Novel Clinically Relevant Genes in Gastrointestinal Stromal Tumors Identified by Exome Sequencing

Sebastian F. Schoppmann; Ursula Vinatzer; Niko Popitsch; Martina Mittlböck; Sandra Liebmann-Reindl; Gerd Jomrich; Berthold Streubel; Peter Birner

doi:10.1158/1078-0432.CCR-12-3863

DOI: 10.1158/1078-0432.CCR-12-3863 Published October 2013

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Figure 1.
Samples of FISH and immunostaining. A, GIST with loss of 1p at FISH. Original magnification ×1,000. B, GIST positive for KIT. Original magnification ×100. C, GIST positive for RAD54L2. Original magnification ×400. D, GIST negative for RAD54L2. Original magnification ×400. E, GIST positive for SYNE2. Original magnification ×400. F, GIST negative for SYNE2. Original magnification ×400. G, GIST positive for DIAPH1. Original magnification ×400. H, GIST negative for DIAPH1. Original magnification ×400.
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Figure 2.
Kaplan–Meier curves of DFS and OS, ticks indicate censored observations. A, DFS according to relative loss of 1p. B, OS according to RAD45L2 expression. C, DFS according to SYNE2 expression. D, OS according to Kit expression. E, DFS according to DIAPH1 expression. F, DFS according to a combination of SYNE2 and DIAPH1 expression (SYNE2/DIAPH1+) versus all other cases (SYNE2/DIAPH1−).

Table 1.

Clinico-pathologic data of 145 GIST patients included into this study

Factor	Number of cases	5 Years disease-free survival rate ± SE	5 years overall survival rate ± SE
Localization		P < 0.001^a	P > 0.05
Gastric	94 (64.8%)	89.8 ± 4.3%	–
Duodenum	6 (4.1%)	100%	–
Small intestine	27 (18.6%)	62.2 ± 10.1%	–
Rectum	3 (2.1%)	100%	–
Other	15 (10.3%)	45.1 ± 14.9%	–
Risk Fletcher		P = 0.001^a	P < 0.001^a
Very low	16 (11%)	87.5 ± 11.7%	100%
Low	50 (34.5%)	93.9 ± 4.2%	95.5 ± 4.4%
Intermediate	32 (22.1%)	79 ± 8.6%	96.4 ± 3.5%
High	47 (32.4%)	54.8 ± 9.7%	73.8 ± 7.8%
Risk Miettinen		P < 0.001^a	P < 0.001^a
None	18 (12.4%)	90 ± 9.5%	100%
Very low	38 (26.2%)	96.3 ± 3.6%	93.8 ± 6.1%
Low	28 (19.3%)	77 ± 10.7%	95.8 ± 4.1%
Moderate	17 (11.7%)	84.6 ± 10%	100%
High	29 (20%)	59.9 ± 12.4%	65.5 ± 9.7%
Insufficient data	15 (10.3%)	45.1 ± 14.9%	83.3 ± 15.2%
Staging UICC		P < 0.00^a	P > 0.05
pT1	19 (13.1%)	90 ± 9.5%	–
pT2	65 (44.8%)	84.5 ± 6.2%	–
pT3	40 (27.6%)	73.6 ± 8.9%	–
pT4	21 (14.5%)	50.5 ± 12.9%	–
Mitotic rate UICC		P = 0.001^a	P < 0.001^a
Low	96 (66.2%)	87.1 ± 4.5%	96.7 ± 2.4%
High	49 (33.8%)	58.9 ± 8.9%	75.8 ± 7.1%
Synchronous metastases		P = 0.003^a	P < 0.001^a
No	131 (90.3%)	80.7 ± 4.3%	94.6 ± 2.7%
Yes	14 (9.7%)	47.3 ± 19%	51.3 ± 12.5%
KIT mutation		P > 0.05	P > 0.05
No	53 (36.6%)	–	–
Yes	92 (63.4%)	–	–
PDGFRA mutation		P > 0.05	P > 0.05
No	124 (85.5%)	–	–
Yes	21 (14.5%)	–	–

↵^aSignificant results after FDR adjustment.

