Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2012  |  Volume : 55  |  Issue : 2  |  Page : 187--191

Human telomerase reverse transcriptase expression in ovarian tumors


Awadh Ali Makhashen Maraei1, Ahmad Zailani Hatta2, Mohd Sidik Shiran3, Geok Chin Tan1,  
1 Department of Pathology and Obstetrics, Universiti Kebangsaan Malaysia, Bandar Tun Razak, Cheras, Kuala Lumpur, Malaysia
2 Department of Gynaecology, Universiti Kebangsaan Malaysia, Bandar Tun Razak, Cheras, Kuala Lumpur, Malaysia
3 Department of Pathology, Universiti Putra Malaysia, Serdang, Malaysia

Correspondence Address:
Geok Chin Tan
Department of Pathology, Universiti Kebangsaan Malaysia, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur
Malaysia

Abstract

Background : Ovarian cancer is the 6 th most common cancer among women. In ovarian tumors, the borderline category is not well defined due to the difficulty in assessing stromal invasion. The World Health Organization (WHO) defined it as tumor that lacks obvious invasion of the stroma with mitotic activity and nuclear abnormalities intermediate between clearly benign and unquestionably malignant. Telomerase is expressed in many human cancers and is hence a potential biomarker for cancer. Immunohistochemical study of anti-human telomerase enzyme reverse transcriptase (hTERT) antibody allows direct visualization of its expression. The aim of this study was to determine the expression of hTERT and serum CA-125 level in ovarian epithelial tumors, and their ability to distinguish borderline tumor from malignancy. Materials and Methods : This was a retrospective study on 68 ovarian epithelial tumors, comprising of 41 cystadenocarcinoma, 22 borderline tumor and five cystadenoma. By immunohistochemistry, hTERT expression was graded as negative (0-10%), focal (11-25%), regional (26-75%) and diffuse (>75%) positivity. Results : hTERT protein expression in ovarian cystadenocarcinoma, borderline tumor and cystadenoma were 71.4%, 59.1% and 0%, respectively. hTERT and CA-125 had a linear relationship with tumor grade and stage. hTERT protein is detected as large granules/speckled in the cytoplasm and nuclei of ovarian tumors. Conclusions : hTERT protein was highly expression in ovarian epithelial carcinoma. However, the difference between carcinoma and borderline tumor was not statistically significant (P-value = 0.51). It is not an independent biomarker to differentiate borderline tumor from malignant tumor. We suggest using the combination of hTERT immunohistochemistry and serum CA-125 to evaluate difficult situations where histological evaluation fails to distinguish malignant from borderline ovarian tumor.



How to cite this article:
Maraei AM, Hatta AZ, Shiran MS, Tan GC. Human telomerase reverse transcriptase expression in ovarian tumors.Indian J Pathol Microbiol 2012;55:187-191


How to cite this URL:
Maraei AM, Hatta AZ, Shiran MS, Tan GC. Human telomerase reverse transcriptase expression in ovarian tumors. Indian J Pathol Microbiol [serial online] 2012 [cited 2020 Oct 26 ];55:187-191
Available from: https://www.ijpmonline.org/text.asp?2012/55/2/187/97865


Full Text

 Introduction



Ovarian cancer ranked 6 th , behind breast, cervix, endometrium and colorectal cancers, in the Malaysian National Cancer Registry in 2003. It consists of 4.1% of all female cancers, with an incident rate of 5.3/100,000 population. [1] This is comparable to developed countries, where epithelial ovarian cancers are the 5 th most common cancers in women. [2]

Most ovarian cancers are classified as epithelial and are believed to have come from the surface of the ovary. [3] The existence and clinical significance of the borderline category in the ovarian tumor is not well defined due to difficulty in assessing stromal invasion. [4] Various terms have been given to borderline ovarian tumor, such as "semi-malignant" tumor, "intermediate" tumor, tumor of borderline malignancy, low-potential malignancy and carcinoma of low-malignant potential. [5],[6]

Various rules have been implemented for the distinction between borderline and malignant ovarian epithelial tumors. If there is unquestionable invasion, the tumor is classified as a carcinoma and if invasion is uncertain, the tumor is classified as borderline. Alternatively, when the atypical epithelium is less than four cells in thickness, it is classified as borderline and when it is four cells or greater, as carcinoma. [7],[8]

In 1999, the combined classification of the International Society of Gynecologic Pathologists and WHO defined borderline epithelial tumor as tumor that lacks obvious invasion of the stroma, with mitotic activity and nuclear abnormalities intermediate between clearly benign and unquestionably malignant tumor. The term "tumor of LMP" became popular. [9] Later, in the year 2003, the term "borderline tumor" was used in the published WHO classification. [10]

