An RNA Interference Lethality Screen of the Human Druggable Genome to Identify Molecular Vulnerabilities in Epithelial Ovarian Cancer

Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 “hits” affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

Prognostic Value and Implication for Chemotherapy Treatment of ABCB1 in Epithelial Ovarian Cancer: A Meta-Analysis

BackgroundChemotherapy resistance is reported to correlate with up-regulation of anti-tumor agent transporter ABCB1 (p-gp) in epithelial ovarian cancer (EOC), but the results remain controversial. To reconcile the results, a systematic review followed by meta-analysis was performed to assess the association between high ABCB1 status or ABCB1 gene variants and overall survival (OS), progression free survival (PFS), and total response rate (TR) in patients with EOC.ResultsThirty-eight retrospective studies of 8607 cases qualified for meta-analysis were identified. Our results suggested that ABCB1 over-expression was significantly associated with unfavorable OS (HR = 1.54; 95% CI, 1.25–1.90), PFS (HR = 1.49; 95% CI, 1.22–1.82) and TR (RR = 0.63; 95% CI, 0.54–0.75). After adjustment for age, clinical stage, residual disease, histological type and tumor grade, high ABCB1 status remained to be a significant risk factor for adverse OS and PFS. Patients with recurrent ABCB1 positivity suffered from poorer OS than those with primary ABCB1 positivity. However, stratified by chemotherapy regimen, inverse correlation between high ABCB1 status and poor OS, PFS and TR were only found in patients underwent platinum-based chemotherapy but not in patients received standard platinum/paclitaxel-based chemotherapy. No evidence was found for any association between ABCB1 gene polymorphisms and OS, PFS or TR.ConclusionHigh ABCB1 status is significantly associated with chemo-resistance and poor prognosis in patients with EOC. Large-scale, prospective studies are needed to assess the clinical value of ABCB1 expression in EOC more accurately.     

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.Despite many advances in the treatment of cancer, early detection and tumor removal remains the best prospect for overcoming disease. Ovarian cancer is an excellent example of the potential prognostic value of early detection because diagnosis at a localized stage has a 5-year survival rate of 93%. However, only 19% of cases are diagnosed at this stage, and by the time the disease has evolved to an advanced stage, the 5-year survival rate drops to 31% (1).Much effort has been expended to find early detection markers of ovarian cancer, and some success has been achieved. Most notable is CA125, the only approved marker for the detection of recurrence of ovarian cancer (2). Other leading targets are mesothelin and HE4, which have been examined by several groups for their efficacy as early detection markers (3–8). Nevertheless, several conditions necessitate the discovery of more specific and sensitive ovarian cancer markers: the heterogeneity of this disease, the ambiguity of its symptoms, its low incidence in the general population, and the low sensitivity and specificity of currently available markers.One of the difficulties in finding markers in blood is the complexity of the plasma/serum proteome, estimated in the tens to hundreds of thousands of proteins, as well as its large range in constituent protein concentrations, which can span 12 orders of magnitude (9). However, along with its easy accessibility, the fact that blood is in contact with virtually every tissue and contains tissue- and tumor-derived proteins makes it a preferred source for disease biomarker discovery.Our previous results (10, 11) and those of others (12–14) using high density, full-length IgG antibody microarrays to validate and discover cancer serum biomarkers demonstrated that this platform is valuable for simultaneously comparing the levels of hundreds of proteins on dozens of serum samples from cancer patients and healthy controls. We confirmed overexpression of CA125, mesothelin, and HE4 in ovarian cancer samples using this high density microarray platform, validating our array methodology for measurement of cancer serum biomarkers and yielding new putative biomarkers for this disease (10, 11).Previously reported approaches are typically limited to a few hundred antibodies. The methodology reported here allows us to exploit the specific advantages of antibodies as high affinity capture reagents to detect differential expression of thousands of tumor biomarkers using a diverse (2 × 10⁸ binding agents) single-chain variable fragment antibody (scFv)¹ library for detection of ovarian cancer markers in serum, tumor cyst fluid, and ascites fluid. Our results build on previous reports of phage display library microarrays to discover autoantibody (15–18) and other protein (12, 19, 20) cancer biomarkers. Our scFv are high affinity capture reagents consisting of the variable regions of human antibody heavy and light chains joined by a flexible linker peptide. These recombinant antibodies are able to recognize a wide variety of antigens, including many previously thought difficult, such as self-antigens and proteins that are not normally immunogenic in animals (21–24). Using a highly diverse recombinant antibody library, one has the ability to overcome the complexity of the serum proteome. It has been calculated that for an immune repertoire to be complete (at least one antibody in the repertoire has reasonable affinity for every epitope possible in nature) it requires a diversity of at least 10⁶ antibodies (25). The reported diversity of our scFv library exceeds this value by 100-fold (21).To enrich for antibodies that differentiate disease status, we performed a selection or panning of the naïve library for proteins that are differentially expressed in cyst fluid, ascites fluid, or serum of cancer patients with respect to healthy serum. We printed this sublibrary on activated hydrogel slides that were queried with three different sets of labeled case and control sera to further select those that discriminate cancer status in a statistically significant manner. Next, we identified some of the targets that bind to the individual scFv using high density nucleic acid programmable protein arrays (NAPPAs) expressing a total of over 7000 proteins. Finally, we validated the effectiveness of the selection process by confirming overexpression of these targets in cancer serum, cyst fluid, and ascites fluid as well as in tumor sections.     

