Molecular Characterization of an Intact p53 Pathway Subtype in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is the most aggressive histological type of epithelial ovarian cancer, which is characterized by a high frequency of somatic TP53 mutations. We performed exome analyses of tumors and matched normal tissues of 34 Japanese patients with HGSOC and observed a substantial number of patients without TP53 mutation (24%, 8/34). Combined with the results of copy number variation analyses, we subdivided the 34 patients with HGSOC into subtypes designated ST1 and ST2. ST1 showed intact p53 pathway and was characterized by fewer somatic mutations and copy number alterations. In contrast, the p53 pathway was impaired in ST2, which is characterized by abundant somatic mutations and copy number alterations. Gene expression profiles combined with analyses using the Gene Ontology resource indicate the involvement of specific biological processes (mitosis and DNA helicase) that are relevant to genomic stability and cancer etiology. In particular we demonstrate the presence of a novel subtype of patients with HGSOC that is characterized by an intact p53 pathway, with limited genomic alterations and specific gene expression profiles.     

Clinical Relevance of Increased Endothelial and Mesothelial Expression of Proangiogenic Proteases and VEGFA in the Omentum of Patients with Metastatic Ovarian High-Grade Serous Carcinoma

Epithelial ovarian cancer (EOC) metastasis to the omentum requires implantation and angiogenesis. We propose that prometastatic changes in the omental endothelium (for angiogenesis) and mesothelium (for implantation) are critical. We investigated the expression of angiogenic proteases [cathepsin D (CD), cathepsin L (CL), and matrix metalloproteinase 2 (MMP2) and MMP9] and vascular endothelial growth factor A (VEGFA) in the mesothelium and endothelium of omentum from patients with EOC with omental metastases and control patients with benign ovarian tumors. Endothelial expression of CL, VEGFA, and MMP9 and mesothelial expression of VEGFA, MMP9, and CD were significantly increased in patients with metastasized EOC. High expression of MMP9 and VEGFA in endothelium and mesothelium and CD in mesothelium was positively associated with poor disease-specific survival (DSS). High MMP9 expression in either endothelium or mesothelium and presence of ascites prospectively showed the greatest risk of shorter DSS [hazard ratio (HR)= 6.16, 95% confidence interval (CI) = 1.76-21.6, P = .0045; HR = 11.42, 95% CI = 2.59-50.35, P = .0013; and HR = 6.35, 95% CI = 2.01-20.1, P = .002, respectively]. High endothelial MMP9 expression and ascites were independent predictors of reduced DSS and overall survival, together resulting in worst patient prognosis. Our data show that omental metastasis of EOC is associated with increased proangiogenic protein expression in the omental endothelium and mesothelium.     

Markers of T Cell Infiltration and Function Associate with Favorable Outcome in Vascularized High-Grade Serous Ovarian Carcinoma

Background

When T cells infiltrate the tumor environment they encounter a myriad of metabolic stressors including hypoxia. Overcoming the limitations imposed by an inadequate tumor vasculature that contributes to these stressors may be a crucial step to immune cells mounting an effective anti-tumor response. We sought to determine whether the functional capacity of tumor infiltrating lymphocytes (TIL) could be influenced by the tumor vasculature and correlated this with survival in patients with ovarian cancer.

Methodology and Principal Findings

In 196 high-grade serous ovarian tumors, we confirmed that the tumor vascularity as measured by the marker CD31 was associated with improved patient disease-specific survival. We also found that tumors positive for markers of TIL (CD8, CD4 and forkhead box P3 (FoxP3)) and T cell function (granzyme B and T-cell restricted intracellular antigen-1 (TIA-1)) correlated significantly with elevated vascularity. In vitro, hypoxic CD8 T cells showed reduced cytolytic activity, secreted less effector cytokines and upregulated autophagy. Survival analysis revealed that patients had a significant improvement in disease-specific survival when FoxP3 expressing cells were present in CD31-high tumors compared to patients with FoxP3 expressing cells in CD31-low tumors [HR: 2.314 (95% CI 1.049–5.106); p = 0.0377]. Patients with high vascular endothelial growth factor (VEGF) expressing tumors containing granzyme B positive cells had improved survival compared to patients with granzyme B positive cells in VEGF-low tumors [HR: 2.522 (95% CI 1.097–5.799); p = 0.0294].

