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ORIGINAL ARTICLE - ENDOCRINE TUMORS
Protein Expression of PTTG1 as a Diagnostic Biomarker in Adrenocortical Carcinoma
Minerva Angélica Romero Arenas, MD, MPH1, Timothy G. Whitsett, PhD2, Anna Aronova, MD3,
Samuel A. Henderson, MD1, Janine LoBello, MD2, Mouhammed Amir Habra, MD1, Elizabeth G. Grubbs, MD1, Jeffrey E. Lee, MD1, Kanishka Sircar, MD1, Rasa Zarnegar, MD3, Theresa Scognamiglio, MD3, Thomas J. Fahey, MD3, Nancy D. Perrier, MD1, and Michael J. Demeure, MD, MBA2
1The University of Texas MD Anderson Cancer Center, Houston, TX; 2Translational Genomics Research Institute, Phoenix, AZ; 3Weill Cornell Medical College, New York, NY
ABSTRACT
Background. Adrenocortical carcinoma (ACC) has a poor prognosis and there is an unmet clinical need for biomarkers to improve both diagnostic and prognostic assessment. Pituitary-tumor transforming gene (PTTG1) has been shown to modulate cancer invasiveness and response to therapy. The potential role of PTTG1 protein levels in ACC has not been previously addressed. We assessed whether increased nuclear protein expression of PTTG1 distinguished ACCs from adrenocortical adenomas (ACAs).
Methods. Patients with ACC or ACA were identified from prospective tissue banks at two independent institutions. Two tissue microarrays (TMAs) consisting of adrenal specimens from 131 patients were constructed and clini- cally annotated. Immunohistochemical analysis for PTTG1 and Ki-67 was performed on each TMA.
Results. TMA-1 (n = 80) contained 20 normal adrenals, 20 ACAs, and 40 ACCs, and the validation, TMA-2 (n = 51), consisted of 10 normal adrenals, 14 ACAs, and 27 ACCs. On TMA-1, nuclear staining of PTTG1 was detected in 12 (31%) ACC specimens, while all ACAs and normal adrenal glands were negative for PTTG1. On TMA- 2, 20 (74%) of the ACC tumors demonstrated PTTG1
nuclear staining of PTTG1, and 13 (93%) ACA and 4 (44%) normal adrenal glands were negative for PTTG1. ACC tumors with increased PTTG1 protein staining had a significantly higher Ki-67 index (p <0.001) than those with lower levels of PTTG1.
Conclusions. Increased nuclear protein expression of PTTG1 was observed in malignant adrenal tumors. PTTG1 correlated with Ki-67 in two independent TMAs. PTTG1 is a promising biologic marker in the evaluation of adrenal tumors.
Adrenocortical carcinoma (ACC) is a rare malignancy with an estimated incidence in the US of 0.5-2 cases per 1 million people each year.1 Patients with ACC are often not diagnosed until the advanced stages, with only 50% pre- senting with potentially resectable tumors and up to 70% presenting with metastatic disease.2 Surgical resection is the mainstay of therapy and the only curative therapy for ACC, with complete resection reported to offer a 40% 5-year survival rate.3 No systemic treatment regimen has been proven consistently effective. Although mitotane is the most commonly used agent, its adjuvant use has not been proven to affect overall survival (OS).4 The recently completed FIRM-ACT trial demonstrated a 23% response rate in patients with metastatic ACC, with an associated median OS of approximately 6 months for patients with advanced ACC who underwent treatment with the combi- nation of mitotane with either doxorubicin, etoposide, and cisplatin, and 2 months when treated with mitotane and streptozocin.5
The Weiss system is the most commonly used histopathologic system in clinical practice.º Even using strictly defined criteria, certain features are prone to
Minerva Angélica Romero Arenas and Timothy G. Whitsett contributed equally to this work.
