—  SYMPOSIUM #26  —

Endometrial Carcinoma: Pathology and Genetics
Moderator: Dr. Michael A. Wells

Section 1 - PTEN: Gatekeeper of the Endometrium

George L. Mutter, MD
Brigham and Women's Hospital
Boston, MA


Overview:
This presentation will integrate complementary pathologic, genetic, and hormonal views of endometrial carcinogenesis, emphasizing mutational inactivation of the PTEN gene as a marker for early lesions, and hormonal effects upon fate of PTEN mutant clones as mediators of ultimate cancer risk.

Endometrial Intraepithelial Neoplasia (EIN), A Diagnosible Monoclonal Precancer
The histologic presentation of premalignant endometrial lesions prone to malignant transformation to endometrioid adenocarcinoma is called EIN. In the past, both generalized hormonal responses and localized premalignant lesions have been lumped under the term "endometrial hyperplasia" subdivided by architectural complexity and cytologic atypia [1]. Although this practice has been widespread, it fails to optimally stratify patients according to those pathologic mechanisms and cancer risks necessary for appropriate therapeutic triaging. Recent molecular studies have provided evidence that the use of the term hyperplasia is conceptually correct for some but not all of these lesions. For these reason, we have chosen to present a practically oriented disease classification in which the hormonal effects of unopposed estrogens (benign hyperplasia) and emergent neoplastic precancerous lesions (endometrial intraepithelial neoplasia (EIN) are separately diagnosed using non-overlapping terminology and discrete criteria (Table 1) [2].

Table I: EIN Diagnostic Criteria. Modified after [3].

EIN Criterion Comments
Architecture Area of Glands greater than Stroma
Cytology Cytology differs between architecturally crowded focus and background, or clearly abnormal.
Size >1 mm Maximum linear dimension exceeds 1mm.
Exclude mimics Benign conditions with overlapping criteria: Basalis, secretory, polyps, repair, etc.
Exclude Cancer Carcinoma if mazelike glands, solid areas, polygonal "mosaic-like" glands, myoinvasion, or significant cribriforming


The risk of developing endometrial cancer, as predicted by an EIN diagnosis are the basis for therapy. Although there are many previous references citing cancer outcomes of EIN patients [4, 5, 6], two studies summarize cancer predictive value of EIN diagnosed by subjective [7] and objective histomorphometric [2] methods. Overall, patients with EIN lesions have an 89-fold increased cancer risk than those without [2].

Molecular Biology of EIN.
Insights into how endometrial precancers behave have been facilitated by application of molecular markers to paraffin embedded human materials. EIN lesions begin as localized monoclonal outgrowths of mutated endometrial cells with a changed cytology and architecture that enables their recognition when compared to the background source polyclonal field [8]. The clonal nature of EIN lesions has been demonstrated by various markers such as nonrandom X chromosome inactivation and clonal propagation of altered microsatellites [8, 9, 10]. Histomorphometric analysis of premalignant endometrial lesions identified by monoclonal growth, and lineage continuity with actual carcinomas that developed in the same patients showed that virtually all precancers have a histologic appearance identical to that seen in histomorphometric studies to increase cancer risk [2]. These histomorphometric features have been incorporated into subjective diagnostic criteria.

Each EIN lesion is the end result of multiple mutations that occur in varying permutations and order of invocation between patients. Within individual patients, those exact genetic alterations present in an EIN lesion are carried forward to the cancer, establishing them as physical progenitors of carcinoma [8, 11]. The clone which comprises an EIN lesion may acquire additional mutations during subsequent clonal expansion, a key element of progression to carcinoma and development of intratumoral heterogeneity [11, 12]. Comparison of the extent and range of genomic damage between premalignant and malignant phases indicates a greater cumulative mutational load in cancers, a feature that must contribute to their differing morphology and behavior. For example, while 55% of EIN lesions have demonstrable inactivating events (mutation and/or deletion) [13] of the PTEN tumor suppressor gene, the proportion rises to 83% in those cancers which follow an EIN lesion [12]. Similarly, for those lesions with microsatellite instability, the burden of altered microsatellite alleles increases between EIN and carcinoma [8, 11].

Several other genes known to be structurally altered in endometrial carcinomas are already abnormal in EIN lesions. Most are somatically acquired rather than inherited defects, as they are intact in the background endometrial tissues. Activating mutations of the KRAS2 cellular oncogene are clonally present in the cells of 16% of EIN lesions [14, 15, 16]. Microsatellite instability caused by defective DNA mismatch repair, is seen in 25-20% of EIN lesions. β-catenin mutations involve 25-30% [17] of endometrial cancers and their premalignant counterparts.

PTEN, a Marker for Endometrioid Carcinogenesis
PTEN, a tumor suppressor located at 10q23 inactivated early in endometrial carcinogenesis, is an informative marker for exploring the premalignant phases of disease. 63% of EIN lesions lack immunohistochemically detectable PTEN protein in a clonal distribution [18]. Despite this very strong association, and the fact that experimental PTEN inactivation in mice leads to a high incidence of endometrial cancer [19], changes in addition to PTEN inactivation must occur before affected cells acquire histopathologic features diagnostic of EIN.

