Gene fusion analysis in renal cell carcinoma by FusionPlex RNA-sequencing and correlations of molecular findings with clinicopathological features.

Translocation renal cell carcinoma (tRCC) affects younger patients and often presents as advanced disease. Accurate diagnosis is required to guide clinical management. Here we evaluate the RNA-sequencing FusionPlex platform with a 115-gene panel including TFE3 and TFEB for tRCC diagnosis and correlate molecular findings with clinicopathological features. We reviewed 996 consecutive RCC cases from our institution over the preceding 7 years and retrieved 17 cases with histological and immunohistochemical features highly suggestive of either TFE3 (n = 16) or TFEB (n = 1). Moderate to strong labeling for TFE3 was present in 15 cases; two cases with weak TFE3 expression were melan-A or cathepsin-K positive. RNA-sequencing detected gene rearrangements in eight cases: PRCC-TFE3 (3), ASPSCR1-TFE3 (2), LUC7L3-TFE3 (1), SFPQ-TFE3 (1), and a novel SETD1B-TFE3 (1). FISH assays of 11 tumors verified six positive cases concordant with FusionPlex analysis results. Two other cases were confirmed by RT-PCR. FusionPlex was superior to FISH by providing precise breakpoints for tRCC-related genes in a single assay and allowing identification of both known and novel fusion partners, thereby facilitating clinicopathological correlations as fusion partners can influence tumor appearance, immunophenotype, and behavior. Cases with partner genes PRCC and novel partner SETD1B were associated with prominent papillary architecture while cases with partner genes ASPSCR1 and LUC7L3 were associated with a predominantly nested/alveolar pattern. The case with SFPQ-TFE3 fusion was characterized by biphasic morphology mimicking TFEB-like translocation RCC. We recommend FusionPlex analysis of RCC in patients under age 50 or when the histologic appearance suggests tRCC.

Molecular Approaches to Diagnosis in Ewing Sarcoma: Targeted RNA Sequencing.

Molecular testing of pathognomonic gene fusions is mandatory for small round cell tumor diagnosis, including Ewing sarcoma which is indeed defined by a variety of chimeric genes. Reference laboratories are increasingly implementing NGS-based techniques to overcome several limitations of conventional singleplex determinations. We have been early adopters of a targeted-RNA sequencing method based on Anchored multiplex PCR, which allows assessing several fusion transcripts simultaneously with previous knowledge of only one partner gene. Here we describe in detail our protocol and tips for nucleic acid extraction, library preparation, sequencing, and reporting of gene fusions.

Multidisciplinary analysis of pediatric T-ALL: 9q34 gene fusions.

T-cell acute lymphoblastic leukemia (T-ALL) is not as frequently reported as the B-cell counterpart (B-ALL), only occurring in about 15% of pediatric cases with a typically heterogeneous etiology. Approximately 8% of childhood T-ALL cases have rearrangements involving the ABL1 tyrosine kinase gene at 9q34.12; although a t(9;22), resulting in a fusion of ABL1 with the BCR gene at 22q11.23 is a common occurrence in B-ALL, it is not a typical finding in T-ALL. A subset of 10 of 40 documented cases of T-ALL analyzed over a 5-year period is presented, each having gene rearrangements within band 9q34 that resulted in fusions other than BCR/ABL1. These cases included fusions involving ABL1, SET (9q34.11), NUP214 (9q34.13), SPTAN1 (9p34.11), and TNRC6B (22q13.1). Among the 10 cases are: six SET/NUP214 fusions, two ABL1/NUP214 fusions (one of which was associated with episomal amplification) and novel SPTAN1/ABL1 and TNRC6B/ABL1 fusions. The evaluations of these clones were each significantly aided by FISH analysis, which directed subsequent microarray and anchored multiplex PCR testing for fusion confirmations.