A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice.

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

A Rare Case of Rhabdoid Pancreatic Carcinoma: Prolonged Disease-Free Survival Following Upfront Resection and Adjuvant Chemotherapy.

The rhabdoid subtype of undifferentiated pancreatic carcinoma is rarely reported. The clinical course of this disease is therefore poorly understood, although it is apparently an aggressive malignancy. We herein discuss the case of a 69-year-old man presenting with a rapidly enlarging mass of the pancreatic body and tail who was diagnosed with locally advanced SMARCB1-deficient undifferentiated pancreatic carcinoma with rhabdoid features, treated with radical resection and adjuvant chemotherapy, and has achieved 18-month disease-free survival ongoing at the time of article publication. We identify and contrast our case with 15 similar tumors reported in the English literature, briefly discuss the biology of this tumor, its relationship to malignant rhabdoid tumors of childhood, the role of SMARCB1 and its parent complex switch/sucrose-non-fermentable chromatin remodeling complex (SWI/SNF) in modulating the behavior of pancreatic malignancy, and the potential therapeutic avenues available for SWI/SNF-mutated malignancies.

Inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1/INI1 protein in a molecular subset of atypical teratoid/rhabdoid tumors.

Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E-10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E-7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated ß-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations.

Primary cutaneous SMARCB1-deficient carcinoma.

BACKGROUND: SMARCB1-deficient malignancies can arise in various sites. We describe a novel primary SMARCB1-deficient carcinoma of skin (SDCS) and characterize SMARCB1 mutations in non-melanoma skin cancers (NMSC). METHODS: Cases underwent immunophenotyping and targeted exome sequencing (MSK-IMPACT) assay interrogating somatic mutations in 468 cancer-related genes. The MSK-IMPACT database from 2014 to 2020 encompassing 55, 000 cases was searched for NMSC with SMARCB1 mutations. RESULTS: SDCS arose on the scalp of an 18-year-old woman showing homozygous SMARCB1 deletion with a LATS2 G963E variant. Another case arose on the temple of a 76-year-old man harboring a SMARCB1 W206* mutation associated with loss of heterozygosity (LOH), 59 concurrent mutations, and a UV mutation signature (UV-MS). Both tumors exhibited INI1 loss, positive CK5/6, p40, p63, and claudin-4 with negative CD34. Of 378 NMSC cases, including 370 carcinomas, 7 SMARCB1-mutated tumors were identified: 3 squamous cell, 3 Merkel cell, and one basal cell carcinoma. Six showed UV-MS. Five INI1-interrogated cases retained protein expression suggesting they were SMARCB1-proficient. CONCLUSIONS: SDCS can be clinically aggressive, harbor SMARCB1 homozygous deletions or truncating SMARCB1 mutations associated with LOH, and can occur with or without UV-MS. Overall, SMARCB1 mutations in NMSC are rare with most being of undetermined significance and associated with retained INI1 and UV-MS.

Recurrent SMARCB1 Mutations Reveal a Nucleosome Acidic Patch Interaction Site That Potentiates mSWI/SNF Complex Chromatin Remodeling.

Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.

The structure of INI1/hSNF5 RPT1 and its interactions with the c-MYC:MAX heterodimer provide insights into the interplay between MYC and the SWI/SNF chromatin remodeling complex.

c-MYC and the SWI/SNF chromatin remodeling complex act as master regulators of transcription, and play a key role in human cancer. Although they are known to interact, the molecular details of their interaction are lacking. We have determined the structure of the RPT1 region of the INI1/hSNF5/BAF47/SMARCB1 subunit of the SWI/SNF complex that acts as a c-MYC-binding domain, and have localized the interaction regions on both INI1 and on the c-MYC:MAX heterodimer. c-MYC interacts with a highly conserved groove on INI1, while INI1 binds to the c-MYC helix-loop-helix region. The binding site overlaps with the c-MYC DNA-binding region, and we show that binding of INI1 and E-box DNA to c-MYC:MAX are mutually exclusive.

SMARCB1/INI1/BAF47- deficient pleural malignant mesothelioma with rhabdoid features.