@ Society of Surgical Oncology 2017
First Received: 22 March 2017
N. D. Perrier, MD e-mail: NPerrier@mdanderson.org
individual interpretation and provide diagnostic challenges. Thus, in addition to histological parameters, molecular techniques have been introduced to help distinguish adrenocortical adenomas (ACAs) from ACCs. Proliferation markers such as Ki-67, for example, may help distinguish ACAs from ACCs;7,8 however, especially as the oncoge- nesis of adrenal tumors is not fully understood, additional markers to help distinguish ACCs from ACAs could pro- vide significant clinical utility.
Pituitary-tumor transforming gene (PTTG1/securin) encodes securin, a protein that prevents separins from promoting sister chromatid separation.9,10 PTTG1 is highly expressed across a number of tumor types, and has been shown to modulate cancer-related angiogenesis, metastasis, and therapeutic response.11,12 The finding of elevated expression of PTTG1 in malignant tissues compared with normal tissues has prompted exploration of its potential role in cancer diagnosis and prognosis. Expression of PTTG1 was found to distinguish benign from malignant pheochromocytomas13 and has been associated with more aggressive gastric, esophageal, and brain tumors.14-16 PTTG1 gene expression correlates with poor survival in ACC, and vorinostat decreased securin levels and inhibited cell growth in vitro.17 The potential role of PTTG1 protein levels in ACC has not been previously addressed.
In this study, we conducted a tissue microarray (TMA)- based analysis of PTTG1 nuclear protein levels across normal adrenal tissue, ACA, and ACC. Differential PTTG1 expression levels between ACC and ACA were validated using publicly available messenger RNA (mRNA) expression data. Lastly, PTTG1 protein levels in ACC were correlated with clinical features.
MATERIALS AND METHODS
This research was approved by the Institutional Review Board of each of the collaborating institutions.
Clinical Samples
Patients who underwent ACC, ACA, or non-adrenal pathology (normal adrenal glands resected en bloc as part of an operation for non-adrenal disease, most typically as part of radical nephrectomy for an adjacent renal cancer) were identified from prospective tissue banks at two independent institutions. Clinical information was retrieved from the patients’ medical record including, but not limited to, de-identified demographic data (age, sex, and race/ ethnicity), laboratory data, medical history, family history, disease status, treatment response, pathology report, and survival duration.
Histopathologic Analysis
Samples were procured from formalin-fixed paraffin- embedded (FFPE) tissue blocks stored in the Department of Pathology at each institution. Hematoxylin and eosin (H&E)-stained slides were reviewed by a pathologist (SH and KS for TMA-1, and TS and AA for TMA-2) from each case to confirm the diagnosis from the adrenalectomy as ACC, ACA, or normal. Ki-67 staining was graded and Weiss scores were assigned to the ACC specimens.18 Tumor and normal adrenal tissue sites were selected on each donor FFPE block and circled with a marking pen, from which tissue cores (two 1.0 mm cores for TMA-1, and three 0.6 mm cores for TMA-2) were taken and transferred into the recipient TMA block by using the Beecher Manual Tissue Microarrayer Model MTA-1 (Beecher Instruments, Inc., Sun Prairie, WI, USA). Two TMAs were constructed (one per clinical institution) using paraffin cassette blocks, with the distance ratio between each core, row, and column set at 1.5 mm. A tissue marker was included in each block to aid in the orientation of the block according to the supplied template map.
Immunohistochemistry
Immunohistochemical (IHC) analysis for PTTG1 was performed on each TMA at the research institution. TMA blocks were sectioned at 5-um thickness using water flotation for tissue section transfer, and dried overnight at room temperature. The slides were dewaxed, rehydrated, and antigen retrieved on-line on the BondMax™M auto- stainer (Leica Microsystems, Inc., Bannockburn, IL, USA). All TMA slides were subjected to heat-induced epitope retrieval using an EDTA-based retrieval solution for 20 min. Endogenous peroxidase was blocked. Both TMAs were incubated for 30 min with PTTG1 (Abcam, Cam- bridge, MA, USA) at 1 ug/ml for TMA-1 and 5 ug/ml for TMA-2, because the lower concentration did not provide good staining on TMA-2. The lowest possible antibody concentration was used. The sections were visualized using the Bond™M Polymer Refine Detection kit (Leica Microsystems, Inc.), with diaminobenzidine chromogen as substrate. A scoring system for each chromophore com- prising of staining intensity and extensiveness captured the outcome-0, negative; 1, weak; 2 moderate; 3, strong19_ and was determined by a board-certified pathologist (JL) blinded to the diagnoses and clinical outcomes.
