Application of TFE3 Immunophenotypic and TFE3 mRNA Expressions in Diagnosis and Prognostication of Adrenal Cortical Neoplasms and Distinction From Kidney Tumors
Xingen Wang, MD, ** Chi-Sing Ng, FRCPath,¿ Weihua Yin, MD, and Li Liang, PhD*
Abstract: We explored the application of TFE3 immunostaining and TFE3 mRNA expression in the differential diagnosis and prognostication of adrenal cortical tumors and distinction of the latter from clear cell renal cell carcinoma (ccRCC) which show significant morphologic overlap. TFE3 immunostaining was per- formed on a large cohort of samples including 40 adrenal cortex tissues, 95 adrenocortical adenoma (ACA), 11 adrenocortical carcinoma (ACC), 53 ccRCC, and 18 pheochromocytomas. TFE3 was compared with other immunomarkers melan-A, inhibin-«, synaptophysin, chromogranin A, CAIX and CD10. One hundred percent normal adrenal cortices and 94% ACA were strongly and diffusely stained for TFE3 while no ACC showed diffuse staining. TFE3 is thus useful in distinguishing ACA from ACC. TFE3 is also useful in separating ACC from ccRCC as 64% ACC showed partial, while only 7% of ccRCC showed partial TFE3 staining. Only 1 pheochromocytoma showed focal weak TFE3 staining. Results also demonstrated superiority of TFE3 over other com- monly used immunomarkers. TFE3 gene rearrangement testing by fluorescence in situ hybridization showed no rearrangement in 6 TFE3 positive adrenal tumors. TFE3 mRNA were analyzed by the Cancer Genome Atlas database and we found TFE3 mRNA expression correlated with overall patient survival in ACC. Our study showed usefulness of TFE3 in distinguishing ACA from ACC, and ACC from ccRCC. TFE3 is superior over other commonly used immunomarkers for adrenal tumors. In addition, decreased TFE3 immunoexpression and TFE3 mRNA expression may carry poor prognostic implication in adrenal tumors.
Key Words: TFE3, adrenal cortical adenoma, adrenal cortical carcinoma, pheochromocytoma, clear cell renal cell carcinoma
(Appl Immunohistochem Mol Morphol 2023;31:9-16)
From the *Department of Pathology, Nanfang Hospital and Basic Medical College, Southern Medical University, Guangzhou; Department of pathology, Peking University Shenzhen Hospital, Shenzhen, China; and ¿Department of Pathology, St. Teresa’s Hospital, Mong Kok, Hong Kong.
X.W. and C .- S.N. are co-first authors and contributed equally to this work.
The study was supported by “San-Ming” Project of Medicine in Shenzhen (NO. SZSM20182088, Weihua Yin).
The authors declare no conflict of interest.
Reprints: Li Liang, PhD, Department of Pathology, Nanfang Hospital and Basic Medical College, Southern Medical University, Guangzhou 510650, China (e-mail: liliang_1691@sina.com).
Copyright @ 2022 Wolters Kluwer Health, Inc. All rights reserved.
