By Author
  By Title
  By Keywords

October 2014, Volume 64, Issue 10

Original Article

Differential expression of Phophatase and Tensin Homologue in normal, hyperplastic and neoplastic endometrium

Fouzia Shaikh  ( Ziauddin University Medical College, Karachi. )
Shahnaz Imdad Kehar  ( Basic Medical Sciences Institute, JPMC, Karachi. )
Summayya Shawana  ( Bahria University Medical and Dental College, Karachi. )

Abstract

Objectives: To observe the differential expression of phophatase and tensin homologue in normal proliferative, hyperplastic and malignant endometrial lesions.
Methods: The retrospective study was based on the analysis of endometrial samples, both hysterectomies and curettage, received at the department of pathology Basic Medical Sciences Institute at the Jinnah Postgraduate Medical Centre, Karachi, from January 1, 2006 to December 31, 2010. A total of 55 endometrial samples were analysed for morphological features and results of immunohistochemical staining.
Results: Of the 55 samples, 25 (45.45%) were malignant endometrial lesions, 6 (10.9%) complex hyperplasias with atypia, 14 (25.45%) complex hyperplasias without atypia hyperplasia, 6 (10.9%) simple hyperplasias without atypia, and 4 (7.27%) normal proliferative endometrium. Among malignant endometrial lesions, 12 (48%) showed complete loss of phophotase and tensin homologue expression out of which majority were endometroid adenocarcinoma. Five (83.3%) cases of complex hyperplasias with atypia and 9 (64.28%) cases of complex hyperplasia without atypia showed complete loss of or diminished expression of phophotase and tensin homologue.
Conclusion: Loss of phophotase and tensin homologue expression was seen in a significant number of well differentiated endometrial adenocarcinomas and complex hyperplasias with atypia suggesting loss of PTEN expression as an early event in endometrial carcinogenesis.
Keywords: Endometrial carcinoma, Hyperplasia, PTEN expression, Atypia, Early event. (JPMA 64: 1103; 2014).