Oncomine Data Analysis
mRNA expression of PTTG1 was performed using the Oncomine database,20 and the Giordano Adrenal 2 dataset was employed. In this dataset, gene expression was
determined by a high-density oligonucleotide array (Hu- man Genome U133 Plus 2.0; Affymetrix, Santa Clara, CA, USA), as previously described.21 Expression of PTTG1 in normal adrenal (n = 10), ACA (n = 22), and ACC (n = 33) is expressed as log2 median-centered intensity, with differences between histologic groups determined by Student’s t test in Oncomine. A p value < 0.05 was con- sidered statistically significant.
Statistics
Correlation between PTTG1 IHC score and clinical outcomes was determined using Pearson correlation coef- ficients, with a p value of 0.05 regarded as statistically significant. Differential Ki-67 index between PTTG1 absent (IHC 0) versus PTTG1 present (IHC score 1-3) was determined using Student’s t test, with a p value < 0.05 considered statistically significant. ACC detection rates for PTTG1 expression (IHC score 0 vs. 1-3) were computed and summarized using frequencies and percentages for each tissue microarray (TMA-1 and TMA-2). Assessments of accuracy of ACC diagnosis by PTTG1 expression were determined using sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) statis- tics, along with corresponding 95% exact confidence intervals (CIs).
OS was available for patients in TMA-1 and computed from the date of diagnosis to the deceased date; patients alive at the last follow-up date were censored. The Kaplan- Meier method was used to estimate OS, and the log-rank test was used to assess differences between groups. In addition, the association between OS and groups of interest was determined using univariate Cox proportional hazards regression models. All statistical analyses were performed using SAS 9.4 for Windows (Copyright @ 2002-2012 by SAS Institute Inc., Cary, NC, USA).
RESULTS
The first TMA (TMA-1, n = 80) included samples from 20 normal adrenal, 20 ACA, and 40 ACC specimens (Table 1). TMA-1 consisted of patients in a 50:50 female- to-male ratio, with a median age of 56 years. Fifty-eight percent of patients with ACC represented on the array were still alive at the time of last follow-up. The second TMA (TMA-2, n = 51) was used as a validation set and con- sisted of 10 normal adrenals, 14 ACAs, and 27 ACCs (Table 2). TMA-2 consisted of a 51:49 female-to-male ratio, with a median age of 53 years. Thirty-six percent of patients with ACC represented on TMA-2 were still alive at the time of last follow-up.
The protein expression of PTTG1 was determined by IHC on both TMA-1 and TMA-2. Figure 1 shows repre- sentative PTTG1 staining of normal adrenals, ACAs, and ACCs on TMA-1 and TMA-2. On TMA-1, nuclear protein expression of PTTG1 was detected in 31% of ACC spec- imens, whereas it was not detected in any of the ACA or normal adrenal gland specimens (Table 3). On TMA-2, nuclear protein expression of PTTG1 was observed in 74% of ACC tumors, and detected in 1 (7%) of the ACA specimens and 5 (56%) of the normal adrenal gland spec- imens (Table 4). Figure 1 demonstrates representative images of the PTTG1 staining from each of the adrenal sample groups in the two TMAs. Measures of diagnostic accuracy (ability to detect ACC vs. ACA) for TMA-1 and TMA-2 were as follows: sensitivity of 0.31 and 0.74, specificity of 1 and 0.93, PPV of 1 and 0.95, and NPV of 0.43 and 0.65, respectively.
