We report the clinicopathologic features of EWSR1-rearranged renal neoplasia from our institution. A retrospective cohort of 39 EWSR1-rearranged renal tumors was identified using fluorescence in situ hybridization (FISH) and RNA-based next generation sequencing (NGS). A final diagnosis of Ewing sarcoma (EWS) was established in 34 of 39 cases (87%), with the remaining cases diagnosed as desmoplastic small round cell tumor (DSRCT; n = 2), sclerosing epithelioid fibrosarcoma (SEF; n = 2), and thyroid-like follicular renal cell carcinoma (TLFRCC; n = 1). Fusion partners identified in EWS included FLI1 (n = 17) and ERG (n = 2). WT1 (n = 2), CREB3L1 (n = 1) and CREB3L2 (n = 1), and PATZ1 (n = 1) fusions were found in DSRCT, SEF, and TLFRCC, respectively. The mean age at EWS diagnosis was 31.4 years (range 6-73), with a similar sex distribution (18 females, 16 males), and a mean tumor size of 10.7 cm (range 3-24 cm). Both DSRCT cases occurred in males aged 6 and 29 years, diagnosed on renal biopsy and brain metastasis, respectively. The SEF cases involved primary tumors in 22-year-old and 43-year-old females. The one case of TLFRCC was identified in a 41-year-old female that underwent radical nephrectomy. Cases with available immunohistochemistry showed most EWS tumors (24/26, 92%) expressed a combination of CD99, FLI1, and ERG, while both SEF cases were positive for MUC4. Our results highlight the importance of molecular testing in providing an integrated diagnosis and are informative regarding the spectrum of renal neoplasia that harbor EWSR1 rearrangements, including EWS, DSRCT, SEF, and TLFRCC as these tumors can exhibit significant clinicopathologic heterogeneity.

OBJECTIVE:In endometrial cancer (EC), pretreatment identification of insensitivity to platinum-based chemotherapy (PbCT) may improve survival. We hypothesized that transcriptome profiling would identify seminal genes associated with chemoresistance. METHODS:We identified 209 EC cases with robust mRNA data from The Cancer Genome Atlas (TCGA) database. Cases with stage III/IV EC or stage I/II plus myometrial invasion ≥ 66% were categorized as clinicopathologic high risk (CPHR); the remainder were clinicopathologic low risk (CPLR). We assessed expression of genes (excluding POLE variants) associated with chemoresistance. Expression data were normalized, log2 transformed, and Pearson correlated with 5-year progression-free survival (PFS). PFS rates were compared using log-rank tests; continuous variables were compared using t-tests. RESULTS:Composite expression scores for chemoresistance-associated genes correlated with 5-year PFS rates: low scores, 84.4%; intermediate, 74.7%; and high, 23.0%. High expression of transcription factor CCNA2/E2F1 significantly correlated with increased expression of DNA damage repair, cell cycle, and antiapoptosis genes (majority, R > 0.80). Independent of most traditional risk factors, low vs high CCNA2 + E2F1 expression stratified patients by 5-year PFS (89% vs 50%), endometrioid and serous histologies, TCGA subgroups, and clinicopathologic risk (all P < .001). Low vs high CCNA2 + E2F1 expression also significantly affected 5-year PFS for CPLR (89% vs 59%) and CPHR (87% vs 39%) cases. CONCLUSION:CPHR or CPLR-plus-recurrence cases with low CCNA2 + E2F1 expression likely have PbCT sensitivity; analogous cases with high CCNA2 + E2F1 expression likely have PbCT insensitivity. Pretreatment transcriptome profiling potentially can determine PbCT insensitivity and identify multiple alternative therapeutic targets, thereby sparing patients platinum-associated toxicities and disease progression.