Introduction

Endometrial carcinoma is one of the most common invasive tumours of the female genital tract and accounts for about 7% of all invasive cancers in women.1 It is the 5th most common cancer of women worldwide.2
An earlier retrospective statistical study showed that endometrial carcinoma with 86 out of 2223 cases formed 3.87% of all malignant neoplasms in females.3 A collective Pakistani cancer registry (1994-2011) reported 707 cases of malignancies of corpus uteri contributing 3.02% of all neoplasms in females above 18 years of age.4 A hospital-based registry showed that in 2010, out of 76 malignancies of the corpus uteri 65 (85.52%) were endometrial adenocarcinomas.5
Endometrial carcinomas have been classified into two main types; type 1 and type 2, on the basis of light microscopic appearance, clinical behaviour and epidemiology. Type 1 and type 2 tumours carry mutations of independent set of genes.
Endometroid (type 1) endometrial carcinoma is often preceded by a characteristic histopathologic lesion designated endometrial hyperplasia. Endometrial hyperplasia is usually associated with prolonged unopposed oestrogen stimulation and is characterised by increased gland-to-stroma ratio and abnormalities of epithelial growth relative to normal endometrium.
The much debated World Health Organisation (WHO) classification of endometrial hyperplasia divides it into 4 types depending on architectural changes and cellular atypia.6 These are: Simple hyperplasia without atypia; Complex hyperplasia without atypia; Simple hyperplasia with atypia; and Complex hyperplasia with atypia.
Atypical hyperplasia has long been considered a precursor lesion for endometrial carcinoma. However endometrial hyperplasias are among the most commonly over-diagnosed lesions in surgical pathology.7 Whether hyperplasia without atypia poses a potential risk for developing into endometrial carcinoma is still not clear. However, prolonged unopposed oestrogen exposure is seen to confer a 2-10 fold increased risk for endometrial carcinoma.8-10
In cases of non-atypical hyperplasias, the recommended treatment is cyclical progestins therapy, whereas in patients with atypical hyperplasias hysterectomy is the recommendation. If, however, the patient is young and wishes to conceive, high-dose progestin therapy may be considered as an option. In one study, 16 out of 17 patients diagnosed as having atypical endometrial hyperplasia showed regression of the lesion with progestin therapy and concluded that treatment of atypical hyperplasia and well-differentiated carcinoma of the endometrium with progestins appears to be a safe alternative to hysterectomy in women under the age of 40.11
Common genetic changes in endometroid endometrial carcinoma include microsatellite instability and mutations of PTEN, K-ras and b-catenin genes along with others.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumour suppressor gene is located at chromosome 10q23. It encodes a 55-KD protein with tyrosine kinase function. PTEN suppressor gene acts at the G1/S checkpoint of the cell cycle. The encoded protein has both lipid and protein phosphatase activity. It enables apoptosis through an AKT-dependent mechanism. PTEN acts in opposition to P13KCA to control levels of phosphorylated AKT. PTEN mutation results in increased P13KCA activity and thus leads to increased AKT phosphorylation. PTEN also phosphorylates FAK which plays a major role in a transcription regulatory signalling system. FAK is activated by integrin and growth factors and induces focal adhesions, cytoskeleton formation, cellular spreading, invasion and migration.
PTEN mutation is considered to be the most common and an early genetic defect in endometroid endometrial carcinoma. Inactivation of the PTEN gene has been seen in majority (about 83%) of cases of endometrial carcinoma preceded by a pre-malignant phase.12 PTEN mutation has been documented in endometrial hyperplasia with and without atypia.  Multiple studies have suggested loss of PTEN function as an early event in the pathogenesis of endometrial carcinoma.12-15 However, a few studies showed contradicting results.16,17 It should be emphasised, however, that different clones of antibody were used in these studies. PTEN mutations have been detected in both atypical hyperplasias associated with the development of adenocarcinoma and those that did not progress to carcinoma. It has been suggested that although PTEN null phenotype in endometrial hyperplasias does not necessarily predict an increase in the incidence of carcinoma in subsequent follow-ups, but the absence of PTEN null phenotype alone may predict a benign follow-up.13 In the last two decades numerous researches have been carried out worldwide to find out ways for early diagnosis of endometrial carcinomas and proper therapeutic interventions. In Pakistan very limited researches have been carried out in this regard. It was therefore decided to carry out the current study to observe the differential expression of PTEN in different endometrial lesions ranging from normal proliferative endometrium to malignant endometrial tumours.

Materials and Methods

The retrospective analytical study was based on the analysis of endometrial samples, both hysterectomies and curettage, received at the Department of Pathology, Basic Medical Sciences Institute at the Jinnah Postgraduate Medical Centre, Karachi, from January 1, 2006 to December 31, 2010.
Over the five-year study period, there had been 303 endometrial lesions from which 55 were selected for the study which were analysed for morphological features and results of immunohistochemical staining. Poorly fixed tissue, inadequate material and samples of foreign nationals and Pakistanis living abroad for more than 10 years were excluded. Haematoxylin and eosin (H&E) stained slides were reviewed to confirm the diagnosis. The most representative section was used for immunohistochemical analysis.
Anti-PTEN (clone 6H2.1), mouse monoclonal antibody procured from Millipore was used in all immunohistochemical analysis. Antigen detection was done using HiDef detection horseradish peroxidase (HRP) polymer system kit (ready to use) procured from Cell Marque. Endometrial stroma was taken as internal positive control while phosphate buffer solution (PBS) substituted primary antibody for negative control. Sections of approximately 5µm were cut on to poly L-lysine coated slides and were deparrafinised and rehydrated. Antigen retrieval was achieved by steamer method using citrate buffer. Slides were allowed to cool for 20 minutes and were then placed in ultraviolet (UV) block for 5 minutes. Tissues were covered with primary antibody at dilution 1:50 and were incubated for 1 hour at room temperature. Slides were then incubated first with Amplifier and then with HRP polymer for 10 minutes. Chromogen was applied for 20 minutes and all the slides were counter-stained with haematoxylin, dehydrated and mounted. Between each step the slides were washed with PBS.
The intensity of staining was graded as no staining (0), weak staining (1+), moderate staining (2+) and strong staining (3+).

