George L. Mutter

Molecular and Pathologic Aspects of Endometrial Carcinogenesis

Endometrial cancer is the most common gynecological malignancy, with 41,000 new cases projected in the United States for 2006. Two different clinicopathologic subtypes are recognized: the estrogen-related (type I, endometrioid) and the non-estrogen-related types (type II, nonendometrioid such as papillary serous and clear cell). The morphologic differences in these cancers are mirrored in their molecular genetic profile with type I showing defects in DNA-mismatch repair and mutations in PTEN, K-ras, and beta-catenin, and type II showing aneuploidy and p53 mutations. This article reviews the genetic aspects of endometrial carcinogenesis and progression. We will define the precursor lesion of type I endometrioid cancer and the role of genetics and estrogen in its progression.

Immunohistochemical Evidence of Loss of PTEN Expression in Primary Ductal Adenocarcinomas of the Breast

Germline mutations in PTEN, encoding a dual-specificity phosphatase on 10q23.3, cause Cowden syndrome (CS), which is characterized by a high risk of breast and thyroid cancers. Loss of heterozygosity of 10q22-24 markers and somatic PTEN mutations have been found to a greater or lesser extent in a variety of sporadic component and noncomponent cancers of CS. Among several series of sporadic breast carcinomas, the frequency of loss of flanking markers around PTEN is approximately 30 to 40%, and the somatic intragenic PTEN mutation frequency is <5%. In this study, we analyzed PTEN expression in 33 sporadic primary breast carcinoma samples using immunohistochemistry and correlated this to structural studies at the molecular level. Normal mammary tissue had a distinctive pattern of expression: myoepithelial cells uniformly showed strong PTEN expression. The PTEN protein level in mammary epithelial cells was variable. Ductal hyperplasia with and without atypia exhibited higher PTEN protein levels than normal mammary epithelial cells. Among the 33 carcinoma samples, 5 (15%) were immunohistochemically PTEN-negative; 6 (18%) had reduced staining, and the rest were PTEN-positive. In the PTEN-positive tumors as well as in normal epithelium, the protein was localized in the cytoplasm and in the nucleus (or nuclear membrane). Among the immunostain negative group, all had hemizygous PTEN deletion but no structural alteration of the remaining allele. Thus, in these cases, an epigenetic phenomenon such as hypermethylation, -ecreased protein synthesis or increased protein degradation may be involved. In the cases with reduced staining, 5 of 6 had hemizygous PTEN deletion and 1 did not have any structural abnormality. Finally, clinicopathological features were analyzed against PTEN protein expression. Three of the 5 PTEN immunostain-negative carcinomas were also both estrogen and progesterone receptor-negative, whereas only 5 of 22 of the PTEN-positive group were double receptor-negative. The significance of this last observation requires further study.

Differential Nuclear and Cytoplasmic Expression of PTEN in Normal Thyroid Tissue, and Benign and Malignant Epithelial Thyroid Tumors

Germline mutations in PTEN (MMAC1/TEP1) are found in patients with Cowden syndrome, a familial cancer syndrome which is characterized by a high risk of breast and thyroid neoplasia. Although somatic intragenic PTEN mutations have rarely been found in benign and malignant sporadic thyroid tumors, loss of heterozygosity (LOH) has been reported in up to one fourth of follicular thyroid adenomas (FAs) and carcinomas. In this study, we examined PTEN expression in 139 sporadic nonmedullary thyroid tumors (55 FA, 27 follicular thyroid carcinomas, 35 papillary thyroid carcinomas, and 22 undifferentiated thyroid carcinomas) using immunohistochemistry and correlated this to the results of LOH studies. Normal follicular thyroid cells showed a strong to moderate nuclear or nuclear membrane signal although the cytoplasmic staining was less strong. In FAs the neoplastic nuclei had less intense PTEN staining, although the cytoplasmic PTEN-staining intensity did not differ significantly from that observed in normal follicular cells. In thyroid carcinomas as a group, nuclear PTEN immunostaining was mostly weak in comparison with normal thyroid follicular cells and FAs. The cytoplasmic staining was more intense than the nuclear staining in 35 to 49% of carcinomas, depending on the histological type. Among 81 informative tumors assessed for LOH, there seemed to be an associative trend between decreased nuclear and cytoplasmic staining and 10q23 LOH (P = 0.003, P = 0.008, respectively). These data support a role for PTEN in the pathogenesis of follicular thyroid tumors.