D ifferentiation of kidney tumors from adrenocortical tumors are problematic, because of their morphologic overlap.1 The problem is greater in small biopsies2,3 of retroperitoneal or renal upper pole lesions, a common scenario in patients with previous history of renal cell carcinoma (RCC) or adrenocortical carcinoma (ACC),2 where distinction is important because of the different prognostic and therapeutic strategies. A panel of multiple antibodies is often used to make the distinction.4,5
Adrenal tumors are common and most are benign, nonfunctional and detected incidentally. They are reported to be found in 1% to 2% of population on imaging examination.6-8 ACC are rare and aggressive with incidence of 0.7 to 2 cases per million people per year.9-11 The histologic distinction of ACC from adrenocortical adenoma (ACA) can be challenging and the golden diagnostic standard of distant metastasis in ACC is not realistic in clinical practice. There- fore, histologic scoring systems have been devised for the distinction and the Weiss score is most commonly used.12-14 However, distinguishing noninvasive low-grade ACC with a low Weiss score from ACA remains a diagnostic challenge, especially in small lesions, borderline cases and ACC variants such as oncocytic ACC.14,15 Immunohistochemistry has been resought to but only few immunohistochemical markers have been reported to be useful.4,5
In our routine practice, we serendipitously noticed strong TFE3 immunostaining of normal adrenal cortex. TFE3 high immunoexpression in adrenal glandular and medium expression in kidney tubular cells is reported in the Human Protein Atlas.16 TFE3 is a basic-helix-loop- helix-zipper domain-containing protein that binds pE3 sites in regulatory elements in the immunoglobulin heavy chain gene.17 TFE3 plays significant role in autophagy and lysosomal biogenesis and is involved in the patho- genesis of some human neoplasms,18-20 including Xp11.2 translocation RCC, alveolar soft part sarcoma, some perivascular epithelioid cell tumors, and epithelioid hemangioendothelioma.21-24 TFE3 immunoexpression has been regarded as a surrogate for TFE3 gene re- arrangement. However, TFE3 staining without TFE3 gene rearrangement were found in granular cell tumor, desmoid-type fibrosis, solid pseudopapillary neoplasms of pancreas and ovarian sclerosing stromal tumors.25-28 There are also studies suggesting a minor role of TFE3 immunohistochemistry in the diagnosis of TFE3- rearranged tumor.29,30
In this study, we performed TFE3 immunostaining on 95 ACA, 11 ACC, 53 clear cell renal cell carcinoma (ccRCC), 18 pheochromocytomas, and 40 samples of adrenal cortex and compared with staining with other commonly used markers melan-A, inhibin-a, synapto- physin, chromogranin A, CAIX, and CD10 in the differ- ential diagnoses. Six TFE-positive cases, including 3 strongly diffusely positive ACA and 3 moderately partially positive ACC, were analyzed for TFE3 gene rearrange- ment by fluorescence in situ hybridization (FISH). Fur- thermore, TFE3 mRNA expression in ACC were analyzed using the Cancer Genome Atlas (TCGA) data by applying the online tool GEPIA2.
MATERIALS AND METHODS
Case Selection
Forty samples from surgical specimens of normal adrenal cortex, 95 ACA, 11 ACC, 18 pheochromocytoma, and 53 ccRCC within the period 2014 to 2021 were studied. Hematoxylin and eosin and immunostained slides were reevaluated by 3 senior pathologists and diagnoses were confirmed. The clinical data of patients and tumor sizes were retrieved from the patients’ records. This study was approved by the Research Ethics Committee of the Peking University Shenzhen Hospital and the Southern Medical University.
Immunohistochemical Analysis
Representative paraffin-embedded tissue blocks were retrieved for hematoxylin and eosin staining. TFE3 (1:100, Clone MRQ-37; Maixin Biotechnology Ltd.) and other markers immunostaining were performed on 4 um thick paraffin tissue sections using an automated immunostainer (Ventana Benchmark XT auto-stainer; Ventana Medical Systems Inc.) after deparaffinization, rehydration, and antigen retrieval of the slides and then counterstained with hematoxylin. Multiple normal tissue microarrays and samples of TFE3 translocation RCC were used as negative and positive control respectively.
The percentage of tumor cells with TFE3 nuclear staining were assessed. Staining percentage is rated as follows: negative when no tumor cells were stained or weak staining in <1% tumor cells. Staining of 1% to 10% of tumor cells was focal. Staining of 10% to 75% tumor cells was partial and more than 75% diffuse. Staining in- tensity was graded as weak when barely visible, moderate when readily visible and strong when easily visible. The different patterns and intensities of staining were com- pared statistically using the x2 test.
