Targeted Deep Sequencing Reveals Unrecognized KIT Mutation Coexistent with NF1 Deficiency in GISTs

Figures & data

Figure 1 Case screening flow chart.

Notes: A total of 576 GISTs cases in our center (Xiangya Hospital) from May 2017 to Oct 2019 were screened. One hundred and eighty-five patients with no available genetic data of routine Sanger sequencing and 11 patients lost to follow-up were all excluded. Twenty-nine patients with poor quality FFPE were also excluded. Finally, 23 cases of 52 wild-types KIT/PDGFRA GISTs were included (negative for exons 9,11,13,14, and 17 in KIT and exon 12, 14, and 18 in PDGFRA using Sanger sequencing). A total of 136 GISTs cases between May 2017 to May 2020 in the Gene+ database and the 131 cases of GISTs from the MSKCC database were also included in our study.

Table 1 Clinical Characteristics of GIST with NF1 Mutation

Clinical Features	mNF1 and mKIT	mNF1 and wtKIT	P-value (Methods)
Number of patients (N)	12	14
Age at diagnosis (years)			0.6665 (Mann–Whitney)
Mean ±SD	65.50±2.12	44.00±22.63
Median	65.5(36–68)	44.00(28–94)
NA (N)	2	8
Gender (N)			0.2466 (Chi-square)
Female	5	9
Male	7	5
NA	0	0
Location (N)			0.4389 (Chi-square)
Duodenum	1	1
Small Bowel	3	9
Stomach	4	3
Intraabdominal	0	1
NA	4	0

Abbreviations: NA, not available; N, number.

Table 2 The Mutation Types and Their Differences Between mNF1 with mKIT and mNF1 with wtKIT

mNF1 Subtype	mKIT	wtKIT	P-value
Frameshift	6	9	0.4642
Missense	5	4	0.5229
Nonsense	3	3	0.8548

Note: No differences of the type of NF1 mutation between with KIT mutation and without KIT mutation.

Table 3 Concurrent Mutation of NF1 and KIT in GISTs Patients

Patient	mNF1				mKIT
	cHGVS	Exon	Abundance		cHGVS	Exon	Abundance
1^#	c.239_245delATCTCTC c.1017CT[2>1] c.6792C>A	3 9 45	10% 3.60% 7.30%		c.2828G>T	21	4.30%
2^#	c.1799T>A	16	38.40%		c.2466T>A	17	39.90%
3^#	c.4009C>T	30	3.50%		c.2466T>A	17	30.50%
4^##	c.3805_3808dupGACT	28	2.10%		c.1676T>G	11	39.90%
5^##	c.7926dupT	54	53.50%		c.1669_1674delTGGAAG	11	33.10%
6^##	c.5691T>G	38	2.20%		c.1669T>A	11	40.80%
7^##	c.1004A>G	9	40.70%		c.1504_1509dupGCCTAT	9	49.90%
8^##	c.2033C>T	18	49.60%		c.1669_1674delTGGAAG	11	11.50%
9^##	c.1799T>A	16	38.80%		c.2466T>A	17	38.60%
10^##	c.2033C>T	18	50.20%		c.1676_1684delTTGTTGAGG	11	34.30%
11^###	c.331A>T	4	NA		c.698G>C c.1657_1662delTATGAA	4 11	NA NA
12^###	c.4960_4967delTTTAAAAC	37	NA		c.1668_1683delinsACAA c.2007_2009delinsGAT	11 13	NA NA

Notes: The detailed information of mutated NF1 gene and concurrent KIT mutation in each of the 12 GISTs cases; ^#Xiangya Hospital; ^##Gene+ database; ^###MSKCC database.

Abbreviations: NA, not available; cHGVS, ctDNA Human Genome Variation Society.

Figure 2 The mutation loci and subtypes within the NF1 gene and their corresponding pathogenicity in GISTs.

Notes:^#Xiangya Hospital; ^##Gene+ database; ^###MSKCC database; ±, Neutral; –, Deleterious; Red symbols of ^{#, ##} and ^###represent n=2.

Table 4 NF1 Concurrent with KIT Mutation

Genes	Chinese (n,%)		American (n,%)	P-value (Chi-square)
	Xiangya	Gene+	MSKCC
mNF1 and mKIT	3(13.0%)	7(5.2%)	2(1.5%)	0.034442
mNF1 and wtKIT	2(8.7%)	4(2.9%)	8(6.1%)

Figure 3 KIT mutations loci and subtype.

Notes: The distribution of various mutation subtypes identified by NGS within the exons of KIT gene; ^#Xiangya Hospital; ^##Gene+ database; ^###MSKCC database; ±, Neutral; –, Deleterious; Red symbols of ^## and ^###represent n=2.

Abbreviations: TM, transmembrane domain; JM, juxtamembrane domain; N, number.

Figure 4 The genomic landscapes of skin neurofibroma and GISTs in 3 cases of type I neurofibromatosis.

Notes: (A–C) represent individual cases 1–3, respectively. In panel A (case 1) and panel B (case 2), compared with the lesions of skin neurofibromas and micro-GISTs (≤1 cm) that merely harbor mNF1, GISTs lesions (>2 cm) have NF1 aberration with a concurrent mKIT. In all three cases, the number of gene mutations is gradually increasing with the neurofibromas lesions to micro-GISTs or/and mini-GISTs to clinic GISTs.