Identification of dual RAS pathway mutations
Using Whole Exome Sequencing of tumor tissue and blood, we identified several somatic mutations in this patient (Table 1). Of these mutations is the c.38 G>T(p.Gly13Val) hotspot mutation in the HRAS gene (Supplementary Figure 1). HRAS is a GTPase Proto-Oncogene that is located on chromosome 11(11p15.5). GTP-bound “on” state Ras proteins convert extracellular stimuli into intracellular signaling cascades, which eventually produce changes in cellular functions; this signaling stops when Ras-bound GTP is hydrolyzed to GDP as a consequence of another signaling cascade. Thus, in normal cells, Ras proteins function as molecular regulators for critical changes in cellular activities, such as cell proliferation and survival, and their appropriate regulation is crucial to maintaining the homeostasis of cells and, eventually, the entire organism. While in case of mutated RAS gene, especially in the codons 12, 13, and 61, results in reduced intrinsic GTPase activity, that in turn leads to permanent activation of RAS genes and downstream signaling pathways, thereby mediating malignant transformation10,11.
In addition to the HRAS mutation, we identified another mutation in the same pathway which is the c.1742 A>T (p.Asn581Ile) in the BRAF gene. BRAF represents one of the most frequently mutated protein kinase genes in the human tumors12. BRAF gene encodes Serine/Threonine Kinase and is located on chromosome 7(7q34). There are three classes of BRAF mutations that were identified, they are grouped based on the kinase activity, dependence on RAS, and dimerization.
Class 1 consists of BRAF V600 mutants, and classes II and III are non-V600 BRAF mutations. In our patient, we identified c.1742A>T, a missense class III BRAF mutation in addition to the c.1742 A>T mutation in the HRAS gene, both are deleterious hotspot mutations involved in the RAS signaling pathway (Table 1).
The IGV coverage for the BRAF and HRAS mutations detected in our patient is 44.8% and 52% respectively with 0 detections in the blood that confirm being somatic mutations (Supplementary Figure 2).
Clinical validation
The sample was sent for clinical validation. The outcome of the molecular examination confirmed the presence of the mutation c.3551 G> A (p. Gly1184Glu) in the codon 1184 of exon 23 of theALK gene with a frequency of 16.06%. The presence of the mutation c.1621 A> C (p. Met541Leu) in codon 541 of exon 10 of the KIT gene with a frequency of 50.23%. The presence of the mutation c.1416 A> T (p. Gln472His) in codon 472 of exon 11 of the KDR gene with a frequency of 60.22%. The presence of the mutation c.1742 A> T (p. Asn581Ile) in codon 581 of exon 15 of the BRAF gene with a frequency of 41.13%. The presence of the canonical mutation c.2651 A> T (p.Glu884Val) in codon 884 of exon 15 of the RET gene with the frequency of 11.43%. The presence of the canonical mutation c.38 G> T (p. Gly13Val) in codon 13 of exon 2 of theHRAS gene with a frequency of 50.35%. The presence of the canonical mutation c.767 A> T (p.Gln256Leu) in codon 256 of exon6 of the SMAD4 gene. And the presence of the canonical mutation c.215 C> A (p. Thr72Lys) in codon 72 of exon 2 of the SMARCB1 gene.
The number of mapped Reads is 379,632 reads and the mean depth is 1,491, On target is 97.40%.