Immunohistochemical expression of GPNMB in TFEB-rearranged and TFEB-amplified renal cell carcinomas

Introduction

TFEB-altered renal cell carcinoma (RCC) is an umbrella term for RCCs that either harbor gene fusions involving the TFEB transcription factor (TFEB-rearranged RCCs) or exhibit amplification of the 6p21 locus containing TFEB, leading to its overexpression (TFEB-amplified RCCs). The term was introduced by the Genitourinary Pathology Society (GUPS) in 2021 [1] and it was later adopted by the World Health Organization (WHO) 2022 classification of renal cell tumors (5th Edition) [2]. TFEB-altered RCCs display a wide range of morphological features and immunohistochemical profiles, often overlapping with other RCC subtypes. This variability poses significant diagnostic challenges in routine practice. Consequently, there is an ongoing need for reliable ancillary tests to aid in identifying cases that warrant molecular testing.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) has been reported as a sensitive immunohistochemical screening marker for TFEB- and TFE3-altered RCCs, as well as for several renal tumor types demonstrating TSC1, TSC2, or MTOR mutations [3–5]. However, in one study, false-negative or equivocal GPNMB results were observed in 13% of FISH-confirmed TFE3/TFEB altered cases [5]. In the current study, we examined the GPNMB expression by immunohistochemistry (IHC) in a multi-institutional cohort of TFEB-altered RCCs, including both TFEB-rearranged and TFEB-amplified tumors that were confirmed by molecular evaluation and/or fluorescence in situ hybridization (FISH).

Materials and methods

Results

A total of 25 TFEB-altered RCCs, including 16 TFEB-amplified RCCs and nine TFEB-rearranged RCCs, were included in the study. Clinicopathologic data are summarized in Supplementary Table 2. Patients were 12 males and 13 females, ranging in age from 11 to 85 years (mean 62.5; median 73 years). Mean tumor size was 6 cm (range 2.2–13; median 4.5 cm).

Discussion

TFEB-rearranged and TFEB-amplified RCCs are currently classified under the broader category of TFEB-altered RCCs [2]. In both RCC subtypes, TFEB gene fusion or amplification leads to overexpression of the native TFEB protein. This overexpressed TFEB functions as an aberrant transcription factor, driving the expression of numerous downstream targets, including genes typically regulated by the related microphthalmia translocation family member, MITF [2]. However, distinct clinical differences between TFEB-amplified RCC and TFEB-rearranged RCC have become increasingly evident [7].

TFEB-rearranged RCC was first described by Argani et al. in 2001 as a pediatric renal neoplasm characterized by distinctive epithelioid morphology, unique immunohistochemical profile, and a specific cytogenetic background [8]. TFEB-rearranged RCC was incorporated into the 2016 WHO Tumor Classification as a tumor characterized by distinctive biphasic morphology, occurring not only in pediatric patients but also in adults, and typically exhibiting non-aggressive behavior [9]. In 2014, Peckova et al., followed by other research groups, described RCCs with amplification of the TFEB gene, also leading to TFEB overexpression [7, 10–12]. Studies have shown that TFEB-amplified RCC primarily occurs in older patients compared to TFEB-rearranged RCC. Importantly, TFEB-amplified RCC is frequently associated with aggressive clinical behavior. The morphology of TFEB-amplified and TFEB-rearranged RCC can however overlap, as well as with other more common renal neoplasms [13], which poses diagnostic challenges in routine practice.

The IHC screening of renal tumors for translocation-type RCCs, particularly for TFEB-altered RCC, remains challenging, because of their rarity and the imperfections of the currently available markers. Both TFEB-rearranged and TFEB-amplified RCCs exhibit reduced expression of epithelial immunohistochemical markers (i.e., cytokeratin and epithelial membrane antigen) [13]. TFEB-altered RCCs typically show aberrant expression of melanocytic markers, particularly melan A, and cathepsin K. However, this expression tends to be more variable in TFEB-amplified RCCs compared to TFEB-rearranged counterparts [7] and a significant number of TFEB-amplified RCCs can be entirely negative for melanocytic markers, or may show limited or negative expression for cathepsin K, as supported by our findings. Recently, TRIM63 was identified as a cancer-specific biomarker in MiTF RCC; its expression can be evaluated by RNA in situ hybridization (RNA-ISH). Studies have documented TRIM63 RNA-ISH and TFE3/TFEB FISH results concordant [14]. Reportedly, TRIM63 can improve the diagnostic accuracy of recognizing MiTF RCC in an adequate histologic background and distinguish them from other renal cell tumors [15]. Mannan et al. suggested that the TRIM63 RNA ISH assay could help identify cases harboring TFE3 or TFEB rearrangements that are associated with false-negative or equivocal TFE3/TFEB FISH results [16].

