Material and Method: The study included 48 patients who had been diagnosed with pituitary adenomas and had clinical follow-ups. Nonneoplastic pituitary tissues were obtained from autopsy specimens (n=20). Immunohistochemistry for TERT antibody was performed. Both the nuclear and cytoplasmic expression of TERT antibody was noted, and total combined TERT staining was evaluated according to nuclear and cytoplasmic stainings.
Results: TERT expression did not differ between neoplastic and nonneoplastic pituitary tissues. Neither total (combined nuclear and cytoplasmic) TERT nor nuclear TERT expression revealed any statistically significant relationship with any of the clinicopathological features. Higher cytoplasmic TERT expression was observed in adenomas with recurrence than adenomas without recurrence (p=0.035).
Conclusion: This study introduces the notion that immunohistochemical expression of TERT does not differ in neoplastic and nonneoplastic pituitary tissues. Pituitary adenomas with cytoplasmic immunohistochemical expression of TERT have significantly higher rates of recurrence. Further studies, including combined methods of immunohistochemistry and molecular analyses in larger groups, may reveal applicable results for the clinical significance of TERT in pituitary adenomas.
Pituitary adenomas can be classified according to size as microadenomas (≤10 mm) or macroadenomas (>10 mm) and according to radiological appearance as invasive, noninvasive, or aggressive-invasive[4]. The World Health Organization[5] currently classifies pituitary adenomas based on the immunohistochemical demonstration of produced and expressed hormones with clinical reflections. However, the most recent classification of pituitary adenomas is based on an immunohistochemical panel consisting of immune profiling of adenohypophyseal hormones by monoclonal antibodies, cell-specific transcription factors, and low-molecular-weight keratin (CAM5.2), Ki-67, and p53[6,7].
Although pituitary adenomas have benign histomorphological features, some of these tumours may present in an aggressive manner by invasion of surrounding tissues, recurrences, and resistance to medical therapies[4,8]. Thus, the WHO classification defined these tumours as invasive pituitary adenomas with increased mitotic activity, a Ki-67 proliferation index of >3%, and extensive p53 immune staining, namely, atypical adenomas'. To predict the behaviour of these tumours, many studies have been performed by investigating various markers related to chromosomal alterations, microRNAs (miRNAs), proliferation markers, oncogenes, tumour suppressor genes, angiogenesis, cell adhesion, growth factors, and their receptors[1,4,9-12]. Such studies show that none of these markers may predict the behaviour of these tumours alone, but combinations of fibroblast growth factor receptor 4 (FGFR4), matrix metalloproteinases (MMPs), particularly MMP2 and MMP9, Ki-67, p53, pituitary tumour transforming gene (PTTG), and deletions in chromosome 11p seem to have benefits for predicting the aggressiveness of pituitary adenomas[1].
Moreover, investigations continue into several biomarkers other than the suggested panel mentioned above[13,14]. One of these markers is telomerase reverse transcriptase (TERT). Telomerase is a ribonucleic protein complex that includes a catalytic subunit TERT (telomerase associated protein 2) and an RNA component (TERC), and it maintains telomere homeostasis and chromosomal integrity[15]. Telomeres are located at the end and inner sides of chromosomes, and shortening of these nucleotide sequences in cell divisions induces apoptosis or cell senescence. On the other hand, lengthening of the telomeres results in prevention of cell replication and thus is assumed to be a part of tumorigenesis, particularly by expression or activation of telomerase[16]. TERT expression is suppressed in normal adult somatic tissues, but it can be expressed in embryogenic tissues. Reactivation of TERT has been detected in approximately 90% of human cancers[3,17-20]. Thus, TERT activity in pituitary adenomas has been the subject of a few studies that showed contradictory results[3,16,21].
The present study aims to investigate mainly two issues. Initially, the authors aim to investigate whether immunohistochemical expression of TERT differs in neoplastic and nonneoplastic pituitary tissues. Then, the authors will investigate whether TERT expression is related to clinicopathological features of pituitary adenomas such as gender, age at the presentation, tumour size, hormonal activity of the tumour, and recurrence.
