Endometrial intraepithelial neoplasia is associated with polyps and frequently has metaplastic change.

AIMS: Endometrial intraepithelial neoplasia (EIN) is a monoclonal precursor to endometrioid endometrial adenocarcinoma characterized by a geographic cluster of crowded glands with epithelial cytology altered relative to the background. It may demonstrate epithelial metaplastic changes, or arise within polyps, but the frequencies of these features as encountered in practice is unknown. The aim was to report the epithelial differentiation state and polyp context of 83 sequential EIN lesions diagnosed over a 2-year period. METHODS AND RESULTS: EIN is a rare lesion, seen in only 1.4% of endometrial biopsy specimens in a busy hospital-based practice. Of 83 EIN cases, 39 contained metaplastic changes (18% squamous morular, 14% tubal secretory and 5% each of secretory, mucinous or ciliated change). Endometrial polyps were more likely (odds ratio 5.2, P < 0.001) to occur in the endometrial biopsy specimens of women with EIN lesions (43.3%), compared with the background polyp rate (12.9%) of comparable specimens from the same patient population. CONCLUSIONS: Non-endometrioid differentiation and occurrence within polyps are frequent presentations of EIN lesions. Possible mechanisms of polyp association with EIN include: non-shedding of polyp tissue creating a shelter for persistence of pre-existing neoplastic glands, or promotion of premalignant glandular clones by unique polyp stroma.

Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.

BACKGROUND: PTEN tumor suppressor gene mutations are the most frequent genetic lesions in endometrial adenocarcinomas of the endometrioid subtype. Testing the hypothesis that altered PTEN function precedes the appearance of endometrial adenocarcinoma has been difficult, however, partly because of uncertainties in precancer diagnosis. METHODS: Two series of endometrial cancer and precancer (endometrial intraepithelial neoplasia, as diagnosed by computerized morphometric analysis) tissue samples were studied, one for PTEN mutations by the use of denaturing gradient gel electrophoresis and another for PTEN protein expression by immunohistochemistry. Endometria altered by high estrogen levels that are unopposed by progestins-conditions known to increase cancer risk-were also studied by immunohistochemistry. Fisher's exact test was used for statistical analysis. RESULTS: The PTEN mutation rate was 83% (25 of 30) in endometrioid endometrial adenocarcinomas and 55% (16 of 29) in precancers, and the difference in number of mutations was statistically significant (two-sided P =.025). No normal endometria showed PTEN mutations. Although most precancers and cancers had a mutation in only one PTEN allele, endometrioid endometrial adenocarcinomas showed complete loss of PTEN protein expression in 61% (20 of 33) of cases, and 97% (32 of 33) showed at least some diminution in expression. Cancers and most precancers exhibited contiguous groups of PTEN-negative glands, while endometria altered by unopposed estrogens showed isolated PTEN-negative glands. CONCLUSIONS: Loss of PTEN function by mutational or other mechanisms is an early event in endometrial tumorigenesis that may occur in response to known endocrine risk factors and offers an informative immunohistochemical biomarker for premalignant disease. Individual PTEN-negative glands in estrogen-exposed endometria are the earliest recognizable stage of endometrial carcinogenesis. Proliferation into dense clusters that form discrete premalignant lesions follows.

X chromosome inactivation in the normal female genital tract: implications for identification of neoplasia.

Monoclonal proliferative lesions may be identified by X chromosome inactivation skewing relative to normal polyclonal tissues. We have quantitatively analyzed X-inactivation patterns throughout polyclonal uterine tissues to develop interpretive criteria for recognition of monoclonal neoplasms. Six fresh tissue samples (two samples each of cervix, endometrium, and myometrium) were collected from hysterectomy specimens, and the percentage of androgen receptor (HUMARA) marker allele present on inactive X chromosomes was calculated from a PCR assay. Exact balancing yields 50% of the marker on the inactive X, whereas complete skewing shows either 0 or 100%. X inactivation was similar throughout the tissues of each uterus but was significantly different among the 11 women studied. Comparison of differences in X inactivation between pairs of polyclonal tissue samples within each uterus (Xi spread) permitted delineation of cumulative experimental and biological variation of this parameter. Polyclonal-polyclonal Xi spread averaged 10.7 and was independent of the tissue type, sampling site, or the individual studied. Severe baseline skewing of reference polyclonal tissues or contamination of monoclonal tissue by polyclonal cells may reduce the polyclonal-monoclonal Xi spread. The extent of X-inactivation skewing necessary to infer a monoclonal process should exceed the 20 or 27 point spread seen, respectively, between 85 and 95% of polyclonal samples.