CA-125 (cancer antigen 125 or carbohydrate antigen 125) is the most studied biomarker in ovarian cancer. It is a protein that is also known as mucin 16 or MUC16 and is encoded by the MUC16 gene. [11] MUC16 is a member of the mucin family of glycoproteins. [12] Bast et al.[13] isolated this monoclonal antibody in 1981 using a murine monoclonal antibody developed in response to immunological challenge with an ovarian cancer cell line. However, CA-125 lacks its specificity as the marker can be elevated in other benign and malignant conditions like fibroids, endometriosis, menstruation and endometrial cancer. [14]

Telomerase is a ribonucleic acid-protein complex that extends and maintains telomeres. By activation of this enzyme, cells are able to overcome replicative senescence and divide indefinitely. It consists of two major components: human telomerase RNA (hTR), which consists of a 451-base integral RNA providing the template for the synthesis of the human telomeric repeat (TTAGGG)n, and human telomerase reverse transcriptase, anti-human telomerase enzyme reverse transcriptase (hTERT), which is a 127 kDa protein providing catalytic function to replicate the ends of linear DNA. [15],[16],[17] The other components in telomerase include hTEP1 (telomerase associated protein 1), hsp90 and p23. [18],[19] hTERT immortalizes cells by maintaining the telomere end, which contributes to cancer cell formation by increasing the cell's life span. [20] Telomerase has been reported to be associated with many malignant tumors. [17],[21],[22],[23] As it is commonly present in cancer, it could be a potential biomarker for malignant tumors.

Immunohistochemical study of hTERT expression allows direct visualization of protein expression and accurate determination of its localization. The aim of this study was to determine the expression of hTERT in ovarian epithelial tumors and its ability to distinguish ovarian borderline from malignant tumors. We also determined the relationship between hTERT expression and CA-125 with FIGO stage and histological grade in ovarian tumors.

 Materials and Methods



Study Design

This was a retrospective immunohistochemical study of hTERT expression on cases diagnosed as ovarian cystadenoma, borderline tumors and carcinoma. Paraffin-embedded samples were obtained from histopathology archives of the Department of Pathology, University Medical Centre, for a period of 7 years and 7 months from 1st January 2002 to 30th July 2009. This study was approved by the ethics committee of the Faculty of Medicine of our University. The total number of cases studied was 68 (41 ovarian carcinoma, 22 borderline tumor and five cystadenoma). Other information like serum CA-125 level, histological grade, stage, age and race of patients was retrieved from the Integrated Laboratory Management System (ILMS). One representative slide was selected from each case if more than one block was retrieved from the archive.

Immunohistochemistry

All tissue were fixed in 10% neutral buffer formalin and embedded in paraffin wax. Paraffin blocks were sectioned at 4 μm in thickness, mounted on sialinized slides and placed in an oven for 10 min. Sections were deparaffinized by two changes for 3 min each in xylene and subsequently rehydrated in decreasing concentrations of alcohol. Sections were incubated in 3% hydrogen peroxide for 6 min to block endogenous peroxide. For antigen retrieval, the slides were pretreated using citrate buffer pH 6.1 retrieval solution in pressurized heating chamber at 121 o C. The sections were incubated for 30 min at room temperature with mouse monoclonal hTERT antibody (1:80 dilution, from GeneTex® Inc., Irvine, CA, USA).

Sections were then washed three-times with Tris-buffered saline TBS and incubated with detection kit "Chemmate Envision" for 30 min at room temperature, followed by rinsing with TBS three times. Slides were incubated with diaminobenzidine for 7 min at room temperature. Finally, the slides were counterstained with hematoxylin for 1 min, washed under running water, followed by dehydrating in increasing concentrations of alcohol and mounted.

Immunohistochemical Analysis

Positive and negative controls were included in each batch of staining. Normal human pancreatic tissue was used as positive control, as recommended by the manufacturer. The negative control was processed similarly, but omitting the primary antibody. In all cases, the hTERT expressions were evaluated by two independent observers.

Interpretation of Results

The hTERT immunoreactivity was indicated by brown cytoplasmic or/and nuclear staining, and was graded by determining the percentage of immunoreactive cells.

The staining reactivity was graded as follows:

0: negative - 0-10% immunoreactive cells

1: focal positivity - 11-25% immunoreactive cells

2: regional positivity - 26-75% immunoreactive cells

3: diffuse positivity - 75-100% of immunoreactive cells

Statistical Analysis

The result was analyzed using Statistical Package for Social Sciences (SPSS) programme version 12. The relationship between the hTERT expressions and ovarian tumor behavior, histological grade and stage were evaluated using chi-square or Fisher exact test, as appropriate. Any P-value of <0.05 was considered as significant.

 Results



Range of Samples

There were a total of 68 cases of ovarian tumors, consisting of 41 cases of ovarian carcinoma (32 serous, six mucinous, one endometrioid, one clear cell and one mixed), 22 cases borderline tumor (two serous, 18 mucinous and two mixed) and five cystadenoma (four serous and one mucinous).

Demographic Data

The ethnic distribution in this study consisted of 41 (60.4%) cases of Malays, 23 (33.8%) Chinese, two (2.9%) Indians and two (2.9%) others. The average age of ovarian carcinoma, borderline tumor and cystadenoma were 52, 41 and 32 years old, respectively. The youngest patient with ovarian carcinoma was 26 years old and the oldest was 83 years old.

