Secondary EWSR1 gene abnormalities in SMARCB1‐deficient tumors with 22q11‐12 regional deletions: Potential pitfalls in interpreting EWSR1 FISH results

Abstract

SMARCB1 inactivation occurs in a variety of tumors, being caused by various genetic mechanisms. Since SMARCB1 and EWSR1 genes are located close to each other on chromosome 22, larger SMARCB1 deletions may encompass the EWSR1 locus. Herein, we report four cases with SMARCB1-deletions showing concurrent EWSR1 gene abnormalities by FISH, which lead initially to misinterpretations as EWSR1-rearranged tumors. Our study group included various morphologies: a poorly differentiated chordoma, an extrarenal rhabdoid tumor, a myoepithelial carcinoma, and a proximal-type epithelioid sarcoma. All cases showed loss of SMARCB1 (INI1) by immunohistochemistry (IHC) and displayed characteristic histologic features for the diagnoses. The SMARCB1 FISH revealed homozygous or heterozygous deletions in three and one case, respectively. The co-hybridized EWSR1 probes demonstrated either unbalanced split signals or heterozygous deletion in two cases each. The former suggested bona fide rearrangement, while the latter resembled an unbalanced translocation. However, all the FISH patterns were quite complex and distinct from the simple and uniform split signals seen in typical EWSR1 rearrangements. We conclude that in the context of 22q11-12 regional alterations present in SMARCB1-deleted tumors, simultaneous EWSR1 involvement may be misinterpreted as equivalent to EWSR1 rearrangement. A detailed clinicopathologic correlation and supplementing the EWSR1 FISH assay with complementary methodology is mandatory for correct diagnosis.

High sensitivity of FISH analysis in detecting homozygous SMARCB1 deletions in poorly differentiated chordoma: a clinicopathologic and molecular study of nine cases

“…Nine cases of poorly differentiated chordoma (PDC) were identified. One case was previously described in the study by Huang et al There were seven females and two males with ages ranging from 2 to 25 years (mean: 10), including 7 (78%) pediatric and 2 (22%) young adult patients. The anatomic sites comprised clivus ( n = 3), cervical spine ( n = 3), clivus and cervical spine ( n = 1), dura mater ( n = 1) and sacrum ( n = 1).…”

“…The primary antibody used was a mouse monoclonal anti‐BAF47 antibody (1:30; BD bioscience, San Jose, CA). Antigen retrieval, antibody incubation, and chromogen counterstaining were performed in a BenchMark ULTRA automated immunostainer (Ventana, Tucson, AZ) as previously described …”

“…Additionally, recent studies have shown that poorly differentiated chordomas (PDCs) are associated with recurrent deletions encompassing the SMARCB1 locus, resulting in consistent loss of nuclear INI1/SMARCB1 expression and implicating this abnormality as the leading pathogenetic mechanism in these tumors . SMARCB1 (also known as INI1, hsnf5, or BAF47) is an ATP‐dependent core subunit of the switch/sucrose non‐fermenting complexes .…”

Role of Next-Generation Sequencing as a Diagnostic Tool for the Evaluation of Bone and Soft-Tissue Tumors

“…Taking into account that SMARCB1/ INI1 and EWSR1 genes are located close to each other on chromosome 22, larger SMARCB1 deletions may encompass the EWSR1 locus, which can lead to misinterpretation by FISH analysis. This reveals the necessity of NGS in significant subgroups of tumors with EWSR rearrangement [5] . Especially in myxoid soft-tissue tumors, NGS can facilitate a precise diagnosis especially on biopsy material by demonstrating recurrent gene fusions or mutations [79] .…”

“…In addition, conflicting FISH results need to be confirmed by alternative techniques (real-time polymerase chain reaction [RT-PCR] or next-generation sequencing [NGS]). One should also keep in mind that in cases of SMARCB1-related deletions, unbalanced abnormalities in EWSR1 can be detected by FISH [5] . To determine not only the EWSR1 break-apart but also the EWSR1 fusion partner, at least 2 FISH examinations have to be performed.…”