High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Overexpression of p16(INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri.

Cytological screening for cervical cancer or its precursors using Papanicolaou's smear test (Pap test) has been highly efficient to reduce the morbidity and mortality of cervical cancer. However, evaluation of the Pap test relies on subjective diagnostic parameters and is affected by a high rate of false-positive and false-negative results. More objective diagnostic parameters to identify truly dysplastic or neoplastic cells in cervical smears as well as in cervical biopsy samples would therefore avoid insecurity for many patients and the high screening costs associated with repeated testing. Cervical dysplasia is induced by persistent infections through high-risk types of human papillomaviruses (HPVs). Outgrowth of dysplastic lesions is triggered by increasing expression of two viral oncogenes, E6 and E7, which both interact with various cell cycle-regulating proteins. Among these is the retinoblastoma gene product pRB, which is inactivated by E7. pRB inhibits transcription of the cyclin-dependent kinase inhibitor gene p16(INK4a). Increasing expression of the viral oncogenes in dysplastic cervical cells might thus be reflected by increased expression of p16(INK4a). In line with this hypothesis, we observed marked overexpression of p16(INK4a) in all cervical intraepithelial neoplasm (CIN) I lesions (n = 47) except those associated with low-risk HPV types (n = 7), all CIN II lesions (n = 32), all CIN III lesions (n = 60) and 58 of 60 invasive cervical cancers. In contrast, no detectable expression of p16(INK4a) was observed in normal cervical epithelium (n = 42), inflammatory lesions (n = 48) and low-grade cervical lesions (CIN I) associated with low-risk HPV types (n = 7). Dysplastic cells could also be identified in cervical smears using a specific p16(INK4a) monoclonal antibody. These data demonstrate that p16(INK4a) is a specific biomarker to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears.

The endometrium in breast cancer patients on tamoxifen.

We restudied histologically and immunohistochemically 17 endometrial carcinomas, 2 malignant mixed tumors and 180 endometria with benign changes during or after tamoxifen therapy. The carcinomas were subtyped according to the 1994 WHO-classification. Endometrial biopsies were taken only if the endometrial thickness was > 8 mm sonographically, when a polyp was seen, or for postmenopausal bleeding. About half of the endometrial specimens showed simple or cystic atrophy, 55-76% had cystic-atrophic polyps or regressive hyperplasia. Depending upon the dose of tamoxifen, 7-19% (30 mg) to 27-36% (20 mg) showed moderate glandular proliferation. 20-33% had foci of mucinous, clear cell or serous-papillary metaplasia. 68-70% revealed diffuse extensive fibrosis of the endometrial stroma. None of 11 patients biopsied before starting tamoxifen therapy had advanced endometrial glandular proliferation in the second endometrial biopsy after tamoxifen treatment. None of the 19 endometrial neoplasms after tamoxifen therapy was of the endometrioid type: 11 were mucinous adenocarcinomas, 4 clear cell carcinomas, 2 serous-papillary carcinomas, one carcinosarcoma and one malignant Mullerian mixed tumor. The reasons for discrepancies between suspicious sonograms and endometrial atrophy are discussed.

Changes in the endometrium caused by endogenous hormonal dysfunction.

The endometrium responds precisely and predictably to every hormonal dysfunction that may result from absent, deficient or excessive hormone production caused by an arrest of follicular maturation at different stages. Anovulatory failures may be due to arrest of follicular development and may cause endometrial atrophy or deficient proliferation. Arrest of follicular regression may result in irregular proliferation or various degrees of hyperplasia. Ovulatory failures may be due to arrest of corpus luteum maturation and followed by deficient secretion with dissociated or coordinated delay in differentiation. Arrest of corpus luteum regression may cause secretory hypertrophy. Both, anovulatory and ovulatory functional disturbances, are followed by prolonged and irregular endometrial shedding with necrosis, since regular dissociation of endometrial stromal cells is impaired. Almost all such functional deviations may cause infertility.

Histopathology of functional and neoplastic changes in cervix and endometrium.