Results

The 55 samples selected for immunohistochemistry, included 4 (7.27%) normal proliferative endometrium, 6 (10.9%) simple hyperplasia without atypia, 14 (25.45%) complex hyperplasias without atypia, 6 (10.9%) complex hyperplasias with atypia and 25 (45.45%) endometrial carcinomas.
The age range for 148 (48.84%) of simple hyperplasia without atypia among the overall 303 cases was 21-70 years with most of the patients belonging to the 41-50 years age. Similar data was noted for all the other three types as well (Table-1).


The nuclear intensity of immunoexpression of PTEN in normal, hyperplastic and neoplastic endometrial samples was noted separately (Table-2).

Complete loss of PTEN expression was observed in 12 (48%) of malignant endometrial tumours. Four (66.6%) cases of complex hyperplasia with atypia showed complete loss of PTEN expression (Figure-1).

In case of hyperplasias without atypia, 5 (35.6%) cases of complex and 3 (50%) of simple hyperplasia showed moderate to strong PTEN staining while 8 (57%) cases of complex and 3 (50%) cases of simple hyperplasia showed weak staining with PTEN. Three (75%) cases of normal proliferative endometrium showed moderate, while 1 (25%) showed strong nuclear staining for PTEN.
Of the malignant endometrial tumours, 13 (52%) showed positive staining for PTEN. However, the intensity of staining was strong (3+) in only 1 (4%) of these cases, while the remaining 12 (48%) cases showed weak to moderate staining with PTEN (Figure-2).

Moreover, these cases included 2 (8%) spindle cell tumours, 2 (8%) poorly differentiated adenocarcinomas and 2 (8%) papillary serous carcinomas.

Table-3 compares the intensity of PTEN immunoreactivity which was also noted according to the degree of differentiation in endometrial adenocarcinomas. Out of 18 cases of well-differentiated adenocarcinomas, 11(61.1%) showed complete loss of PTEN expression and none showed strong staining with PTEN. There was a single case of moderately differentiated adenocarcinomas which showed complete (100%) loss of PTEN expression. Three (75%) cases of poorly differentiated tumours showed moderate staining, while 1 (25%) showed strong staining for PTEN.

Discussion

The present study attempted differential expression of PTEN to observe its intensity of staining in overtly malignant endometrial lesions as well as in hyperplasias with and without atypia and normal proliferative endometrium.
The mean age for malignant endometrial tumours was found to be 57 years and majority (67.56%) belonged to the 6th and 7th decade of life. Our findings correspond to the figures documented in the national cancer registry report (1994-2011) which showed that about 66% of patients with uterine cancers belonged to the 6th and 7th decades of life.4 Similar findings were observed in another study where 61.62% of patients with endometrial carcinomas belonged to the age range of 51 to 70 years.3
In the present study all cases of endometroid endometrial adenocarcinoma showed either loss of or diminished expression of PTEN, with 61.11% showing complete loss of staining with PTEN. These findings are in accordance with different studies conducted worldwide.12,18,19 However, a few other studies showed a comparatively lower percentage of endometroid endometrial adenocarcinomas with complete loss of PTEN expression,15,16,20,21 while one reported no loss of PTEN expression.22 This difference may be due to the discrepancy in sample size or the fact that polyclonal anti-PTEN antibody (28H6) was used in this study. Weak to moderate staining with PTEN was seen in 38.8% cases which may be due to the fact that in the early stages the cells may express PTEN to counter the abnormal proliferation. It has also been suggested that a mechanism, other than PTEN mutation, may also contribute to the progression of endometrial carcinoma.20,23
Six cases of endometroid endometrial carcinoma with complete loss of PTEN expression also showed, in the adjacent areas, foci of potential pre-malignant lesions in the form of complex hyperplasias with and without atypia with loss of PTEN expression in more than 50% of nuclei, probably representing an early event in the progression to carcinoma.
The 2 papillary serous carcinomas in the present study showed moderate to intense expression of PTEN. This was an expected finding as non-endometroid (type II) endometrial carcinomas have been shown to be associated with p53 rather than PTEN mutation.24 Different studies have shown that 80% and 75% of non-endometroid endometrial carcinomas had intense staining with PTEN.12,18
The two poorly differentiated adenocarcinomas showed moderate staining with PTEN. This finding is similar to that of another study in which high expression of PTEN in Grade 3 endometrial carcinoma was observed and it was suggested that PTEN protein may have been induced to counter the aggressive growth of the poorly differentiated carcinoma.17 A study showed that PTEN mutation was associated with early stage and more favourable survival and it was suggested that mutation of genes other than PTEN may be involved in case of more aggressive malignancies.23
Immunohistochemistry was done on 6 cases of complex hyperplasia with atypia, out of which 4 showed complete loss of PTEN expression, 1 each showed moderate and 1 weak staining with PTEN. These figures correspond to other studies where loss of PTEN in 64% and 56% of complex hyperplasia with atypia was observed respectively.13,19 Other studies showed loss of PTEN expression in only 25% and 20% cases of complex hyperplasia with atypia respectively16,25 and it was proposed that using a polyclonal antibody might be the cause for these lower figures.
Out of 6 cases of simple hyperplasias without atypia, all showed weak to moderate staining with PTEN. It was observed in one study that 60% cases of simple hyperplasia without atypia showed weak to moderate PTEN staining.19 In other studies, none of the hyperplasias without atypia showed loss of PTEN expression.13
All four normal proliferative endometrial samples showed moderate to strong PTEN staining. A study related to the expression of PTEN during different phases of the menstrual cycle found that with the regeneration of the functional layer of endometrium during the proliferative phase, PTEN signals became widespread in epithelial and stromal compartments.26