FISH
Three TFE3 diffuse of positive ACA, 3 TFE3 partial of positive ACC, and 2 Mit family translation RCC (as control) were analyzed by FISH using com- mercial break-apart probes for TFE3 (Xp11.2). TFE3 gene translocation was positive when at least 15% of one hundred scored nuclei shows a split signal pattern.
10 | www.appliedimmunohist.com
The Cancer Genome Atlas (TCGA) Database Analysis
TCGA gene expression data for 77 ACC, 523 ccRCC, 66 kidney chromophobe cell carcinomas (chrRCC), 286 kidney papillary renal cell carcinomas (PRCC), and 182 pheochromocytomas/paragangliomas were analyzed using the online platform GEPIA2 (http:// gepia2.cancer-pku.cn) and matched with TCGA normal data and GTEx (Genotype-Tissue Expression) data. Sta- tistical methods for analysis of variance were used for differential gene expression analysis. Log2FC cutoff was set as 1 and q value cutoff was set as 0.01. Survival data were evaluated with Kaplan-Meier analysis with a log- rank test.
RESULTS
Clinicopathologic Features
For ACA, there were 46 males and 49 females. The tumor sizes range from 0.8 to 5 cm, with average of 2.5 cm. The patient ages ranged from 24 to 70 years, with median age of 47 years. For ACC, there were 6 males and 5 females. The tumor sizes ranged from 2 to 13 cm, with average of 5.9 cm. The patient ages ranged from 23 to 41 years, with median age of 33 years. For ccRCC, there were 15 females and 38 males. The ages ranged from 30 to 85 years, with median age of 55 years. The tumor sizes ranged from 1.2 to 12, with average of 4.3 cm. For pheochromocytomas, there were 9 females and 9 males. Patients aged from 26 to 71 years, with median age 33 years. The tumor sizes ranged from 3 to 9 cm, with average of 4.8 cm.
TFE3 Immunoexpression in Adrenal Cortex, Adrenocortical Neoplasms, Pheochromocytoma and Clear Cell Renal Cell Carcinoma
Two hundred and seventeen samples were analyzed for TFE3 expression (40 cases of adrenal cortex, 95 ACA, 11 ACC, 18 pheochromocytoma, and 53 ccRCC). All 40 adrenal cortices (100%) showed strong diffuse staining. 89/ 96 (94%) of ACA showed strong diffuse staining (Fig. 1) and 6/95 (6%) showed moderate partial staining. No diffuse TFE3 staining was found in ACC, pheochromocytoma and ccRCC (Fig. 2). No TFE3 staining were observed on normal adrenal medulla. However, in ACC, 1/11 (9%) showed strong partial, 5/11 (45%) showed moderate partial, 1/11 (9%) showed weak partial, 2/11 (18%) showed moderate focal, and 2/11 (18%) showed weak focal TFE3 staining (Fig. 3). Although 41/53(77%) ccRCC were TFE3 positive, only 4 (8%) showed partial (1 moderate and 3 weak staining intensity) while the remaining 37 (70%) focal (19 moderate and 18 weak staining intensity) staining. There was only 1 of 18 pheochromocytomas being focally weakly for TFE3 positive. There were significant statistical differences in TFE3 immunoexpression between ACA/ ACC and ACC/ccRCC in terms of pattern of expression and intensity of staining.
Copyright @ 2022 Wolters Kluwer Health, Inc. All rights reserved.
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TFE3 Compared With Other Markers in Adrenal Neoplasms
Strong diffuse expression rate is highest for TFE3 in ACA compared with that of other commonly used adrenal markers. The percentage of strong diffuse expression for TFE3, melan-A, inhibin-a, synaptophysin, and chromog- ran A in ACA were 94%, 43%, 15%, 53%, and 0%, re- spectively. In ACC, there was no strong diffuse expression for TFE3 and other markers. One ACC showed diffuse expression of synaptophysin. Sixty-four percent of ACC
showed partial expression of TFE3, compared with focal expression in 10% for melan-A, 45% for inhibin-« and 27% for synaptophysin. All 18 pheochromocytoma showed dif- fuse expression of synaptophysin and chromogranin A, and negative for melan-A and inhibin-a, with 1 case focally weakly positive for TFE3. In ccRCC, 4/53 (7%) showed partial and 37/53 (70%) focal TFE3 expression. They also diffusely expressed CAIX and CD10 (96% and 81%, re- spectively) (Table 1). The average Ki67 proliferation index was 4% and 43% in ACA and ACC, respectively.