Over 90% of all cervical carcinomas originate from hyperplastic reserve cells of the endocervix. When cell cycles of the pluripotent reserve cells are shortened or damaged by hormonal overstimulation or toxic chemicals, DNA repair after viral infection is no longer possible. The immortalized cells may then become malignant with precancerous and carcinomatous differentiation to squamous, adenosquamous or adenocarcinomas. To determine the type of HPV infection is clinically important for estimating prognosis and for planning further therapy. In recent years HPV 18-associated endocervical adenocarcinomas have increased considerably in number as compared with squamous cell carcinomas. In endometrial carcinomas the estrogen-stimulated endometrioid types must be distinguished from the gestagen-stimulated mucinous, clear-cell and serous-papillary types because prognosis as well as operative and adjuvant therapy differ considerably. The endometrioid carcinomas and their preceeding atypical hyperplasia are frequently associated with bcl-2, c-myk and c-ki-ras oncogenes. The mixed Muellerian types, in contrast, and their preceeding mucinous, clear-cell and serous-papillary metaplasias very often show genetic defects, LOH, mutations, amplifications and deletions, especially of p53. The endometrial carcinomas occurring in the reproductive age period are of endometrioid type and frequently present microsatellite instabilities. In rapidly proliferating tissues like the endometrium there appears to be a close correlation between functional and neoplastic changes. A simple explanation for that might be found in the greatly enhanced mitotic activity in a hormonally stimulated endometrium with shortening of cell cycles, leaving insufficient time for DNA repair. Since, however, carcinogenesis is a multi-factorial event, to correlate endometrial function with morphology can contribute only one aspect to the understanding of neoplastic change. In discussing hormonal function it is important to realize that the endocervix and the endometrium differ not only in their cellular structure, but they react antagonistically to hormonal stimulation: excessive (endogenous or exogenous) estrogen results in hyperproliferation of endometrial epithelial cells, but in the cervix in differentiation of the endocervical cells with mucus production. Extensive synthetic gestagens lead to hyperproliferation of endocervical epithelial cells (reserve cells as well as glandular epithelial cells), but in the endometrium cause atrophy (see Table 1).

Lower frequency of allele loss on chromosome 18q in human breast cancer than in colorectal tumors.

Inactivation of tumor suppressor genes is thought to be a critical step in tumorigenesis. The DCC (deleted in colorectal carcinoma) gene, located on the long arm of chromosome 18, has been shown to be frequently deleted in colorectal tumors. To investigate the involvement of allelic deletions on chromosome 18q in breast cancer tumorigenesis we analyzed 28 primary breast tumors and 28 colorectal tumors (24 carcinomas, 4 adenomas) with four different polymorphic DNA markers detecting RFLPs on chromosome 18q. In breast cancer we found loss of heterozygosity (LOH) in 4 of 27 (15%) informative cases whereas 15 of 25 (60%) colorectal tumors showed allelic deletions. In all cases of allelic loss the DCC locus or its proximal vicinity (locus SSAV1) were involved. LOH on chromosome 18q occurs both in breast and colorectal cancer, yet the frequency of these deletions in breast tumors is lower than in colorectal tumors. Moreover, in breast cancer these mutations were only detected in large and undifferentiated tumors.

[Morphologic endometrium changes with tamoxifen]. / Morphologische Endometriumveränderungen unter Tamoxifen.

Women receiving tamoxifen as adjuvant therapy for breast cancer usually develop atrophy of the endometrium. Cystic polyps as well as endocervical and clear-cell metaplasias also frequently arise in the these atrophic endometria. The invasive endometrial carcinomas we have observed in these women have all been until now either mucinous, clear-cell or serous-papillary carcinomas, that is, different from the endometrioid type of adenocarcinomas that arise under estrogenic stimulation. Our histological studies support the assumption that tamoxifen has an antiestrogenic, gestagen-like action on the endometrium; they contradict the premise that tamoxifen therapy exerts an estrogen effect.

Mucinous and clear cell adenocarcinomas of the endometrium in patients receiving antiestrogens (tamoxifen) and gestagens.

In recent years, tamoxifen therapy for breast carcinoma has been associated with endometrial carcinoma with increasing frequency. We describe 10 cases of this association as well as 12 cases of endometrial carcinoma after therapy with synthetic gestagens. All but one of the 22 carcinomas we describe here were of the mucinous or clear cell type, and arose in atrophic endometria containing foci of mucinous (endocervical) and clear cell metaplasia. In the endometrium, the antiestrogenic action of tamoxifen closely resembles the antiproliferative action of synthetic gestagens on the endometrium. In the endocervix, tamoxifen as well as synthetic gestagens, stimulate the endocervical mucosa to proliferate. In the endometrium, both tend to produce mucinous (endocervical) and clear cell metaplasia. A weak estrogenic action of tamoxifen may be expected to augment its own antiestrogenic gestagen-like action. Consequently, the antiestrogenic effect of tamoxifen is most likely responsible for the development of endocervical metaplasia and possibly corresponding endocervical and clear cell types of carcinomas within an atrophic endometrium. We believe that gestagen therapy administered to patients with endometrioid type carcinoma is contraindicated in patients with mucinous adenocarcinoma of the endometrium and that this is another reason to distinguish between these two types of carcinoma.

