Steroid Receptor Coactivator-1 Expression in Pheochromocytoma: Clinicopathologic Correlation and Potential Diagnostic Pitfall
Meng-Jun Xiong, MD* and Adeboye O. Osunkoya, MD **¿ §
Abstract: Pheochromocytoma is a relatively uncommon tumor, and the histomorphologic and biochemical features that may portend malignant behavior have poor overall consensus across various proposed classification systems. Steroid receptor coactivator-1 (SRC-1) is a nuclear protein that mediates transcriptional activity. Current diagnostic applications of SRC-1 are limited, and include distinguishing adrenocortical carcinoma (ACC) from renal cell car- cinoma, and other mimickers. SRC-1 expression in pheochromo- cytoma has not been previously studied. Pheochromocytoma cases were retrieved from our Urological Pathology database and expert consultation files of the senior author, from 2015 to 2019. Clin- icopathological data were obtained. SRC-1 expression was scored systematically. Thirty-eight cases were included, with a female pre- dominance, and a mean age of 52 years (range, 16 to 75 y). Seven patients had heritable mutations including RET (n=3), VHL (2), SDHB (1), and ATM and PDGFRA (1). Two patients developed clinical metastasis, who individually had ATM and PDGFRA mu- tations, and SDHB p.V140F mutation. All heritable tumors were positive for SRC-1, including diffuse/strong staining and intensity in the VHL cases, and diffuse staining with variable intensity in RET cases. Diffuse positivity was seen in most of our heritable cases, providing evidence for a putative link between RET and down- stream SRC-1 signaling. An inverse relationship was observed be- tween SRC-1 expression and Pheochromocytoma of the Adrenal Gland Scaled Score/tumor size, suggesting that SRC-1 phenotype may become muted in pheochromocytomas that have malignant potential. SRC-1 expression in aggressive pheochromocytomas, may also be a potential diagnostic pitfall in view of the fact that these tumors may be misinterpreted as ACC in the primary or metastatic setting.
Key Words: SRC-1, pheochromocytoma, ACC, genetics, mutations, pitfalls
(Appl Immunohistochem Mol Morphol 2019;00:000-000)
Received for publication July 16, 2019; accepted September 15, 2019.
From the Departments of *Pathology; ¡ Urology, Emory University School of Medicine; §Winship Cancer Institute of Emory University, Atlanta; and įDepartment of Pathology, Veterans Affairs Medical Center, Decatur, GA.
Parts of this study were presented at the 2019 USCAP Meeting in National Harbor, MD.
The authors declare no conflict of interest.
Reprints: Adeboye O. Osunkoya, MD, Department of Pathology, Emory University School of Medicine, Suite H174, 1364 Clifton Road, NE, Atlanta, GA 30322 (e-mail: adeboye.osunkoya@emory.edu).
Copyright @ 2019 Wolters Kluwer Health, Inc. All rights reserved.
P heochromocytoma is a relatively uncommon tumor derived from the chromaffin cells of the adrenal medulla. The tumor has been historically named adrenal para- gangliomas as they share the same functionality and histo- morphologic features as extra-adrenal paragangliomas located along the sympathetic chain. Currently, the WHO Classification of Tumors of Endocrine Organs maintains the definition of a malignant pheochromocytoma from the prior third edition, that is, the requirement for the clinical pres- ence of metastasis or recurrence.1 A subtle difference is that a metastasis is more narrowly defined as the presence of tumor outside of the native adrenal organ, such as to lymph nodes. Currently, putative histomorphological and bio- chemical features that reliably predict malignant tumor bi- ology do not have general consensus and are not consistently applied. Several seminal classification schemes have attempted to address this deficit. The largest case series to date examining potential high-grade features of these tumors include comparative studies of adrenal and extra- adrenal paragangliomas by Linnoila et al2 and Grading system for adrenal pheochromocytoma and paraganglioma (GAPP) by Kimura et al.3 A retrospective study of pheo- chromocytoma exclusively, by Thompson,4 examined 100 pheochromocytomas, and developed a scaled score coined Pheochromocytoma of the Adrenal Gland Scaled Score (PASS). Of the 100 cases, a cutoff PASS score of ≥4 cor- rectly identified all patients (n =33) who went on to develop clinical metastasis, and it was observed that all of the pa- tients (comprising half of the cohort of 100) with malignant histology had a score of ≥4.