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FISH
None of 6 TFE3 positive adrenal tumors showed TFE3 gene rearrangement. Both 2 Mit family trans- location RCC tested as control displayed TFE3 gene split pattern (Fig. 4).
TFE3 mRNA Levels in Adrenal Cortex, ACC and 4 Other Tumor Types
We analyzed TFE3 mRNA expression data in 5 tumor types using TCGA database by the online tool GEPIA2. Seventy-seven cases of ACC, 66 chrRCC, 523 ccRCC, 286 PRCC, 182 pheochromocytomas, and para- gangliomas were included. As control, TFE3 mRNA ex- pression in normal tissues adjacent to the tumor or normal adrenal gland and kidney tissue were also analyzed using. TFE3 mRNA expression in normal adrenal gland tissue was higher than kidney tissue. The average TFE3 mRNA
expression in ACC was highest among the 5 types of tu- mors and was thus useful in differentiating ACC from kidney carcinomas and pheochromocytoma and para- ganglioma. Expression level of TFE3 mRNA in ACC was lower than that in normal adrenal gland tissue, which was therefore also useful in identifying ACC in adrenal speci- mens (Fig. 5). In addition, survival analysis showed that the prognosis was significantly worse in ACC patients with TFE3 mRNA low expression than patients with TFE3 mRNA high expression. The 5-year overall survival rate of ACC was about 80% in the TFE3 mRNA high expression group, but <50% in the TFE3 mRNA low expression group (Fig. 5).
DISCUSSION
The distinction between adrenal tumors and renal tumors is enigmatic, because of the significant morpho-
| TABLE 1. IHC Characteristics of Adrenocortical Neoplasms, Pheochromocytoma and Clear Cell Renal Cell Carcinoma | ||||||||
|---|---|---|---|---|---|---|---|---|
| Tumors Type | TFE3 (P <0.001) | Melan-A (P=0.007) | Inhibin-a (P<0.001) | Synaptophysin (P<0.001) | Chromogranin A (P= 0.731) | CAIX | CD10 | Ki67 (Range, Average) (P <0.001) |
| ACA | 89/95 (D, 94%) | 30/69 (D, 43%) | 13/89 (D, 15%) | 44/83 (D, 53%) | 0/91 (D, 0%) | ND | ND | 1%-15%, 4% |
| 6/95 (P, 6%) | 6/69 (F,9%) | 72/89 (F, 81%) | 35/83 (F, 42%) | 2/91 (F, 2%) | ||||
| 0/95 (N, 0%) | 33/69 (N,48%) | 4/89 (N, 4%) | 4/83 (N, 5%) | 89/91 (N, 98%) | ||||
| ACC | 0/11 (D, 0%) | 0/10 (D, 0%) | 0/11 (D, 0%) | 1/11 (D, 9%) | 10/10 (N, 100%) | ND | ND | 3%-90%, 43% |
| 7/11 (P, 64%) | 1/10 (F, 10%) | 5/11(F, 45%) | 3/11 (F, 27%) | |||||
| 4/11 (F, 36%) | 9/10 (N, 90%) | 6/11 (N, 55%) | 7/11 (N, 64%) | |||||
| 0/11 (N, 0%) | ||||||||
| Pheochromocytoma | 0/18 (D, 0%) | 8/8 (N, 100%) | 7/7 (N, 100%) | 18/18 (D, 100%) | 18/18 (D, 100%) | ND | ND | ND |
| 1/18 (F, 6%) | ||||||||
| 17/18 (N, 94%) | ||||||||
| CCRCC | 0/53 (D, 0%) | ND | ND | ND | ND | 51/53 (D, 96%) | 37/46 (D, 81% ) | ND |
| 4/53 (P, 7%) | 2/53 (F, 4%) | 8/46 (F, 17%) | ||||||
| 37/53 (F, 70%) | 0/53 (N, 0%) | 1/46(N, 2%) | ||||||
| 12/53 (N, 23%) | ||||||||
ACA indicates adrenocortical adenoma; ACC, adrenocortical carcinoma; CCRCC, clear cell rental carcinoma; D, diffuse; F, focal; N, negative; ND, not done; P, partial.
