López‐Terrada, Dolores

Context Next-generation sequencing–based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. Objective To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). Design CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. Results Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. Conclusions These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics.

Nodular fasciitis is a benign self‐limited myofibroblastic neoplasm, which usually involves the upper extremities and trunk of young patients. These tumors have been shown to harbor a translocation involving the MYH9 and USP6 genes, leading to overexpression of the latter. We report seven cases of nodular fasciitis with cutaneous presentations. All cases involved the dermis, with six involving the superficial subcutis, and one auricular tumor extending into cartilage. All cases showed USP6 rearrangement by fluorescence in situ hybridization; in two of three cases, the characteristic MYH9‐USP6 fusion was shown by RT‐PCR. All patients underwent conservative resection. Nodular fasciitis is an uncommon mesenchymal neoplasm that can occasionally present in superficial locations and is sometimes mistaken for a malignant process. Molecular testing can be useful to distinguish this entity from other cutaneous spindle cell tumors.

AimsEpithelioid haemangioendothelioma (EHE) is a malignant vascular neoplasm. Subsets have been characterized previously by translocations resulting in either WWTR1–CAMTA1 or YAP1–TFE3 fusion. We sought to develop molecular and immunohistochemical (IHC) assays to aid in the diagnosis and characterization of EHE.Methods and resultsFifty‐two formalin‐fixed, paraffin‐embedded (FFPE) cases diagnosed between 2002 and 2014 were retrieved from the pathology files of our institutions. Reverse transcription–polymerase chain reaction (RT–PCR) assays were optimized to detect WWTR1–CAMTA1 and YAP1–TFE3 fusion transcripts in FFPE tissue and transcription factor E3 (TFE3) protein accumulation was examined by immunohistochemistry (IHC). RNA was extracted from 33 adequate samples, with more recent cases providing a greater yield of high quality RNA. Fourteen of 18 informative cases were positive for WWTR1–CAMTA1 fusion transcripts, four of which showed higher‐grade cytological features termed by some as ‘malignant EHE’. Novel in‐frame fusion transcripts were identified in four cases by direct sequencing. IHC revealed variable nuclear TFE3 staining in six of 17 cases; three with patchy staining showed WWTR1–CAMTA1 fusion. One of 18 informative cases was positive for YAP1–TFE3 fusion and showed strong nuclear TFE3 staining by IHC.ConclusionsThis study confirms the high incidence of WWTR1–CAMTA1 and YAP1–TFE3 rearrangements in EHE and indicates that the staining pattern for TFE3 IHC is critical for specificity.

Constitutively active nuclear factor-κB (NF-κB) is integral to the survival of Hodgkin/Reed-Sternberg cells (H/RS) in Hodgkin lymphoma (HL). To investigate NF-κB pathway proteins in pediatric HL, we utilized a tissue microarray compiled from 102 children enrolled in the Children's Oncology Group intermediate-risk clinical trial AHOD0031 (56 male, 78 Caucasian, median age 15 years (range 1–20 years), 85 nodular sclerosing subtype, 23 Epstein-Barr virus (EBV) positive, and 24 refractory/relapsed disease). We examined the intensity, localization, and pathway correlations of NF-κB pathway proteins (Rel-A/p65, Rel-B, c-Rel, NF-κB1, NF-κB2, IκB-α, IKK-α, IKK-β, IKK-γ/NEMO, NIK, and A20), as well as their associations with EBV status and clinical outcome. NF-κB pathway proteins were overexpressed in pediatric HL patients compared to controls. Patients with EBV− tumors, or with rapid early therapy response, had tightly coordinated regulation of NF-κB pathway proteins, whereas patients with EBV+ tumors, or slow early therapy response, had little coordinated NF-κB pathway regulation. High NIK expression was associated with a slow response to therapy and decreased EFS. Elevated Rel-B, NIK, and the NF-κB inhibitor A20 were associated with decreased EFS in multivariate analysis. These studies suggest a pivotal role for the NF-κB pathway in therapy response and patient survival in Hodgkin lymphoma.