A Review of Principal Studies on the Development and Treatment of Epithelial Ovarian Cancer in the Laying Hen Gallus gallus

Often referred to as the silent killer, ovarian cancer is the most lethal gynecologic malignancy. This disease rarely shows any physical symptoms until late stages and no known biomarkers are available for early detection. Because ovarian cancer is rarely detected early, the physiology behind the initiation, progression, treatment, and prevention of this disease remains largely unclear. Over the past 2 decades, the laying hen has emerged as a model that naturally develops epithelial ovarian cancer that is both pathologically and histologically similar to that of the human form of the disease. Different molecular signatures found in human ovarian cancer have also been identified in chicken ovarian cancer including increased CA125 and elevated E-cadherin expression, among others. Chemoprevention studies conducted in this model have shown that decreased ovulation and inflammation are associated with decreased incidence of ovarian cancer development. The purpose of this article is to review the major studies performed in laying hen model of ovarian cancer and discuss how these studies shape our current understanding of the pathophysiology, prevention and treatment of epithelial ovarian cancer.

Ovarian cancer is the leading cause of death among female gynecologic malignancies, with a 47% 5 y relative survival rate.¹⁵⁴ Early detection of the disease is necessary for decreasing the high mortality rate. However, early detection is difficult due to the lack of known specific biomarkers and clinically detectable symptoms until the tumor reaches at an advanced stage. The disease has multiple subtypes. Epithelial ovarian cancer (EOC) is the most common type of ovarian cancer, accounting for about 90% of all reported cases.^127,164 EOC is commonly subdivided into 5 histotypes: high-grade serous (HGSOC), low-grade serous, mucinous, endometroid (EC), and clear cell. The histotypes differ in terms of tumor cell morphology, severity, systemic effect, and response to treatment. Among the different subtypes, HGSOC accounts for about 70% of cases of EOC observed in women. HGSOC has a higher mitotic index and is a more aggressive form of cancer with a worse prognosis. HGSOC and low-grade serous histotypes exhibit distinctly different presentations of the disease^82,166 and demand different treatment modalities. EC (10% to 20%), mucinous (5% to 20%), and clear cell (3% to 10%) histotypes are less common forms of the disease. The subtypes of EOC also differ in terms of 5 y survival rates of patients; that is, HGSOC (20% to 35%), EC (40% to 63%), mucinous (40% to 69%), and clear cell (35% to 50%).^20,76,148Developing a representative animal model for EOC has been challenging due to the histologic and pathologic differences among different subtypes of EOC. While developing a reliable animal model is challenging due to the vast complexity and limited understanding of the origin of the disease, laying hens naturally develop EOC that is histopathologically very similar to the human form of the disease (Figure 1).¹⁵ All the different human ovarian cancer histotypes have been observed in laying hen ovarian cancer (Figure 2). In addition, the presentation of the disease in chickens is remarkably similar to the human form of the disease, with early-stage ovarian cancer in laying hens having similar precursor lesions as occur in women.¹⁵ The laying hen develops ovarian cancer spontaneously, allowing analysis of early events and investigation into the natural course of the disease, as tumors can be examined as they progress from normal to late-stage ovarian carcinoma. The gross appearance of these stages is shown in Figure 3.Open in a separate windowFigure 1.Gross pathologic presentation of chicken compared with human ovarian cancer. The remarkably similar presentation in hens (A,B) and women (C,D) at the gross anatomic level with profuse abdominal ascites and peritoneal dissemination of metastasis. A) Ascites in abdominal cavity chicken with advanced ovarian cancer (photo credit: DB Hales); (B) Chicken ovarian cancer with extensive peritoneal dissemination of metastasis (photo credit: DB Hales); (C) Distended abdomen from ascites fluid accumulation in woman with ovarian cancer (http://www.pathguy.com/bryanlee/ovca.html) (D) Human ovarian cancer with extensive peritoneal dissemination of metastasis (http://www.pathguy.com/bryanlee/ovca.html).Open in a separate windowFigure 2.Gross anatomic appearance of different stages of ovarian cancer in the chicken The progression from the normal hen ovary to late-stage metastatic ovarian cancer. (A) Normal