Targeted exome sequencing profiles genetic alterations in leiomyosarcoma

Abstract

Leiomyosarcoma (LMS) belongs to the class of genetically complex sarcomas and shows numerous, often non-recurrent chromosomal imbalances and aberrations. We investigated a group of LMS using NGS platform to identify recurrent genetic abnormalities and possible therapeutic targets. Targeted exome sequencing of 230 cancer-associated genes was performed on 35 primary soft tissue and visceral (extra-uterine) LMS. Sequence data were analyzed to identify single nucleotide variants, small insertions/deletions (indels), and copy number alterations. Key alterations were further investigated using FISH assay. The study group included patients with median age of 64 years and median tumor size of 7 cm. The primary sites included retroperitoneal/intra-abdominal, extremity, truncal, and visceral. Thirty-one tumors were high grade LMS, while four were low grade. Losses of chromosomal regions involving key tumor suppressor genes PTEN (10q), RB1 (13q), CDH1 (16q), and TP53 (17p) were the most frequent genetic events. Gains mainly involved chromosome regions 17p11.2 (MYOCD) and 15q25-26 (IGF1R). The most frequent mutations were identified in the TP53 gene in 13 of 35 (37%) cases. FISH analysis showed amplification of the myocardin (MYOCD) gene in 5 of 25 (20%) cases analyzed. None of the four low grade LMS showed losses or mutations of PTEN or TP53 genes. Genetic complexity is the hallmark of LMS with losses of important tumor suppressor genes being a common feature. MYOCD, a key gene associated with smooth muscle differentiation, is amplified in a subset of both retroperitoneal and extremity LMS. Further studies are necessary to investigate the significance of gains/amplifications in the development of these tumors.

Figure 1

A comprehensive profile of the genomic alterations in LMS and associated clinical information with samples sorted by location of tumor. Low grade LMS cases are identified in red font. Copy number alterations were identified in all of the cases. Chromosomal losses were more frequently identified than gains and were most frequently seen in regions of known tumor suppressor genes, such as PTEN (10q23), RB1 (13q14), and TP53 (17p13). An additional region with frequent losses identified was in the gene CDH1 (16q22.1). Gains were most frequently seen in chromosome regions 17p, 15q, 8q, 1q and 3q. The most frequent gains were seen in the 17p11.2 region.

Figure 2

Schematic representation of the TP53 gene showing the sites of mutations identified. Thirteen of the 35 cases (37%) showed presence of TP53 mutations. Overall, five frameshift mutations, 9 missense mutations and 1 in-frame deletion were noted.

Figure 3

Schematic representation of copy number alterations in Case 3 (A) showing gains of 17p11-13 and losses of 10q, 13q, 16q and 17p. (B) H&E image showing typical morphology of the tumor composed of spindle cells in fascicles with cytologic atypia and mitotic activity. (C) FISH for myocardin (MYOCD) gene showing amplification (red signal).