Whole transcriptome sequencing identifies BCOR internal tandem duplication as a common feature of clear cell sarcoma of the kidney

Abstract

Purpose: Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor that is frequently difficult to distinguish among other childhood renal tumors due to its histological heterogeneity. This work evaluates genetic abnormalities carried by a series of CCSK samples by whole transcriptome sequencing (WTS), to identify molecular biomarkers that could improve the diagnostic process.

Methods: WTS was performed on tumor RNA from 8 patients with CCSK. Bioinformatic analysis, with implementation of a pipeline for detection of intragenic rearrangements, was executed. Sanger sequencing and gene expression were evaluated to validate BCOR internal tandem duplication (ITD).

Results: WTS did not identify any shared SNVs, Ins/Del or fusion event. Conversely, analysis of intragenic rearrangements enabled the detection of a breakpoint within BCOR transcript recurrent in all samples. Three different in-frame ITD in exon15 of BCOR, were detected. The presence of the ITD was confirmed on tumor DNA and cDNA, and resulted in overexpression of BCOR.

Conclusions: WTS coupled with specific bioinformatic analysis is able to detect rare genetic events, as intragenic rearrangements. ITD in the last exon of BCOR is recurrent in all CCSK samples analyzed, representing a valuable molecular marker to improve diagnosis of this rare childhood renal tumor.

Keywords: BCOR; CCSK; whole transcriptome sequencing.

Figure 1. BCOR ITD detection by whole transcriptome sequencing

A. Reconstruction of the three BCOR ITD events identified. Through de novo assembly and local realignment of unmapped reads, three different breakpoint regions were found, identifying three different ITD events in exon 15 of BCOR. The nucleotide sequences colored in green represents the wild type sequence while the ones in orange correspond to the duplicated segment. Reads overlapping the breakpoint regions are shown at the bottom. B. PCR amplification of BCOR exon 15 on tumor DNA of 8 CCSK and one negative control. Only CCSK samples carried the ITD (higher molecular weight). Two amplicons, corresponding to the duplicated and WT allele, were present in the 3 female patients. Two of the five male patients (CCSK1 and CCSK4) showed a fainter WT band, due to the presence of normal cells within the tumor tissue. Negative control carried only the WT allele (low weight). C. Chromatograms of the three different ITD breakpoint regions of the high weight bands obtained from amplification of CCSK tumor DNA. D. Amplification of BCOR from cDNA of 8 CCSK and 5 Wilms tumors (W1-W5). All CCSK expressed predominantly the ITD allele, while all Wilms tumors expressed only the WT allele. E. Evaluation of mRNA expression level of BCOR in the CCSK tumors with respect to the Wilms tumors, determined by quantitative RT-PCR. Expression level was normalized on GAPDH and significance (P = 0.004) was estimated with t-test statistic.