chicken ovary showing hierarchal clutch of developing follicles and postovulatory follicle; (B) Stage 1 ovarian cancer, confined to ovary with vascularized follicles; (C) Stage 2/3 ovarian cancer, metastasis locally to peritoneal cavity with ascites; (D) Stage 4 ovarian cancer, late stage with metastasis to lung and liver with extensive ascites (photo credits: DB Hales).Open in a separate windowFigure 3.Histologic subtypes in chicken compared with human ovarian cancers. H and E staining of formalin fixed paraffin embedded tissues from hens with ovarian cancer (A through D) and women (E through G). (A) Chicken clear cell carcinoma; (B) Chicken endometrioid carcinoma; (C) Chicken mucinous adenocarcinoma; (D) Chicken serous papillary adenocarcinoma (photo credits: DB Hales). (E) Human clear cell carcinoma; (F) Human endometrioid carcinoma; (G) Human mucinous cystadenocarcinoma; (H) Human serous adenocarcinoma (https://www.womenshealthsection.com).Over the past 2 decades, the laying hen has emerged as a valuable experimental model for EOC, in addition to other in vivo models such as Patient-Derived Xenografts (PDX) and Genetically Engineered Mouse Models (GEMMs). Comparison of the hen model with other animal models has been reviewed elsewhere.⁷² Modern-day laying hens, such as the white leghorn, have been selected from their ancestor red jungle fowl⁵⁷ for decreased broodiness and persistent ovulation, resulting in approximately one egg per day, if proper nutrition and light-dark cycles are maintained. Daily rupture and consequent repair of the ovarian surface epithelia (OSE) due to the persistent ovulation promotes potential error during rapid DNA replication. This increases the probability of oncogenic mutations, ultimately leading to neoplasia.¹³⁷ Inflammation resulting from continuous ovulation also promotes the natural development of EOC.⁸¹ By the age of 2.5 to 3 y, laying hens have undergone a similar number of ovulations as a perimenopausal woman. The risk of ovarian cancer in white leghorn hens in this time (4%) is similar to the lifetime risk of ovarian cancer in women (0.35% to 8.8%).¹²⁵ By the age of 4 to 6 y, the risk of ovarian cancer in hens rises to 30% to 60%.⁵⁴ The incidence of ovarian carcinoma in the hens, however, depends on the age, genetic strain,⁸⁰ and the egg-laying frequency of the specific breed.⁵⁴ The common white leghorn hen has routinely been employed in chicken ovarian cancer studies. On average, hens are exposed to 17 h of light per day, with lights turned on at 0500 h and turned off at 2200 h. The laying hen model of EOC does present some considerable challenges. Despite its great utility for research, the model is still used mainly by agricultural poultry scientists and a small number of ovarian cancer researchers.Comprehensive and proper vivarium support is required to conduct large-scale cancer prevention studies. Only a few facilities are available for biomedical chicken research, including University of Illinois Urbana-Champaign, Cornell University, Penn State University, NC State, Auburn University, and MS State University. Another difficulty is a lack of available antibodies specific for chicken antigens. Because of the structural dissimilarities between most human proteins and murine antigens to their chicken counterparts, cross-reactivity of available antibodies is also limited. The entire chicken genome was sequenced in 2004;⁷⁸ however, the chromosomal locus of many key genes, such as p53, are still unknown. Overall, humans and chickens share about 60% of genetic commonality, whereas humans and rats share about 88% of their genes. Specific pathway-mutated strains of chickens are not yet available, limiting the ability to study key pathways in carcinogenesis and prevention of cancer using this model. Although all 5 different subtypes of ovarian cancer are present in hens, their most predominant subtype is different from women. Close to 70% of women diagnosed with ovarian cancer have serous EOC, while the predominant subtype reported in hens is endometrioid.¹⁵ However, these comparisons are complicated because observations of cancer in hens consist of both early and late stages of the disease, wherein women, most of the data is from late stage and aggressive ovarian carcinoma.The spontaneous onset of ovarian cancer and the histologic and pathologic similarities to the human form of the disease make laying hens an excellent model for continued research on EOC. To date, a large number of studies have been performed on laying hens. Here we have divided the current studies into 2 groups— (A) studies that have described the molecular presentation of EOC to be similar to that in women; (

