Sample collection and patient characteristics
Of the 1746 NSCLC cases, RET rearrangements were identified in 25 cases (1.43 %) with locally advanced or metastatic NSCLC, of which 20 (80%) were female. The 25 patients have mean age of 53.5 ranging from 27 to 78. NGS testing was performed on their 25 pairs of FFPE tumor tissue and PBL samples. In addition, we performed cfDNA NGS testing on 17 of them to check their RET gene status in cfDNA sample. All the samples passed the histology quality control (HQC) and yielded sufficient amounts of DNA for NGS.
Identification of RET rearrangements using targeted sequencing
In order to identify RET rearrangement of the DNA from patients’ FFPE samples, we designed probes to cover the intron 6 ,7,8,9,10, 11 of RET as well as introns of some well-known RET fusion partners. We identified RET rearrangements and corresponding breakpoints in the sequencing data of these patients. The statistical summary of the rearrangement events are presented in Table 1 and in Figure 1. The breakpoint distribution in RET are shown in Figure 2 . We found that 14 out of 25 patients had an KIF5B-RET fusion, with KIF5B exon15-RET exon12, KIF5B exon23-RET exon12, and KIF5B exon24-RET exon11 detected in 14, 3, and 1 patients respectively. We also identified one novel RET fusion partner PLCE1 and 4 intergenic-breakpoint fusions.
Table 1. Fusion patterns of RET
Fusion Type
|
Counts
|
percent(%)
|
KIF5B-exon15-RET-exon12
|
14
|
56
|
CCDC6-exon1-RET-exon12
|
3
|
12
|
KIF5B-exon23-RET-exon12
|
1
|
4
|
KIF5B-exon24-RET-exon11
|
1
|
4
|
PLCE1-exon20-RET-exon11
|
1
|
4
|
RET-exon11-CCDC6-exon3
|
1
|
4
|
Other
|
4
|
16
|
Mutational profiles of RET fusion-positive NSCLC patients
Genomic alterations were found in 24 (n = 24/25, 96%) samples with a total of 113 alterations identified including variants of nonsynonymous mutations and splicing mutations. The top 20 alterations are chosen to present in Figure 3A. The mutation landscapes of RET fusion-positive NSCLC patients were highly heterogeneous. The median TMB was 2.4 mut/Mb with a range between 0 to 8.4 mut/Mb, which is similar to the TMB of the TCGA NSCLC cohort[35].
We constructed a heatmap to demonstrate the somatic mutations occurring in the tumor tissues of the patients (Figure 3A). TP53 was most commonly altered (n = 10, 42%), followed by SETD2 (n = 4, 17%), CSMD3 (n = 3, 12%), and PTEN (n = 3, 12%). Other genomic alterations with low frequencies are ATM (n = 2, 6%), CACNA1C (n = 2, 8%), CIC (n = 2, 8%), CTCF (n = 2, 8%), DOT1L (n = 2, 8%), FANCA (n = 2, 8%), FANCG (n = 2, 8%),LRP1B (n = 2, 8%), MAP2K4 (n = 2, 8%), NOTCH1(n = 2, 8%), PRKCI (n = 2, 8%), PTPRT (n = 2, 8%) , RB1 (n = 2, 8%), SMAD4 (n = 2, 8%), and SUZ12 (n = 2, 8%). Alterations in DICER1 were identified in one sample (n = 1, 4%). We further compared our results with the MSK-IMPACT study [32], in which we extracted 30 RET fusion positive cases that yielded 81 mutations. Overall, our results were highly consistent with the MSK-IMPACT findings, which showed that TP53 and SETD2 are the most frequently altered genes (Fig. 3B).
We further studied their mutational signatures. We observed that C>T transition occurred most frequently, followed by C>G transversions (Fig. 4). This pattern is consistent with COSMIC signature 84 according to website(https://cancer.sanger.ac.uk/signatures/sbs/sbs84/) that had been found in some cancer samples.
Different driver gene mutations revealed inter-tumor heterogeneity. TP53 mutations in exon 4-8 were observed, and we further elaborately portrayed the TP53 mutation sites on the peptide sequence in a lollipop plot. (Fig. 5).
Copy number aberrations of RET fusion-positive NSCLC patients
Somatic copy number alterations were found in 11 (n = 11/25, 44%) samples. A total of 22 alterations were identified, including gain and loss (Figure 6). CDK4 were most commonly amplified genes (n = 3/11, 27%). Loss of copy number was most frequently observed in FGFR3 (n=4/11, 36%).
PD-L1 expression and Microsatellite Instability (MSI) status of RET fusion-positive NSCLC patients
High (≥50%), intermediate (1–49%), and negative (< 1%) PD-L1 expression was observed in 0/14 (0%), 8/14 (57%), and 6/14(43%) cases, respectively.
MSI status were evaluated in 20 cases. They are all microsatellite stable (MSS).
Concordance in tumor DNA(tDNA) and plasma DNA(ctDNA) sample pairs of RET fusion-positive NSCLC patients
A total of 17 tDNA and ctDNA sample pairs were analyzed. 9 patients are found to have the same breakpoint of RET fusions in both tDNA and ctDNA samples, indicating 52.9% RET fusion could be detected in ctDNA. A total of 111 mutations (snv and indel) were identified, including 90 in tDNA and 64 in plasma ctDNA, and 43 concordant mutations in both tDNA and plasma ctDNA. Seven sample pairs (7/17, 41.2%) had concordant mutations in both tDNA and plasma ctDNA , and the average variant frequency in these plasma ctDNA samples was 8.17%.