Significance

Overall, this data provides a rationale for developing strategies aimed at improving the adaptability and function of TIL to hypoxic tumor conditions.     

Proteomic Discovery of Plasma Protein Biomarkers and Development of Models Predicting Prognosis of High-Grade Serous Ovarian Carcinoma

Ovarian cancer is one of the most lethal female cancers. For accurate prognosis prediction, this study aimed to investigate novel, blood-based prognostic biomarkers for high-grade serous ovarian carcinoma (HGSOC) using mass spectrometry–based proteomics methods. We conducted label-free liquid chromatography–tandem mass spectrometry using frozen plasma samples obtained from patients with newly diagnosed HGSOC (n = 20). Based on progression-free survival (PFS), the samples were divided into two groups: good (PFS ≥18 months) and poor prognosis groups (PFS <18 months). Proteomic profiles were compared between the two groups. Referring to proteomics data that we previously obtained using frozen cancer tissues from chemotherapy-naïve patients with HGSOC, overlapping protein biomarkers were selected as candidate biomarkers. Biomarkers were validated using an independent set of HGSOC plasma samples (n = 202) via enzyme-linked immunosorbent assay (ELISA). To construct models predicting the 18-month PFS rate, we performed stepwise selection based on the area under the receiver operating characteristic curve (AUC) with 5-fold cross-validation. Analysis of differentially expressed proteins in plasma samples revealed that 35 and 61 proteins were upregulated in the good and poor prognosis groups, respectively. Through hierarchical clustering and bioinformatic analyses, GSN, VCAN, SND1, SIGLEC14, CD163, and PRMT1 were selected as candidate biomarkers and were subjected to ELISA. In multivariate analysis, plasma GSN was identified as an independent poor prognostic biomarker for PFS (adjusted hazard ratio, 1.556; 95% confidence interval, 1.073–2.256; p = 0.020). By combining clinical factors and ELISA results, we constructed several models to predict the 18-month PFS rate. A model consisting of four predictors (FIGO stage, residual tumor after surgery, and plasma levels of GSN and VCAN) showed the best predictive performance (mean validated AUC, 0.779). The newly developed model was converted to a nomogram for clinical use. Our study results provided insights into protein biomarkers, which might offer clues for developing therapeutic targets.     

Spatial and Temporal Heterogeneity in High-Grade Serous Ovarian Cancer: A Phylogenetic Analysis

Background

The major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease.

Methods and Findings

Evolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22–46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66–1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy.

Conclusions

This study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.     

Interrogation of Functional Cell-Surface Markers Identifies CD151 Dependency in High-Grade Serous Ovarian Cancer

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Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry

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miR-450a-5p在浆液性卵巢癌中的表达及临床意义

目的:检测miR-450a-5p在浆液性卵巢癌中的表达情况,并分析miR-450a-5p的表达与浆液性卵巢癌临床病理特征之间的关系,探讨其在浆液性卵巢癌发生、发展中的意义。方法:采用实时荧光定量PCR技术,检测101例浆液性卵巢癌组织及对照50例正常输卵管伞端组织中miR-450a-5p的表达情况,并分析miR-450a-5p表达水平与临床病理特征之间的关系。实验数据采用统计学软件进行分析。结果:miR-450a-5p在浆液性卵巢癌组织中的表达量显著低于正常输卵管组织对照组(P0.01)。miR-450a-5p的表达水平与浆液性卵巢癌临床病理特征之间差异均无统计学意义(P0.05)。结论:miR-450a-5p可能作为抑癌基因,在卵巢癌的发生、发展中发挥重要作用,进一步的研究有望为浆液性卵巢癌的早期诊断及个体化治疗提供新的理论依据。     

Predictive Modeling Using a Somatic Mutational Profile in Ovarian High Grade Serous Carcinoma

Purpose

Recent high-throughput sequencing technology has identified numerous somatic mutations across the whole exome in a variety of cancers. In this study, we generate a predictive model employing the whole exome somatic mutational profile of ovarian high-grade serous carcinomas (Ov-HGSCs) obtained from The Cancer Genome Atlas data portal.