[Receptivity of the endometrium: comparison of ultrasound and histologic findings after hormonal stimulation]. / Zur Rezeptivität des Endometriums: Vergleich sonographischer und histologischer Befunde nach hormoneller Stimulation.

On the day of the scheduled embryo transfer, endometrial biopsies were taken from 53 patients of an IVF-programme. There was subsequent failure of fertilisation in all of these patients. The histologic pattern, compared with reflectivity and thickness of the endometrium was determined sonographically on the same day. All patients had been stimulated with hMG/hCG, and the ovaries and the endometrium had been monitored by transvaginal sonography. The reflectivity pattern was divided into grades (A to D according to Smith et al., 1984) and related to histological findings. The distribution of the grades was as follows: 16 patients (30%) grade A, 22 patients (42%) grade B, and 15 patients (28%) grade C. There was no case of grade D in our study. The results of histologic examination for grades A to C were not significantly different from each other. However, in only 37% of grade A, 63% of grade B and 66% of grade C, the endometrial response corresponded to the state of the cycle. Endometrial thickness for the receptive and non-receptive groups was not significantly different (8.8 +/- 0.29 mm vs. 9.13 +/- 0.4 mm). The results show no correlation between histologic findings and endometrial thickness or reflectivity. We therefore conclude, that sonographic determination of these parameters is not helpful in the evaluation of the degree of endometrial response.

The value of immunohistochemistry in the differential diagnosis of endometrial carcinomas.

Endometrial carcinomas may originate from endometrial glandular epithelium and show endometrial differentiation, or from various types of metaplasias developing in the endometrium from pluripotent Müllerian epithelium. They then show endocervical or serous papillary differentiation. Because of their differences in spread, speed of growth and survival rates, it is important to subclassify these endometrial carcinomas. Immunohistochemically, adenocarcinoma with endometrial differentiation including adenoacanthomas and adenosquamous carcinomas can be recognized by their coexpression of cytokeratin 8 and vimentin, and by their negative reaction for CEA. Distinction from adenocarcinomas with mucinous differentiation, including mucoepidermoid adenocarcinomas, is possible by their negative reaction for vimentin and by their positive reaction for CEA. On the other hand, carcinomas with mucinous differentiation primarily located in the endometrium can not be distinguished from those primarily located in the endocervix by immunohistochemistry; that distinction must be made topographically. The same holds true for clear cell carcinomas of both locations. Over the past decade, mucinous adenocarcinomas and clear cell carcinomas originating from the endometrium have increased, whereas adenocarcinomas with endometrial differentiation have become less frequent. This shift is closely related to the altered postmenopausal hormone substitution with the addition of the synthetic gestagens. These apparently stimulate proliferation of endocervical epithelium not only in the endocervix, but also that arising in endocervical metaplasias of the endometrium.

Immunohistochemical studies on uterine tumors. I. Invasive squamous cell carcinomas of the cervix and their precursors.

40 invasive carcinomas and 80 preinvasive lesions of the uterine cervix were studied immunohistochemically; 40 benign lesions served as controls. On histological and immunohistochemical examination, invasive and preinvasive carcinomas were subdivided in the squamous (large cell, ectocervical) type and the reserve cell (small, large or clear cell, endocervical) type. Immunohistochemically, 100% of the invasive and preinvasive squamous cell carcinomas were positive with anticytokeratins 13, 14, 16 and negative with anticytokeratin 8 and anti-CEA. Most of the invasive and preinvasive reserve cell carcinomas showed a coexpression of cytokeratins 13, 14, 16, 8 and CEA. The subdivision of invasive carcinomas of the ecto- and endocervix into squamous cell and reserve cell types made by means of their structural differences is substantiated and re-evaluated by their immunohistochemical reactions. Both types of carcinomas retain the complex pattern of cytokeratins shown by their cells of origin. The reserve cell carcinomas, in addition, acquire a coexpression for CEA that indicates malignant transformation. The subdivision is of clinical importance because both types of carcinomas vary in their mode and speed of invasion and spread and in their association with HPV infection.