Malignant mesothelioma (MM) with rhabdoid features is an MM variant. Fifteen cases have been reported previously, all of which were combined with other types of MM. Herein, we report an autopsy case of pleural MM with monomorphic rhabdoid features. The patient was a 62-year-old male without a history of asbestos exposure. An autopsy revealed a soft, granular tumor that replaced the entire left pleura and had invaded to the diaphragm and lower lobe of the lung. The tumor cells, which had eosinophilic plump cytoplasm and eccentric nuclei, were loosely cohesive. Immunohistochemistry showed that the cells were diffusely positive for calretinin, D2-40, vimentin, CAM5.2, and AE1/AE3; and negative for WT-1, TTF-1, CK7, CEA, desmin, CD34, BCL-2, S100 protein, and p40. Neither homozygous deletion of p16 nor BAP-1 protein loss was observed. Loss of INI1/BAF47 protein, an indicator of malignant rhabdoid tumor, was observed. Therefore, MM with rhabdoid features was confirmed.

A primary extradural malignant rhabdoid tumor at the craniovertebral junction in a 3-year-old boy.

INTRODUCTION: Malignant rhabdoid tumor (MRT) is a highly aggressive childhood neoplasm and mainly presents in kidney and brain. We report the case of a patient with extrarenal extracranial MRT in the craniovertebral junction (CVJ). CASE REPORT: A 3-year-old boy presented with tetraparesis that had rapidly developed for 2 weeks. The tumor was located in the ventral side of the C1 and C2 extradural space and had invaded the lower clivus. The symptom recovered after subtotal resection of the tumor through a lateral approach with removal of bilateral lateral masses at C1 and instrumental occipital-C2 posterior fusion. Definite histological diagnosis of the extradural tumor was difficult; however, a metastatic lesion in the rib showed a proliferation of INI1/SMARCB1-negative spindle and rhabdoid cells, indicating the tumor was MRT. Stereotactic CyberKnife radiotherapy with a marginal dose of 26 Gy contributed to control of local regrowth of the tumor before histopathological confirmation and induction of chemotherapy with ifosfamide, cisplatin, and etoposide. The patient survived for 29 months without local recurrence of the tumor and with independent activity, however then died of multiple metastases. CONCLUSION: Treatment strategies for MRT in the CVJ should include an optimal surgical approach for reduction of tumor volume and stabilization of the spine, followed by high dose chemotherapy. Stereotactic radiotherapy may be useful for local control.

Evaluation of the role of downregulation of SNF5/INI1 core subunit of SWI/SNF complex in clear cell renal cell carcinoma development.

Clear cell renal cell carcinoma (ccRCC) is characterized by stabilization of hypoxia-inducible factor (HIF1), and mutations in von Hippel-Lindau (VHL) gene. Additionally, in about 40% of ccRCC cases the mutation in PBRM1 (POLYBROMO1) gene coding for a non-core subunit of SWI/SNF chromatin remodeling complex was found suggesting potential impairment of this complex function in ccRCC. In this study we assessed the extent to which the core SWI/SNF complex subunit - INI1 (hSNF5/SMARCB1) is affected in ccRCC and whether it has any consequences on the development of this type of cancer. The evaluation of INI1 protein level in samples from 50 patients with diagnosed ccRCC, including three displaying rhabdoid features, showed the INI1 positive staining in rhabdoid cells while the conventional ccRCC cells exhibited reduced INI1 level. This indicated the rhabdoid component of ccRCC as distinct from other known rhabdoid tumors. The reduced INI1 protein level observed in all conventional ccRCC cases used in this study correlated with decreased SMARCB1 gene expression at the transcript level. Consistently, the overexpression of INI1 protein in A498 ccRCC cell line resulted in the elevation of endogenous SMARCB1 transcript level indicating that the INI1-dependent regulatory feedback loop controlling expression of this gene is affected in ccRCC Moreover, the set of INI1 target genes including i.e. CXCL12/CXCR7/CXCR4 chemokine axis was identified to be affected in ccRCC. In summary, we demonstrated that the inactivation of INI1 may be of high importance for ccRCC development and aggressiveness.

Structural Insights into BAF47 and BAF155 Complex Formation.