GPNMB is a transmembrane protein expressed at low levels in most normal tissues. High levels of GPNMB expression have been reported in various tumors, including melanoma, triple-negative breast cancer, and gliomas [17]. Of note, several studies have also highlighted the utility of GPNMB IHC as an ancillary diagnostic tool in specific differential diagnoses, when evaluating a broad morphologic spectrum of tumors with underlying TSC1/TSC2/mTOR alterations, including perivascular epithelioid cell tumor (PEComa)/angiomyolipoma (AML) [18], low-grade oncocytic tumor (LOT) [3, 5], eosinophilic solid and cystic (ESC) RCC [3, 5], eosinophilic vacuolated tumor (EVT) [3, 5], RCC with fibromyomatous stroma (RCC FMS) [19], primary renal haemangioblastoma [20], RCC FMS with hemangioblastoma-like areas, both in a tuberous sclerosis [21] and in sporadic setting [22], subependymal giant cell astrocytoma [23], and FLCN-associated eosinophilic renal tumors [24]. GPNMB overexpression was noted in mouse models of TFE3-rearranged RCC [17], which led to the identification of GPNMB as a proposed marker for MiTF translocation RCC [25]. Salles et al. investigated GPNMB expression across a range of renal tumors, including TFE3- and TFEB-driven RCCs, RCCs associated with TSC1/TSC2/mTOR alterations, and a limited number of other renal neoplasms. They demonstrated GPNMB positivity in 82% of FISH-confirmed TFE3- and TFEB-altered RCCs [3]. However, it is unclear what proportion of their cases represented TFEB-rearranged vs TFEB-amplified RCCs. Whaley et al. documented GPNMB expression in 92% of TFE3-RCCs (24/26), 100% of TFEB-rearranged RCCs (19/19), and 100% of TFEB-amplified RCCs (17/17) [4]. Despite reporting 100% reactivity for GPNMB in TFEB-amplified RCCs they illustrate zonal absence of GPNMB staining in their Fig. 7E–F [4]. Li and Matoso have recently performed a retrospective analysis of GPNMB immunohistochemistry in nearly 1000 surgical pathology specimens [5]. They found that GPNMB was diffusely positive in 94.8% (218/230) of TSC/mTOR-TFE-related neoplasms (including RCCs with TFE3/TFEB rearrangements, PEComas, and other entities). In contrast, 83.3% of non-TSC/mTOR-TFE-related neoplasms were negative for GPNMB. In their study, TFE3 or TFEB rearrangement, or TFEB amplification was identified by FISH in 46 cases. In the FISH-confirmed cases, GPNMB immunohistochemistry was positive in 40/46 tumors (87%), negative in three cases (6.5%; two with TFEB amplification and one with TFE3 rearrangement), and patchy or equivocal in an additional three RCCs (6.5%; two with TFEB amplification and one with TFE3 rearrangement). Overall, false-negative or equivocal GPNMB results were observed in 13% of FISH-confirmed cases, including four cases with TFEB amplification, and two TFE3-rearranged RCCs [5].

Focal or weak GPNMB staining has been also reported in other renal tumors, for example in a small subset of chromophobe RCCs, among the common renal types [3]. Some authors have thus cautioned that non-diffuse or focal GPNMB staining should be interpreted as nonspecific or negative, and only strong, diffuse cytoplasmic expression should be considered truly positive [5, 19]. However, fully recognizing the limitations of any individual stain, it is important to stress the need of appropriate use of IHC panels in the context of a given morphology and specific differential diagnosis. When considering a specific diagnosis of TFEB-altered RCC, one should also take into account the findings of other markers, such as Melan A and Cathepsin K, and limited/absent cytokeratin expression, particularly in limited biopsy specimens. Importantly, such IHC screening-based diagnosis requires confirmatory genomic assays, including FISH.

Our findings show that interpreting GPNMB staining in TFEB-amplified RCCs may be problematic when applying the criteria proposed by authors of previous studies (strong, diffuse), as a significant number of our cases (50% TFEB-amplified RCCs) did not exhibit diffuse GPNMB staining pattern. Consequently, we particularly recommend caution when assessing GPNMB reactivity in limited tissue samples, such as needle biopsies from renal masses. Interpretation of GPNMB immunohistochemistry can also be challenging and inconsistent when, for example, the staining is diffuse but weak, or strongly positive only in individual scattered cells [5]. Even a negative GPNMB staining does not completely exclude the diagnosis of TFEB amplified RCC and requires correlation with the IHC panel results and the morphology. Our study along with previous finding [5] indicate that GPNMB expression may well be affected by the specific type of molecular alteration, including differences in translocation partners, mutation sites, or underlying mechanisms (e.g., translocation vs. amplification), as well as technical issues, such as tissue fixation and antibody dilutions. Even with the limitations of its sensitivity and specificity and the variability of staining in some cases, GPNMB provides a useful and cost-effective screening tool for further diagnostic workup.

Conclusion

We found that GPNMB was positive in all evaluated TFEB-altered RCCs by IHC, supporting its potential role as a sensitive screening marker. While TFEB-rearranged RCCs typically demonstrated strong, diffuse GPNMB immunoreactivity, TFEB-amplified RCCs showed more often focal or patchy staining of lower intensity. This variability highlights the need for cautious interpretation, considering the morphologic findings and the other IHC stains, particularly in limited biopsy samples. Despite these limitations, our findings support the routine use of GPNMB as an ancillary screening tool in RCC diagnostics.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

Conceptualization: KP. Methodology: KP, RA. Formal analysis and investigation: KP, PG, PSte. Writing—original draft preparation: KP, PG, PSte. Writing—review and editing: KP, KT, OO, RA. Funding acquisition: MH, MM, KP. Resources: LK, JR, KT, JIL, BR, LS, ZS, ON, AM, JS, ES, LB, JSk, PStr, AB-V, TP. All authors have read and approved the final manuscript.

Funding

Open access publishing supported by the institutions participating in the CzechELib Transformative Agreement. This work was supported by the Cooperatio Program, research area SURG, Kristyna Pivovarcikova, and the Institutional Research Fund FN 00669806, Milan Hora.

Data availability

Additional data supporting study findings are available from the corresponding author on request.

Declarations