[New cytogenetic classification of precancerous lesions and carcinomas of the endometrium: possibilities and limits of immunohistochemical differentiation]. / Die neue cytogenetische Klassifikation der Präcancerosen und Carcinome des Endometrium: Möglichkeiten und Grenzen der immunhistochemischen Differenzierung.

Various grades of adenomatous hyperplasia represent precancerous states leading to the most common type of adenocarcinoma with endometrial differentiation. Grades 1 and 2 are characterized by architectural abnormalities only (complex hyperplasia). They rarely progress to carcinoma. Grade 3 in addition is characterized by cytological atypicalities (atypical hyperplasia) and shows a much higher progression rate into invasive carcinoma. Endometrial carcinomas may originate from endometrial glandular epithelium and show endometrial differentiation, or from various types of metaplasias developing in the endometrium from pluripotent Müllerian epithelium. They then show endocervical or serous papillary differentiation. Because of their differences in spread, speed of growth and survival rates, it is important to subclassify these endometrial carcinomas. Immunohistochemically, adenocarcinomas with endometrial differentiation including adenoacanthomas and adenosquamous carcinomas can be recognized by their coexpression of cytokeratin 8 and vimentin, and by their negative reaction for CEA. Distinction from adenocarcinomas with mucinous differentiation, including muceopidermoid adenocarcinomas, is possible by their negative reaction for vimentin and by their positive reaction for CEA. On the other hand, carcinomas with mucinous differentiation primarily located in the endometrium can not be distinguished from those primarily located in the endocervix by immunohistochemistry; that distinction must be made topographically. The same holds true for clear cell carcinomas of both locations. Over the past decade, mucinous adenocarcinomas and clear cell carcinomas originating from the endometrium have increased, whereas adenocarcinomas with endometrial differentiation have become less frequent. This shift is closely related to the altered postmenopausal hormone substitution with the addition of the synthetic gestagens. These apparently stimulate proliferation of endocervical epithelium not only in the endocervix, but also that arising in endocervical metaplasias of the endometrium.

[Role of steroid hormones and related biological substances in the etiology of endometrial carcinomas]. / Stellenwert der Steroidhormone und biologisch verwandter Substanzen in der Atiologie des Endometriumcarcinoms.

Synthetical oestrogens, as well as gestagens may cause metaplastic changes of the endometrial epithelia. While squamous and tubal metaplasia are most often due to oestrogenic stimulation and develop in hyperplastic endometria, mucinous and clear cell metaplasia are stimulated by gestagens and tamoxifen. Tamoxifen seems to act gestagen-like on the endometrium. We present data of 31 patients, who were treated with tamoxifen for breast cancer disease. 3 of these 31 developed endometrial adenocarcinoma.

The histogenetic origin of cervical mesonephric hyperplasia and mesonephric adenocarcinoma of the uterine cervix studied with immunohistochemical methods.

Forty-four cases of mesonephric hyperplasia (MH) and two adenocarcinomas arising from mesonephric remnants (MA) in the cervix were compared immunohistochemically with 10 embryonic and fetal mesonephric tissues. The mesonephric cells retained their pattern for intermediate filaments during ontogenesis, as well as in the mature, hyperplastic, and neoplastic states: they expressed cytokeratin 8, cytokeratin 13, and vimentin, the two latter in variable amounts. In embryonic mesonephric tissues, cytokeratin was absent, whereas the staining for vimentin was intense. Fetal mesonephric cells stained for cytokeratin 13 and vimentin, but that staining diminished as maturation progressed. All MH and MA expressed cytokeratin 8, whereas only 20-30% of the cells in MH and 10-20% of carcinomatous mesonephric cells showed positive reactions with anti-cytokeratin 13 and anti-vimentin. CEA was always negative in cells of mesonephric origin. We regard these results to be important, since the reactions with anti-CEA and anti-vimentin enable one to distinguish cervical adenocarcinomas of mesonephric origin from those of endocervical origin, the latter being CEA-positive and vimentin-negative. Clinical studies revealed that approximately 75% of the patients with MH had used oral contraceptives for several years, 46% had precancerous lesions of the cervix, and in 62% the cervical mucosa showed adenomatous microglandular hyperplasia. We believe that hyperplasia of mesonephric remnants in the cervix may occur more often in patients with disturbed hormonal balance. However, the lack of a control population does not enable us to advance this hypothesis with assurance.