Sarcomatoid renal cell tumor harboring a novel BYSL::TFEB fusion with concurrent TFEB amplification

Transcription factor EB (TFEB)‐rearranged renal cell carcinoma (RCC) exhibits diverse gene fusion patterns and heterogeneous clinicopathologic features. Rare TFEB‐amplified RCCs have been described recently and are associated with a more aggressive clinical course. Herein, we report a case of an 86‐year‐old man with a solid 9.2‐cm kidney tumor that showed a diffuse high‐grade sarcomatoid morphology. The tumor demonstrated a novel BYSL::TFEB fusion containing exons 1–2 of the BYSL gene fused to exons 3–10 of TFEB via next‐generation sequencing by using NextSeq sequencer. Fluorescence in situ hybridization (FISH) studies displayed concurrent high‐copy number TFEB amplification in two distinct patterns, a balanced increase of 5′ and 3′ copies, and solely increased 5′ copies, and mouse double minute 2 (MDM2) gene amplification by using TFEB (6p21.1) dual‐color break‐apart probe and MDM2 FISH probe. Notably, the tumor showed a distinctive immunoprofile with overexpressions of TFEB, epithelial membrane antigen, Cathepsin K, and PDL‐1 (SP263). FISH test for transcription factor binding to IGHM enhancer 3 (TFE3) was negative for rearrangement and corresponding immunonegativity of TFE3. These findings not only expand the repertoire of known TFEB fusion partners implicated in tumorigenesis, but also may provide novel information for target therapy.


| INTRODUCTION
The evolving classification of renal tumors is based on integration of histopathology, immunohistochemistry, and underlying molecular pro- TFEB plays an important role in cellular processes such as autophagy-lysosomal biogenesis, energy homeostasis, metabolism and stress response. 2 It modulates various signaling pathways including mTORC1, ERK, Wnt, calcium, and AKT. Alterations of the TFEB gene have been reported in a variety of human cancers such as pancreas, colon, breast, lung and skin cancers. Although the molecular mechanisms of TFE3 and TFEB in RCC tumorigenesis are not well understood, promotor substitution involving transcription factors seems to be the key molecular event. 3 TFE3-and TFEB-rearranged RCCs account for approximately 40% of pediatric RCCs and up to 5% of adult sporadic RCCs. 4 The molecular events in TFEB-altered RCC include TFEB gene fusion and amplification resulting in TFEB overexpression. 5 TFEB-amplified RCCs typically occur in older patients and are associated with a higher tumor grade and advanced tumor stage. In general, TFEB-rearranged RCCs have an indolent clinical behavior, while TFEB-amplified and TFE3-rearranged RCCs are relatively more aggressive, with prognosis similar to that of ccRCC. All of these tumors display remarkable histologic heterogeneity overlapping with other RCC subtypes without an unequivocal genotype-phenotype correlation. 6 Molecular and cytogenetic testing is often required for an accurate distinction. Most TFEBrearranged RCCs show a distinctive biphasic morphology composed of nests of large and small epithelioid cells clustering around homogeneous eosinophilic basement membrane materials. 7 The tumor cells show low expression of epithelial markers (cytokeratin and EMA), along with variable expression of TFE3/TFEB downstream melanocytic markers HMB45 and Melan A (even more variable within TFEBamplified RCCs), and cysteine protease Cathepsin K. 6 Cathepsin K is a reliable and predictive biomarker for translocation RCCs (tRCCs), and was not observed in the most common adult RCCs. 8,9 Compared to epithelial neoplasm, Cathepsin K is a not as specific a marker in spindled mesenchymal lesions as it is in epithelial tumor, including peritumoral and desmoplastic stroma. 10 It is well known that TFEB-altered RCC can lose expression of PAX8. 11 To date, fewer than 100 cases of TFEB-rearranged RCC have been reported, and even rarer (<5 cases) were cases of RCCs with concurrent TFEB rearrangement and amplification. 6,8,[12][13][14][15] Sarcomatoid differentiation in TFEB-altered RCC is an unusual phenomenon as seen in our case. Sarcomatoid RCCs are often associated with poor therapeutic response and survival. However, current data indicated a benefit from checkpoint inhibitor therapy. 14 Herein, we report a case of an elderly man with a sarcomatoid     copies was shown by equally increased 5 0 -probe and 3 0 -probe signals ( Figure 3b1). An unbalanced pattern was depicted as the loss of the 3 0 TFEB probe signals with retention of the 5 0 -probe signals (Figure 3b2,   b3). Within the tumor, multiple hybridization signals (6 to >20) for the MDM2 probe were seen in 88 out of 100 nuclei analyzed, with a 12q15/CEP12 ratio of 3.14. This finding is consistent with amplification of the MDM2 gene. In adipose tissue adjacent to the tumor, the 12q15/CEP12 ratio was 0.98, indicating a negative result ( Figure 3C).