Mammalian BAF complexes are a subfamily of SWI/SNF ATP-dependent chromatin remodelers that dynamically modulate chromatin structure to regulate fundamental cellular processes including gene transcription, cell cycle control, and DNA damage response. So far, many distinct BAF complexes have been identified with polymorphic assemblies of up to 15 subunits from 29 genes. The evolutionarily conserved BRG1/BRM, BAF47, and BAF155/BAF170 form a stable complex that carries out essential chromatin remodeling activity and therefore have been regarded as the core components of BAF complex. Here, we first confirmed that SWIRM domain of BAF155 is responsible for its interaction with BAF47 and then narrowed down the SWIRM-binding region in BAF47 to the Repeat 1 (RPT1) domain. We further presented the high-resolution crystal structure of SWIRM/RPT1 complex. Extensive mutagenesis experiments together with isothermal titration calorimetry and NMR titrations were performed to corroborate the interactions observed in crystal structure. Overall, we demonstrated that BAF155 SWIRM is a modular domain involved in BAF47 interaction, which is functionally distinct from other characterized SWIRM domains that possess DNA binding activity.

Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.

MYC interaction with the tumor suppressive SWI/SNF complex member INI1 regulates transcription and cellular transformation.

MYC is a key driver of cellular transformation and is deregulated in most human cancers. Studies of MYC and its interactors have provided mechanistic insight into its role as a regulator of gene transcription. MYC has been previously linked to chromatin regulation through its interaction with INI1 (SMARCB1/hSNF5/BAF47), a core member of the SWI/SNF chromatin remodeling complex. INI1 is a potent tumor suppressor that is inactivated in several types of cancers, most prominently as the hallmark alteration in pediatric malignant rhabdoid tumors. However, the molecular and functional interaction of MYC and INI1 remains unclear. Here, we characterize the MYC-INI1 interaction in mammalian cells, mapping their minimal binding domains to functionally significant regions of MYC (leucine zipper) and INI1 (repeat motifs), and demonstrating that the interaction does not interfere with MYC-MAX interaction. Protein-protein interaction network analysis expands the MYC-INI1 interaction to the SWI/SNF complex and a larger network of chromatin regulatory complexes. Genome-wide analysis reveals that the DNA-binding regions and target genes of INI1 significantly overlap with those of MYC. In an INI1-deficient rhabdoid tumor system, we observe that with re-expression of INI1, MYC and INI1 bind to common target genes and have opposing effects on gene expression. Functionally, INI1 re-expression suppresses cell proliferation and MYC-potentiated transformation. Our findings thus establish the antagonistic roles of the INI1 and MYC transcriptional regulators in mediating cellular and oncogenic functions.

Atypical teratoid/rhabdoid tumors-current concepts, advances in biology, and potential future therapies.

Atypical teratoid/rhabdoid tumor (AT/RT) is the most common malignant CNS tumor of children below 6 months of age. The majority of AT/RTs demonstrate genomic alterations in SMARCB1 (INI1, SNF5, BAF47) or, to a lesser extent, SMARCA4 (BRG1) of the SWItch/sucrose nonfermentable chromatin remodeling complex. Recent transcription and methylation profiling studies suggest the existence of molecular subgroups. Thus, at the root of these seemingly enigmatic tumors lies a network of factors related to epigenetic regulation, which is not yet completely understood. While conventional-type chemotherapy may have significant survival benefit for certain patients, it remains to be determined which patients will eventually prove resistant to chemotherapy and thus need novel therapeutic strategies. Elucidation of the molecular consequences of a disturbed epigenome has led to the identification of a series of transduction cascades, which may be targeted for therapy. Among these are the pathways of cyclin D1/cyclin-dependent kinases 4 and 6, Hedgehog/GLI1, Wnt/ß-catenin, enhancer of zeste homolog 2, and aurora kinase A, among others. Compounds specifically targeting these pathways or agents that alter the epigenetic state of the cell are currently being evaluated in preclinical settings and in experimental clinical trials for AT/RT.

Epithelioid Sarcoma: Opportunities for Biology-Driven Targeted Therapy.