Uteri of women with endometrial carcinoma contain a histopathological spectrum of monoclonal putative precancers, some with microsatellite instability.

We have tested the hypothesis that endometrial precancers persist in uteri of patients with endometrial carcinoma and are monoclonal. Twenty-two hysterectomies with both well-differentiated endometrial adenocarcinoma and adjacent (normal or abnormal) noncancerous endometrium underwent successful clonal analysis using a PCR assay for nonrandom X chromosome inactivation. Monoclonal lesions included endometrial carcinoma, endometrial polyps, and atypical endometrial hyperplasias, whereas normal and anovulatory endometrium were polyclonal. Comparison of the specific X chromosome copy preferentially inactivated by the matched monoclonal cancers and associated monoclonal lesions allowed us to exclude polyps, but not endometrial hyperplasias, as potential precancers. The repetitive genetic marker (HUMARA) for X inactivation was altered in some cancers, permitting identification of microsatellite instability (RER+). Two patients with RER+ cancers also had adjacent RER+ hyperplasias. The seven monoclonal and two RER+ hyperplasias had focal or diffuse cytological atypia, a feature previously associated with risk for endometrial cancer. We conclude that: (a) putative endometrial precancers and cancers share a monoclonal growth pattern; (b) cancers with microsatellite instability may acquire this feature as precancers; and (c) monoclonal endometrial precancers have the morphology of hyperplasias, which vary in the extent of cytological atypia and degree of architectural complexity.

Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer.

Progressive microsatellite changes in replication error positive (RER+) endometrium were used to reconstruct evolutionary stages of nonfamilial adenocarcinoma. RER+ putative endometrial precancers (atypical endometrial hyperplasias) progress to RER+ carcinomas, which retain some of the altered microsatellites acquired in earlier precursor stages. The RER+ phenotype may provide a specific marker for early-stage endometrial neoplasms that cannot be resolved by routine histopathology and may be a useful tool to stratify stages in the evolution of RER+ tumors.

Molecular identification of latent precancers in histologically normal endometrium.

Discovery of somatically mutated cells in human tissues has been less frequent than would be predicted by in vitro mutational rates. We analyzed the PTEN tumor suppressor gene as an early marker for endometrial carcinogenesis, and we show that 43% of histologically normal premenopausal endometria contain rare glands that fail to express PTEN protein because of mutation and/or deletion. These persist between menstrual cycles. Histopathology of PTEN-null glands is initially unremarkable, but with progression, they form distinctive high-density clusters. These data are consistent with a progression model in which initial mutation is not rate limiting.

Linking gene expression patterns to therapeutic groups in breast cancer.

A major objective of current cancer research is to develop a detailed molecular characterization of tumor cells and tissues that is linked to clinical information. Toward this end, we have identified approximately one-quarter of all genes that were aberrantly expressed in a breast cancer cell line using differential display. The cancer cells lost the expression of many genes involved in cell adhesion, communication, and maintenance of cell shape, while they gained the expression of many synthetic and metabolic enzymes important for cell proliferation. High-density, membrane-based hybridization arrays were used to study mRNA expression patterns of these genes in cultured cells and archived tumor tissue. Cluster analysis was then used to identify groups of genes, the expression patterns of which correlated with clinical information. Two clusters of genes, represented by p53 and maspin, had expression patterns that strongly associated with estrogen receptor status. A third cluster that included HSP-90 tended to be associated with clinical tumor stage, whereas a forth cluster that included keratin 14 tended to be associated with tumor size. Expression levels of these clinically relevant gene clusters allowed breast tumors to be grouped into distinct categories. Gene expression fingerprints that include these four gene clusters have the potential to improve prognostic accuracy and therapeutic outcomes for breast cancer patients.

Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.

Somatic mutations in mtDNA have recently been identified in colorectal tumours. Studies of oncocytic tumours have led to hypotheses which propose that defects in oxidative phosphorylation may result in a compensatory increase in mitochondrial replication and/or gene expression. Mutational analysis of mtDNA in thyroid neoplasia, which is characterised by increased numbers of mitochondria and is also one of the most common sites of oncocytic tumours. has been limited to date. Using the recently developed technique of two-dimensional gene scanning, we have successfully examined 21 cases of thyroid tumours, six cases of non-neoplastic thyroid pathology, 30 population controls, nine foetal thyroid tissues and nine foetal tissues of non-thyroid origin, either kidney or liver. We have identified three different somatic mutations (23%) in papillary thyroid carcinomas. In addition, we have found significant differential distributions of mtDNA sequence variants between thyroid carcinomas and controls. Interestingly, these variants appear to be more frequent in the genes which encode complex I of the mitochondrial electron transport chain compared to normal population controls. These findings suggest first, that somatic mtDNA mutations may be involved in thyroid tumorigenesis and second, that the accumulation of certain non-somatic variants may be related to tumour progression in the thyroid.

A compendium of gene expression in normal human tissues.

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Changes in endometrial PTEN expression throughout the human menstrual cycle.

Frequent mutation of the PTEN tumor suppressor gene in endometrial adenocarcinoma has led to the prediction that its product, a phosphatase that regulates the cell cycle, apoptosis, and possibly cell adhesion, is functionally active within normal endometrial tissues. We examined PTEN expression in normal human endometrium during response to changing physiological levels of steroid hormones. PTEN ribonucleic acid levels, assessed by RT-PCR, increase severalfold in secretory compared to proliferative endometrium. This suggested that progesterone, a known antineoplastic factor for endometrial adenocarcinoma, increases PTEN levels. Immunohistochemistry with an anti-PTEN monoclonal antibody displayed a complex pattern of coordinate stromal and epithelial expression. Early in the menstrual cycle under the dominant influence of estrogens, the proliferative endometrium shows ubiquitous cytoplasmic and nuclear PTEN expression. After 3-4 days of progesterone exposure, glandular epithelium of early secretory endometrium maintains cytoplasmic PTEN protein in an apical distribution offset by expanding PTEN-free basal secretory vacuoles. By the midsecretory phase, epithelial PTEN is exhausted, but increases dramatically in the cytoplasm of stromal cells undergoing decidual change. We conclude that stromal and epithelial compartments contribute to the hormone-driven changes in endometrial PTEN expression and infer that abnormal hormonal conditions may, in turn, disrupt normal patterns of PTEN expression in this tissue.

Multinucleated atypia of the vulva. Report of a distinct entity not associated with human papillomavirus.

The vulvar mucosa often demonstrates epithelial nuclear atypia in association with reactive and inflammatory conditions. These nuclear changes are usually mild and can be readily distinguished from vulvar intraepithelial neoplasia (VIN) and human papillomavirus (HPV)-related lesions. In a recent survey of vulvar biopsies in reproductive-aged women, we identified 12 cases of an unusual pattern of atypia associated with multinucleated epithelial cells but lacking the usual stigmata of reactive changes, condyloma, or VIN. The average age of the patients with multinucleated atypia of the vulva (MAV) was 37 years. The multinucleated cells were commonly in the lower to middle epithelial layers and contained between two and 10 nuclei, often with prominent nucleoli. In contrast to condyloma and VIN, there was no surface atypia, and the multinucleated cells lacked hyperchromasia, irregularity, or variation in nuclear size. No significant inflammation or identifiable infectious process was present, and none of the patients had received any topical treatment other than mild corticosteroids. Two of the patients had a history of VIN at a noncontiguous site. None of the 12 cases contained HPV DNA by either in situ hybridization or polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. This is in contrast to 49 of 65 women with VIN and 21 of 26 with condyloma who had HPV demonstrable by the PCR method (p < 0.00001). Immunoperoxidase stains for herpes types I and II were also negative in all the cases. Thus, MAV appears to be a distinct entity occurring in relatively young women; when it is not associated with condyloma or VIN, MAV is not related to HPV. As the morphologic features may overlap with both condyloma and VIN, it is important that MAV not be confused with these lesions or vice versa. It is not known whether MAV is a risk factor for VIN, represents an exaggerated reactive response, or is an entity with a distinct origin.

