Genotyping cancer-associated genes in chordoma identifies mutations in oncogenes and areas of chromosomal loss involving CDKN2A, PTEN, and SMARCB1

Abstract

The molecular mechanisms underlying chordoma pathogenesis are unknown. We therefore sought to identify novel mutations to better understand chordoma biology and to potentially identify therapeutic targets. Given the relatively high costs of whole genome sequencing, we performed a focused genetic analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometer (Sequenom iPLEX genotyping). We tested 865 hotspot mutations in 111 oncogenes and selected tumor suppressor genes (OncoMap v. 3.0) of 45 human chordoma tumor samples. Of the analyzed samples, seven were identified with at least one mutation. Six of these were from fresh frozen samples, and one was from a paraffin embedded sample. These observations were validated using an independent platform using homogeneous mass extend MALDI-TOF (Sequenom hME Genotyping). These genetic alterations include: ALK (A877S), CTNNB1 (T41A), NRAS (Q61R), PIK3CA (E545K), PTEN (R130), CDKN2A (R58*), and SMARCB1 (R40*). This study reports on the largest comprehensive mutational analysis of chordomas performed to date. To focus on mutations that have the greatest chance of clinical relevance, we tested only oncogenes and tumor suppressor genes that have been previously implicated in the tumorigenesis of more common malignancies. We identified rare genetic changes that may have functional significance to the underlying biology and potential therapeutics for chordomas. Mutations in CDKN2A and PTEN occurred in areas of chromosomal copy loss. When this data is paired with the studies showing 18 of 21 chordoma samples displaying copy loss at the locus for CDKN2A, 17 of 21 chordoma samples displaying copy loss at PTEN, and 3 of 4 chordoma samples displaying deletion at the SMARCB1 locus, we can infer that a loss of heterozygosity at these three loci may play a significant role in chordoma pathogenesis.

Figure 1. Mass Spectrophotometry Plot: Observing a Mutation in SMARCB1.

PCR products differ in mass depending on whether the probe extension detects a cytosine (C) or thymine (T). The figure in the right shows mass spec plot of an allele in SMARCB1 displaying a peak at both C and T. The left panel shows that most samples tested were homozygous with C, and 2 samples were heterozygous.

Figure 2. Selected View of aCGH Analyses (Chromosomes 9 and 10) from Two Cases.

X-axis measures location along chromosome 9 (Panel A) or 10 (Panel B). Y-axis measures relative probe count. A: aCGH showing heterozyogous copy loss at CDKN2A; B: aCGH showing heterozyogous copy loss at PTEN.