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logic overlap, common clinical locations and scenarios of local recurrence, invasion or metastasis in patients with previous renal or adrenal carcinoma. The difficulty is compounded by the frequent use of small biopsies on retroperitoneal or upper pole kidney lesions where the task of separating renal from adrenal tumors is onerous.1-3 Immunohistochemistry using melan-A, inhibin-«, syn- aptophysin, SF-1 and the renal cell markers CAIX, CD10 and hkIM-1 has been applied.4,5 One study reported sen- sitivity of melan-A, inhibin-a, synaptophysin in adrenal cortical lesions as 86%, 86%, and 59%, respectively.5 In our study, the overall sensitivity of melan-A, inhibin-a, synaptophysin for ACA were 52%, 96%, and 95%, re- spectively. Though this overall sensitivity of im- munostaining for inhibin-a and synaptophysin for diagnosing ACA is appealing, staining was diffuse in only 15% inhibin-a and 53% synaptophysin for ACA. In con- trast, staining for TFE3 in ACA was diffuse and strong in 94% in our series. TFE3 also showed partial staining in 64% ACC while no ACC stained for melan-A and inhibin- a though 1 (9%) ACC showed diffuse staining for syn- aptophysin. Thus, we demonstrated that TFE3 is a supe- rior immunomarker for diagnosing adrenocortical tumors. We also demonstrated significant statistical differences in
TFE3 staining patterns and intensities in ACC and ccRCC. TFE3 partial staining in ACC was present in 7/11 (64%) and only in 4/53 (7%) ccRCC. TFE3 staining in- tensity was strong in 1/11 (9%) and moderate in 6/11 (55%) in ACC. In ccRCC, no case showed strong TFE3 staining and only 20/53 (38%) showed moderate staining. TFE3 is thus a useful marker for distinguishing ACC from ccRCC. The use of renal cell markers CAIX and CD10 which are positive only in RCC (96% for CAIX and 81% for CD10) further enhances diagnostic accuracy. We recommend a simple antibody panel of TFE3 and CAIX for differential diagnosis of adrenal cortical tumors and ccRCC. Chro- mogranin A staining was strongly and diffusely positive in 18/18 (100%) pheochromocytoma, and chromogranin A and may be included in the working panel if the latter were also a diagnostic concern.