CA-125 versus Tumor Behavior, Histological Grade and FIGO Stage

Serum CA-125 level showed a linear relationship with tumor behavior, histological grade and FIGO stage. The average serum CA-125 level was highest in ovarian carcinoma (1098 U/mL), followed by borderline tumor (104 U/mL) and cystadenomas (20 U/mL). The average serum CA-125 level in different stages of FIGO were 144 U/mL in stage 1, 667 U/mL in stage 2, 1472 U/mL in stage 3 and 2613 U/mL in stage 4, while in tumor grades they were 184 U/mL (grade 1), 1018 U/mL (grade 2) and 1454 U/mL (grade 3) [Figure 1].{Figure 1}

hTERT Expression versus Tumor Behavior

hTERT expression was observed in the cytoplasm at perinuclear location as coarse dot-like immunoreactivity [Figure 2]. The comparison of hTERT expression in ovarian epithelial tumors is shown in [Table 1]. Twenty-nine (29/41, 70.7%) of the cases of ovarian carcinoma were positive toward hTERT immunoreactivity, while in borderline tumor, 13 (13/22, 59.1%) of the cases were positive toward hTERT. None of the ovarian cystadenoma (0/5, 0%) was immunoreactive toward hTERT. The difference of hTERT expression between malignant and borderline tumor was not statistically significant (P=0.51) [Table 1].{Figure 2}{Table 1}

hTERT Expression versus Tumor Grade

None (0/6, 0%) of the grade 1 ovarian carcinoma was positive toward hTERT. In contrast, 11 of the 16 cases (11/16, 68.8%) in grade 2 and 18 of the 19 (18/19, 94.7%) cases in grade 3 were positive [Figure 1].

hTERT Expression versus FIGO Stage

Eight of the ovarian carcinoma (8/16, 50%) in stage 1 were positive toward hTERT, three cases (3/4, 75%) in stage 2, 11 cases (10/13, 76.9%) in stage 3 and eight cases (8/8, 100%) in stage 4. The difference between hTERT expression in stage 1 with other stages was statistically significant (P = 0.032); however, when hTERT expression was compared between stage 4 and other stages, it was not significant (P = 0.08) [Figure 1].

 Discussion



hTERT protein was seen as large granules/speckled in the cytoplasm and nuclei of ovarian tumors. This finding is consistent with that of Kyo et al.,[24] who demonstrated that hTERT is expressed not only in the nuclei but also in the cytoplasm of cancer cells. They also performed Western blot analyses, showing hTERT in both nuclear and cytoplasmic extracts. They suggested that cytoplasmic hTERT is unphosphorylated and inactive and that phosphorylation of hTERT correlates with translocation from the cytoplasm to the nucleus. [24]

In our study, hTERT protein was highly expressed in the ovarian epithelial carcinoma, similar to previous studies. [25],[26] However, the differences between carcinoma and borderline tumor were not statistically significant (P-value = 0.51). It was consistently negative in all benign ovarian tumors. The difference between borderline and benign tumors was statistically significant (P-value = 0.04).

In this study, we showed that hTERT protein expression increases with tumor grade and stage. Therefore, higher hTERT protein expression might present as a poor prognostic factor in cancer. In addition, coarse granular staining of hTERT immunoreactivity was observed mostly in high-grade ovarian cystadenocarcinoma. This characteristic staining pattern might indicate a high-grade tumor or a poorer prognosis.

hTERT expression was reported in some studies to be exclusive to malignant ovarian epithelial tumors and was not expressed in benign and borderline tumors. [27] However, some studies were similar to ours, showing hTERT expression in both borderline and malignant tumors. [28] Patients with borderline tumor (41 years old) were on an average 10 years older than those with benign tumor (32 years old). Meanwhile, patients with malignant tumor (52 years old) were 10 years older than those with borderline tumor.

CA-125 is a biomarker for detection, response to treatment and prediction of prognosis for ovarian cancer. In this experiment, we found that the CA-125 level increases in a linear pattern from benign to malignant ovarian tumors. It also correlates well with the increase in stage and grade of the tumor.

 Conclusion



In summary, this study shows that hTERT expression was present in both the cytoplasm and the nuclei of ovarian tumor. hTERT immunohistochemical expression and serum CA-125 had a linear relationship with grade and stage of malignant ovarian tumor. Highest hTERT expression was observed in malignant tumor. However, it is not an independent biomarker to differentiate borderline tumor from malignant tumor. We suggest using the combination of hTERT immunohistochemistry and serum CA-125 to evaluate difficult situations where histological evaluation fails to distinguish malignant from borderline ovarian tumor.

 Acknowledgment



This study was funded by the Faculty of Medicine, Universiti Kebangsaan Malaysia (FF-098-2008). We would like to thank Wirda Indah Binti Farouk for her expert technical assistance.

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