Introduction
Cushing’s disease, caused primarily by pituitary corticotroph adenomas, is associated with substantial morbidity and mortality, mainly due to cardiovascular and infectious complications [30]. In most patients with Cushing’s disease, transsphenoidal tumour removal can induce remission (~80% for microadenomas, ~60% for macroadenomas) [31], but about 20% of patients have tumour recurrence in the long term [3,5,13,16,43,45]. These patients require second-line treatment, such as neurosurgical procedures, radiation therapy, or chemotherapy with temozolomide or other agents [32,35]. However, choosing a particular second-line treatment remains difficult due to a lack of reliable clinical or ancillary criteria.
In patients with pituitary tumours, the expression of O-6-methylguanine-DNA methyltransferase (MGMT) is a prognostic factor of treatment outcomes: low MGMT tumour expression has been linked to a high recurrence risk of these tumours [9,11,22]. MGMT is a DNA repair enzyme that restores mutagenic changes at position O-6 of methylguanine [18]. Loss of MGMT expression increases carcinogenic risk with many tumour types such as gliomas, lymphomas, or breast cancers [14]. In the central nervous system, MGMT is typically expressed by normal tissue, but primary brain tumours often lack MGMT expression [39].
MGMT expression is also important from a therapeutic standpoint because this enzyme reduces the effectiveness of alkylating chemotherapeutics, including temozolomide [38,40]. This type of treatment acts via induction of mutations at position O6 of guanine [17]. Consequently, low MGMT expression has been also associated with a good response to temozolomide treatment [1,4,21,24,41].
Assessment of MGMT expression in corticotroph pituitary adenomas can help identify patients who are most likely to benefit from temozolomide treatment. However, current evidence on the role of MGMT expression in corticotroph adenomas is scarce. Therefore, we analysed the relationship of MGMT expression in corticotroph tumours with clinical and pathological variables among patients who underwent transsphenoidal pituitary surgery.
Material and methods
Study design and cohort
We included all consecutive patients with Cushing’s disease, who underwent transsphenoidal surgery between 2012 and 2018 due to corticotroph pituitary adenomas. There were 72 patients who fulfilled the inclusion criteria (83% female; mean age ±SD: 44.15 ±15.15 years). All operations were done by the same neurosurgeon according to the same protocol. Details of all operative procedures were taken from operative records. A direct transnasal transsphenoidal microscopic approach was performed for selective resection of pituitary adenoma. The study was retrospective, but all data were acquired prospectively during the study period. The study was approved by the local Bioethics Committee.
Clinical and ancillary assessments
The diagnosis of Cushing’s disease was made according to standard criteria [29]. Tumour size was measured on magnetic resonance images (GE Signa, 1.5 Tesla) in the coronal, sagittal, and axial dimensions. The greatest of the three was taken as the maximum tumour diameter, in line with the RECIST criteria. A macroadenoma was defined as a tumour with a maximum dimension ≥ 10 mm. Tumour invasiveness of macroadenomas was assessed according to the Knosp scale [19].
Histopathological assessments
Histopathological examinations of specimens obtained during surgery were performed in accordance with the 2017 WHO classification [23]. Tissue samples, fixed in 10% buffered formalin and embedded in paraffin, were routinely stained with haematoxylin and eosin. Immunohistochemical staining was performed according to the labelled EnVision Flex Visualization System (K8000, Dako/Agilent) with 3,3’-diaminobenzidine as the chromogen. Slides with paraffin-embedded tumour samples were immunolabeled with antibodies against the following antigens: growth hormone (cat. no. M5-1328-P1), prolactin (cat. no. M5-1362-P1), ACTH (cat. no. M5-1452-P1), -TSH (cat. no. M5-1453-P1), -FSH (cat. no. M5-1449-P1), -LH (cat. no. MS-1448-P1), and MGMT (clone MT 23.2, cat. no. MA3-16537); all antibodies from Thermo Fisher Scientific. Moreover, the antibodies against the glycoprotein -subunit (cat. no. MCA 1026, Bio-Rad, UK), p53 protein (cat. no. IR 616, DO-7), and Ki-67 (MIB-1 clone, cat. no. IR626), purchased from DAKO/Agilent, were used. MGMT expression was graded in four categories: < 25%, 25-50%, 50-75%, and > 75% of cells; the Ki-67 labelling index was graded in three categories: < 3%, 3-10%, and > 10% positive nuclei; p53 expression was graded in three categories: < 5%, 5-50%, and > 50% positive nuclei; and mitotic index was graded in two categories: ≤ 2 and > 2 mitoses per 10 high-power fields.