hMLH1 Promoter Hypermethylation Is an Early Event in Human Endometrial Tumorigenesis

It has recently been suggested that silencing of the hMLH1 gene by promoter hypermethylation is the mechanism underlying the presence of the microsatellite instability (MSI) phenotype in sporadic colon and endometrial carcinomas. To determine whether hMLH1 promoter hypermethylation is a relatively early event in endometrial tumorigenesis we evaluated endometrial hyperplasia (EH) characterized as simple, complex, and atypical (the direct precursor of endometrial carcinoma) for hMLH1 aberrant methylation. In addition, we studied the hMLH1, hMSH2, hMSH3, and hMSH6 promoter methylation and MSI status of those endometrial carcinomas with synchronous hyperplasias and those without them. We found that 11 of 12 (91%) cases of endometrial carcinoma (EC) displaying MSI had hMLH1 promoter hypermethylation, whereas aberrant methylation of any of the other mismatch repair genes was not observed. All 15 cases of EC without MSI were unmethylated at hMLH1. Abnormal methylation of hMLH1 was also present in 8 of 116 (7%) cases of EH and was restricted primarily to the atypical endometrial hyperplasia (AEH) type with coexisting endometrial carcinoma. In this set, half of EH methylated at hMLH1 displayed MSI, whereas none of the unmethylated EH had MSI. Our data suggest that hypermethylation of hMLH1 can be an early event in the pathogenesis of EC, preceding the development of an apparent MSI phenotype in a subset of cases.

Craniofacial abnormalities induced by ectopic expression of the homeobox gene Hox-1.1 in transgenic mice

Hox-1.1 is a murine homeobox-containing gene expressed in a time- and cell-specific manner during embryogenesis. We have generated transgenic mice that ectopically express Hox-1.1 from the chicken beta-actin promoter. In these mice Hox-1.1 expression was changed to an almost ubiquitous pattern. Ectopic expression of Hox-1.1 leads to death of the transgenic animals shortly after birth and is associated with multiple craniofacial anomalies, such as cleft palate, open eyes at birth, and nonfused pinnae. This phenotype is similar to the effects seen after systemic administration of retinoic acid during gestation. This suggests that retinoic acid embryopathy and the specific developmental defects caused by ectopic expression of a potential developmental control gene share a common pathogenic mechanism.

The spectrum of endometrial pathology induced by progesterone receptor modulators

Progesterone receptor modulators (PRM) are hormonally active drugs effective in the management of endometriosis and uterine leiomyomata. The endometrial effects of progestin blockade by PRMs in premenopausal women are currently being evaluated in several clinical trials, but few pathologists have had access to these materials and published information of the histological changes is scanty. Eighty-four endometrial specimens from women receiving one of four different PRMs were reviewed by a panel of seven experienced gynecologic pathologists to develop consensus observations and interpretive recommendations as part of an NIH-sponsored workshop. Although the pathologists were blinded to agent, dose, and exposure interval, the review was intended to provide an overview of the breadth of possible findings, and a venue to describe unique features. Endometrial histology included inactive and normal-appearing cycling endometrium. Overtly premalignant lesions (atypical hyperplasia or EIN) were not seen. In a subset of cases, asymmetry of stromal and epithelial growth resulted in prominent cystically dilated glands with admixed estrogen (mitotic) and progestin (secretory) epithelial effects of a type not encountered in contemporary clinical practice. The variety of endometrial appearances suggested that findings might differ by agent and dose over time according to relationships that must be specified for each agent. The constellation of changes seen in those endometria with cystically dilated glands is so novel that new terminology and diagnostic criteria are required for pathologists to recognize them. The panel has designated these changes as PRM-associated endometrial changes (PAEC). Additional follow-up studies will be needed to fully define their natural history and relationship to specific agents and administration regimens.

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.

Endometrial precancer diagnosis by histopathology, clonal analysis, and computerized morphometry.