Conclusion

The loss of PTEN expression was seen in a significant number of well-differentiated endometrial adenocarcinomas and hyperplasias with atypia, suggesting that loss of PTEN expression might be an early event in endometrial carcinogenesis. Moreover, loss of PTEN expression was also observed in foci of hyperplasias adjacent to the areas with frank carcinoma. Cases belonging to the category of hyperplasias without atypia showing loss of PTEN could not be followed to ascertain their outcome. After mutiple studies conducted worldwide suggesting PTEN-loss to be an effective marker for detecting early pre-malignant endometrial lesions, PTEN is still a marker for research purposes only. Therefore, further, preferably prospective, studies with proper long-term follow-up of patients, are recommended to assess the final outcomes of hyperplastic lesions showing PTEN-loss and to assess the efficacy of PTEN-loss as a marker for early detection of endometrial pre-malignant lesions. It is also recommended that patients with endometrial hyperplasias showing loss of PTEN expression should be followed up regularly for the development of endometrial carcinoma.

References

1. Crum CP. The female genital tract. In: Robin and Cotran: Pathologic basis of Disease. 8th ed. Bangladesh and Pakistan: Elsevier, 2009; pp 1085-8.
2. Jemal A, Segal R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics 2007. CA Cancer J Clin 2007; 57: 43-66.
3. Ahmed Z, Azad NS, Yaqoob N, Husain A, Ahsan A, Khan AN, etal. Frequency of primary solid malignant neoplasms in both sexes as seen in our practice. J Ayub Med Coll 2007; 19: 53-5.
4. Collective cancer registry report (1994-2011). Shaukat Khanum Cancer Hospital and Research Centre, pp 6.
5. Annual cancer registry report 2010. Shaukat Khanum Cancer Hospital and Research Centre, p19.
6. Kurman RJ, Norris HJ. Endometrial hyperplasia and related cellular changes. In: Kurman RJ, ed. Blaustein\'s pathology of the female genital tract. 4th ed. New York: Springer-Verlag, 1994; 411-37.
7. Silverberg SG. The 1999 long course on pathology of the uterine corpus and cervix. Problems in the differential diagnosis of endometrial hyperplasia and carcinoma. Mod Pathol 2000; 13: 309-27.
8. Parrazini F, LaVecchia C, Bocciolone L, Franceschi S. The epidemiology of endometrial cancer. Gynecol Oncol 1991; 41: 1-16.
9. Shapiro S, Kelly JP, Rosenberg L. Risk of localized and widespread endometrial cancer in relation to recent and discontinued use of conjugated estrogens. N Engl J Med 1985; 313: 969-72.
10. Zelenuich-Jacquotte A, Akhmedkhanov A, Kato I. Postmenopausal endogenous oestrogens and risk of endometrial cancer: results of a prospective study. Br J Cancer 2001; 84: 975-81.
11. Randall TC, Kurman RJ. Progestin treatment of atypical hyperplasia and well differentiated carcinoma of the endometrium in women under age 40. Obstet Gynecol 1997; 90: 434-40.
12. Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Baak JP, Lees JA, et al. Altered PTEN expression as a diagnostic marker for earliest endometrial precancers. J Natl Cancer Inst 2000; 92: 924-30.