A Targeted Mass Spectrometry Strategy for Developing Proteomic Biomarkers: A Case Study of Epithelial Ovarian Cancer

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Highlights

• •Rigorous experimental design and data analysis for large-scale SRM studies.
• •Plasma-based biomarker signature combined with CA125 for ovarian cancer detection.
• •Broadly applicable strategy for the development of diagnostic biomarker assays.

     

Validation of LRG1 as a Potential Biomarker for Detection of Epithelial Ovarian Cancer by a Blinded Study

Background

Leucine-rich alpha-2-glycoprotein (LRG1) was found to be differentially expressed in sera from patients with Epithelial Ovarian Cancer (EOC). The aim of this study is to investigate the performance of LRG1 for detection of EOC, including early stage EOC, and to evaluate if LRG1 can complement CA125 in order to improve EOC detection using two independent blinded sample sets.

Methods and Results

Serum LRG1 and CA125 were measured by immunoassays. All assays were performed blinded to clinical data. Using the two independent sample sets (156 participants for sample set 1, and 233 for sample set 2), LRG1 was differentially expressed in EOC cases as compared to healthy, surgical, and benign controls, and its performance was not affected by the conditions of blood collection. The areas under the ROC curve (AUC) for LRG1 in differentiating EOC cases from non-cases were 0.797 and 0.786 for sample set 1 and 2. For differentiating EOC cases from healthy controls, the AUC values for LRG1 were 0.792 and 0.794. At a fixed specificity of 95%, LRG1 detects 52%, and 53.5% of EOC cases from healthy controls for sample set 1 and 2. When combining LRG1 and CA125, the AUC value increased to 0.927, which was improved compared to CA125 (AUC=0.916) (p=0.008) alone in distinguishing EOC cases from non-cases. More importantly, LRG1 also showed potential performance in differentiating early stage EOC from non-cases with an AUC of 0.715 for sample set 1, and 0.690 for sample set 2. The combination of LRG1 and CA125 resulted in an AUC of 0.838, which outperforms CA125 (AUC=0.785) (p=0.018) in detecting early stage EOC cases from non-cases using the larger sample set.

Conclusions

LRG1 could be a useful biomarker alone or in combination with CA125 for the diagnosis of ovarian cancer.     

动态监测上皮性卵巢癌患者血清CA125水平对疗效及预后的评估价值

目的:通过观察卵巢上皮性癌患者术前、术后及化疗后CA125水平变化,探讨其水平变化对患者预后的评价价值。方法:选取2012年3月至2013年2月间医院诊治的卵巢上皮性癌患者85例为研究对象,所有患者均接受规范的卵巢癌分期手术或卵巢癌细胞减灭术,术后常规进行系统化疗。比较手术及化疗后患者血清CA125水平的变化,分析患者血清CA125水平动态变化、转阴时间与患者预后关系。结果:治疗后,患者CA125水平呈明显下降趋势,差异具有统计学意义(P0.05)。T1/2≤20 d患者存活时间明显长于T1/220 d的患者,差异具有统计学意义(P0.05)。CA125转阴时间≤8周的患者存活时间长于CA125转阴时间8周的患者,差异具有统计学意义(P0.05)。其复发率明显低于CA125转阴时间8周的患者,复发时间长于CA125转阴时间8周的患者,差异具有统计学意义(P0.05)。结论:通过检测血清CA125水平变化可以对卵巢上皮性癌患者预后进行较为准确的评价。     

Focal Therapy: A New Paradigm for the Treatment of Prostate Cancer

Focal therapy has been proposed in recent years as a means of bridging the gap between radical prostatectomy and active surveillance for treatment of prostate cancer. The rationale for focal therapy comes from its success in treating other malignancies. One of the challenges in applying such an approach to the treatment of prostate cancer has been the multifocal nature of the disease. This review addresses the selection of potentially ideal candidates for focal therapy and discusses which modalities are currently being used and proposed for focal therapy. Setting and meeting guidelines for oncologic efficacy is a challenge we must embrace to safely deliver this potentially revolutionary approach to treating men with prostate cancer.Key words: Focal therapy, Photodynamic therapy, Prostatic neoplasms, Prostate-specific antigen, Prostatectomy, Ultrasound, high-intensity focused, transrectal, CryosurgeryWith the advent of prostate-specific antigen (PSA) screening there has been a stage migration, with radical prostatectomy (RP) being performed with increasing frequency in men with low-risk disease.¹ Whole gland treatment of prostate cancer carries a significant risk of incontinence and sexual dysfunction. Even in the most experienced centers, the rate of potency following RP is approximately 60%.^2–4 Stage migration has led many to recommend active surveillance (AS) as a means to decrease the number of men who may be overtreated; however, AS has been slow to gain acceptance in the United States.An analysis of over 5300 men from the Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) National Prostate Cancer Registry⁵ showed that only 7% of men with clinically localized prostate cancer chose AS as an initial option. Aside from the anxiety that stems from not treating a diagnosed cancer, the greater difficulty with AS lies in selection of candidates and appropriate parameters for surveillance, allowing prompt intervention without compromising cure rates.Focal therapy has been proposed in recent years as a means of bridging the gap between whole gland treatment and AS. Many believe that for patients with low-risk disease, focal therapy is the ideal option for maximizing quality of life by avoiding the effects of whole gland radiation or surgery while alleviating the anxiety and uncertainty of AS. The definition of focal therapy itself is not well established and includes lesion-targeted therapy (LAT), hemiablative therapy (HAT), or subtotal gland therapy (STAT), sparing at least 1 neurovascular bundle.⁶The rationale for focal therapy comes from its success in treating other malignancies. In breast cancer treatment, for example, radical mastectomy has been replaced in many instances by local excision and Mohs surgery has led to less radical surgery for the treatment of melanoma.⁷ In our own field, the push for nephron-sparing surgery has led to the favoring of partial nephrectomy in tumors less than 7 cm, with oncologic outcomes similar to those of radical nephrectomy.⁸The challenge in applying such an approach to the treatment of prostate cancer has been the multifocal nature of prostate cancer and the fact that most cancers are detected without identifying a lesion on palpation or imaging studies.^9,10In this review, we revisit the current status of focal therapy in the treatment of prostate cancer. We discuss whether there are ideal candidates for focal therapy; we then discuss how these candidates should be selected. We review which modalities are currently being used and proposed for focal therapy. Finally, we discuss potential definitions of successful treatment. As this article shows, there are still many aspects of focal therapy that are yet to be defined, that warrant a great need for further research.     