Management of endometrial precancers is compromised by longstanding debate over the natural history of endometrial hyperplasias and inconsistencies in their diagnosis. The recent demonstration that some hyperplasias, like cancers, are phenotypically monoclonal is useful in recognizing biological precancers. A clonal analysis has been undertaken of a series of 93 endometrial tissues and their morphology has been evaluated by subjective diagnostic classification and computerized morphometric analysis. A pathologists diagnosis of atypical endometrial hyperplasia was highly associated with monoclonal growth. Both microsatellite‐stable and microsatellite‐unstable precancers were classified as atypical hyperplasias, indicating overlapping morphologies for these two groups. Diagnosis of non‐atypical endometrial hyperplasias was not reproducible and identified a group of lesions equally likely to be monoclonal as polyclonal. Computerized morphometry resolved these lesions into monoclonal and polyclonal subgroups with a high degree of accuracy and reproducibility. The predictive value of morphometry was dominated by that fraction of the sample which consisted of stroma (volume percentage stroma). This can be measured manually and used to predict monoclonality when below the threshold value of 55%. This study shows that morphometric analysis reproducibly and precisely identifies monoclonal endometrial precancers from histological sections. It may serve, furthermore, to classify accurately lesions judged by pathologists as indeterminate (non‐atypical hyperplasias). The material from this study (available at www.endometrium.org from March 1, 2000) and precisely defined architectural diagnostic criteria provide new tools for diagnostic standardization of endometrial precancers. Copyright

Combined Total Genome Loss of Heterozygosity Scan of Breast Cancer Stroma and Epithelium Reveals Multiplicity of Stromal Targets

Recent breast cancer studies have highlighted the importance of interactions between cancer epithelium and tumor stroma. Recently, the focus of solid tumor investigations has shifted from mutations in carcinomatous epithelium to disturbances of tissue organization in cancer. The genetic basis of this microenvironment, however, remains to be clarified. To begin to resolve this problem, a total genome loss of heterozygosity (LOH) scan was done on epithelial and stromal DNA from 134 sporadic invasive breast carcinomas. In addition to detecting more frequent LOH at three loci in stroma than in epithelium, we found strong evidence that LOH frequencies were significantly elevated in specific regions of each chromosome. We detected 57 markers, which were preferentially lost either in stroma (n = 38) or epithelium (n = 19), relative to the background LOH frequencies on their respective chromosomes. This multiplicity of stromal cell LOH, and hence loss of genetic material, provides a possible mechanism for interpatient variation in host-stromal response to invading adenocarcinoma cells. This is consistent with a model in which initial, random LOH occurs equally among epithelium and stroma, but subsequent clonal selection is driven by factors, which appear to be distinctly different between malignant epithelial and surrounding stromal cells. Genetic alterations in stroma did not mimic those in epithelium, but they could play a different and parallel role in carcinogenesis and tumor progression, probably by modifying some features specific to breast cancer.

Molecular pathogenesis of uterine smooth muscle tumors from transcriptional profiling

Uterine smooth muscle tumors range from the very common benign leiomyoma to the uncommon, but frequently lethal, leiomyosarcoma. Morphological and clinical differences between these tumors are presumed to result from differences in gene expression. To test this hypothesis, RNAs from four normal uterine myometria, seven uterine leiomyomas, and nine uterine leiomyosarcomas were profiled using microarrays of oligonucleotides representing about 7,000 unique probe sets. RNAs whose levels distinguished any of the three sample types were selected by analysis of variance (ANOVA). The 153 (2.2% of the total) probe sets representing 146 unique genes with the highest test statistic selected for further analysis met minimum ratio and range thresholds between groups. Cluster analysis distinguished benign and malignant samples at the first node, and myometrium and leiomyoma were resolved in a secondary node. Downregulation of specific genes in uterine leiomyosarcoma was the most common pattern of differential gene expression selected by the three‐way ANOVA. Four extrauterine leiomyosarcomas had profiles most similar to that of the uterine leiomyosarcomas. Functional analysis of the 146 genes did not reveal any strong biological theme. These genes were distributed throughout the genome, but there was slight overrepresentation of genes on 1p and 2q. These genes define a tumor signature for uterine smooth muscle neoplasia, and they suggest that the molecular pathways in leiomyoma and leiomyosarcoma are distinct.