WES analysis of sequence nucleotide polymorphism and variations in a low grade MEC patient completely responding gefitinib
To study the somatic mutations related to the tumorigenesis and gefitinib response of MEC, we obtained whole-exome sequencing data from the frozen fresh sputum and whole blood samples from a low grad MEC patient (Li, Zhang et al. 2017). This 10-year patient was admitted to our hospital in 2012 and successfully responded to the gefitinib treatment. No relapse has been observed until now.
A total of 46,979,124 and 47,415,894 sequencing reads were generated for the sputum and blood samples, respectively. Over 97% of the targeted exon regions was covered for both samples, and 74% and 76% of targeted bases showed > 10-fold coverage for in the sputum and whole blood samples, respectively (Table S1).
Non-synonymous somatic mutations occur in tumor suppressors with a higher frequency than in oncogenes
Using the criteria described in Methods section, we identified 53 candidate somatic mutations in 50 genes in the sputum of the patient (Table S2). The mutations comprised 34 non-synonymous SNVs, 10 synonymous SNVs, 3 frameshift insertion/deletion, 3 essential splice sites, 1 stopgain, as well as 2 variations without annotations and 2 noncoding RNA mutations. Two mutations were identified in each of the following three genes, TTN (p.A4284T, p.A3465T), PGM5 (p.G215S, p.I227V) and DEAF1 (p.Y300Y, p.P299L). Five out of the six mutations were nonsynonymous mutations. A total of 12 of the 50 somatic mutation-containing genes were annotated as oncogenes and tumor-suppressor genes in the CancerMine database, which were significantly enriched among all genes (p value, 1.11e-5, hypergeometric test). Non-synonymous mutations were found in two oncogenes ADAM28 and TTN, and five tumor-suppressor genes ARRDC3, MRPL48, NPAS3, EPB41L3 and RANBP2, showing much higher frequency in tumor suppressor genes (Table 1). Additionally, nonsynonymous mutation was also observed in PRAMEF15, a member in the preferentially expressed antigen in melanoma (PRAME) family (Oberthuer, Hero et al. 2004, Epping, Wang et al. 2005, Field, Decatur et al. 2016, Hermes, Kewitz et al. 2016).
Genes harboring candidate somatic mutations were subjected to functional clustering to analyze the potential biological roles that these mutations preferentially affect. In the biological process terms of GO analysis, they only enriched in DNA-dependent transcription function (p-value greater than 0.05). In the KEGG pathway analysis, they were mostly enriched in apoptosis (RIPK1, SPTA1 and ACTG1), followed by RNA transport (RANBP2, POM121L2 and SNORD3A). Two genes (EPB41L3 and ACTG1) were enriched in tight junction (Fig. 1A).
Non-synonymous somatic mutations were enriched in the loss and gain of Thr, Ser or Tyr
Gefitinib is an ATP analogue and well-known for its effective inhibition of the constitutively active kinase activity of the EGFR mutants (Lynch, Bell et al. 2004, Paez, Janne et al. 2004, Nyati, Morgan et al. 2006). Our recent study has shown that gefitinib inhibits the phosphorylation of multiple signaling kinases in the JAK-STAT and MAPK/ERK signaling pathways that is activated by the CRTC1-MAML2 fusion in H292 cells (Wu, He et al. 2019). We hypothesized that MEC patients might harbor phosphorylation site mutations that could sensitize some tumor-related proteins to gefitinib treatment in this patient. To test this hypothesis, we investigated the non-synonymous somatic mutations involved in the loss and gain of the protein phosphorylation amino acids Thr, Ser or Tyr. Among the 34 non-synonymous somatic mutations, we found 5 caused the gain of phosphorylation amino acids including A3636T and A4284T in TTN (Rhabdomyosarcoma Antigen MU-RMS-40.14), K135T in PRAMEF15, G215S in PGM5 (Phosphoglucomutase 5), T367S in DNAH11 (Dynein Axonemal Heavy Chain 11) and P888S in RALGAPB (Ral GTPase Activating Protein Non-Catalytic Beta Subunit), and 3 caused the loss including Y76F in ARL6 (ADP Ribosylation Factor Like GTPase 6), T3219I in RANBP2 (RAN Binding Protein 2;RAN is a small GTP-binding protein of the RAS superfamily) and S880P in UBN1 (Ubinuclein 1, required for SAHF formation (Table 2). This significant high frequency of the loss (p-value, 0.023, probability test) and gain (p-value, 0.089, probability test) of the three phosphorylation amino acids in the non-synonymous somatic mutations in this studied patient might explain a part of his complete response to gefitinib.
Comparison of genes containing somatic mutations identified from this study and those from a previous study
We next compared the somatic mutations identified in this study with those identified in 18 pairs of samples from MEC patients (Kang, Tan et al. 2016). Among the 50 somatic mutation genes identified from the low-grade MEC patient in this study, 5 of them were overlapped with the reported 705 genes (Kang, Tan et al. 2016). The overlapped genes include ADAM28, DYSF, GP2, PPP2R5B, TTN, as shown in Fig. 1B. Among these overlapped somatic mutation genes, ADAM28, TTN and PPP2R5B were detected in two intermediate grade patients, and DYSF, GP2 were detected in two low grade patients in the published data.