Sporadic microsatellite instability is specific to neoplastic and preneoplastic endometrial tissues.

Microsatellite instability is a frequent (13%-24%) finding in sporadic endometrial adenocarcinoma and its precursor lesions, but most studies are limited to patients who already have malignant or premalignant endometrial disease. We performed retrospective testing for microsatellite instability in women in whom cancers showing microsatellite instability developed later and prospective testing in randomly selected normal and anovular endometrial biopsy specimens. Microsatellite instability in cancer-bearing biopsy specimens accurately reflected that seen in matched malignant tissues obtained at hysterectomy. In 1 patient, microsatellite instability developed in a scanty sample of fragmented endometrial tissues 7 years before the onset of endometrial cancer. Prospective testing for microsatellite instability in the endometria of women unselected for subsequent appearance of endometrial cancer showed a very low rate of microsatellite instability. Only 1 endometrial specimen showing microsatellite instability was found among 75 anovulatory endometrial specimens, and none were found in 377 normal endometrial specimens and 46 polyps examined. Microsatellite instability may precede the onset of histologically diagnosed carcinoma but is rare in randomly sampled histologically normal endometrial tissues.

Diagnosis of premalignant endometrial disease.

Modern molecular methods for precancer diagnosis have expanded the range of detectable disease to a preclinical level and provided material for histopathological correlation. The precancer scenario begins with sporadic acquisition of rare PTEN mutation bearing glands, which are morphologically unremarkable, and progresses to discrete foci of cytologically altered glands, readily visible on routinely stained sections. Clinical outcome studies of women with endometrial lesions have established threshold diagnostic features that confer increased cancer risk. This class of high risk lesions has been designated endometrial intraepithelial neoplasia (EIN). EIN is diagnosed by presence of cytological demarcation, crowded gland architecture, minimum size of 1mm, and careful exclusion of mimics. Most EIN lesions have been diagnosed as atypical endometrial hyperplasias in the World Health Organisation system. Specialised molecular and morphometric analyses have been extremely useful in redefining clinically relevant premalignant endometrial disease, but translation to improved patient care requires the informed participation of pathologists.

Phytoestrogen supplementation and endometrial cancer.

BACKGROUND: Phytoestrogens are increasingly used by patients as "natural" alternatives to hormone replacement. Attention in scientific and lay literature has focused on their potential to prevent menopausal symptoms, bone loss, heart disease, or breast cancer. Less is known about effects on the endometrium, specifically, whether prolonged exposure to phytoestrogens could promote hyperplasia or neoplasia, as does unopposed estrogen. CASE: We report the case of a woman diagnosed with grade 1 endometrioid adenocarcinoma of the endometrium whose history was notable for extensive use of supplemental phytoestrogens. CONCLUSION: The effects of phytoestrogens on endometrial tissue are not known. Given their increasing popularity and availability in concentrated form as dietary supplements, additional research is warranted before we can counsel our patients regarding the safety of such supplements.

Evaluation of gene deletions by quantitative polymerase chain reaction. Experience with the alpha-thalassemia model.

In order to evaluate the feasibility of using quantitative polymerase chain reaction (PCR) to evaluate gene dosage, we developed an assay to detect alpha-globin genes, which are frequently deleted in alpha-thalassemia patients. In this quantitative assay alpha-1 and alpha-2 globin consensus regions are coamplified by one oligonucleotide pair, along with a second primer pair targeting a single-copy reference gene, namely, tumor necrosis factor alpha, or TNF-alpha. A series of DNA samples titrating alpha-globin against TNF-alpha DNA have a strong linear relationship between template ratios and product ratios (r > 0.98). Minimal sequence divergence (91% homology) between alpha-1 and alpha-2, internal to the identical primer annealing sites, results in a lower amplification efficiency for alpha-1, to 94% of alpha-2 for each cycle. Furthermore, when applied to a variety of individual DNA samples, the signal ratios of alpha-globin to TNF-alpha were far more variable than previously observed for titrated control DNA. We conclude that DNA isolates from different individuals may have idiosyncratic changes in amplification efficiency owing to polymorphic sequence variation and/or variable presence of unidentified contaminants. Despite these potentially confounding factors, however, we were able to identify by quantitative PCR a single gene deletion later confirmed by Southern blot analysis in 20 individual DNA samples.

