Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor–positive cancers (34.5%; P = 0.32), 17 of 75 HER-2–positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a “tumorigenic” signature defined using CD44+/CD24− breast tumor–initiating stem cell–like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell–like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets.
doi:10.1158/0008-5472.CAN-08-3441
PMCID: PMC2737191  PMID: 19435916
20.  Targeting the Epidermal Growth Factor Receptor in Epithelial Ovarian Cancer: Current Knowledge and Future Challenges 
Journal of Oncology  2009;2010:568938.
The epidermal growth factor receptor is overexpressed in up to 60% of ovarian epithelial malignancies. EGFR regulates complex cellular events due to the large number of ligands, dimerization partners, and diverse signaling pathways engaged. In ovarian cancer, EGFR activation is associated with increased malignant tumor phenotype and poorer patient outcome. However, unlike some other EGFR-positive solid tumors, treatment of ovarian tumors with anti-EGFR agents has induced minimal response. While the amount of information regarding EGFR-mediated signaling is considerable, current data provides little insight for the lack of efficacy of anti-EGFR agents in ovarian cancer. More comprehensive, systematic, and well-defined approaches are needed to dissect the roles that EGFR plays in the complex signaling processes in ovarian cancer as well as to identify biomarkers that can accurately predict sensitivity toward EGFR-targeted therapeutic agents. This new knowledge could facilitate the development of rational combinatorial therapies to sensitize tumor cells toward EGFR-targeted therapies.
doi:10.1155/2010/568938
PMCID: PMC2796463  PMID: 20037743
21.  An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer 
Cancer research  2008;68(15):6084-6091.
Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy–based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor–positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor–positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor–positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor–positive breast cancer.
doi:10.1158/0008-5472.CAN-07-6854
PMCID: PMC2680495  PMID: 18676830
22.  RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays 
BMC Bioinformatics  2008;9:555.
Background
Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest.
Results
In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape.
Conclusion
The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis.
doi:10.1186/1471-2105-9-555
PMCID: PMC2639439  PMID: 19102773
23.  A Prospective Study of Reasons for Prolonged Hospitalizations on a General Medicine Teaching Service 
BACKGROUND
Delays in the care of hospitalized patients may lead to increased length of stay, iatrogenic complications, and costs. No study has characterized delays among general medicine inpatients in the current prospective payment era of care.
OBJECTIVE
To quantify and characterize delays in care which prolong hospitalizations for general medicine inpatients.
DESIGN
Prospective survey of senior residents.
SETTING
Urban tertiary care university-affiliated teaching hospital.
PARTICIPANTS
Sixteen senior residents were surveyed regarding 2,831 patient-days.
INTERVENTIONS
None.
MEASUREMENTS AND MAIN RESULTS
Data were collected on 97.6% (2,762) of patient-days eligible for evaluation. Three hundred seventy-three patient-days (13.5% of all hospital days) were judged unnecessary for acute inpatient care, and occurred because of delays in needed services. Sixty-three percent of these unnecessary days were due to nonmedical service delays and 37% were due to medical service delays. The vast majority of nonmedical service delays (84%) were due to difficulty finding a bed in a skilled nursing facility. Medical service delays were most often due to postponement of procedures (54%) and diagnostic test performance (21%) or interpretation (10%), and were significantly more common on weekend days (relative risk [RR], 1.49; P=.02). Indeed, nearly one fourth of unnecessary patient-days (24% overall, 88 patient-days) involved an inability to access medical services on a weekend day (Saturday or Sunday).
CONCLUSIONS
At our institution, a substantial number of hospital days were judged unnecessary for acute inpatient care and were attributable to delays in medical and nonmedical services. Future work is needed to develop and investigate measures to decrease delays.
doi:10.1111/j.1525-1497.2005.40269.x
PMCID: PMC1490052  PMID: 15836542
delays; hospital care; quality improvement; hospitalist; length of stay
24.  Characteristics of women undergoing repeat induced abortion 
Background
Although repeat induced abortion is common, data concerning characteristics of women undergoing this procedure are lacking. We conducted this study to identify the characteristics, including history of physical abuse by a male partner and history of sexual abuse, of women who present for repeat induced abortion.