The histopathologic diagnosis of ACC is challenging even for experienced pathologists. The distant metastasis criteria are difficulty to apply in most clinical situations. Distinction of ACC from ACA is very important because of different therapeutic and prognostic implications. The widely used Weiss scoring system for ACC diagnosis may be equivocal for localized small lesions, borderline cases, and ACC variants.12-15,31 Our study found that TFE3
A
8
Expression-log2(TPM+1)
4
2
0
ACC (num(T)=77; num(N)=128)
KICH (num(T)=66; num(N)=53)
KIRC (num(T)=523; num(N)=100)
KIRP (num(T)=286; num(N)=60)
PCPG (num(T)=182; num(N)=3)
B
Overall Survival
1.0
Low TFE3 Group
High TFE3 Group
Logrank p=0.0037
0.8
HR(high)=0.31
p(HR)=0.0056 n(high)=38
Percent survival
0.6
n(low)=38
0.4
0.2
0.0
0
50
100
150
Months
immunohistochemical staining in ACA was strongly and diffusely positive in 94% cases. Contrary to, no ACC strong diffuse positive staining. TFE3 was only partially or focally expressed in ACC with staining intensity in most ACC being moderate or weak. Differential TFE3 staining intensities and patterns thus useful in separating ACC from ACA. This also suggests that decrease in im- munoexpression of TFE3 in adrenal tumors may portend malignant transformation. The underlying molecular mechanism and clinical implications are worth further investigations. Our results also showed that the Ki67 proliferation index in ACC is significantly higher than that of ACA, as reported in previously studies.32
TFE3 strong nuclear immunoreactivity is found in neoplasms bearing TFE3 gene fusions including Xp11.2
translocation RCC, alveolar soft part sarcoma, epithelioid hemangioendothelioma, and a subset of perivascular epi- thelioid tumors.21-24 The antibody against TFE3 C-terminus binding site was believed to be sensitive and specific for tu- mors with TFE3 gene rearrangement.33 Over time, a variety of tumors without TFE3 gene rearrangement were found to be TFE3 immunopositive including solid pseudopapillary neoplasm of pancreas, ovarian sclerosing stromal tumor, clear cell sarcoma, melanoma, desmoid-type fibrosis, and granular cell tumor.25-28,34-38 Our results expanded this list by demonstrating that ACAs and ACCs are immunoreactive for TFE3 without TFE3 gene rearrangement. furthermore, our study first reported that normal adrenal cortex tissue but not adrenal medulla tissue shows strong nuclear immunor- eactive for TFE3. Awareness of this is very important, when
evaluating TFE3 translocation RCC involvement of adrenal gland, where testing for TFE3 rearrangement is mandatory.
For further diagnostic and prognostication criteria, we analyzed mRNA expression of TFE3 in five tumors types including 77 ACC, 66 chrRCC, 523 ccRCC, 286 PRCC, 182 pheochromocytomas, and paragangliomas, using the TCGA data and online tool GEPIA2. We found that the mRNA expression of TFE3 in ACC was higher than pheochromocytoma and tumors of kidney and mRNA expression of TFE3 was significantly decreased in ACC compared with normal adrenal gland tissue. De- creased TFE3 mRNA expression may therefore portend malignant transformation in the adrenal tumors. Fur- thermore, the prognosis was significantly worse in ACC patients with TFE3 mRNA low expression than with TFE3 mRNA high expression. Five-year overall survival rate of patients with ACC was about 80% in the TFE3 mRNA high expression group, but <50% in the TFE3 mRNA low expression group. TFE3 mRNA expression level is thus useful for prognostication of ACC.
Our study first demonstrated in a large sample co- hort that there is diffuse nuclear TFE3 staining in normal adrenal cortex and adrenal cortical adenoma without TFE3 gene rearrangement. TFE3 immunoexpression is useful in the right clinical setting in differentiating ACA from ACC and ACC from ccRCC. In addition, TFE3 mRNA decrease in adrenal cortical neoplasm suggest malignant transformation, and TFE3 mRNA is a prog- nostic marker in ACC. The latter implies a tumor sup- pressor role of TFE3 in adrenocortical oncogenesis.
ACKNOWLEDGMENTS
The authors thank Yuwei, Aiping Zheng, Miqing Wang, Dr Xiaoxin Shi (Peking University Shenzhen Hos- pital) for technical assistance and immunohistochemistry work. The authors also thank Yvonne Chan for meticulous typing of the manuscript.
REFERENCES
1. Nappi O, Mills SE, Swanson PE, et al. Clear cell tumors of unknown nature and origin: a systematic approach to diagnosis. Semin Diagn Pathol. 1997;14:164-174.
2. Renshaw AA, Richie JP. Subtypes of renal cell carcinoma: different onset and sites of metastatic disease. Am J Clin Pathol. 1999;111: 539-543.
