Coming into focus: the nonovarian origins of ovarian cancer.

BACKGROUND: The traditional view of epithelial ovarian cancer asserts that all tumor subtypes share a common origin in the ovarian surface epithelium (OSE) DESIGN: A literature review was carried out to summarize the emerging understanding of extraovarian sources of epithelial ovarian carcinomas. RESULTS: Historically, there were no diagnostic criteria for documenting the origin of ovarian epithelial carcinomas. Moreover, there are no normal epithelial tissues in the ovary with morphologic similarities to these tumors. In fact, no precursor lesions have ever been reproducibly identified in the ovary. However, there is a strong correlation between extrauterine Müllerian tissue and the development of ovarian carcinomas, tumors of low malignant potential, and cystadenomas. The most recent support for this hypothesis comes from the careful analysis of risk-reducing bilateral salpingo-oopherectomy specimens from BRCA1 or BRCA2 mutation carriers. These studies showed that a significant majority of high-grade serous ovarian carcinomas, the most common subtype, arise from the fallopian tube fimbriae rather than the OSE. CONCLUSIONS: Mounting evidence indicates that the vast majority of epithelial ovarian carcinomas are not ovarian in origin. Extrauterine Müllerian epithelium from various sites in the reproductive tract likely accounts for the diverse morphology and behavior of these tumors.

BRCA genes: lessons learned from experimental and clinical cancer.

Advances in the study of BRCA1 and BRCA2 gene functions have relied on the development of animal models for seeking to explore further what we have learned from the human disease. Specifically, mouse models of a 'triple-negative' breast cancer (utilizing conditional knockout of BRCA1 and p53 in the breast), of an endometrioid ovarian cancer (based on oncogenic kras and loss of function of pten), and of anatomic and functional consequences of BRCA1 mutations in granulosa cells, have led to further inquiry into the pathogenesis and therapeutic consequences of genetic alterations. A striking susceptibility of these murine malignancies to platinum drugs has emerged, providing further confidence in their relevance to the human disease. In addition to these models, the pathogenesis of high-grade serous disease derived from risk-reducing surgeries in mutation carriers has pointed to a role of mutations in p53 commonly encountered in tubal intraepithelial carcinomas.

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis.

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). In this study we report the creation of a novel mouse model in which GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in Purkinje cells (PCs). GRP78-depleted PCs activate UPR including the induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2alpha phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding in which an abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78-null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

Increased chromosomal stability in cultures of ovarian tumours of low malignant potential compared to cystadenomas.

Cell cultures of ovarian cystadenomas transfected with SV40 large T antigen are not immortal because they invariably reach a phenomenon called crisis, which is triggered in part by telomere attrition. Recovery from crisis may be an integral component of the malignant transformation process. We reported earlier that such ovarian cystadenoma cell cultures undergo severe changes in DNA ploidy as they approach crisis and that such changes are an important determinant of crisis independent of telomere attrition. Here, we show that in sharp contrast to these benign ovarian tumours, the DNA content of ovarian tumours of low malignant potential (LMP) was remarkably stable as they approached crisis, suggesting that telomere attrition was the main determinant of this mortality checkpoint. Lack of a ploidy-based crisis was not due to loss of expression of a functional SV40 large T antigen protein. We conclude that ovarian LMP tumours are characterised by increased numerical chromosomal stability compared to cystadenomas. This might account for the fact that most LMP tumours are diploid or near diploid in vivo. This fundamental difference in chromosomal stability between ovarian cystadenomas and LMP tumours also suggests potential differences in predisposition to progression to malignancy between these two ovarian tumour subtypes.

The receptor tyrosine kinase EphB4 is overexpressed in ovarian cancer, provides survival signals and predicts poor outcome.

EphB4 is a member of the largest family of transmembrane receptor tyrosine kinases and plays critical roles in axonal pathfinding and blood vessel maturation. We wanted to determine the biological role of EphB4 in ovarian cancer. We studied the expression of EphB4 in seven normal ovarian specimens and 85 invasive ovarian carcinomas by immunohistochemistry. EphB4 expression was largely absent in normal ovarian surface epithelium, but was expressed in 86% of ovarian cancers. EphB4 expression was significantly associated with advanced stage of disease and the presence of ascites. Overexpression of EphB4 predicted poor survival in both univariate and multivariate analyses. We also studied the biological significance of EphB4 expression in ovarian tumour cells lines in vitro and in vivo. All five malignant ovarian tumour cell lines tested expressed higher levels of EphB4 compared with the two benign cell lines. Treatment of malignant, but not benign, ovarian tumour cell lines with progesterone, but not oestrogen, led to a 90% reduction in EphB4 levels that was associated with 50% reduction in cell survival. Inhibition of EphB4 expression by specific siRNA or antisense oligonucleotides significantly inhibited tumour cell viability by inducing apoptosis via activation of caspase-8, and also inhibited tumour cell invasion and migration. Furthermore, EphB4 antisense significantly inhibited growth of ovarian tumour xenografts and tumour microvasculature in vivo. Inhibition of EphB4 may hence have prognostic and therapeutic utility in ovarian carcinoma.

Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

Collection of buccal cell DNA in seventh-grade children using water and a toothbrush.

We developed a simple and effective method for collecting a large quantity of buccal cell DNA in school-based studies of seventh-grade and older children. Seventh-grade students at schools in Wuhan, China brushed each buccal surface with a soft toothbrush and then rinsed with 10 ml of water. We added 5 ml of 99% ethanol to preserve the sample. Among 1563 samples transported at room temperature over 1 week and then stored for 13-14 months at -70 degrees C before extraction, using a modified Gentra Puregene protocol, the median total DNA yield was 108 microg, range of 14 to 416 microg. We assayed every 20th sample (n = 77) for NAT2 by the PCR, and all samples gave a 1093-bp product. From the 1563 samples, we obtained a result for single nucleotide polymorphisms in the interleukin-13 gene (at +2044) by RFLP-PCR on 98.8% and in the promoter of the myeloperoxidase gene (at -463) by real-time PCR on 99.7%. A water-rinse method, that we used among 12th-grade students in Southern California, gave a lower total DNA yield than the toothbrush rinse (median of 17 microg) and a slightly reduced ability to generate a PCR product. However, 26 of 27 water-rinse samples gave a result for two genes, albumin and CYP1A1, using real-time PCR methods. We did not quantify human, versus bacterial, DNA in our samples. However, given the amounts of total DNA required for genotyping, a sample with the median yield of 108 microg should suffice for approximately 2160 genotypes by RFLP-PCR methods or five times as many by real-time PCR. We recommend the toothbrush-rinse method, combined with a modified Gentra Puregene DNA extraction protocol, for large-scale, in-person collections of buccal cell DNA in children. The method requires only inexpensive, readily available materials and produces a large quantity of high-quality DNA for PCR analyses.

Ovarian tumor development: insights from ovarian embryogenesis.

The study of ovarian embryogenesis can provide important clues about the etiology and development of the different subtypes of ovarian neoplasms. The coelomic epithelium, also called germinal epithelium, was once thought to represent the site of origin of most cellular elements present in the adult ovary. However, recent observations at the morphological, functional, and molecular biological levels strongly suggest that this epithelium plays little or no role in ovarian development. The same observations provide strong support for an important role of the components of the fetal excretory system. These conclusions weaken the hypothesis that the coelomic epithelium is the site of origin of ovarian epithelial tumors. Knowledge of the origin and maturation of germ cells can shed light on several clinico-pathological characteristics of germ cells tumors, including their occasional extra-gonadal origin and differences in the biological behavior of ovarian versus testicular lesions. Knowledge of the mechanisms of regulation of mitotic and meiotic activity during ovarian germ cell maturation can provide insights into the molecular genetic determinants of germ cell neoplasms. The elucidation of molecular pathways actively involved in controlling gonadal differentiation may shed further light into our understanding of the relationship between aberrant differentiation and predisposition to gonadal cancers.

Suppression of tumorigenicity in human ovarian cancer cell lines is controlled by a 2 cM fragment in chromosomal region 6q24-q25.

Multiple distinct regions of chromosome 6 are frequently affected by losses of heterozygosity in primary human ovarian carcinomas. We introduced a normal human chromosome 6 into HEY and SKOV-3 ovarian carcinoma cell lines using microcell-mediated chromosome transfer techniques to further investigate the role of this chromosome in ovarian tumorigenesis. The exogenous chromosome was stably propagated in the recipient cells based on fluorescence in situ hybridization (FISH) analyses with a chromosome 6 painting probe. The tumorigenicity of HEY and SKOV-3 cells was completely suppressed after transfer of chromosome 6, but not after transfer of a chromosome 11q13-qter fragment used as control. Using 46 polymorphic microsatellite markers, the region bounded by D6S1649 and D6S1564 was found to be commonly deleted in HEY: chromosome 6 tumorigenic revertant clones. The boundaries of the commonly deleted region could be further narrowed down to a 2 cM (based on the Whitehead genetic map) or 0.36 megabase (based on gdb mapping data) region between D6S1637 and D6S1564 after transferring the exogenous chromosome from revertants into mouse L cells and performing allelic deletion mapping studies against this mouse background. We conclude that this region contains a tumor suppressor gene important for the control of ovarian tumor development.