In vitro cell line data has suggested that the tumor suppressor functions of PTEN, including G1 arrest and enabling of apoptosis, are mediated by a cascade which maintains the putative downstream factor Akt in a dephosphorylated state [20, 21]. Although PTEN presents itself as a major determinant of Akt-mediated apoptosis and G1 arrest these are, however, basic cellular functions controlled by a complex web of regulatory pathways that probably include elements outside the PTEN-Akt axis. The finding that p27 and cyclin D1 do not necessarily behave according to a simple linear model aligned with PTEN and Akt [22] suggests that these downstream events are indirect or subject to modification.

Latent Precancers: a Preclinical Phase of Disease Identified by the PTEN Biomarker
Initially, somatically acquired endometrial gland mutations in the PTEN tumor suppressor gene are not accompanied by any cytologic or architectural modifications evident at the light microscopic level. This "latent precancer" phase is subclinical in every respect, falling below the threshold of detection using routine diagnostic methods, and without a greatly increased prospective cancer risk. 43% of normal premenopausal naturally cycling women have small numbers of these immunohistochemically detected PTEN deficient endometrial glands, which when microdissected bear acquired mutations and deletions of the PTEN gene itself [18]. Progression from this stage to carcinoma must be extremely inefficient, as the lifetime risk of endometrial cancer is only 2.6% [23]. These first events of endometrial carcinogenesis occur with such sufficient frequency that they can be considered a feature of "normal" endometrial biology rather than part of a pathologic state. In the latent phase, mutated cells may participate in successive endometrial regeneration over the course of many menstrual cycles, persisting for years as discernible clones, and demonstrate normal morphogenesis in conjunction with associated stroma [18].

Cancer Risk Modulation is Codetermined by Latent Precancer Fate
Hormonal and genetic mechanisms are linked in the very earliest stages of endometrial carcinogenesis through the selective effects of hormones upon genetically defective compared to intact endometrial cells.

Hormonal environment is one systemic factor which may modulate physiologic demand for PTEN protein, thereby defining a shifting normal baseline against which the functional implications of PTEN loss must be measured. Normal PTEN expression increases in endometrial glands during the estrogenic follicular phase of the menstrual cycle [24], and declines dramatically upon introduction of the antiestrogenic hormone progesterone. A rapidly dividing estrogen stimulated endometrial gland has a greater PTEN requirement than a quiescent progesterone exposed non mitotic gland, and it is reasonable to conclude that these settings would respond differently to loss of PTEN function. Consistent with this notion is the fact that the primary epidemiologic risk factor for PTEN-deficient endometrial carcinomas (endometrioid histologic subtype) is protracted estrogen exposure [25].

Under conditions of a normal monthly menstrual cycle progesterone exposures are insufficient to ablate latent precancers, only 17% of which disappear a year later [18]. If the dose and duration of progestins are increased to therapeutic levels, PTEN mutant latent precancers undergo a 90% rate of involution, thereby resetting the carcinogenesis "clock" [26]. These events are all inapparent at the level of routine histology.

Comments on Use of PTEN Immunohistochemistry.
PTEN tumor suppressor inactivation permits visual delineation of affected EIN lesions by routine immunohistochemistry. One third of EIN lesions express normal levels of the PTEN protein, making it relatively insensitive to render decisions regarding individual patients. Of equal concern, frequent inactivation of this marker at a preclinical stage means that demonstration of isolated PTEN-null glands in the absence of otherwise diagnostic EIN cannot be considered a high cancer risk state [18]. It may, however, have some value when localizing lesions are found to be PTEN null, and thus likely EIN, in otherwise difficult or equivocal diagnostic situations such as within endometrial polyps [27] or secretory endometrium. Lesions that express PTEN, the normal state for endometrial tissues, are non-informative. The assay requires use of appropriate reagents that have been validated in paraffin sections against mutational data (antibody 6h2.1), applied to freshly cut sections following antigen retrieval [18].

References:
  1. Scully RE, Bonfiglio TA, Kurman RJ, Silverberg SG, Wilkinson EJ. Uterine corpus. Histological Typing of Female Genital Tract Tumors. New York: Springer-Verlag, 1994: 13-31.

  2. Baak JPA, Mutter GL, Robboy S et al. In endometrial hyperplasias, the molecular-genetics and morphometry-based EIN classification more accurately predicts cancer-progression than the WHO94. Cancer 2005; 103(11):2304-2312.

  3. Silverberg SG, Mutter GL, Kurman RJ, Kubik-Huch RA, Nogales F, Tavassoli FA. Tumors of the uterine corpus: epithelial tumors and related lesions. In: Tavassoli FA, Stratton MR, editors. WHO Classification of Tumors: Pathology and Genetics of Tumors of the Breast and Female Genital Organs. Lyon, France: IARC Press, 2003: 221-232.