Mutations in the KIT and PDGFRA proto-oncogenes are key drivers in gastrointestinal stromal tumors (GIST) and guide targeted therapy. While DNA-based next-generation sequencing (NGS) is standard for mutation detection, RNA sequencing (RNA-Seq) may offer complementary advantages. This study evaluates RNA-Seq performance and presents molecular epidemiologic data from a large DNA-sequenced GIST cohort. RNA-Seq was performed on 24 GIST cases previously analyzed by DNA-based NGS (16 KIT mutants, 5 PDGFRA mutants, 3 wild-type) using the Agilent SureSelectXT RNA Direct Library Prep Kit and an in-house analysis pipeline. RNA-Seq was successful in 21 cases, identifying 13 of 14 KIT mutations and all PDGFRA mutations, including single nucleotide variants (SNV), insertions, and in-frame deletions (3-27 bp). One complex 45 bp deletion-insertion was not detected, though low-level evidence (<10% of reads) was present. Separately, DNA-based NGS results from 579 GIST cases were reviewed to assess mutation prevalence and clinicopathologic associations. Mutations were found in 83.2% of cases (403 KIT, 79 PDGFRA), with secondary KIT mutations in 6.0%. Mutation site correlated with tumor location and patient age; secondary mutations were more frequent in non-gastric tumors. RNA-Seq demonstrates high accuracy for detecting clinically relevant KIT and PDGFRA mutations and may complement DNA-based profiling. The DNA cohort provides broader context for mutation prevalence and patterns in clinical practice.

Bone and soft tissue sarcomas harboring EWSR1::NFATC2 and FUS::NFATC2 fusions (NFATC2-rearranged sarcomas) are a recently defined entity with a morphologic spectrum and clinical behavior that are not fully elucidated. We studied 32 such sarcomas that occurred in 21 male and 11 female patients. The EWSR1::NFATC2 fusion was found in tumors of 25 patients (17 men and 8 women; median age, 40 years; range, 14-78 years), 16 of which arose in soft tissue, while 8 originated in the bone. Morphologically, they showed relatively consistent morphologic features yet variable degrees of cytologic atypia, mitotic rates, and necrosis. Follow-up (19 patients; median, 22 months; range, 1-70 months) demonstrated local recurrence in 3 patients, while distant metastases occurred in 6 patients. Two patients died of disease, 4 were alive with disease, and 13 were alive without evidence of disease. In contrast, the FUS::NFATC2 fusion was exclusively seen in osseous tumors, which occurred in 7 patients (4 men and 3 women; median age, 32 years; range, 4-62 years). Further, FUS::NFATC2 tumors showed significant morphologic heterogeneity. Follow-up (6 patients; median, 18 months; range, 13-60 months) demonstrated local recurrence in 2 patients and lung metastases in 2 patients. At the last follow-up, 3 patients were alive with disease, while 3 patients were alive without evidence of disease. Using a 2-tiered grading scheme based on cytologic atypia, mitotic rate, and necrosis, patients with low-grade tumors experienced significantly fewer adverse events than those with high-grade tumors (P = .026); however, estimated metastasis-free survival was not statistically significant due to our limited sample size. Overall, our study expands on the morphologic spectrum of NFATC2-rearranged sarcomas and highlights the clinicopathologic, molecular, and genetic differences between the EWSR1- and FUS-rearranged tumors. Although additional long-term follow-up data are required, our study further suggests that a subset of these sarcomas have a protracted clinical course, while high-grade morphologic features such as atypia, mitotic activity, and necrosis may correlate with worse behavior.

BACKGROUND:Biphenotypic sinonasal sarcoma (BSNS) typically demonstrates expression of both neural and myogenic markers while harboring PAX3 gene fusions. Reported fusion partners include MAML3, NCOA1, NCOA2, FOXO1, FOXO6, WWTR1, and YAP1. Herein, we report two additional cases of BSNS harboring PAX3::FOXO6 gene fusions to aid in variant classification. CASE PRESENTATION:Patient 1 was a 37-year-old female and patient 2 was 56-year-old male with BSNS involving the right nasal cavity and left middle turbinate, respectively. Both sarcomas were unencapsulated infiltrative spindle cell neoplasms that demonstrated morphologic features of BSNS. Case 1 demonstrated variable aberrant nuclear accumulation of beta-catenin protein and lacked expression of SMA and S100 protein. Positive reactions were observed with SMA and S100 protein in case 2. RNA sequencing identified PAX3::FOXO6 (exon 7::exon 2) in both cases. CONCLUSION:The sinonasal location, morphology, and immunophenotype allow for discrimination against competing entities in most cases. In select cases genetic evaluation becomes necessary and to further aid in variant classification and to provide additional support for this rare finding we provide two examples of BSNS harboring PAX3::FOXO6.