Methods

A total of 311 patients were included for modeling overall survival (OS) and 259 patients were included for modeling progression free survival (PFS) in an analysis of 509 genes. The model was validated with complete leave-one-out cross-validation involving re-selecting genes for each iteration of the cross-validation procedure. Cross-validated Kaplan-Meier curves were generated. Cross-validated time dependent receiver operating characteristic (ROC) curves were computed and the area under the curve (AUC) values were calculated from the ROC curves to estimate the predictive accuracy of the survival risk models.

Results

There was a significant difference in OS between the high-risk group (median, 28.1 months) and the low-risk group (median, 61.5 months) (permutated p-value <0.001). For PFS, there was also a significant difference in PFS between the high-risk group (10.9 months) and the low-risk group (22.3 months) (permutated p-value <0.001). Cross-validated AUC values were 0.807 for the OS and 0.747 for the PFS based on a defined landmark time t = 36 months. In comparisons between a predictive model containing only gene variables and a combined model containing both gene variables and clinical covariates, the predictive model containing gene variables without clinical covariates were effective and high AUC values for both OS and PFS were observed.

Conclusions

We designed a predictive model using a somatic mutation profile obtained from high-throughput genomic sequencing data in Ov-HGSC samples that may represent a new strategy for applying high-throughput sequencing data to clinical practice.     

The Genomic Landscape of TP53 and p53 Annotated High Grade Ovarian Serous Carcinomas from a Defined Founder Population Associated with Patient Outcome

High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.     

IPI59: An Actionable Biomarker to Improve Treatment Response in Serous Ovarian Carcinoma Patients

Despite improvements in operative management and therapies, overall survival rates in advanced ovarian cancer have remained largely unchanged over the past three decades. Although it is possible to identify high-risk patients following surgery, the knowledge does not provide information about the genomic aberrations conferring risk, or the implications for treatment. To address these challenges, we developed an integrative pathway-index model and applied it to messenger RNA expression from 458 patients with serous ovarian carcinoma from the Cancer Genome Atlas project. The biomarker derived from this approach, IPI59, contains 59 genes from six pathways. As we demonstrate using independent datasets from six studies, IPI59 is strongly associated with overall and progression-free survival, and also identifies high-risk patients who may benefit from enhanced adjuvant therapy.     

Mutations in p53 cDNA sequence introduced by retroviral vector

The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors.     

Eotaxin-1在浆液性卵巢癌组织中的表达及临床意义研究

目的:研究嗜酸性粒细胞趋化因子1(Eotaxin-1)在浆液性卵巢癌组织中的表达及临床病理意义。方法:收集2013年4月至2014年5月于我院妇产科手术切除的60例浆液性卵巢癌及对应癌旁组织,采用免疫组织化学染色检测Eotaxin-1表达,分析Eotaxin-1蛋白与肿瘤临床病理资料之间的相关性。结果:浆液性卵巢癌组织中Eotaxin-1蛋白表达水平较对应癌旁组织显著升高(P0.05),浆液性卵巢癌组织中高表达Eotaxin-1蛋白与恶性组织病理分级、淋巴结转移及高TNM分期呈显著正相关(P0.05)。结论:Eotaxin-1蛋白在浆液性卵巢癌组织中表达上调,并与肿瘤恶性临床病理特征有关;Eotaxin-1可能成为浆液性卵巢癌早期诊断的重要标志物和生物靶向治疗的有效靶点之一,具有广阔的临床应用前景。     

Mutations in the p53 homolog p63: allele-specific developmental syndromes in humans

p63 is the most recently discovered but most ancient member of the p53 family. In marked contrast to p53, p63 is highly expressed in embryonic ectoderm and in the basal, regenerative layers of many epithelial tissues in the adult. The p63-knockout mouse dies at birth and lacks limbs, epidermis, prostate, breast and urothelial tissues, apparently owing to the loss of stem cells required for these tissues. Significantly, several dominant human syndromes involving limb development and/or ectodermal dysplasia have been mapped to chromosome 3q27 and ultimately the gene encoding p63. The heterozygous p63mutations are distinct for each of the syndromes and are thought to act through both dominant-negative and gain-of-function mechanisms rather than a loss-of-function haploinsufficiency. The allele specificity of these syndromes offers unique molecular insights into the poorly understood actions of p63 in limb development, ectodermal-mesodermal interactions and stem cell maintenance.