To further confirm the difference in PTTG1 expression between ACC, ACA, and normal adrenal glands, mRNA expression was determined from a publicly available microarray database dataset. The Giordano Adrenal 2 dataset, consisting of 10 normal adrenal, 22 ACA, and 33 ACC specimens was employed.21 As shown in Fig. 2, the mRNA expression level of PTTG1 was significantly higher in ACC tumors compared with normal adrenal glands (p <0.00001), whereas the expression of PTTG1 was comparable between ACA and normal adrenal glands.
Lastly, we explored correlations between nuclear PTTG1 protein expression and clinical parameters. On both TMAs, elevated nuclear protein expression of PTTG1 (IHC score of 2 or 3) was significantly associated with higher Ki- 67 index (p = 0.0008 and p = 0.0053, respectively) compared with lower PTTG1 IHC scores (0 or 1) (Fig. 3). PTTG1 nuclear protein expression did not correlate with TP53 protein expression, tumor size, age at diagnosis, Weiss score, or metastasis on either of the TMAs. In TMA- 1, OS was assessed for ACC patients by PTTG1 IHC score (0 vs. 1-3). The median OS for patients in both groups was 84.3 months. There was no significant association between OS and PTTG1 expression [hazard ratio (HR) 0.74, 95% CI 0.23-2.37; p = 0.62] or between OS and Ki-67 (HR 1.44, 95% CI 0.80-2.60; p = 0.23).
DISCUSSION
In this study, we demonstrated that nuclear PTTG1 protein expression, assessed by IHC, could distinguish ACCs from ACAs and normal adrenal glands. In two independent TMAs, nuclear expression of PTTG1 was observed in 31 and 75% of ACC tumor specimens, and in 0 and 7% of ACA specimens. Elevated nuclear expression (IHC score 2-3) was observed in 16 and 59% of ACC
| Variable | Pathology | Total | ||
|---|---|---|---|---|
| Adenoma | Normal | ACC | ||
| Sex | ||||
| Male | 9 (45%) | 13 (65%) | 18 (45%) | 40 (50%) |
| Female | 11 (55%) | 7 (35%) | 22 (65%) | 40 (50%) |
| Race/ethnicity | ||||
| White | 15(75%) | 13 (65%) | 32 (80%) | 60 (75%) |
| Hispanic | 4 (20%) | 5 (25%) | 7 (18%) | 16 (20%) |
| Other | 1 (5%) | 2 (10%) | 1 (2%) | 4 (5%) |
| Vital status | ||||
| Alive | 19 (95%) | 6 (30%) | 23 (58%) | 48 (60%) |
| Dead | – | 14 (70%) | 15 (37%) | 29 (36%) |
| Median age at ADX | 51 (31-83) | 60 (39-85) | 53 (20-87) | 55 (20-87) |
| Follow-up in months | 10 (0-73) | 10 (1-51) | 42 (0-143) | 22 (0-143) |
| Laterality | ||||
| Left | 12 | 10 | 18 | 40 |
| Right | 8 | 10 | 22 | 40 |
| Mitotane therapy | ||||
| Yes | N/A | N/A | 16 | |
| No | 7 | |||
| Total | 20 | 20 | 40 | 80 |
| Variable | Pathology | Total | ||
|---|---|---|---|---|
| Adenoma | Normal | ACC | ||
| Sex | ||||
| Male | 7 (50%) | 5 (50%) | 13(48%) | 25 (49%) |
| Female | 7 (50%) | 5 (50%) | 14 (52%) | 26 (51%) |
| Race/ethnicity | ||||
| White | 6 (43%) | 4 (40%) | 9 (33%) | 19 (37%) |
| Hispanic | 1 (7%) | – | – | 1 (2%) |
| Other | 7 (50%) | 6 (60%) | 18 (67%) | 31 (61%) |
| Vital status | ||||
| Alive | 14 (100%) | 9 (90%) | 8 (36%) | 31 (67%) |
| Dead | – | 1 (10%) | 13 (59%) | 14 (30%) |
| Median age at ADX | 47 (21-73) | 57 (37-73) | 53 (19-77) | 53 (19-77) |
| Follow-up in months | 33 (1-121) | 18 (1-171) | 16 (0-246) | 21 (0-246) |
| Laterality | ||||
| Left | 7 | 6 | 14 | 27 |
| Right | 7 | 4 | 11 | 22 |
| Mitotane therapy | ||||
| Yes | N/A | N/A | 8 | |
| No | 5 | |||