Methods
We surveyed a consecutive series of women presenting for initial or repeat pregnancy termination to a regional provider of abortion services for a wide geographic area in southwestern Ontario between August 1998 and May 1999. Self-reported demographic characteristics, attitudes and practices regarding contraception, history of relationship violence, history of sexual abuse or coercion, and related variables were assessed as potential correlates of repeat induced abortion. We used χ2 tests for linear trend to examine characteristics of women undergoing a first, second, or third or subsequent abortion. We analyzed significant correlates of repeat abortion using stepwise multivariate multinomial logistic regression to identify factors uniquely associated with repeat abortion.
Results
Of the 1221 women approached, 1145 (93.8%) consented to participate. Data regarding first versus repeat abortion were available for 1127 women. A total of 68.2%, 23.1% and 8.7% of the women were seeking a first, second, or third or subsequent abortion respectively. Adjusted odds ratios for undergoing repeat versus a first abortion increased significantly with increased age (second abortion: 1.08, 95% confidence interval [CI] 1.04–1.09; third or subsequent abortion: 1.11, 95% CI 1.07–1.15), oral contraceptive use at the time of conception (second abortion: 2.17, 95% CI 1.52–3.09; third or subsequent abortion: 2.60, 95% CI 1.51–4.46), history of physical abuse by a male partner (second abortion: 2.04, 95% CI 1.39–3.01; third or subsequent abortion: 2.78, 95% CI 1.62–4.79), history of sexual abuse or violence (second abortion: 1.58, 95% CI 1.11–2.25; third or subsequent abortion: 2.53, 95% CI 1.50–4.28), history of sexually transmitted disease (second abortion: 1.50, 95% CI 0.98–2.29; third or subsequent abortion: 2.26, 95% CI 1.28–4.02) and being born outside Canada (second abortion: 1.83, 95% CI 1.19–2.79; third or subsequent abortion: 1.75, 95% CI 0.90–3.41).
Interpretation
Among other factors, a history of physical or sexual abuse was associated with repeat induced abortion. Presentation for repeat abortion may be an important indication to screen for a current or past history of relationship violence and sexual abuse.
doi:10.1503/cmaj.1040341
PMCID: PMC550633  PMID: 15738488
25.  Risk of Major Hemorrhage for Outpatients Treated with Warfarin 
OBJECTIVE
To determine the incidence of major hemorrhage among outpatients started on warfarin therapy after the recommendation in 1986 for reduced-intensity anticoagulation therapy was made, and to identify baseline patient characteristics that predict those patients who will have a major hemorrhage.
DESIGN
Retrospective cohort study.
SETTING
A university-affiliated Veterans Affairs Medical Center.
PATIENTS
Five hundred seventy-nine patients who were discharged from the hospital after being started on warfarin therapy.
MEASUREMENTS AND MAIN RESULTS
The primary outcome variable was major hemorrhage. In our cohort of 579 patients, there were 40 first-time major hemorrhages with only one fatal bleed. The cumulative incidence was 7% at 1 year. The average monthly incidence of major hemorrhage was 0.82% during the first 3 months of treatment and decreased to 0.36% thereafter. Three independent predictors of major hemorrhage were identified: a history of alcohol abuse, chronic renal insufficiency, and a previous gastrointestinal bleed. Age, comorbidities, medications known to influence prothrombin levels, and baseline laboratory values were not associated with major hemorrhage.
CONCLUSIONS
The incidence of major hemorrhage in this population of outpatients treated with warfarin was lower than previous estimates of major hemorrhage measured before the recommendation for reduced-intensity anticoagulation therapy was made, but still higher than estimates reported from clinical trials. Alcohol abuse, chronic renal insufficiency, and a previous gastrointestinal bleed were associated with increased risk of major hemorrhage.
doi:10.1046/j.1525-1497.1998.00096.x
PMCID: PMC1496955  PMID: 9613886
major hemorrhage; warfarin; alcohol abuse; chronic renal insufficiency; gastrointestinal hemorrhage

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