Clinicopathological Criteria
Tumours were grouped according to the preoperative
radiological size and the secreted hormone(s) that caused
clinical signs. Tumours sized ≤10 mm were classified as
microadenomas, whereas tumours sized >10 mm were
classified as macroadenomas. Tumours were grouped
according to the clinical symptoms and dominant immune
reaction of secreted hormones as in the following (since
adenohypophyseal transcription factors could not be
performed, the classification was performed according to
the status of adenohypophyseal hormones):
- GH producing/expressing adenomas: Somatotroph
adenomas
- PRL producing/expressing adenomas: Lactotroph
adenomas
- FSH-LH producing/expressing adenomas: Gonadotroph
adenomas
- ACTH producing/expressing adenomas: Corticotroph
adenomas
- TSH producing/expressing adenomas: Thyrotroph
adenomas
- GH and PRL producing/expressing adenomas: Mixed
somatotroph and lactotroph adenomas
- Nonproducing adenomas: Null cell adenomas
The clinical features considered in the statistical analysis included age at the time of diagnosis, sex (male or female), tumour size (≤10 mm, >10 mm) and recurrence (absent/ present).
Immunohistochemistry
Immunostaining for the TERT antibody was performed
with a fully automated immunohistochemistry and in
situ hybridization (IHC/ISH) staining machine (Ventana
BenchMark XT, USA). The following primary antibody at the indicated dilutions was used for TERT immune staining
(TERT polyclonal antibody, unconjugated, 1:100; Bioss,
USA, Catalogue No. bs-1411R). A single pathologist (N.C.)
who was blinded to the clinical assessments of each case
evaluated the expression in tissues with a Nikon Eclipse 80i
microscope. Both nuclear and cytoplasmic expression of
TERT antibody was noted (22) and scored. Then, a total
(combined nuclear and cytoplasmic) score was obtained
from the sum of cytoplasmic and nuclear scores. The
scoring schema is presented below.
The scoring of cytoplasmic TERT expression:
- Cytoplasmic score 0: Cytoplasmic staining in <10% of
the tissue (Figure 2A),
- Cytoplasmic score 1: Mild cytoplasmic staining in ≥10%
of the tissue (Figure 2B),
- Cytoplasmic score 2: Moderate cytoplasmic staining in
≥10% of the tissue (Figure 2C),
- Cytoplasmic score 3: Significant cytoplasmic staining in
≥10% of the tissue (Figure 2D).
The scoring of nuclear TERT expression:
- N uclear score 0: Nuclear staining in <10% of the tissue,
- N uclear score 1: Dot-like nuclear staining in ≥10% of
the tissue (Figure 3A),
- N uclear score 2: Complete nuclear staining in ≥10% of
the tissue (Figure 3B).
Total (combined nuclear and cytoplasmic) TERT expression:
- N egative for TERT expression: The sum of nuclear and
cytoplasmic scores <2,
- Positive for TERT expression: The sum of nuclear and
cytoplasmic scores ≥2 (Figures 3C-3D).
Statistical Analysis
Statistical analysis was carried out using SPSS v20.0
software (IBM SPSS, Inc., Chicago, IL, USA). Appropriate
chi-square tests (Pearson, Yates, or Fisher) were used to
compare the total TERT expression with clinicopathological
features such as tissue type, gender, tumour size, hormonal
tumour type, and recurrence. The Mann-Whitney U test
and Kruskal-Wallis test were used in the comparisons
of numerical data (nuclear/cytoplasmic TERT score
with tissue type, gender, tumour size, hormonal tumour
type, and recurrence). A p value of <0.05 was considered
statistically significant.
Table I: Clinicopathological features of patients
When the patients were grouped according to tumour size, 8 (16.7%) had microadenomas, while the tumour size was larger than 10 mm in 40 (83.3%) of the cases. Among all of the cases, 4 (8.3%) of the tumours recurred after initial surgery, whereas 44 (91.7%) did not. The mean follow-up period for the patients was 57.5±31.07 months. All of the patients were alive.