Ultrastructural examination was performed with a Philips CM120 BioTWIN transmission electron microscope. Corticotroph adenomas were classified on the basis of commonly accepted ultrastructural features as densely granulated or sparsely granulated.
Hormonal assessments and remission criteria
Immediate postoperative remission was ascertained when the nadir serum cortisol concentration at 06:00 h was ≤ 2.5 µg/dl on the first or second postoperative day, as described previously [47]. We used the following criteria for remission at six months after surgery: clinical and biochemical evidence of adrenal insufficiency or, when adrenal function was preserved, biochemical evidence of cortisolaemia: urinary free cortisol, morning serum cortisol, and plasma ACTH levels within their respective reference ranges; preservation of the circadian rhythm of serum cortisol; and serum cortisol after overnight dexamethasone suppression test ≤ 1.8 µg/dl.
ACTH concentrations were measured with an immunoradiometric assay (ELSA-ACTH, CIS Bio International, Gif-sur-Yvette Cedex, France; analytical sensitivity, 2 pg/ml; reference range, 10-60 pg/ml). Serum cortisol concentrations were measured with an electrochemiluminescence immunoassay (Elecsys 2010, Roche Diagnostics; analytical sensitivity, 0.02 µg/dl; reference range, 6.2-19.4 µg/dl).
Statistical analysis
Descriptive statistics were used in accordance with data distribution. The Mann-Whitney test was used for comparisons between two groups. The Jonckheere-Terpstra trend test or analysis of variance (ANOVA) were used to study the relationship between the expression of MGMT (categories) and continuous variables. Relationships between categorical variables were tested with the 2 test or the Fisher exact test. Univariate and multivariate logistic regressions were used to find significant predictors of remission. Dependent variables were treated as ordered in the models. A value of p < 0.05 was considered statistically significant. All calculations were completed in IBM SPSS software (version 25).
Results
Patients
Among 72 patients included in the study remission was ascertained in 44 (61%) patients immediately after surgery, and in 47 (65%) patients at 6 months. The remaining cohort characteristics are shown in Table I.
MGMT expression and its relationship with clinicopathological variables
MGMT expression was > 75% in half of all tumours, < 25% in 14 tumours, and 25-50% or 50-75% in 11 tumours, respectively. Table II shows the expression of the remaining histopathological markers.
Tumours in men had a lower MGMT expression that those in women (p = 0.004). There was no significant relationship between MGMT expression and age (p = 0.217).
Higher MGMT expression was associated with a lower tumour diameter (p = 0.001; Fig. 1) and lower ACTH concentrations at 8:00 (p = 0.002; Fig. 2). There was no significant relationship between MGMT expression and morning serum cortisol concentrations (p = 0.177) or Knosp grade in corticotroph macroadenomas (p = 0.466). Higher MGMT expression was related to lower p53 expression (p = 0.026) and higher frequency of DG-ACTH tumours on electron microscopy (p = 0.009). There was a tendency toward a negative relationship of MGMT expression with the expressions of Ki-67 (p = 0.099) and the number of mitotic figures (p = 0.096).
Among all the histopathological markers analysed, only MGMT expression was significantly related to early clinical remission (p = 0.005; Table III), which was also confirmed after an adjustment for the ultrastructure category in stepwise logistic regression (p = 0.043). This relationship remained a statistical trend after adjustment for tumour size (p = 0.074, Table IV).
Discussion
This study confirmed that MGMT is a promising prognostic marker for patients undergoing surgical removal of corticotroph pituitary adenomas. We observed that immediate remission was more likely in patients with corticotroph tumours with high MGMT expression. Moreover, higher MGMT expression was associated with lower tumour aggressiveness assessed in ancillary studies.
Our cohort comprised patients with typical features of Cushing’s disease. The mean age and sex ratio in our cohort were similar to previous studies among patients with corticotroph tumours [29,30]. The remission rate in our cohort (~60%) was below the median from over 40 studies (~80%), probably because the proportion of macroadenomas among our patients was high [33].