13. Quddus MR, Ologun BA, Sung CJ, Steinhoff MM, Lawrence WD. Utility of PTEN expression of endometrial surface epithelial changes and underlying atypical endometrial hyperplasia. Int J Gynecol Pathol 2009; 28: 471-6.
14. Maxwell GL, Risinger JI, Gumbs C, Shaw H, Bentley RC, Barelt JC,etal . Mutations of the PTEN tumor suppressor gene in endometrial hyperplasias. Cancer Res 1998; 58: 2500-3.
15. Taranger-Charpin C, Carpentier S, Dales JP, Garcia S, Djemli A, Andrac L, etal. Immunohistochemical expression of PTEN antigen: a new tool for diagnosis of early endometrial neoplasia. Bull Acad Natl Med 2004; 188: 415-27.
16. Sarmadi S, Izadi-Mood N, Sotodeh K, Tavangar SM. Altered PTEN expression; a diagnostic marker for differentiating normal, hyperplastic and neoplastic endometrium. Diagn Pathol 2009; 4: 41.
17. Kimura F, Watanabe J, Hata H, Fujisawa T, Kamata Y, Nishimurai Y, etal. PTEN immunohistochemical expression is suppressed in G1 endometroid adenocarcinoma of the uterine corpus. J Cancer Res Clin Oncol 2004; 130: 161-8.
18. An HJ, Lee YH, Cho NH, Shim JY, Lee C, Kim SJ. Alteration of PTEN expression in endometrial carcinoma is associated with down regulation of cyclin dependent kinase inhibitor p27. Histopathology 2002; 41: 437-45.
19. Tantbirojn P, Triratanachat S, Trivijitsilp P, Niruthisard S. Detection of PTEN immunoreactivity in endometrial hyperplasia and adenocarcinoma. J Med Assoc Thai 2008; 91: 1161-4.
20. Kanamori Y, Kigawa J, Itamochi H, Shimada M, Takahashi M, Kamazawa S, etal. Correlation between loss of PTEN expression and Akt phosphorylation in endometrial carcinoma. Clin Cancer Res 2001; 7: 892-8.
21. Terakawa N, Kanamori Y, Yoshida S. Loss of PTEN expression followed by Akt phosphorylation is a poor prognostic factor for patients with endometrial cancer. Endocr Relat Cancer 2003; 10: 203-8.
22. Cirpan T, Terek MC, Mgoyi L, Zekioglu O,  Iscan O, Ozsaran A. Immunohistochemical evaluation of PTEN protein in patients with endometrial intraepithelial neoplasia compared to endometrial adenocarcinoma and proliferative phase endometrium. Eur J Gynecol Oncol 2006; 27: 389-92.
23. Risinger JI, Hayes K, Maxwell GL, Carney ME, Dodge RK, Barret JC, etal. PTEN mutation in endometrial cancers is associated with favourable clinical and pathological characteristics. Clin Cancer Res 1998; 4: 3005-10.
24. Sherman ME. The 1999 long course on pathology of the uterine corpus and cervix: Theories of endometrial carcinogenesis: A multidisciplinary approach. Mod Pathol 2000; 13: 295-308.
25. Kapucuoglu N, Aktepe F, Kaia H, Bircan S, Karahan N, Cins M. Immunohistochemical expression of PTEN in normal, hyperplastic and malignant endometrium and it\'s correlation with hormone receptors bcl-2, bax and apoptotic index. Pathol Res Pract 2007; 203: 153-62.
26. Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Eng C. Changes in endometrial PTEN expression throughout the human menstrual cycle. J Clin Endocrinol Metab 2000; 85: 2334-88.

Journal of the Pakistan Medical Association has agreed to receive and publish manuscripts in accordance with the principles of the following committees: