Sequential molecular changes and dynamic oxidative stress in high-grade serous ovarian carcinogenesis

The mechanism of high-grade serous ovarian cancer (HGSC) development remains elusive. This review outlines recent advances in the understanding of sequential molecular changes associated with the development of HGSC, as well as describes oxidative stress-induced genomic instability and carcinogenesis. This article reviews the English language literature between 2005 and 2017. Clinicopathological features analysis provides a sequential progression of fallopian tubal epithelium to precursor lesions to type 2 HGSC. HGSC may develop over a long time after incessant ovulation and repeated retrograde menstruation via stepwise accumulation of genetic alterations, including PAX2, ALDH1A1, STMN1, EZH2 and CCNE1, which confer positive selection of cells with growth advantages through acquiring driver mutations such as BRCA1/2, p53 or PTEN/PIK3CA. Haemoglobin and iron-induced oxidative stress leads to the emergence of genetic alterations in fallopian tubal epithelium via increased DNA damage and impaired DNA repair. Serous tubal intraepithelial carcinoma (STIC), the likely precursor of HGSC, may be susceptible to DNA double-strand breaks, exhibit DNA replication stress and increase genomic instability. The induction of genomic instability is considered to be a driving mechanism of reactive oxygen species (ROS)-induced carcinogenesis. HGSC exemplifies the view of stepwise cancer development. We describe how genetic alterations emerge during HGSC carcinogenesis related to oxidative stress.     

Expression of Cell Competition Markers at the Interface between p53 Signature and Normal Epithelium in the Human Fallopian Tube

There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 – 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 – 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

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.     

Cytologic studies of the fallopian tube in patients undergoing salpingo-oophorectomy

Background

Mounting evidence suggests the fallopian tube as the origin for ovarian high grade serous carcinoma (HGSC). We attempted to identify the tubal cytological features that allow us to distinguish malignant from benign conditions.

Methods

Tubal specimens (n = 56) were collected from patients who underwent bilateral salpingo-oophorectomy (BSO) due to various clinical indications. A standard procedure to collect fallopian tube brushings from freshly received surgical specimens was developed. Cytological diagnoses were classified into three categories: benign, atypical, and suspicious for malignancy/malignant. Cytological variables of individual cells and epithelia were subjected to statistical analysis. The fallopian tube histology was used as diagnostic reference for confirmation of cytology diagnosis.

Results

Among the 56 fallopian tube specimens, 2 (3.7 %) showed inadequate cellularity preventing further evaluation, 11 (20.4 %) were diagnosed as malignant or suspicious of malignancy, 7 were atypical, and 36 were benign. The presence of three dimensional clusters (p < 0.0001, Fisher’s Exact Test), or prominent nucleoli (p = 0.0252, Fisher Exact test) was highly correlated with the diagnosis of malignancy. The suspicious malignant/malignant cytological diagnosis was also highly correlated with presence of HGSC with or without serous tubal intraepithelial carcinoma (STIC).

Conclusions

Tubal cytology may be useful for ovarian cancer screening and early detection.

     

New insights on the pathogenesis of ovarian carcinoma: molecular basis and clinical implications

Ovarian carcinoma can be subdivided into two categories termed type I and type II. Type I tumours, usually having an indolent clinical behaviour, are often detected in early stage, and rarely harbour p53 gene mutations. Each histological type has a distinct molecular profile with mutations of genes involved in different signalling transduction pathways, such as KRAS, BRAF, CTNNB1, PTEN, PIK3CA and ARID1A. Type II tumours, accounting for 75% of the cases, have a very aggressive biological behaviour, are usually in advanced stage at presentation, harbour p53 gene mutations in 80% of the cases, and sometimes have alterations of homologous recombination (HR). Both type I and type II tumours arise from extra-ovarian precursors. Serous carcinomas derive from tubal epithelium, endometrioid and clear cell carcinomas from endometrial tissue, and mucinous and Brenner tumours from transitional epithelial cells located near the tubo-peritoneal junction. These new concepts on the pathogenesis of ovarian carcinoma could deeply modify both the preventive approach in women with germ-line BRCA(1) or BRCA(2) mutations and the treatment of patients with advanced or recurrent disease. For instance, BRAF inhibitors could be used in low-grade serous carcinomas, PIK3CA inhibitors could be employed in clear cell carcinoma, and poly (ADP-ribose) polymerase inhibitors could be used not only in hereditary ovarian carcinoma but also in non-hereditary, high-grade serous ovarian carcinoma which sometimes shows defective HR.     