The sex ratio of normal and manipulated human sperm quantitated by the polymerase chain reaction.

OBJECTIVE: To establish the primary sex ratio, the relative abundance of X and Y chromosome-bearing sperm, in unselected sperm and in sperm selected by swim-up or Sephadex filtration (SpermPrep column; Fertility Technologies, Inc., Natick, MA). This was done to evaluate the possibility that these semen manipulations change the primary sex ratio. DESIGN: Ninety-eight unmanipulated semen samples were analyzed for sex chromosome content using quantitative polymerase chain reaction (PCR). A smaller number of samples was analyzed before and after either swim-up or Sephadex filtration. RESULTS: The mean percentage of all sex chromosomes identified as a Y chromosome in unmanipulated semen samples ranged from 41.9% to 56.7%, with a mean average of 50.3%. There was no significant change in sex chromosome composition after either swim-up (n = 17) or column filtration (n = 20). CONCLUSIONS: The chromosome compositions of semen samples from a large number of men have equal numbers of X and Y. Swim-up and SpermPrep filtration do not appear to alter the primary sex ratio.

The effect of consecutive day inseminations on semen characteristics in an intrauterine insemination program.

This study demonstrates a statistically significant decrease in semen volume, sperm concentration, and sperm motility in samples obtained on the 2nd day of consecutive day inseminations in an IUI program. This diminution in semen characteristics persists despite sperm washing. The effects of a second ejaculation on semen parameters in oligospermic and asthenospermic men were mixed. Thus, in general, sperm-washing procedures cannot overcome the natural reduction in semen quality produced by frequent ejaculations. Clinicians may wish to use this information in timing IUI cycle inseminations.

Role of imprinting in abnormal human development.

Parental-specific differences in the expression of certain genes (imprinting), may be implicated in the pathogenesis of anomalous gestations, but only a minority manifest themselves as malformation syndromes. Delayed or lost gestations are much more frequent sequelae, as are those disorganized to such an extent that they are usually classified as neoplastic rather than developmental processes. Expression levels from imprinted loci are dependent not only on the number of genomic alleles present and their structural integrity, but also on their specific parental origin. Anomalous expression of imprinted genes during development is sometimes caused by imbalanced representation of maternal and paternal contributions, 'uniparental disomy'. Uniparental parthenogenetic or androgenetic gestations form ovarian teratomas or complete hydatidiform moles, respectively--examples of an arrested developmental program. Uniparental disomy of individual chromosomes or portions thereof has been associated with developmental delay or gestational loss. The phenotype of hemizygous mutation or deletion of imprinted genes is modified by the parental origin of the mutant copy, with dichotomous syndromes defined by parental inheritance, as in the Prader-Willi and Angelman syndromes. Lastly, failure of the imprinting process itself, 'loss of imprinting', may quantitatively alter expression levels of normally imprinted transforming or tumor-suppressing genes, thereby increasing risk for developmental tumors such as Wilms' tumor or choriocarcinoma.

Advances in the molecular biology of gestational trophoblastic disease.

Gestational trophoblastic diseases are a heterogeneous pool of clinically and histopathologically defined entities with two clinically relevant features: reproductive failure and a high neoplastic potential. Here we review recent advances in understanding the biology and natural history of the most common form of trophoblastic disease, hydatidiform mole, with an emphasis on the clinical implications for patient management. There are no reliable genetic markers for predicting which subset of moles will behave aggressively, nor are there molecular diagnostic methods at present that offer advances over traditional histopathology. The predominant genetic finding in complete and partial hydatidiform moles is an imbalance of parental chromosomes, completely androgenetic (paternal) in the former and an extra paternal haploid set in the latter. This lack or imbalance of a maternal genomic contribution probably changes the gene expression since there is no evidence of gene mutation in these lesions.