Comparisons of TERT Expression in Neoplastic and
Nonneoplastic Pituitary Tissues
TERT expression did not significantly differ between
neoplastic and nonneoplastic pituitary tissues. According
to these results, TERT expression was present in 4 (20%) of
20 nonneoplastic autopsy tissues, whereas it was expressed
in 16 (33.3%) of 48 neoplastic tissues. Despite the absence
of statistical significance, mean ranks of cytoplasmic and
nuclear TERT expression was higher in neoplastic tissues
than in nonneoplastic autopsy tissues (p>0.05) (Table II).
Table II: Comparisons of TERT expression in neoplastic and nonneoplastic pituitary tissues
Comparisons of Total (Combined Nuclear and
Cytoplasmic) TERT Expression with Clinicopathological
Features
The results of the comparisons of clinicopathological
features with total (combined nuclear and cytoplasmic)
TERT expression are presented in Table III. TERT
expression did not reveal any statistically significant
relationship with any of the clinicopathological features.
TERT positivity was present in 30.8% (4/13) of somatotroph
adenomas, in 25.0% (2/8) of lactotroph adenomas, in
33.3% (1/3) of gonadotroph adenomas, in 40% (2/5) of
corticotroph adenomas, in none (0/1) of the thyrotroph
adenomas, in 33.3% (3/9) of mixed somatotroph and
lactotroph adenomas, and finally, in 44.4% (4/9) of null
cell adenomas. TERT staining was defined in 50.0% (4/8)
of microadenomas, while it was defined as 30.0% (12/40) of macroadenomas. Total TERT expression was observed
in 75.0% (3/4) of the patients with recurrence, whereas
staining was present in 29.5% (13/44) of the patients
without recurrence.
Comparisons of Nuclear TERT Expression with
Clinicopathological Features
The results of the comparisons of clinicopathological
features with nuclear TERT expression are presented
in Table IV. Nuclear TERT expression did not reveal
any statistically significant relationship with any of the
clinicopathological features. However, mean ranks of
nuclear TERT expression were higher in males than in
females, in mixed somatotroph and lactotroph adenomas,
in lactotroph adenomas, and in adenomas sized >10 mm
(p>0.05).
Comparisons of Cytoplasmic TERT Expression with
Clinicopathological Features
The results of the comparisons of clinicopathological
features with cytoplasmic TERT expression are presented
in Table IV. Cytoplasmic TERT expression had a
significant relationship with recurrence (p=0.035); namely, higher cytoplasmic TERT expression was observed in some
adenomas with recurrence but not in adenomas without
recurrence. Cytoplasmic TERT expression was observed in
3 of 4 patients who had recurred. Nuclear TERT expression
was not observed in any of the patients with recurrence. Two
of these 3 patients were male and 1 was female. Tumours
were sized >10 mm in 2 of the patients and sized ≤10 mm
in 1 patient. Among these 3 patients, hormonal type was
corticotroph adenoma in 1 of the patients, gonadotroph
adenoma in 1, and null cell adenoma in 1. The follow-up
period ranged between 37 months and 256 months.
There was no significant relationship between cytoplasmic TERT expression and clinicopathological features other than recurrence. Although there was no significant relationship, mean ranks of cytoplasmic TERT expression were higher in males than in females, in corticotroph and lactotroph adenomas than in other types, and in microadenomas than in macroadenomas (p>0.05).
Telomerase activity in neoplastic and in nonneoplastic pituitary tissues has been the subject of a few previous studies using various methods of polymerase chain reaction (PCR)[14,21]. The mentioned previous studies have reported that there is no difference in telomerase activity and telomere length between neoplastic and nonneoplastic pituitary tissues. It has been suggested that this may be elucidated by the low mitotic activity of pituitary adenomas[14]. In agreement with previous studies, the present study showed no significant difference in total TERT expression in neoplastic and nonneoplastic pituitary tissues. Although TERT expression was evaluated by immunohistochemistry targeting only TERT and not TERC in the present study and the comparisons showed a lack of statistical significance (p>0.05), TERT expression rates were higher in neoplastic tissues than in nonneoplastic tissues. The most accurate state of telomerase activity and telomere length in pituitary tumours/nonneoplastic pituitary tissues should be investigated in larger study groups by combined synchronous detection methods.