The cell of origin of ovarian epithelial tumors and the ovarian surface epithelium dogma: does the emperor have no clothes?

The widely favored hypothesis that ovarian epithelial tumors arise from the mesothelial cell layer lining the ovarian surface fails to explain the resemblance of these tumors to those arising in organs that are embryologically derived from the Müllerian ducts such as fallopian tubes, endometrium, and endocervix. In addition, this theory cannot account for the fact that tumors that are morphologically identical to ovarian carcinomas can sometimes be found outside the ovary. A suggestion is made that components of the secondary Müllerian system, which include paraovarian/paratubal cysts, rete ovarii, endosalpingiosis, endometriosis, and endomucinosis, merit some consideration as to their possible role in ovarian tumorigenesis.

Satellite DNA hypomethylation vs. overall genomic hypomethylation in ovarian epithelial tumors of different malignant potential.

Rearrangements in heterochromatin in the vicinity of the centromeres of chromosomes 1 and 16 are frequent in many types of cancer, including ovarian epithelial carcinomas. Satellite 2 DNA is the main sequence in the unusually long heterochromatin region adjacent to the centromere of each of these chromosomes. Rearrangements in these regions and hypomethylation of satellite 2 DNA are a characteristic feature of patients with a rare recessive genetic disease, ICF (immunodeficiency, centromeric region instability, and facial anomalies). In all normal tissues of postnatal somatic origin, satellite 2 DNA is highly methylated. We examined satellite 2 DNA methylation in ovarian tumors of different malignant potential, namely, ovarian cystadenomas, low malignant potential (LMP) tumors, and epithelial carcinomas. Most of the carcinomas and LMP tumors exhibited hypomethylation in satellite 2 DNA of both chromosomes 1 and 16. A comparison of methylation of these sequences in the three types of ovarian neoplasms demonstrated that there was a statistically significant correlation between the extent of this satellite DNA hypomethylation and the degree of malignancy (P<0.01). Also, there was a statistically significant association (P<0.005) between genome-wide hypomethylation and undermethylation of satellite 2 DNA among these 17 tumors. In addition, we found abnormal hypomethylation of satellite alpha DNA in the centromere of chromosome 1 in many of these tumors. Our findings are consistent with the hypothesis that one of the ways that genome-wide hypomethylation facilitates tumor development is that it often includes satellite hypomethylation which might predispose cells to structural and numerical chromosomal aberrations. Several of the proteins that bind to pericentromeric heterochromatin are known to be sensitive to the methylation status of their target sequences and so could be among the sensors for detecting abnormal demethylation and mediating effects on chromosome structure and stability.

Differences in p16 gene methylation and expression in benign and malignant ovarian tumors.

OBJECTIVE: The aim of this study was to test the hypothesis that DNA methylation is important for silencing the p16 tumor suppressor gene in ovarian epithelial tumors and to compare the prevalence of this mechanism among different ovarian epithelial tumor subtypes. METHOD: Methylation-specific PCR was used to analyze the p16 gene for DNA methylation in 20 ovarian cystadenomas, 15 low malignant potential (LMP) tumors, and 37 carcinomas. p16 expression was determined immunohistochemically in 58 of these tumors (16 cystadenomas, 13 LMP tumors, 29 carcinomas). Differences in methylation or expression rates between specific tumor subgroups were examined by Fisher's exact test. RESULTS: Fragments from the distal promoter and beginning of the first exon of the p16 gene were both methylated in 5 of 15 (33%) LMP tumors compared to 2 of 37 (5%) carcinomas (P = 0. 02). Those sites were also methylated in 5 of 20 (25%) cystadenomas. Lack of p16 expression was present in 7 of 16 cystadenomas, 4 of 13 LMP tumors, and 22 of 29 carcinomas (P [LMPs versus carcinomas] = 0. 01) and correlated with methylation changes in LMP tumors (P = 0.05). p16 expression was correlated with mucinous differentiation in cystadenomas (P = 0.001). CONCLUSION: p16 silencing may be important for the development of ovarian carcinomas and a subset of LMP tumors. Changes in DNA methylation may be more important for inactivation of this gene (and perhaps other tumor suppressor genes) in LMP tumors, which lack many of the alternative mechanisms present in carcinomas. p16 expression is primarily related to mucinous differentiation in cystadenomas.