Expression of Serum Amyloid A in Human Ovarian Epithelial Tumors: Implication for a Role in Ovarian Tumorigenesis

Serum amyloid A (SAA) is an acute phase protein which is expressed primarily in the liver as a part of the systemic response to various injuries and inflammatory stimuli; its expression in ovarian tumors has not been described. Here, we investigated the expression of SAA in human benign and malignant ovarian epithelial tumors. Non-radioactive in situ hybridization applied on ovarian paraffin tissue sections revealed mostly negative SAA mRNA expression in normal surface epithelium. Expression was increased gradually as epithelial cells progressed through benign and borderline adenomas to primary and metastatic adenocarcinomas. Similar expression pattern of the SAA protein was observed by immunohistochemical staining. RT-PCR analysis confirmed the overexpression of the SAA1 and SAA4 genes in ovarian carcinomas compared with normal ovarian tissues. In addition, strong expression of SAA mRNA and protein was found in the ovarian carcinoma cell line OVCAR-3. Finally, patients with ovarian carcinoma had high SAA serum levels, which strongly correlated with high levels of CA-125 and C-reactive protein. Enhanced expression of SAA in ovarian carcinomas may play a role in ovarian tumorigenesis and may have therapeutic application. (J Histochem Cytochem 58:1015–1023, 2010)     

Screening and Identification of a Specific Binding Peptide to Ovarian Cancer Cells from a Phage-Displayed Peptide Library

To select specific binding peptides for imaging and detection of human ovarian cancer. The phage 12-mer peptide library was used to select specific phage clones to ovarian cancer cells. After four rounds of biopanning, the binding specificity of randomly selected phage clones to ovarian cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). DNA sequencing and homology analysis were performed on specifically bound phages. The binding ability of the selected peptides to SKOV3 cells was confirmed by fluorescence microscopy and flow cytometry. After four rounds of optimized biological panning, phage recovery was 34-fold higher than that of the first round, and the specific phage clones bound to SKOV3 cells were significantly enriched. A total of 32 positive phage clones were preliminarily identified by ELISA from 54 randomly selected clones, and the positive rate was 59.3%. S36 was identified as the clone with best affinity to SKOV3 cells via fluorescence microscopy and flow cytometry. A representative clone of OSP2, S36 is expected to be an effective probe for diagnosis and treatment of ovarian cancer.

     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.     

Assessment of a Novel Approach to Identify Trichiasis Cases Using Community Treatment Assistants in Tanzania

Background

Simple surgical intervention advocated by the World Health Organization can alleviate trachomatous trichiasis (TT) and prevent subsequent blindness. A large backlog of TT cases remain unidentified and untreated. To increase identification and referral of TT cases, a novel approach using standard screening questions, a card, and simple training for Community Treatment Assistants (CTAs) to use during Mass Drug Administration (MDA) was developed and evaluated in Kongwa District, a trachoma-endemic area of central Tanzania.

Methodology/Principal Findings

A community randomized trial was conducted in 36 communities during MDA. CTAs in intervention villages received an additional half-day of training and a TT screening card in addition to the training received by CTAs in villages assigned to usual care. All MDA participants 15 years and older were screened for TT, and senior TT graders confirmed case status by evaluating all screened-positive cases. A random sample of those screened negative for TT and those who did not present at MDA were also evaluated by the master graders. Intervention CTAs identified 5.6 times as many cases (n = 50) as those assigned to usual care (n = 9, p < 0.05). While specificity was above 90% for both groups, the sensitivity for the novel screening tool was 31.2% compared to 5.6% for the usual care group (p < 0.05).