Steroid receptor coactivator-1 (SRC-1) has emerged as a novel immunohistochemical marker. This nuclear marker is also known as nuclear receptor coactivator 1, and binds to nuclear receptors that mediate transcriptional activity with a variety of nuclear binding partners including progesterone receptor, estrogen receptor, glucocorticoid receptors, reti- noids, thyroid hormone, and prostanoids.5 SRC-1 also forms coactivation complexes with transcriptional factors such as STAT3, STAT5A, STAT5B, and STAT6.6 The diagnostic applications of SRC-1 have not been thoroughly investigated. SRC-1 has been demonstrated to be a sensitive marker for adrenocortical carcinoma (ACC).7 Furthermore, SRC-1 has high sensitivity as a marker of adrenal origin in the 80% to 90% range for normal adrenal, adenoma, and ACC, excluding oncocytic adrenal tumors (sensitivity of 50%), but has a low specificity. Thus, a triple combination
panel including 2 of 3 markers such as CAIX, PAX8, or RCC and SRC-1 or Melan A reliably distinguishes meta- static renal cell carcinoma to the adrenal from ACC.8 Our study is the first to investigate SRC expression in pheo- chromocytoma, with emphasis on correlation with clin- icopathologic and prognostic features.
MATERIALS AND METHODS
A search was made through our Urologic Pathology files and expert consultation files of the senior author for cases of pheochromocytoma from 2015 to 2019. Only cases with available tissue blocks were included in the study. Extra- adrenal paragangliomas were excluded. All tumors were re- reviewed and clinicopathologic parameters were documented, including results of clinical/genetic work-up for heritable diseases performed by the patients’ physicians/genetic coun- selors. Immunohistochemical stain for SRC-1 (cell signaling; monoclonal antibody 128E7; 1:100 dilution) was performed on a representative tissue block of all cases, with appropriate controls. In all tumors, the immunohistochemical stains were scored in a stepwise fashion. Positive staining was defined as nuclear staining in viable tumor cells. The pattern of positive staining was scored as focal or diffuse, and the intensity was scored dichotomously as weak or strong. Results of im- munohistochemical stains for SDHB previously performed on cases during sign-out was documented. This study was completed following the guidelines and with approval from the Institutional Review Board of our institution.
RESULTS
Thirty-eight cases of pheochromocytoma were included in this study. There were 26 female and 12 male patients with a mean age of 52 years (range, 16 to 75 y). The most common presenting symptoms, with overlap in some cases, included: hypertension (n=16), headache (6), palpitations (8), and an incidental finding on imaging for other conditions (18). Over 50% of our cohort under 40 years of age (n=5/9) had a heritable mutation, and the heritable mutations encountered were: RET (n=3), VHL (2), SDHBp.V140F (1), and ATM and PDGFRA (1). Four patients had bilateral disease. Two patients, both of whom harbored a heritable mutation, in ATM and PDGFRA, and the other patient in SDHB p. V140F, developed clinical metastasis at 8 years and 14 years of follow-up, respectively. About 40% of patients were loss to follow-up by 6 months. Just over 50% (20/38) of patients reached at least the 1-year benchmark, of which 6 patients (15%) had at least 3 years of follow-up, and the 2 patients who developed clinical metastasis were actively followed for 10 and 16 years. No patients died of their primary tumor in our cohort at last follow-up.