Grading of epithelioid mesothelioma (EM) has recently been reported to be predictive of survival. We investigated whether RNA expression profiles and immunohistochemical (IHC) markers could refine EG grading and potentially identify therapeutic targets. Forty-seven pleural mesothelioma biopsies were studied, including 6 sarcomatoid/desmoplastic mesothelioma (SM), 6 biphasic mesothelioma (BM), and 35 EM. We subclassified EM cases into low-(LG) and high-grade (HG) based on WHO classification. FFPE slides were used for RNA expression and IHC (BAP1, EZH2 and PD-L1) studies. RNA expression was profiled by nCounter PanCancer Pathways Panel (NanoString, 770 genes). In 35 EM, 14 were classified as EM-HG and 21 as EM-LG. RNA expression heat mapping demonstrated that EM-LG predominantly cluster together while EM-HG exhibit more heterogeneity with some cases exhibiting proximity to SM/BM. SM/BM demonstrated a noticeable different IHC profile compared to EM-LG: fewer BAP1 loss (25% vs 86%), higher expression of PD-L1 (mean 30.1%, range 0-80% vs mean 1.3%, 0-10%) and higher EZH2 (mean H score 136.3, 10-250 vs mean 57.1, 5-150). Similarly to EM-LG, EM-HG exhibited low PD-L1 expression with a mean TPS of 1.2%, suggesting limited clinical utility for PD-L1 IHC for EM-LG and EM-HG. A non-statistically significant trend for higher EZH2 expression in EM-HG (mean H score 88.3, 60-140) compared to EM-LG was seen. In conclusion, in addition to supporting histology grading of EM, our findings highlight that EM-HG cases display more heterogeneous expression profiles than EM-LG with some features in between EM-LG and SM/BM. Further data is needed to explore the potential clinical utility EZH2 IHC in the clinical management of pleural mesothelioma.

An amplicon-based targeted next-generation sequencing (NGS) assay for the detection of gene fusions in sarcomas was developed, validated, and implemented. This assay can detect fusions in targeted regions of 138 genes and BCOR internal tandem duplications. This study reviews our experience with testing on the first 652 patients analyzed. Gene fusions were detected in 238 (36.5%) of 652 cases, including 83 distinct fusions in the 238 fusion-positive cases, 10 of which had not been previously described. Among the 238 fusion-positive cases, the results assisted in establishing a diagnosis for 137 (58%) cases, confirmed a suspected diagnosis in 66 (28%) cases, changed a suspected diagnosis in 25 (10%) cases, and were novel fusions with unknown clinical significance in 10 (4%) cases. Twenty-six cases had gene fusions (ALK, ROS1, NTRK1, NTRK3, and COL1A1::PDGFB) for which there are targetable therapies. BCOR internal tandem duplications were identified in 6 (1.2%) of 485 patients. Among the 138 genes in the panel, 66 were involved in one or more fusions, and 72 were not involved in any fusions. There was little overlap between the genes involved as 5'-partners (31 different genes) and 3'-partners (37 different genes). This study shows the clinical utility of a next-generation sequencing gene fusion detection assay for the diagnosis and treatment of sarcomas.