3. Oliveira AM, Erickson LA, Burgart LJ, et al. Differentiation of primary and metastatic clear cell tumors in the liver by in situ hybridization for albumin messenger RNA. Am J Surg Pathol. 2000;24:177-182.
4. Li HM, Hes O, MacLennan GT, et al. Immunohistochemical distinction of metastases of renal cell carcinoma to the adrenal from primary adrenal nodules, including oncocytic tumor. Virchows Arch. 2015;466:581-588.
5. Sangoi AR, Fujiwara M, West RB, et al. Immunohistochemical distinction of primary adrenal cortical lesions from metastatic clear cell renal cell carcinoma: a study of 248 cases. Am J Surg Pathol. 2011;35:678-686.
6. Lerario AM, Moraitis A, Hammer GD. Genetics and epigenetics of adrenocortical tumors. Mol Cell Endocrinol. 2014;386:67-84.
7. Karkhofs TMA, Verhoeven RHA, Vanderzwan JM, et al. Adreno- cortical carcinoma: a population based study on incidence and survival in Netherlands since 1993. Eur J Cancer. 2013;49: 2579-2586.
8. Mansmann G, Lau J, Balk E, et al. The clinically inapparent adrenal mass: update in diagnosis and management. Endocr Rev. 2004;25: 309-340.
9. Berruti A, Grisanti S, Pulzer A, et al. Long-term outcomes of adjuvant mitotane therapy in patients with radically resected adrenocortical carcinoma. J Clin Endocrinol Metab. 2017;102:1358-1365.
10. Cawood TJ, Hunt PJ, O’shea D, et al. Recommended evaluation of adrenal incidentalomas is costly, has high false-positive rates and confers a risk of fatal cancer that is similar to the risk of the adrenal lesion becoming malignant; time for a rethink? Eur J Endocrinol. 2009;161:513-527.
11. Sharma E, Dahal S, Sharma P, et al. The characteristics and trends in adrenocortical carcinoma: a United States population based study. J Clin Med Res. 2018;10:636-640.
12. Lau SK, Weiss LM. The Weiss system for evaluating adrenocortical neoplasms: 25 years later. Hum Pathol. 2009;40:757-768.
13. Jain M, Kapoors, Mishra A, et al. Weiss criteria in large adrenocortical tumors. A validation study. Indian J Pathol Micro- biol. 2010;53:222-226.
14. Papetti M, Live R, Duregon E, et al. The Weiss score and beyond- histopathology for adrenocortical carcinoma. Horm Cancer. 2011;6: 333-340.
15. Ciaramella PD, Vertemati M, Petrella D, et al. Analysis of histological and immunohistochemical patterns of benign and malignant adrenocortical tumors by computerized morphometry. Pathol Res Pr. 2017;213:815-823.
16. TFE3 protein expression. The Human Protein Atlas. Available at: https://www.proteinatlas.org/ENSG00000068323-TFE3/tissue. Assecced September 15, 2021.
17. Henthorn PS, Stewart CC, Kadesch T, et al. The gene encoding human TFE3, a transcription factor that binds the immunoglobulin heavy-chain enhancer, maps to Xp11.22. Genomics. 1991;11: 374-378.
18. Artandi SE, Merrell K, Avitahl N, et al. TFE3 contains two activation domains, one acidic and the other proline-rich, that synergistically activate transcription. Nucleic Acids Res. 1995;23: 3865-3871.
19. Raben N, Puertollano R. TFEB and TFE3: linking lysosomes to cellular adaptation to stress. Annu Rev Cell Dev Biol. 2016;32:255-278.
20. Martina JA, Diab HI, Brady OA, et al. TFEB and TFE3 are novel components of the integrated stress response. EMBO J. 2016;35: 479-495.
21. Weterman MA, Wilbrink M, Geurts van Kessel A. Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11; q21)-positive papillary renal cell carcinomas. Proc Natl Acad Sci USA. 1996;93:15294-15298.