Mean tumor size was 4.7 cm (range, 1.5 to 14.5 cm). Two cases (5%) were malignant with clinical metastasis. PASS score of each case was documented. Clinicopatho- logical features are summarized in Table 1. Twenty-five of 38 (66%) cases had diffuse and strong SRC-1 expression, whereas 12/38 (34%) cases had either diffuse weak/focal SRC-1 expression, and 1 case was negative (Figs. 1-11). All 7 cases with genetic mutations showed positive SRC-1
| Sex | Presenting Symptoms | Site | |||
|---|---|---|---|---|---|
| Male | 12 | Hypertension | 16 | Unilateral | 34 |
| Female | 26 | Headache | 6 | Bilateral | 4 |
| Age (y) | Palpitations | 8 | Metastasis | ||
| Range | 16-75 | Incidental on imaging | 18 | Yes | 2 |
| Mean | 52 | Genetics | No | 36 | |
| Median | 58 | RET | 3 | Follow-up (y (y) | |
| ≥40 | 29 | VHL | 2 | <0.5 | 15 |
| Heritable | 2 | SDHB p. V140F | 1 | 0.5-1 | 5 |
| <40 | 9 | ATM and PDGFRA | 1 | >1 | 18 |
| Heritable | 5 | >3 | 6 | ||
| 10 | 1 | ||||
| 16 | 1 | ||||
expression, 5/7 (71%) of which had a diffuse pattern. All VHL cases exhibited diffuse pattern-strong expression of SRC-1, whereas RET cases showed diffuse positivity, but ranged in staining intensity. Mean tumor size and PASS scores showed an inverse correlation with SRC-1 expression as follows: focal positivity subset was 6.8 cm (PASS=3.6), diffuse pattern-weak intensity subset was 4.2 cm (PASS=3.5), and diffuse pattern strong intensity subset was 4.2 cm (PASS=2.7). The correlation is summarized in Table 2 and Figure 12. Immunohistochemical staining for SDHB was previously performed in 29/38 (76%) of our cohort, and exhibited loss of expression (interpreted as SDHB mutated) in only 1 patient that had metastatic disease at presentation. The patient was alive at last follow-up (16 y).
DISCUSSION
Comparison of Our Cohort’s Clinicopathologic Characteristics to Prior Literature
Pheochromocytoma is a relatively uncommon tumor, and histomorphologic predictors of malignant potential
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are poorly defined. Part of the challenge is that the path- obiology of this tumor is not well elucidated. The clin- icopathological characteristics of our patient cohort corroborate with those reported in the literature.
It has been observed that patients who have an as- sociated syndrome or genetic mutation tend to present younger, and more frequently with bilateral disease. We observed that 5/9 (56%) patients in our cohort under the age of 40 had heritable mutations. In addition, 4 bilateral tu- mors all occurred in patients with heritable mutations (MEN2B in 2 cases, MEN2A in 1 case, and VHL in 1 case). Heritable mutations linked to pheochromocytoma most commonly include: RET protooncogene (in MEN2A and MEN2B autosomal dominant inherited disorders), VHL, NF1, and SDH family (SDHB, SDHC, and SDHD).9 Our study group also encompassed this heterogeneity, and
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reflects the evolving thought that a significant number of these tumors are linked to heritable mutations. RET (n=3), VHL (2), SDHB (1), and ATM and PDGFRA (1) were encountered in 7 of 38 (18%) heritable mutated cases. It should, however, be noted that in view of the fact that the hereditary screening was done by the patients’ clinicians/ genetic counselors, the issue of potentially undiagnosed patients with heritable mutations may exist in this cohort (including expert consultation cases), and the general pheochromocytoma patient population.
Of particular note, SDHB mutations have been shown to be associated with increased incidence of ag- gressive disease. Kimura et al3 observed that in their cohort of 163 tumors, including 40 metastatic tumors, that there
was loss of SDHB in 13 (8%) moderately differentiated or poorly differentiated tumors and 10 of the 13 (77%) pa- tients had metastasis. In our cohort, immunohistochemical staining for SDHB was performed during sign-out in 29/38 (76%) cases, and exhibited loss of expression (interpreted as SDHB mutated) in only 1 patient that had metastatic disease at presentation. The patient was alive at last follow- up, 16 years postoperative. The patient’s mutation oc- curred at the short arm of the SDHB gene converting a Valine to Phenylalanine (p.V140F), which was reported as a “likely pathogenic variant.” SDHB has a high proportion of genetic variation compared with SDHD, which has more limited polymorphisms in such as G12S, H50R, and S68S. SDHB germ-line mutated tumors also have a high frequency of recurrence, metastasis potential, or extra- adrenal presentation.10 Another study, by Neumann et al11
observed that half of the 12 patients with SDHB mutations presented with extra-adrenal disease, as seen our patient. Our SDHB mutated case showed a diffuse-strong pattern of SRC-1 staining. The only other case in our cohort that developed metastasis had an ATM and PDGFRA muta- tion, which is novel. The patient had a reported history of an incidentally discovered pheochromocytoma 8 years prior, and presented with widely metastatic disease seeding the peri-adrenal adipose tissue, peritoneum and adjacent capsular surfaces of the liver and kidney. She was alive at last follow-up, 10 years postoperative. This patient had a remote reported history of breast cancer, and it is notable that individually, an ATM mutation is associated with an increased risk for breast cancer, whereas a PDGFRA mu- tation is frequently observed in gastrointestinal stromal tumors. This patient did not have any other history of
malignancy. Our ATM and PDGFRA mutated case showed a focal-positive pattern of SRC-1 staining.