Molecularly defined renal cell carcinomas include TFE3-rearranged renal cell carcinoma (TFE3-RCC) and TFEB-altered renal cell carcinoma (TFEB-RCC). There is significant morphologic and immunophenotypic overlap between these entities and common renal tumors, such that molecular testing is often required to make the diagnosis. Herein, we reviewed our reference laboratory experience pertaining to TFE3 and TFEB FISH testing, targeted next generation RNA sequencing (NGS), and GPNMB immunohistochemistry (IHC). Most FISH testing (2963/3543, 83.6%) was performed on renal tumors. TFE3 FISH showed rearrangements in 449 of 2467 specimens (18.2%), including 281 (of 1887, 14.9%) renal tumors. TFEB FISH identified an abnormality in 107 of 1076 (9.9%) renal tumors, including 52 (of 107, 48.6%) rearrangements, 41 (of 107, 38.3%) amplifications, or 14 (of 107, 13.1%) with both rearrangements and amplifications. More specifically, TFE3-rearranged, TFEB-rearranged, TFEB-amplified, and TFEB-rearranged/amplified renal tumors occurred in females in 54%, 69.6%, 39.1%, and 40% of cases, respectively. The pediatric and young adult population (aged ≤21 years) included 44 (of 121, 36.3%) TFE3-RCC and 9 (of 50, 18%) TFEB-rearranged RCC. TFE3-RCC fusion partners included RBM10, NONO, ASPSCR1, FUBP1, SFPQ, MAPK1IP1L, and PRCC. TFEB-rearranged RCC fusion partners SYNRG and BYSL were identified. Diffuse GPNMB expression was seen in 92% of TFE3-RCC (24/26; median H-score 275), 100% of TFEB-rearranged RCC (19/19; median H-score 300), and 100% of TFEB-amplified RCC (17/17; 240). Finally, our cohort included 5 eosinophilic TFEB-amplified RCCs with non-focal keratin 20 expression. This large series of TFE3-RCC and TFEB-RCC provides population data regarding these rare tumors and demonstrates the clinical value of targeted FISH strategies. Our results suggest that GPNMB IHC is an effective screen for TFE3-RCC and TFEB-RCC. Additionally, we report a RCC harboring a novel SYNRG::TFEB fusion.

The feasibility of circulating tumor (ct)-DNA assays in first-approach pan-cancer genomic profiling is not well established. Furthermore, low ctDNA levels limit assay sensitivity, which challenges adaptation to clinical genomic profiling. In this study, a 33-gene next-generation sequencing-based ctDNA panel was validated, and these issues were investigated using real-world clinical data. The cohorts included 123 patients who underwent first-approach ctDNA testing, and 48 patients for whom matched tissue was tested at the same time-point. The overall ctDNA assay failure rate was 0%. Insufficient tumor tissue was the main reason for liquid biopsy (69%). The most common primary cancer profiled was lung (39.0%), followed by colon (13.8%), bile duct (8.9%), pancreas (8.1%), and breast and prostate (each 4.1%). Tier I variants were detected in 33.3% of patients, and Tier I or II variants were detected in 65.0% (including 54.5% cholangiocarcinomas, in which tissue biopsy may be challenging due to anatomic location). Compared with matched tissue, ctDNA showed 76% sensitivity for Tier I variants. Actionable variants were increased by 14.3% with ctDNA versus tissue testing alone. ctDNA results preceded tissue results by an average of 21 days. High feasibility, actionability, and sensitivity support ctDNA assays as a potential first-line genomic test, especially in specific tumor types for advanced tumors with insufficient or unavailable tissue.