22. Joyama S, Ueda T, Shimizu K, et al. Chromosome rearrangement at 17q25 and xp11.2 in alveolar soft-part sarcoma: a case report and review of the literature. Cancer. 1999;86:1246-1250.
23. Argani P, Aulmann S, Karanjawala Z, et al. Melanotic Xp11 translocation renal cancers: a distinctive neoplasm with overlapping features of PEComa, carcinoma, and melanoma. Am J Surg Pathol. 2009;33:609-619.
24. Antonescu CR, Loarer FL, Mosquera JM, et al. Novel YAP1-TFE3 fusion defines a distinct subset of epithelioid hemangioendothelioma. Genes Chromosomes Cancer. 2013;52:775-784.
25. Harrison G, Hemmerich A, Guy C, et al. Overexpression of SOX11 and TFE3 in solid-pseudopapillary neoplasms of the pancreas. Am J Clin Pathol. 2017;149:67-75.
26. Liu Y, Zheng Q, Wang C, et al. Granular cell tumors overexpress TFE3 without gene rearrangement: Evaluation of immunohisto- chemistry and break-apart FISH in 45 cases. Oncol Lett. 2019;18: 6355-6360.
27. Park CK, Kim HS. Clinicopathological characteristics of ovarian sclerosing stromal tumor with an emphasis on TFE3 overexpression. Anticancer Res. 2017;37:5441-5447.
28. Zhou LT, Xu HM, Zhou J, et al. Nuclear TFE3 expression is a diagnostic marker for desmoid-type fibromatosis. Diag Pathol. 2019;14:34.
29. Sharain RF, Gown AM, Greipp PT, et al. Immunohistochemistry for TFE3 lacks specificity and sensitivity in the diagnosis of TFE3-
rearranged neoplasms: a comparative, 2 laboratory study. Hum Pathol. 2019;87:65-74.
30. Flucke U, Vogels Rob JC, Somerhausen Ni de SA, et al. Epithelioid hemangioendothelioma: clinicopathologic, immunohistochemical, and molecular genetic analysis of 39 cases. Diagn Pathol. 2014;9:131.
31. Weiss LM. Comparative histologic study of 43 metastasizing and non-metastasizing adrenocortical tumors. Am J Surg Pathol. 1984;8: 163-169.
32. Mete O, Gucer H, Kefeli M, et al. Diagnostic and prognostic biomarkers of adrenal cortical carcinoma. Am J Surg Pathol. 2018;42:201-213.
33. Argani P, Lal P, Hutchinson B, et al. Aberrant nuclear immunor- eactivity for TFE3 in neoplasms with TFE3 gene fusions: a sensitive and specific immunohistochemical assay. Am J Surg Pathol. 2003;27: 750-761.
34. Williams A, Bartle G, Sumathi VP, et al. Detection of ASPLITFE3 fusion transcripts and the TFE3 antigen in formalin-fixed, paraffin-
embedded tissue in a series of 18 cases of alveolar soft part sarcoma: useful diagnostic tools in cases with unusual histological features. Virchows Arch. 2011;258:291-300.
35. Chamberlain BK, Mcclain CM, Gonzalez RS, et al. Alveolar soft part sarcoma and granular cell tumor: an immunohistochemical comparison study. Hum Pathol. 2014;45:1039-1044.
36. Dickson BC, Brooks JS, Pasha TL, et al. TFE3 expression in tumors of the microphthalmia-associated transcription factor family. Int J Surg Pathol. 2011;19:26-30.
37. Tsuji K, Ishikawa Y, Imamura T. Technique for differentiating alveolar soft part sarcoma from other tumors in paraffin-embedded tissue: comparison of immunohistochemistry for TFE3 and CD147 and of reverse transcription polymerase chain reaction for AS- PSCR1-TFE3 fusion transcript. Hum Pathol. 2012;43:356-363.
38. Jiang Y, Xie J, Wang B, et al. TFE3 is a diagnostic marker for solid pseudopapillary neoplasms of the pancreas. Hum Pathol. 2018;81: 166-175.