The effects of telomerase activity and telomere lengths on clinicopathological features in pituitary adenomas have been evaluated in several studies[3,14,16,21,23-25]. Some of these papers have concluded that telomerase activity and telomere length do not have any impact on the clinical course of pituitary adenomas based on investigations of telomerase activity and telomere length by various types of polymerase chain reactions[14,16,21,25]. On the other hand, some of the previous studies have suggested that TERT expression is associated with an aggressive clinical course, particularly recurrences and invasiveness[3,23,24]. Harada et al.[24] have reported telomerase activity by using Southern blotting and reverse transcriptase-chain reaction in a pituitary carcinoma evolving in a background of an initially telomerase-negative PRL-producing benign adenoma. Yoshino et al.[23] have reported telomerase activity via PCR-based telomeric repeat amplification protocol (TRAP) assay and PCR enzyme-linked immunosorbent assay (ELISA) in 13% of adenomas with invasive features. Ortiz-Plata et al.[3] have examined telomerase activity by TERT immunohistochemistry and have reported that telomerase activity could be a marker for cellular proliferation, angiogenesis and hormonal activity in pituitary adenomas. However, the authors did not inform either cellular localisation or the degree of TERT expression in their study. In the present study, we could not determine any significant relationship between total TERT expression and clinicopathological features, including presentation age, gender, tumour size, recurrence, and hormonal type. But, we could observe that cytoplasmic staining with TERT polyclonal antibody is significantly related to tumour recurrence, as in the case reported by Harada et al.[24]. Also, total TERT expression was present in 75% of pituitary adenomas with recurrence without any significance. Although the present study has limitations, including the immunohistochemical evaluation of only the TERT catalytic subunit of telomerase protein complex but not TERC or any detections of mutational status, the authors present the significant increase of recurrence in pituitary adenomas with cytoplasmic TERT expression.
According to the current proposals, a panel of some biomarkers, including PTTG1 and MMPs (MMP1), may predict the aggressive behaviour of pituitary adenomas[1]. It is said that higher levels of electron transport system (ETS) transcription factors induce MMP1 expression and cause tumour invasion in pituitary adenomas[1]. One of the most recent reports investigating the TERT expression in malignant melanoma has concluded that mutations in TERT promoter create additional binding sites for ETS transcription factors, particularly ETS1, and finally activates the mitogen-activated protein kinase (MAPK) pathway and cell proliferation[19]. PTTG1 is a member of the securin family and is highly expressed in hormonesecreting invasive pituitary adenomas[1]. Another recent study observing the relationship between PTTG1 and TERT expressions in human mesenchymal stem cells has reported that overexpression of TERT induces PTTG1 expression, and this interaction between TERT and PTTG1 is mediated by Ku70, which is a heterodimeric protein involved in maintenance of telomeres, in an increase in the cell cycle, autophagy, and self-renewal[26].
Consideration of distinct results of the previous studies and the present study about telomerase activity and telomere length in pituitary adenomas and the recent observations revealing interactions of TERT with PTTG1 and ETS1 may require further studies. Such studies should investigate these interactions in pituitary adenomas and may contribute detailed data in the issue of TERT expression as a prognostic predictor in pituitary adenomas.
In conclusion, the present study posits that immunohistochemical expression of TERT does not significantly differ in neoplastic and nonneoplastic pituitary tissues. In addition, pituitary adenomas with cytoplasmic immunohistochemical expression of TERT have significantly higher rates of recurrence. Further studies, including combined methods of immunohistochemistry and molecular analyses in larger groups, may reveal applicable results for the clinical significance of telomerase activity and telomere length in pituitary adenomas.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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