Epithelioid sarcoma (ES) is a soft tissue sarcoma of children and young adults for which the preferred treatment for localized disease is wide surgical resection. Medical management is to a great extent undefined, and therefore for patients with regional and distal metastases, the development of targeted therapies is greatly desired. In this review, we will summarize clinically relevant biomarkers (e.g., SMARCB1, CA125, dysadherin, and others) with respect to targeted therapeutic opportunities. We will also examine the role of EGFR, mTOR, and polykinase inhibitors (e.g., sunitinib) in the management of local and disseminated disease. Toward building a consortium of pharmaceutical, academic, and non-profit collaborators, we will discuss the state of resources for investigating ES with respect to cell line resources, tissue banks, and registries so that a roadmap can be developed toward effective biology-driven therapies.

Utility of characteristic 'Weak to Absent' INI1/SMARCB1/BAF47 expression in diagnosis of synovial sarcomas.

Recently, very few studies have shown value of immunohistochemical (IHC) expression of INI1/SMARCB1 in diagnosis of synovial sarcomas (SSs). This study was aimed at testing reproducibility and utility of this finding. Sixty-eight SSs and 147 other tumours, in the form of various biopsies, were tested for IHC expression of INI1. Twenty-six SSs were further confirmed with positive SS18 rearrangement. Forty monophasic spindle cell type (58.8%), 13 biphasic (19.1%), 12 poorly differentiated (17.6%) and three calcifying SSs (4.4%) were positive for epithelial membrane antigen (EMA) (46/62) (74.1%), pan cytokeratin (AE1/AE3) (31/47) (65.9%), cytokeratin (CK7) (20/31) (64.5%), BCL2 (62/66) (93.9%), MIC2 (61/63) (96.8%), transducin-like enhancer of split 1 (TLE1) (29/31) (93.5%) and CK19 (14/24) (58.3%). INI1 expression was 'weak to absent' in 60/68 (88.2%) SSs; in 1/3 atypical ossifying fibromyxoid tumours (AOFMTs) and in 3/10 (30%) malignant peripheral nerve sheath tumours (MPNSTs) of various types. INI1 was completely absent in 10/10 (100%) epithelioid sarcomas (ESs), 4/4 (100%) malignant rhabdoid tumours, single paediatric undifferentiated sarcoma, 5/19 (26.3%) myoepithelial carcinomas and in 2/4 (50%) epithelioid-subtype of MPNSTs. Remaining 100 tumours, including 12 Ewing sarcomas, 15 carcinomas, eight solitary fibrous tumours (SFT), seven extraskeletal myxoid chondrosarcomas, three fibrosarcomas and other tumours retained INI1 expression. A unique 'weak to absent' IHC expression of INI1 is highly sensitive (88.2%) and specific (97.3%) for a SS, irrespective of its subtypes and types of biopsies. This can be considered useful in diagnosing SSs, especially in settings lacking molecular and/or cytogenetic analysis. A similar INI1 expression is shared by certain AOFMTs and MPNSTs.

Pathology and diagnosis of SMARCB1-deficient tumors.

Malignant rhabdoid tumor (MRT) can occur in the kidney, central nervous system, or extracranial/extrarenal locations and is characterized by alterations in the SMARCB1 gene. The tumors occur in infants and young children and confer a poor prognosis requiring aggressive therapeutic interventions to improve the chances for survival. MRTs pose a diagnostic challenge, as they display heterogeneous histopathologic features and differentiate along multiple lineages. The identification of alterations in the SMARCB1 gene in MRT using immunohistochemical (IHC) staining has lead to improved diagnosis of MRT as well as the discovery of the loss of SMARCB1 expression in some non-MRTs. Whether loss of SMARCB1 plays a pathogenic role in nonrhabdoid tumors remains to be determined; however, most of these tumors lack the clinical and other molecular features of MRT. We review the histopathologic features of MRT and the importance and significance of loss of expression of SMARCB1 in both MRT and nonrhabdoid tumors.

Non-rhabdoid pediatric SMARCB1-deficient tumors: overlap between chordomas and malignant rhabdoid tumors?