  4. Baak JPA, Nauta J, Wisse-Brekelmans E, Bezemer P. Architectural and nuclear morphometrical features together are more important prognosticators in endometrial hyperplasias than nuclear morphometrical features alone. J Pathol 1988; 154:335-341.

  5. Dunton C, Baak J, Palazzo J, van Diest P, McHugh M, Widra E. Use of computerized morphometric analyses of endometrial hyperplasias in the prediction of coexistent cancer. Am J Obstet Gynecol 1996; 174:1518-1521.

  6. Orbo A, Baak JP, Kleivan I et al. Computerised morphometrical analysis in endometrial hyperplasia for the prediction of cancer development. A long-term retrospective study from northern Norway. J Clin Pathol 2000; 53(9):697-703.

  7. Hecht JL, Ince TA, Baak JP, Baker HE, Ogden MW, Mutter GL. Prediction of endometrial carcinoma by subjective endometrial intraepithelial neoplasia diagnosis. Mod Pathol 2005; 18:324-330.

  8. Mutter GL, Baak JPA, Crum CP, Richart RM, Ferenczy A, Faquin WC. Endometrial precancer diagnosis by histopathology, clonal analysis, and computerized morphometry. J Pathol 2000; 190:462-469.

  9. Jovanovic AS, Boynton KA, Mutter GL. Uteri of women with endometrial carcinoma contain a histopathologic spectrum of monoclonal putative precancers, some with microsatellite instability. Cancer Res 1996; 56:1917-1921.

  10. Mutter GL, Chaponot M, Fletcher J. A PCR assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers. Am J Pathol 1995; 146:501-508.

  11. Mutter GL, Boynton KA, Faquin WC, Ruiz RE, Jovanovic AS. Allelotype mapping of unstable microsatellites establishes direct lineage continuity between endometrial precancers and cancer. Cancer Res 1996; 56:4483-4486.

  12. Mutter GL, Lin MC, Fitzgerald JT et al. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst 2000; 92:924-930.

  13. Esteller M, Catasus L, Matias-Guiu X et al. hMLH1 Promoter Hypermethylation Is an Early Event in Human Endometrial Tumorigenesis. Am J Pathol 1999; 155(5):1767-1772.

  14. Enomoto T, Inoue M, Perantoni A et al. K-ras activation in premalignant and malignant epithelial lesions of the human uterus. Cancer Res 1991; 51:5304-5314.

  15. Duggan BD, Felix JC, Muderspach LI, Tsao J-L, Shibata DK. Early mutational activation of the c-Ki-ras oncogene in endometrial carcinoma. Cancer Res 1994; 54:1604-1607.

  16. Mutter GL, Wada H, Faquin W, Enomoto T. K-ras mutations appear in the premalignant phase of both microsatellite stable and unstable endometrial carcinogenesis. Mol Pathol 1999; 52:257-262.

  17. Matias-Guiu X, Catasus L, Bussaglia E et al. Molecular pathology of endometrial hyperplasia and carcinoma. Hum Pathol 2001; 32(6):569-577.

  18. Mutter GL, Ince TA, Baak JPA, Kust G, Zhou X, Eng C. Molecular identification of latent precancers in histologically normal endometrium. Cancer Res 2001; 61:4311-4314.

  19. Stambolic V, Tsao MS, Macpherson D, Suzuki A, Chapman WB, Mak TW. High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten+/- mice. Cancer Res 2000; 60(13):3605-3611.

  20. Weng LP, Smith WM, Dahia P, Ziebold U, Lees J, Eng C. PTEN supresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death. Cancer Res 1999; 59:5808-5814.

  21. Li J, Simpson L, Takahashi M et al. The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res 1998; 58(24):5667-5672.

  22. Kurose K, Zhou X, Araki T, Cannistra S, Maher E, Eng C. Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am J Pathol 2001; 158:1895-1898.

  23. Ries LAG, Eisner MP, Kosary CL et al. SEER Cancer Statistics Review, 1975-2002, National Cancer Institute. Bethesda, MD. http://seer.cancer.gov/csr/1975_2002/ . 2005.

  24. Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Ziebold U, Eng C. Changes in endometrial PTEN expression throughout the human menstrual cycle. J Clin Endocrinol Metab 2000; 85:2334-2338.

  25. Parazzini F, La Vecchia C, Bocciolone L, Franceschi S. The epidemiology of endometrial cancer. Gynecol Oncol 1991; 41:1-16.

  26. Zheng W, Baker HE, Mutter GL. Involution of PTEN-Null Endometrial Glands with Progestin Therapy. Gynecol Oncol 2004; 92:1008-1013.

  27. Hecht JL, Pinkus JL, Pinkus GS. Enhanced detection of atypical hyperplasia in endometrial polyps by PTEN expression. Applied Immunohistochem & Mol Morphology 2004; 12(1):36-39.