Targeted next-generation sequencing of CIC-DUX4 soft tissue sarcomas demonstrates low mutational burden and recurrent chromosome 1p loss

Abstract

Gene fusions between CIC and DUX4 define a rare class of soft tissue sarcomas poorly understood at the molecular level. Previous karyotyping and fluorescence in situ hybridization studies support chromosome 8 trisomy as a recurrent alteration; however, other driving alterations are largely unknown. Thus, we analyzed 11 formalin-fixed, paraffin-embedded CIC-DUX4 sarcoma tissue samples (including 3 sample pairs) using targeted Ion Torrent-based multiplexed polymerase chain reaction next-generation sequencing to characterize potential somatic driver alterations in 409 genes. Although we did not identify recurrent somatic mutations (point mutations or insertions/deletions), copy number analysis showed recurrent, broad copy number alterations, including gain of chromosome 8 and loss of 1p. In one sample pair (untreated primary and local recurrence resections), we identified similar copy number profiles and a somatic ARID1A R963X nonsense mutation exclusively in the local recurrence sample. In another sample pair (pre- and post-radiation treatment specimens), we observed single-copy loss of chromosome 7q exclusively in the posttreatment recurrence sample, supporting it as an acquired event after radiation treatment. In the last sample pair (near-concurrent, postchemotherapy primary and distant metastasis), molecular profiles were highly concordant, consistent with limited intertumoral heterogeneity. In summary, next-generation sequencing identified limited somatic driver mutations in CIC-DUX4 sarcomas. However, we identified novel, recurrent copy number alterations, including chromosome 1p, which is also the locus of ARID1A. Additional functional work and assessment of larger cohorts are needed to determine the biological and clinical significance of the alterations identified herein.

Keywords: ARID1A; CIC-DUX4; FFPE tissue; Next-generation sequencing; Soft tissue sarcoma.

Fig. 1. Histology of CIC-DUX4 sarcomas subjected to next generation sequencing (NGS)

Low (A, C & E) and high power (B, D & F) hematoxylin and eosin stained (H&E) stained sections from three CIC-DUX4 sarcomas, Sample 1 (A&B), Sample 2 (C&D) and Sample 6A (E&F), subjected to NGS. Tumors show typical small round cell morphology, geographic necrosis, coarse chromatin, and focal extracellular myxoid matrix, with Sample 1 showing more pleomorphic histology. Original magnifications 10× (A, C & E) and 40× (B, D & F).

Fig. 2. Somatic copy-number profiles of CIC-DUX4 sarcomas generated by targeted next generation sequencing (NGS)

Somatic, autosomal copy number profiles are presented for the 11 CIC-DUX4 sarcoma samples from 8 cases assessed by NGS. Gene-level copy number estimates are shown for all target genes with >= 3 amplicons across samples. Colors correspond to log2 copy number ratios (tumor to composite normal) as indicated in legend. Samples 5A&B represent a pre-treatment tumor and a post-radiation metastasis/local recurrence from the same patient. Samples 6A&B represent a primary tumor and near concurrent untreated brain metastasis. Samples 7A&7B are a primary tumor and rapid local recurrence.

Fig. 3. Recurrent copy number alterations (CNAs) identified by next generation sequencing (NGS) of CIC-DUX4 sarcomas

A. Log₂ copy number profiles (tumor to composite normal) for selected chromosomes from the genome wide plots in Figure 2 are shown. Gains and losses are shown in red and blue, respectively, with lighter shades indicating lower level alterations. Genes are indicated below the copy number profiles with ARID1A (chr 1p) MYC (chr 8q) and ETV4 (chr 17q) bolded and indicated by dashed green lines. B. Confirmation of ARID1A copy number loss by quantitative PCR (qPCR). Genomic DNA was assessed by qPCR in triplicate for ARID1A copy number normalized to the average of three reference genes without CNA by NGS (FBXW7, DNMT3A, and IGF1R) from samples with available DNA. Normalized ARID1A copy number ratio was calibrated to an unrelated benign FFPE genomic DNA sample as the calibrator control (Con.). Mean + S.E. are shown.