Somatic alterations in the tumor suppressor gene SMARCB1 were first described in the malignant rhabdoid tumor (MRT) of infancy. Since then, SMARCB1 alterations have been found in other tumors, forming a varied group of SMARCB1-deficient tumors, which sometimes shares overlapping immunohistochemical and histological findings. Thus, the diagnosis is challenging. We report two cases of pediatric SMARCB1-deficient tumors from the clivus that illustrate the diagnostic difficulties. Both cases were strongly positive for epithelial markers associated with loss of BAF47 (INI1) expression, and were negative for S100 and CD34. Molecular analyses of the SMARCB1 gene found a deletion of all nine exons in both cases. In the first case, a 5-year-old girl presented with a thoracic metastasis of a clival tumor, which was diagnosed as MRT and treated accordingly. The morphological findings and the expression of brachyury would favor the diagnosis of a poorly differentiated chordoma. The second case was a quickly fatal clival tumor in a 2-year-old boy: This tumor was morphologically undifferentiated and raises the problem of differential diagnosis between an MRT, a malignant myoepithelial tumor, or an undifferentiated chordoma due to the location and the expression of brachyury. Studies of biological signatures, such as transcriptome profiling, could help to understand the apparent overlap between these tumors.

The SWI/SNF tumor suppressor complex: Regulation of promoter nucleosomes and beyond.

Nucleosomes, octamers of histones wrapped in 147 bp of DNA, are the basic unit of chromatin. In eukaryotic cells, the placement of nucleosomes along the genome is highly organized, and modulation of this ordered arrangement contributes to regulation of gene expression. The SWI/SNF complex utilizes the energy of ATP hydrolysis to mobilize nucleosomes and remodel chromatin structure. Recently, the complex has also been implicated in oncogenesis as genes encoding multiple SWI/SNF subunits have been found mutated at high frequency across a wide spectrum of cancers. Given that epigenetic aberrations are now characterized as a hallmark of human cancer, hypotheses have been put forth that the SWI/SNF complex inhibits tumor formation by regulating key chromatin functions. To understand how the SWI/SNF complex contributes to nucleosome organization in vivo we performed a genome-wide study in mammalian cells. We found that inactivation of SWI/SNF subunits leads to disruptions of specific nucleosome patterning and a loss of nucleosome occupancy at a large number of promoters. These findings define a direct relationship between the SWI/SNF complex, chromatin structure, and transcriptional regulation. In this extra view, we discuss our findings, their relevance to gene regulation, and possible links to the tumor suppression activities of the SWI/SNF complex.

Immunohistochemical validation of INI1/SMARCB1 in a spectrum of musculoskeletal tumors: an experience at a Tertiary Cancer Referral Centre.

The purpose of this study was to evaluate and validate immunohistochemical (IHC) expression of INI1/SMARCB1 in various musculoskeletal tumors in the light of the established literature. Twenty-seven cases of epithelioid sarcoma (ES); 4 of extrarenal rhabdoid tumor (ERRT) of soft tissue and 97 other tumors, including 16 cases of synovial sarcoma (SS), were evaluated for IHC expression of INI1 on formalin-fixed, paraffin-embedded tissue sections of various biopsies. Out of 128 tumors, INI1/SMARCB1 staining was completely lacking in cases of ES (23/27) (85.1%), ERRTs (4/4) (100%), myoepithelial tumors (4/14) (28.5%) and in (1/16) (6.2%) cases of SS. Fourteen out of 15 SSs displayed a reduced staining pattern. Other 67 studied tumors were INI1-positive. Sensitivity for complete INI1 negativity in ES was 85.1%, and specificity with respect to its differentials, excluding ERRTs, was 94.8%. Complete lack of INI1 immunostaining in most ESs indicates its value as a diagnostic marker for ESs, including those occurring at rare sites; in ERRTs and in some myoepithelial tumors, within an appropriate clinicopathological context, in all kinds of biopsies. ES, at least in some cases, is immunohistochemically the most closely related tumor to an ERRT. A unique pattern of reduced INI1 expression in a SS is useful during triage of some cases for molecular testing. Its expression should be interpreted in the tumor cells, rather than intermixed stromal cells and or inflammatory cells that retain INI1 expression.