Genetics and biology of gynecologic cancer.

Gynecologic epithelial tumors can be grouped into two major categories depending on whether they are derived embryologically from squamous epithelium of the urogenital sinus or from müllerian ducts. Ovarian carcinomas appear morphologically similar to those arising in müllerian-derived organs, and molecular genetic defects present in tumors from these different sources appear to reflect more their histologic subtypes than their organ of origin. The possibility that ovarian epithelial tumors arise from remnants of müllerian ducts in the vicinity of the ovary therefore merits further investigation. Recent advances in our understanding of the state of clonality of various gynecologic tumors, of the influence of age and ovulatory activity on their genetic characteristics, and of their overall molecular genetic features, provide important clues about their initial underlying mechanisms. Novel strategies based on these advances are being tested for their potential utility in treating and monitoring gynecologic tumors.

Detection of ovarian cancer cells: comparison of a telomerase assay and cytologic examination.

BACKGROUND: Telomerase is an enzyme essential for the normal replication of chromosomes. Telomerase activity is absent in most somatic cells in adults, but it is usually expressed in cancer cells, including ovarian carcinoma cells. Our principal goal was to compare the sensitivity of a telomerase assay, i.e., the telomeric repeat amplification protocol (TRAP) assay, with that of cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian carcinoma. METHODS: TRAP assays and cytologic examinations were performed on peritoneal washings and ascitic fluids from 42 patients with active ovarian carcinoma. Control specimens included washings from 29 patients with benign ovarian diseases and ascitic fluids from 14 patients with liver failure. We also evaluated the stability of telomerase in ascitic fluids left unprocessed at room temperature as well as the ability of the TRAP assay to detect cancer cells in mixtures containing large numbers of normal cells. RESULTS: Specimens from 37 (88%) of the 42 patients with ovarian carcinoma tested positive for telomerase. Cytologic examination detected cancer cells in only 27 of the telomerase-positive specimens (i.e., in specimens from 64% of the 42 patients). This difference of 24% (95% confidence interval = 17%-30%) in sensitivity between the two tests was statistically significant (two-sided P = .002). Specimens from five of the patients with ovarian carcinoma were cytologically negative and telomerase negative. All 43 control specimens were cytologically negative, but the TRAP assay detected telomerase in two of them. Telomerase activity was detected in unprocessed samples left at room temperature for 5 days and in mixtures containing a small number of cancer cells and a 2000- to 10000-fold excess of normal cells. CONCLUSIONS: Assaying for telomerase is more sensitive than cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian carcinoma.

Biology of ovarian cancer.

Although ovarian carcinomas are thought to arise from the ovarian surface mesothelial layer, the possibility that they develop from müllerian remnants within paraovarian tissues merits further consideration. Molecular genetic studies suggest that ovarian cystadenomas, low malignant potential tumors, and carcinomas are not part of a disease continuum but do represent separate disease entities. Recent advances in our understanding of the molecular genetic changes associated with ovarian epithelial tumor development can be summarized in a working genetic model for ovarian tumorigenesis, which can provide a framework for further studies. Certain molecular changes, such as telomerase expression and alterations in DNA methylation levels, are associated with both tumors of low malignant potential and carcinomas but not with cystadenomas. Mutations in the p53 gene and the development of multiple losses of heterozygosity are specific for the malignant phenotype. The nature of the specific chromosomes affected by the latter losses in a given tumor dictates its biologic aggressiveness.

Telomerase activity in benign and malignant epithelial ovarian tumors.