Conclusions/Significance

CTAs appear to be viable resources for the identification of TT cases. Additional training and use of a TT screening card significantly increased the ability of CTAs to recognize and refer TT cases during MDA; however, further efforts are needed to improve case detection and reduce the number of false positive cases.     

Application of a Drug-Induced Apoptosis Assay to Identify Treatment Strategies in Recurrent or Metastatic Breast Cancer

BackgroundA drug-induced apoptosis assay has been developed to determine which chemotherapy drugs or regimens can produce higher cell killing in vitro. This study was done to determine if this assay could be performed in patients with recurrent or metastatic breast cancer patients, to characterize the patterns of drug-induced apoptosis, and to evaluate the clinical utility of the assay. A secondary goal was to correlate assay use with clinical outcomes.MethodsIn a prospective, non-blinded, multi institutional controlled trial, 30 evaluable patients with recurrent or metastatic breast cancer who were treated with chemotherapy had tumor samples submitted for the MiCK drug-induced apoptosis assay. After receiving results within 72 hours after biopsy, physicians could use the test to determine therapy (users), or elect to not use the test (non-users).ResultsThe assay was able to characterize drug-induced apoptosis in tumor specimens from breast cancer patients and identified which drugs or combinations gave highest levels of apoptosis. Patterns of drug activity were also analyzed in triple negative breast cancer. Different drugs from a single class of agents often produced significantly different amounts of apoptosis. Physician frequently (73%) used the assay to help select chemotherapy treatments in patients, Patients whose physicians were users had a higher response (CR+PR) rate compared to non-users (38.1% vs 0%, p = 0.04) and a higher disease control (CR+PR+Stable) rate (81% vs 25%, p<0.01). Time to relapse was longer in users 7.4 mo compared to non-users 2.2 mo (p<0.01).ConclusionsThe MiCK assay can be performed in breast cancer specimens, and results are often used by physicians in breast cancer patients with recurrent or metastatic disease. These results from a good laboratory phase II study can be the basis for a future larger prospective multicenter study to more definitively establish the value of the assay.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00901264","term_id":"NCT00901264"}}NCT00901264     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

The Role of BRCA Status on the Prognosis of Patients with Epithelial Ovarian Cancer: A Systematic Review of the Literature with a Meta-Analysis

Objective

The role of BRCA dysfunction on the prognosis of patients with epithelial ovarian cancer (EOCs) remains controversial. This systematic review tried to assess the role of BRCA dysfunction, including BRCA1/2 germline, somatic mutations, low BRCA1 protein/mRNA expression or BRCA1 promoter methylation, as prognostic factor in EOCs.

Methods

Studies were selected for analysis if they provided an independent assessment of BRCA status and prognosis in EOC. To make it possible to aggregate survival results of the published studies, their methodology was assessed using a modified quality scale.

Results

Of 35 evaluable studies, 23 identified BRCA dysfucntion status as a favourable prognostic factor. No significant differences were detected in the global score of quality assessment. The aggregated hazard ratio (HR) of overall survival (OS) of 34 evaluable studies suggested that BRCA dysfunction status had a favourable impact on OS (HR = 0.69, 95% CI 0.61–0.79), and when these studies were categorised into BRCA1/2 mutation and low protein/mRNA expression of BRCA1 subgroups, all of them demonstrated positive results (HR = 0.67, 95% CI: 0.57–0.78; HR = 0.62, 95% CI: 0.51–0.75; and HR = 0.51, 95% CI: 0.33–0.78, respectively), except for the subgroup of BRCA1 promoter methylation (HR = 1.59, 95% CI: 0.72–3.50). The meta-analysis of progression-free survival (PFS), which included 18 evaluable studies, demonstrated that BRCA dysfunction status was associated with a longer PFS in EOC (HR = 0.69, 95% CI: 0.63–0.76).

Conclusions

Patients with BRCA dysfunction status tend to have a better outcome, but further prospective clinical studies comparing the different BRCA statuses in EOC is urgently needed to specifically define the most effective treatment for the separate patient groups.