利用cDNA微阵列-CGH筛选支气管上皮细胞恶性转化中的扩增基因

目的:基因组不稳定是导致肺癌发生与发展的重要分子机理之一。本研究旨在筛选支气管上皮细胞恶性转化过程中拷贝数扩增的基因。方法:利用业已建立的支气管上皮细胞体外恶性转化模型,通过cDNA微阵列-CGH技术对支气管上皮来源的永生化细胞和癌变细胞的基因拷贝数进行了检测,并对部分结果进行了实时PCR验证。结果:永生化BEP2D细胞染色体中的某些区域存在不同程度的扩增,包括5q31.3、9q32-33.1、14q22.2-23.1、19p13.12-13.13、20q13.12-13.31;恶性转化BERP35T2细胞染色体中的扩增区域集中在1p12-13.1、5q33.1、5q31.3、9q32、19p13.12-13.13;5q31.3、9q32、19q13.12-13.13是以上2种细胞系中的共同扩增区域。共检测到201个基因的拷贝数发生扩增,其中PCNA、TP53及GADD45A基因的异常扩增已经实时PCR进一步验证。结论:在支气管上皮细胞恶性转化过程中,病毒与低剂量辐射的双重作用使得某些重要基因的拷贝数发生扩增,因基因剂量增加而导致某些癌基因高表达可能是细胞恶性转化的重要机制之一。     

Chromosome 3 anomalies investigated by genome wide SNP analysis of benign, low malignant potential and low grade ovarian serous tumours

Ovarian carcinomas exhibit extensive heterogeneity, and their etiology remains unknown. Histological and genetic evidence has led to the proposal that low grade ovarian serous carcinomas (LGOSC) have a different etiology than high grade carcinomas (HGOSC), arising from serous tumours of low malignant potential (LMP). Common regions of chromosome (chr) 3 loss have been observed in all types of serous ovarian tumours, including benign, suggesting that these regions contain genes important in the development of all ovarian serous carcinomas. A high-density genome-wide genotyping bead array technology, which assayed >600,000 markers, was applied to a panel of serous benign and LMP tumours and a small set of LGOSC, to characterize somatic events associated with the most indolent forms of ovarian disease. The genomic patterns inferred were related to TP53, KRAS and BRAF mutations. An increasing frequency of genomic anomalies was observed with pathology of disease: 3/22 (13.6%) benign cases, 40/53 (75.5%) LMP cases and 10/11 (90.9%) LGOSC cases. Low frequencies of chr3 anomalies occurred in all tumour types. Runs of homozygosity were most commonly observed on chr3, with the 3p12-p11 candidate tumour suppressor region the most frequently homozygous region in the genome. An LMP harboured a homozygous deletion on chr6 which created a GOPC-ROS1 fusion gene, previously reported as oncogenic in other cancer types. Somatic TP53, KRAS and BRAF mutations were not observed in benign tumours. KRAS-mutation positive LMP cases displayed significantly more chromosomal aberrations than BRAF-mutation positive or KRAS and BRAF mutation negative cases. Gain of 12p, which harbours the KRAS gene, was particularly evident. A pathology review reclassified all TP53-mutation positive LGOSC cases, some of which acquired a HGOSC status. Taken together, our results support the view that LGOSC could arise from serous benign and LMP tumours, but does not exclude the possibility that HGOSC may derive from LMP tumours.     

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.     

Chloroxine overrides DNA damage tolerance to restore platinum sensitivity in high-grade serous ovarian cancer