| Fluorescence in situ hybridization
In contrast, FISH test for TFE3 was negative for gene rearrangement (data not shown).
After the radical nephrectomy, essentially no adjuvant chemotherapy or radiation therapy was recommended by the oncologists for the patient treatment. After 8 months, he remained stable without evidence of recurrence. Interestingly, TFEB fusion and TFEB amplification seem mutually exclusive in most cases. As a rare finding, fewer than five cases with F I G U R E 3 Molecular and cytogenetic findings. Next-gene sequencing (NGS) showed a novel in-frame BYSL-TFEB fusion gene (A). Fluorescence in situ hybridization (FISH) studies with dualcolor break-apart probes covering chromosome 6p21.1 locus were used (B). Balanced TFEB amplification with equally increased 5 0 -probe and 3 0 -probe signals (b1). Unbalanced pattern with loss of 3 0probe signals and retention of 5 0 -probe signals (b2,b3). A possible third pattern of coexisting balanced TFEB amplification (> two yellow-colored signals) with unbalanced TFEB rearrangement/deletion (b2). MDM2 FISH analysis (C) shows MDM2 amplification in the tumor cells (c1) and normal copy numbers of MDM2 in adipose tissue adjacent to the tumor (c2).
both functional domain-retained TFEB fusion and TFEB (6p21. 1) amplification have been reported. 6,16,17 In the current report, we identified the first case of a sarcomatoid renal tumor harboring a novel expression. 18 Molecularly, sRCC is discrete from the parent ccRCC in that it has fewer deletions at 3p, a lower rate of two-hit loss for VHL and PBRM1 and higher mutation rates in PTEN, TP53, and RELN genes.
In the present case, no other masses or malignancies were identified on extensive clinical evaluation and imaging studies, supporting the diagnosis of a primary kidney tumor. Also, our case was immunoreactive for Cathepsin K, strongly supporting the diagnosis of tRCC. 9 Furthermore, our sarcoma fusion NGS panel did not reveal any other sarcoma fusion genes, and multiple sarcoma immunomarkers were negative. Interestingly, the existence of an extrarenal RCC, including MiT family tRCC, in the kidney's proximity but without an identifiable renal primary was an uncommon clinical scenario in a recent study. 19 Taken together, these findings favor the diagnosis of a primary tRCC.
Our case differs molecularly from previously reported TFEB-  Figure 2L), which has been considered negative staining in prior studies. 25 No TFE3 gene rearrangement was identified by FISH test (data not shown). The coexisting TFEB fusion and amplification events in our case, therefore, could be sequential or independent. As both are implicated in tumorigeneses, we cannot definitively delineate the individual role of these two events in the oncogenic process.
In this study, both the TFEB and BYSL genes are located on These genes, therefore, will be amplified in parallel with the TFEB gene and may be responsible for the aggressive behavior of the TFEBaltered tumors. CCND3 gene overexpression was prominent in tumorigenesis and aggressiveness. 26 MDFI overexpression was implicated in muscle fiber-type transformation in myoblasts, which could be responsible for the focal rhabdoid phenotype found in our case. 27 Another significant genetic alteration in our case was the amplification of MDM2 gene. The MDM2 gene encodes an ubiquitin-protein ligase acting as a negative regulator of the p53 tumor suppressor.
MDM2 gene amplification has been commonly observed in well-and de-differentiated liposarcomas, other sarcomas, and in conventional/ sarcomatoid RCC. 28 The discovery of MDM2 gene amplification in the present case prompted us to rule out liposarcoma. Extensive tissue sampling, including the adipose tissue adjacent to the tumor, showed no evidence for liposarcoma by FISH test. Combined with the absence of TFEB gene alterations in liposarcomas, we believed the likelihood of a diagnosis of liposarcoma was low.
Given that RCCs with sarcomatoid features may benefit from immune checkpoint inhibitor-based therapy, 14 immunohistochemical staining for PD-L1 (SP263) was performed. We found membranous staining of PD-L1 (SP263) of the tumor cells, similar to that reported in other TFEB-rearranged RCCs. 14 This result could offer therapeutic options in this case.

| CONCLUSION
In summary, we report a case of sarcomatoid RCC in an elderly patient carrying a novel BYSL::TFEB fusion, with concurrent TFEB and MDM2 gene amplification, and with a unique immunoprofile. Our findings expand the morphologic and molecular spectrum of TFEB-altered RCCs, and provide a better understanding of the role TFEB alterations play in RCC tumorigenesis. A detailed molecular analysis certainly aids in a more accurate diagnosis and potentially contributes to the management of these malignancies.