| Total | 14 | 10 | 25 | 49 |
TMA-1
TMA-2
Normal IHC Score 0
ACA IHC Score 0
ACC
IHC Score 3
| IHC score NUC | Normal (n = 20) | ACA (n = 20) | ACC (n = 39) |
|---|---|---|---|
| 0 | 20 (100%) | 20 (100%) | 27 (69%) |
| 1 | 0 | 0 | 6 (15%) |
| 2 | 0 | 0 | 1 (3%) |
| 3 | 0 | 0 | 5 (13%) |
| IHC score NUC | Normal (n = 9) | ACA (n = 14) | ACC (n = 27) |
|---|---|---|---|
| 0 | 4 (44%) | 13 (93%) | 7 (26%) |
| 1 | 5 (56%) | 1 (7%) | 4 (15%) |
| 2 | 0 | 0 | 8 (29.5%) |
| 3 | 0 | 0 | 8 (29.5%) |
tumor specimens, whereas ACA and normal adrenal did not demonstrate elevated nuclear expression of PTTG1. The potential of using PTTG1 in distinguishing ACCs from ACAs is suggested by the measures of diagnostic accuracy; however, this would be better supported if the studies could be repeated with IHC at the same concentration. Through a query of a publicly available microarray database, we further observed that the mRNA levels of PTTG1 were
TMA-2 (bottom). A high-powered magnification (×20) of the ACC samples are included in the panels on the right. IHC immunohisto- chemistry, PTTG1 pituitary-tumor transforming gene, TMAs tissue microarray, ACC adrenocortical carcinoma
significantly higher in ACCs compared with ACAs and normal adrenal specimens. Lastly, our data showed a cor- relation between increased PTTG1 staining and Ki-67 proliferation index, a known prognostic indicator in ACC.
Overexpression of PTTG1 has been observed across a number of tumor types, and is associated with poor prog- nosis, migratory potential, and therapeutic resistance.22 In this study, nuclear protein expression of PTTG1 was ele- vated in up to 16-59% of ACC specimens across two separate TMAs, by IHC. While the relatively small sample size limits conclusions regarding differences in relative PTTG1 expression across our two TMAs, it is possible that differences in PTTG1 staining between the two TMAs could be the result of differences in tissue banking, the higher proportion of deceased patients in the TMA-2 group, the different antibody concentrations that were optimized for each of the two TMAs, or differences in the ACC tumors themselves. Given the rarity of disease, combining tissue samples from different tissue banks was necessary to maximize numbers for validation of data. Optimization of the antibody concentration was required as the concentration used on TMA-1 did not provide good staining on TMA-2, although it was clear that normal and ACA samples never showed strong protein staining of PTTG1. Elevated nuclear protein expression of the 29 normal adrenal samples and 34 ACA specimens across the two TMAs was not observed.
2.0
**
·
1.5
PTTG1-Log2
median-centered intensity
1.0
0.5
·
·
·
0
Normal
ACA
ACC
Overexpression of PTTG1 in tumor tissues compared with normal tissues has been observed in numerous tumor types.22 In pheochromocytomas, PTTG1 protein expression distinguished benign from malignant tumors.13 While PTTG1 protein can be detected in both nuclear and cytosolic fractions, our data suggest that nuclear staining is more predictive of ACC versus ACA/normal adrenal. In brain tumors, Salehi et al. found that nuclear protein expression of PTTG1 was higher than cytoplasmic PTTG1 in high-grade tumors.16 Thus, nuclear protein expression of PTTG1 might be associated with aggressive, high-grade ACC tumors.