BACKGROUND: Ovarian epithelial tumors include benign lesions lacking invasive and metastatic abilities (cystadenomas) in addition to malignant lesions (carcinomas). An intermediate category, called tumors of low malignant potential (LMP), is also recognized. The merit of this classification is being challenged because the clinical behavior of LMP tumors appears closer to that of cystadenomas than to that of carcinomas. PURPOSE: To verify our hypothesis that the expression of the enzyme telomerase distinguishes these two categories of ovarian epithelial tumors, we examined and compared such expression in ovarian cystadenomas and carcinomas. By examining the expression of telomerase in LMP tumors, we then sought to determine if these tumors were more closely related to cystadenomas or to carcinomas with regard to telomerase expression. METHODS: We examined a total of 64 consecutive ovarian tumors subdivided into 20 carcinomas, 17 LMP tumors, and 27 cystadenomas. We subsequently discarded three of the 27 cystadenomas because of the presence of admixed normal ovarian stroma in those specimens. Tumor subtyping was done without knowledge of the telomerase results, and telomerase assays were likewise interpreted without knowledge of tumor types. Telomerase activity was determined by use of the TRAP (i.e., telomeric repeat amplification protocol) assay. Differences between the proportions of tumors expressing this enzyme in each subgroup were evaluated by use of Fisher's exact test (two-sided). RESULTS: Telomerase activity was detected in all 20 carcinomas and in all 17 LMP tumors examined. In contrast, it was not detected in 19 of the 24 cystadenomas. These differences between rates of telomerase expression in either carcinomas or LMP tumors and those in cystadenomas were statistically significant (P<.0001). All five of the telomerase-positive cystadenomas belonged to a variant called papillary cystadenomas, whereas none of the telomerase-negative cystadenomas belonged to this variant (P<.0001). CONCLUSIONS AND IMPLICATIONS: The presence of telomerase expression in ovarian LMP tumors supports the merit of continuing to separate these tumors from cystadenomas, in spite of their apparent benign clinical course. The finding of telomerase expression in papillary cystadenomas suggests that such tumors may be mechanistically related to LMP tumors and should perhaps be reclassified as variants of LMP tumors. Lack of telomerase expression in ovarian cystadenomas raises questions about the alleged immortality of these tumors because expression of this enzyme is thought to be essential for continuous growth in adult tumors.

Alterations in DNA methylation are early, but not initial, events in ovarian tumorigenesis.

We compared global levels of DNA methylation as well as methylation of a specific locus (MyoD1) in ovarian cystadenomas, ovarian tumours of low malignant potential (LMP) and ovarian carcinomas to investigate the association between changes in DNA methylation and ovarian tumour development. As we realized that cystadenomas showed different methylation patterns from both LMP tumours and carcinomas, we verified their monoclonal origin as a means of confirming their true neoplastic nature. High-pressure liquid chromatographic (HPLC) analyses showed that global methylation levels in LMP tumours and carcinomas were 21% and 25% lower than in cystadenomas respectively (P = 0.0001 by one-way variance analysis). Changes in the methylation status of the MyoD1 locus were not seen in any of ten cystadenomas analysed but were present in five of ten LMP tumours and in five of ten carcinomas (P = 0.03). These findings suggest that alterations in DNA methylation are absent (or at least not as extensive) in ovarian cystadenomas, but are present in LMP tumours, the phenotypic features of which are intermediate between those of benign and malignant ovarian tumours. The results also emphasize the merit of distinguishing ovarian LMP tumours from cystadenomas, in spite of their similar clinical characteristics.

Establishment of long-term in vitro cultures of human ovarian cystadenomas and LMP tumors and examination of their spectrum of expression of matrix-degrading proteinases.

OBJECTIVES: To obtain long-term cultures of ovarian cystadenomas and ovarian tumors of low malignant potential (LMP) displaying gene expression patterns similar to those found in vivo and test the hypothesis that such cultures would express different levels of matrix-degrading proteinases than cultured ovarian carcinomas. METHODS: Transfection with an adenoviral expression vector for simian virus 40 (SV40) large T antigen was used to establish long-term cultures of the above tumors. Levels of expression of various genes were evaluated using molecular biological and immunohistochemical approaches. Zymography and reverse zymography were used to examine the activity of various metalloproteinases and plasminogen activators (PA). Two-sided P values for differences in plasminogen activator expression between different cell types were evaluated by Fisher's exact test. RESULTS: Long-term cultures derived from cystadenomas and LMP tumors were obtained which formed colonies on semisolid supports, but were not tumorigenic in nude mice. The cultured cells expressed keratin, estrogen receptor, gonadotropin receptors, BRCA1, and originated from monoclonal populations. There was no apparent association between the malignant phenotype and the expression of either matrix metalloproteinases or tissue inhibitors of metalloproteinases. However, a correlation was seen between this phenotype and expression of urokinase (uPA) and tissue type (tPA) plasminogen activators (P = 0.08 and 0.02 respectively). CONCLUSIONS: The above cell strains provide a useful model for investigating various aspects of the biology of benign ovarian tumors, including their response to steroid and gonadotropin hormones, and the role of specific proteinases in the acquisition of invasive and metastatic abilities.