Our study provides evidence on the expression of MGMT among a non-selected cohort of patients with corticotroph adenomas. Half of all corticotroph tumours in our study had high MGMT expression (> 75% of cells). This finding strongly suggests that temozolomide would not be appropriate as a first-line treatment. Temozolomide seems better as a second-line treatment, after surgery, because recurring tumours tend to have low MGMT expression and are more susceptible to this type of chemotherapy [32]. In contrast to our study, Salehi et al. found that nearly a half of corticotroph pituitary adenomas had low MGMT expression (< 10% of cells) [36]. In that study, however, a third of all tumours were Crooke cell adenomas, which are more aggressive than other corticotroph tumours. In another study, nearly 80% of corticotroph pituitary tumours had low MGMT expression, but the scoring method was different than in our study [45].
In line with previous research, we did not find any significant relationship between MGMT expression and age [9]. We found that MGMT expression was lower in men than in women. Similarly, in the study of Salehi et al., MGMT expression > 25% was seen among women only, although those authors concluded that there was no relationship between MGMT expression and sex [41]. In contrast, this relationship was not significant for other pituitary tumour studies [9]. We also found that higher MGMT expression was significantly associated with lower tumour diameter and hormone production. This finding may be specific for ACTH-producing adenomas [41] because it has not been reported in other pituitary tumours [9]. MGMT expression was not related to cavernous sinus invasion among our patients. Other factors, such as mediators of angiogenesis, might be implicated [25].
We found that increased MGMT expression was associated with a low expression of established histopathological markers of aggressiveness of pituitary tumours. In our cohort, higher MGMT expression was related to lower Ki-67 expression, which is associated with increased invasiveness of pituitary adenomas [27,33]. Similarly, higher MGMT expression was found in tumours that had low p53 expression and a lower number of mitotic figures, which both indicate a low tumour proliferation rate [15,26]. Moreover, higher MGMT expression was more likely in densely granulated tumours, which tend to be microadenomas with a low recurrence rate [10]. We confirmed previous reports that high MGMT expression was a favourable indicator of remission after surgery. In our study, MGMT expression was the only histopathological marker that significantly predicted remission.
Our findings support the use of temozolomide after failure of surgical treatment when MGMT expression is low. Although most corticotroph adenomas seem to have high MGMT expression, tumours that recur after surgery are more likely to have low MGMT expression and therefore be susceptible to temozolomide [41,49]. Several groups showed that temozolomide was effective among patients with aggressive pituitary tumours, including corticotroph adenomas [1,4,7,8,12,19,21,24,41]. Based on such findings, the European Society of Endocrinology recommends temozolomide monotherapy as the first-line chemotherapy for aggressive pituitary tumours or carcinomas after failure of standard therapies [35]. Because the response to temozolomide among patients with recurrent pituitary tumours in not universal, we need to find additional prognostic factors. It seems that MGMT might be such a factor. Several reports indicated that low MGMT expression is associated with a favourable response to temozolomide treatment [25]. One group suggested that MGMT expression in < 50% of cells was a predictor of successful temozolomide treatment among patients with atypical pituitary adenomas or pituitary carcinomas [2]. However, in a study among 7 patients with aggressive pituitary tumours, MGMT tumour expression did not predict the outcomes of temozolomide treatment [6]. From a practical standpoint, it seems that one trial course of temozolomide could help establish its efficacy, because first-course non-responders are unlikely to respond to further courses [7,36].
The limitations of our study need to be mentioned. First, the design was retrospective, which could bias data collection. However, the data were taken from a prospectively collected database maintained in our department. Moreover, all surgeries and assessments were done according to the same protocol. Second, we could not investigate how MGMT expression was related to the outcomes of temozolomide treatment.
The strengths of our work include a relatively large number of patients with corticotroph pituitary adenomas. Additionally, we assessed the relationship between MGMT tumour expression and other proliferation markers and laboratory examinations.
In conclusion, our investigation confirmed the value of MGMT expression in predicting the outcomes of transsphenoidal surgery for Cushing’s disease. Moreover, we observed that low MGMT expression was associated with increased aggressiveness of corticotroph tumours. Temozolomide seems to be a suitable treatment for recurring and invasive corticotroph tumours, but more studies are needed to establish the criteria for its use in Cushing’s disease.
Acknowledgements
The authors thank the clinical staff and patients who participated in the study. Medical editorial assistance was provided by Proper Medical Writing; funding for this support was provided by the Medical University of Warsaw.
Funding
The study was supported by the Warsaw Medical University and Military Institute of Medicine, Warsaw, Poland.
Data availability
Data are available upon request.
Ethical approval
The study was approved by the local Bioethics Committee and conducted in accordance with the International Conference on Harmonisation Guideline for Good Clinical Practice and the ethical principles of the Declaration of Helsinki.
Disclosure
The authors declare no conflict of interest.
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