High-grade serous cancer (HGSC) accounts for ~67% of all ovarian cancer deaths. Although initially sensitive to platinum chemotherapy, resistance is inevitable and there is an unmet clinical need for novel therapies that can circumvent this event. We performed a drug screen with 1177 FDA-approved drugs and identified the hydroxyquinoline drug, chloroxine. In extensive validation experiments, chloroxine restored sensitivity to both cisplatin and carboplatin, demonstrating broad synergy in our range of experimental models of platinum-resistant HGSC. Synergy was independent of chloroxine’s predicted ionophore activity and did not relate to platinum uptake as measured by atomic absorption spectroscopy. Further mechanistic investigation revealed that chloroxine overrides DNA damage tolerance in platinum-resistant HGSC. Co-treatment with carboplatin and chloroxine (but not either drug alone) caused an increase in γH2AX expression, followed by a reduction in platinum-induced RAD51 foci. Moreover, this unrepaired DNA damage was associated with p53 stabilisation, cell cycle re-entry and triggering of caspase 3/7-mediated cell death. Finally, in our platinum-resistant, intraperitoneal in vivo model, treatment with carboplatin alone resulted in a transient tumour response followed by tumour regrowth. In contrast, treatment with chloroxine and carboplatin combined, was able to maintain tumour volume at baseline for over 4 months. In conclusion, our novel results show that chloroxine facilitates platinum-induced DNA damage to restore platinum sensitivity in HGSC. Since chloroxine is already licensed, this exciting combination therapy could now be rapidly translated for patient benefit.Subject terms: Ovarian cancer, High-throughput screening     

Distinct functions for WRN and TP53 in a shared pathway of cellular response to 1-beta-D-arabinofuranosylcytosine and bleomycin

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-beta-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway.     

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.     

Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection.

We have previously shown that p53 mutations are associated with cisplatin resistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely elucidated; in particular, the biological mechanisms behind the acquired drug-resistant p53-mutant phenotype were not clearly explained. Thus, in this study, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (270 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexistent in the cisplatin-sensitive parental cell line (IGROV-1) or were induced by drug (genotoxic) treatment. For this purpose, an allele-specific PCR approach was used. Primers carrying the desired mutations (T-->A codon 270, C-->T codon 282) in the 3' terminus, and the corresponding wild-type primers were used to amplify genomic DNA from the original IGROV-1 cell line used to select the mutant IGROV-1/Pt 1. To increase sensitivity, we hybridized blots of the PCRs with the radiolabeled PCR fragment from IGROV-1/Pt 1. Amplification was obtained for IGROV-1 DNA with the mutated allele-specific primers, indicating the preexistence of a mutated population in the IGROV-1 cell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%. Single-strand conformation polymorphism and allele-specific PCR analysis of the IGROV-1/Pt 0.1 cells, which are less resistant to cisplatin than IGROV-1/Pt 1 cells and which carry both mutant and wild-type p53 alleles with a wild-type predominance, suggested a progressive selection of the mutant population by cisplatin treatment. This is the first observation that indicates that a subpopulation of p53 mutant cells can occasionally be selected by cisplatin treatment. Thus, considering the susceptibility to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, the selection process resulting in emergence of p53 mutant tumors is a possible origin of resistance of ovarian carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.     

High throughput interrogation of somatic mutations in high grade serous cancer of the ovary

Background

Epithelial ovarian cancer is the most lethal of all gynecologic malignancies, and high grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. The objective of this study was to determine the frequency and types of point somatic mutations in HGSC using a mutation detection protocol called OncoMap that employs mass spectrometric-based genotyping technology.

Methodology/Principal Findings

The Center for Cancer Genome Discovery (CCGD) Program at the Dana-Farber Cancer Institute (DFCI) has adapted a high-throughput genotyping platform to determine the mutation status of a large panel of known cancer genes. The mutation detection protocol, termed OncoMap has been expanded to detect more than 1000 mutations in 112 oncogenes in formalin-fixed paraffin-embedded (FFPE) tissue samples. We performed OncoMap on a set of 203 FFPE advanced staged HGSC specimens. We isolated genomic DNA from these samples, and after a battery of quality assurance tests, ran each of these samples on the OncoMap v3 platform. 56% (113/203) tumor samples harbored candidate mutations. Sixty-five samples had single mutations (32%) while the remaining samples had ≥2 mutations (24%). 196 candidate mutation calls were made in 50 genes. The most common somatic oncogene mutations were found in EGFR, KRAS, PDGRFα, KIT, and PIK3CA. Other mutations found in additional genes were found at lower frequencies (<3%).

Conclusions/Significance

Sequenom analysis using OncoMap on DNA extracted from FFPE ovarian cancer samples is feasible and leads to the detection of potentially druggable mutations. Screening HGSC for somatic mutations in oncogenes may lead to additional therapies for this patient population.     

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.