Current clinical methodology uses a number of histolog- ical and molecular features to distinguish ACCs from ACAs, with the Weiss system being the most widely accepted.18
Subjectivity of certain histological parameters and cases with biologic and clinical behavior defying Weiss score have prompted investigations into other parameters for distin- guishing ACCs from ACAs. Ki-67, a well-established marker of cell proliferation, has been used as a diagnostic marker in adrenal tumors.8 In our study, elevated nuclear protein expression of PTTG1 was only highly expressed in ACCs compared with ACAs, and positively correlated with Ki-67 index. Both Ki-67 and PTTG1 have been proposed as markers to distinguish malignant from benign thyroid tumors.23 In addition, higher Ki-67 has recently been asso- ciated with shorter ACC patient survival.24 We previously demonstrated that elevated PTTG1 mRNA expression sig- nificantly correlated with shorter median ACC patient survival.17 It is a limitation of this study that the available survival data were insufficient to demonstrate a survival difference based on PTTG1 expression. A larger study looking at PTTG1 protein expression and ACC patient sur- vival should be completed. This study is also based on a series of surgical specimens, therefore it is weighted toward earlier-stage tumors or cases with resectable oligometastatic disease. Thus, these patients may experience a better prog- nosis than those with more advanced, unresectable ACC. Nonetheless, protein expression of PTTG1 on a breast cancer TMA was associated with aggressive disease and shorter survival.25
In addition to having diagnostic potential in adrenal tumors, expression of PTTG1 may have therapeutic impli- cations. The association of PTTG1 expression with therapeutic response has been observed in several tumor types.22 PTTG1 expression was associated with gefitinib resistance in cancer cell lines in vitro,26 and depletion of PTTG1 sensitized colon cancer cell lines to apoptosis induced by fisetin.27 Our group demonstrated that vorinostat,
PTTG1 protein expression positively correlates with Ki67 index in ACC.
A
80
p=0.0053
60
Ki67 Index
40
20
0
0-1
2-3
PTTG1 IHC Score
B 40
p=0.0008
30
Ki67 Index
20
10
0
0-1
2-3
PTTG1 IHC Score
a histone deacetylase inhibitor, could suppress protein levels of PTTG1 in ACC cell lines and suppress cell growth.17 Thus, PTTG1 overexpression may have a prognostic value, and suppression of PTTG1 expression could have thera- peutic implications for patients with ACC and other cancers.
CONCLUSIONS
Increased nuclear protein expression of PTTG1 was observed in ACC samples using immunohistochemistry in two independent TMAs. Elevated nuclear protein expres- sion of PTTG1 was only observed in ACC and never in ACA or normal adrenal glands. Overexpression of PTTG1 in ACC samples compared with ACA and normal adrenal glands was confirmed in mRNA expression data. Further- more, ACC samples with increased nuclear PTTG1 staining demonstrated higher Ki-67 proliferation index. PTTG1 may have clinical value as a prognostic marker distinguishing ACC from ACA.
ACKNOWLEDGMENTS The authors would like to acknowledge Ms. Denái R. Milton for her review of the statistical analyses. Support for Minerva A. Romero Arenas was provided in part by the Golfers Against Cancer and the Dupre Research Fellowship in Surgical Endocrinology. The authors would like to acknowledge support provided by the ATAC Research Fund and the Kristen’s Legacy Fund.
DISCLOSURES Michael J. Demeure is a paid consultant and has received research support from Arbutus Biopharma Corporation. Minerva Angélica Romero Arenas, Timothy G.Whitsett, Anna Aro- nova, Samuel A. Henderson, Janine LoBello, Mouhammed Amir Habra, Elizabeth G. Grubbs, Jeffrey E. Lee, Kanishka Sircar, Rasa Zarnegar, Theresa Scognamiglio, Thomas J. Fahey, and Nancy D. Perrier have no conflicts of interest to disclose.
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