SRC-1 Expression in Pheochromocytoma
To date, this is the first reported large novel inves- tigation of SRC-1 expression in pheochromocytoma. We observed that the overwhelming majority of our pheo- chromocytoma cases exhibited nuclear reactivity for SRC-1, and that the background normal adrenal cortical tissue served as a positive internal control. Diffuse pattern with strong intensity staining was present in 25/38 (66%) of our cases, whereas 12/38 (34%) had either diffuse weak or focal staining, and 1 case was negative. From a biological standpoint, SRC-1 is also known as Nuclear receptor co- activator 1, and it is a common transcriptional coactivator
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| SRC-1 Expression | Mean Tumor Size (cm) | PASS Score |
|---|---|---|
| Focal (n=6) | 6.8 | 3.6 |
| Diffuse, weak (n=6) | 4.2 | 3.5 |
| Diffuse, strong (n =25) | 4.2 | 2.7 |
Mean tumor size and PASS scores showed an inverse correlation with SRC-1 expression. Only 1 case was negative for SRC-1, and therefore not included in the number (n) here and Figure 12.
central to several steroidogenesis pathways that include those of the adrenal gland. It was recently suggested that SRC-1 is useful in distinguishing metastatic tumors of po- tential adrenal primary (ACC) from a renal primary.8 The authors utilized a triple combination panel including 2 of 3 markers such as CAIX, PAX8, or RCC in addition to SRC- 1 or Melan A, in the distinction between metastatic renal cell carcinoma to the adrenal from ACC.8 However, based on the findings in our study, SRC-1 would not be helpful in a panel to differentiate ACC from pheochromocytoma. It would therefore be important to exclude ACC with addi- tional immunohistochemical stains (eg, inhibin, S-100, Melan-A, HMB-45, etc.).
An inverse relationship was observed between SRC-1 expression and PASS score/tumor size in a subset of patients in our study, suggesting that the SRC-1 phenotype may become muted in pheochromocytomas that have malignant potential. The larger tumor size observed in our focal- staining group (mean size=6.8 cm) could be skewed by the relatively few cases we had in this subset (n=6), or those with diffuse with weak staining (n =6). This is comparison to the subset exhibiting diffuse pattern and strong positivity (n=25) that showed a smaller mean size of 4.2 cm.
CONCLUSIONS
This is the first and largest study to analyze SRC-1 expression in pheochromocytoma. We observed that diffuse positivity was seen in most of our heritable cases, and the
8
7
Mean PCT size (cm)
PASS Score
6
6.8
5
4
4.2
4.2
3
3.6
3.5
2
2.7
1
0
Focal (n=6)
Diffuse, weak (n=6)
Diffuse, strong (n=25)
SRC-1 Expression
majority of tumors also exhibited diffuse strong nuclear positivity. In a subset of patients, an inverse relationship was observed between SRC-1 expression and PASS score/tumor size, suggesting that SRC-1 phenotype may become mutated in pheochromocytomas that have malignant potential, however this finding warrants further investigation. Finally, SRC-1 expression in pheochromocytoma is a potential di- agnostic pitfall in patients with an adrenal mass presumed to be ACC from the clinical/radiologic and pathologic stand- points, in the primary and metastatic setting.
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