BACKGROUND:The few reported patients with pathogenic IRF8 variants have manifested 2 distinct phenotypes: (1) an autosomal recessive severe immunodeficiency with significant neutrophilia and absence of or significant decrease in monocytes and dendritic cells and (2) a dominant-negative form with only a decrease in conventional type 2 dendritic cells (cDC2s) and susceptibility to mycobacterial disease. OBJECTIVES:Genetic testing of a child with persistent EBV viremia identified a novel IRF8 variant: c.1279dupT (p.∗427Leuext∗42). The variant was also found in his mother, who was subsequently diagnosed with a human papillomavirus-positive tumor. We sought to examine the pathogenicity of the identified IRF8 variant and its phenotypic and functional characteristics. METHODS:Immunophenotypic and functional flow cytometry, natural killer cell cytotoxicity, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, T-cell receptor Vβ spectratyping, Sanger sequencing, RNA-sequencing, Olink proteomics, immunoblotting, molecular cloning, dual-luciferase reporter assay, immunofluorescence microscopy, and image analysis. RESULTS:The 42 amino acid C-terminal extension of the mutant IRF8 (∼4 kDa heavier than wild type) impaired IRF8 nuclear localization in a dominant-negative manner and inhibited IRF1/IRF8-mediated transcriptional activities. Both patients had a decrease in plasmacytoid dendritic cells (pDCs) and in cDC1s, a mild neutrophilia and a mild monocytosis. Their existing pDCs had impaired IFN-α production. On TLR engagement, the production of IL-1β, IL-6, IL-10, and IL-12 by their monocytes and of IL-12 by their myeloid DCs were within normal limits. Natural killer cell development and cytolytic activity were essentially normal. RNA-sequencing and proteomic approaches bolstered the phenotypic and functional findings. CONCLUSIONS:This study defines the pathogenic nature of the c.1279dupT (p.∗427Leuext∗42) IRF8 variant, determines its dominant-negative mechanism of action, and broadens the existing phenotype of human IRF8 immunodeficiency.

Various cutaneous adnexal neoplasms have been associated with gene fusions as the main driver of their histogenesis, most of which showed poroid or hidradenomatous differentiation or represented other tumors of sweat glands. Recently, we encountered two cases of primitive basaloid neoplasms of predominantly folliculogenic/pilosebaceous origin, both surprisingly harboring recurrent EWSR1 rearrangements. Both tumors presented in young patients of either sex (1 F; 17 years; 1 M; 37 years) and involved the dermis and subcutaneous tissue of the abdominal wall (case 1) and the buttock (case 2). Histologically, the tumors were composed of primitive, basaloid cells with areas of keratinization (case 1) or foci of sebaceous differentiation (case 2) and stained strongly with keratins, p40 (case 1) or p63 (case 2), while lacking significant CD99 expression in both cases. Case 1 harbored an EWSR1::FLI1 fusion while case 2 harbored an EWSR1::PBX3 fusion. Clinical follow-up was available for case 2 and showed no evidence of disease at 15 months of follow-up. Methylation profiling showed case 1 to cluster with cutaneous squamous cell carcinoma, while case 2 was independent but positioned close to salivary gland epithelial-myoepithelial carcinoma. These findings expand on the histopathologic and molecular genetic features of basaloid tumors with apparent adnexal differentiation and raise awareness to carefully interpret and correlate molecular findings with morphology and immunophenotype to avoid misinterpretation as a mesenchymal neoplasm.

Fluorescence in situ hybridization (FISH) using a break-apart probe (BAP) design is a rapid, clinically useful method for targeted evaluation of gene rearrangements in formalin-fixed, paraffin-embedded tumors. Although clinically validated BAP FISH assays usually yield unequivocal positive or negative results, rare tumors yield equivocal FISH results. This study had two aims: to summarize typical and atypical BAP FISH results on 56,584 formalin-fixed, paraffin-embedded solid tumors over approximately one decade of clinical testing; and to investigate the clinical utility of RNA sequencing (RNA-seq) for tumors with equivocal FISH results. Of 8586 (15.2%) cases with abnormal FISH results reported, 748 (8.7%) were equivocal. RNA-seq was performed on 113 tumors, and oncogenic fusions involving the gene of interest were detected in 46 of 113 tumors (40.7%). Of the 106 tumors with equivocal FISH results, RNA-seq detected a fusion involving the expected gene target in 37 of 62 (59.7%) tumors with isolated probe signals corresponding to the active side of the gene region but only 4 of 44 (9.1%) tumors with other atypical signal patterns. This study provides a useful framework for categorizing atypical BAP FISH results and demonstrates the clinical utility of follow-up RNA-seq testing on tumors with equivocal FISH results.