Table 2.

Copy number variations assessed by DNA array (n = 29)

Chromosome arm	Type of CNV	Cases (n/%; 95% CI)	ROI Start bp	ROI End bp	Size (Mb)
1p	Loss	13 (45%; 95% CI, 28–63%)	5657217	16851502	11.2
3p	Loss	4 (19%; 95% CI, 6–31%)	47478496	50156251	2.7
13q	Loss	5 (17%; 95% CI, 8–35%)	45307552	49798504	4.5^b
14q	Loss^a	17 (59%; 95% CI, 41–75%)	26370859	28652135	2.3
			41327139	46436189	5.1
			54142190	106897379	52.8
15q	Loss	7 (24%; 95% CI, 12–42%)	49340635	56142902	6.8
22q	Loss	11 (38%; 95% CI, 23–56%)	28772816	51134186	22.4
1q	Gain	3 (10%; 95% CI, 5–33%)	118649839	249224376	232.4
4q	Gain	6 (21%; 95% CI, 10–38%)	52920476	56180478	3.3^c
5q	Gain	8 (28%; 95% CI, 15–46%)	120645755	180645101	60.0
7p	Gain	3 (10%; 95% CI, 5–33%)	7058423	62706404	55.7
11q	Gain	4 (19%; 95% CI, 6–31%)	78846125	134944770	56.1
12p	Gain	3 (10%; 95% CI, 5–33%)	479074	15635796	15.2

ROI (region of interest) denotes minimal region of overlap, genome annotations applied in data analysis refer to the human reference assembly GRCh37/hg19.

↵^aThe minimal region of overlap on chromosome 14 comprised 3 regions with a total size of 60.2 Mb.
↵^bRB1 is located within the minimal region of overlap.
↵^cKIT and PDGFRA are located within the minimal region of overlap, respectively.

Table 3.

Results of exome sequencing in 13 patients

Region of interest	Type defined by microarray	Number of genes with variants	Genes with recurrent variations	Number of cases (95% CI)	Potentially oncogenic (according to Cancer genes)	PubMed Search: Gene AND GIST	Potential oncogenic according to Gene Database
1p	Loss	48
			GPR153	4 (31%; 95% CI, 13–58%)	No	No	No
			LOC440563	4 (31%; 95% CI, 13–58%)	No	No	No
			PRAMEF19	4 (31%; 95% CI, 13–58%)	No	No	No
			CROCC	5 (39%; 95% CI, 18–65%)	No	No	Yes
			PRAMEF1	6 (46%; 95% CI, 23–71%)	No	No	Yes
			HNRNPCL1	7 (54%; 95% CI, 29–77%)	No	No	Yes
3p	Loss	33
			ALS2CL	3 (23%; 95% CI, 8–50%)	No	No	Yes
			COL7A1	3 (23%; 95% CI, 8–50%)	No	No	No
			LAMB2	3 (23%; 95% CI, 8–50%)	No	No	Yes
			MST1	3 (23%; 95% CI, 8–50%)	No	No	Yes
			*RBM5*^a	3 (23%; 95% CI, 8–50%)	Yes	No	Yes
			*RAD54L2*^a	4 (31%; 95% CI, 13–58%)	Yes	No	Yes
13q	Loss	9
			FAM194B	4 (31%; 95% CI, 13–58%)	No	No	Yes
			*RB1*^a	3 (23%; 95% CI, 8–50%)	Yes	Yes	Yes
14q	Loss	64
			*SYNE2*^a,^b	3 (23%; 95% CI, 8–50%)	No	No	Yes
			GALC	3 (23%; 95% CI, 8–50%)	No	No	No
			DYNC1H1	5 (39%; 95% CI, 18–65%)	No	No	No
			AHNAK2	6 (46%; 95% CI, 23–71%)	No	No	No
15q	Loss	12
			MYO5A	3 (23%; 95% CI, 8–50%)	No	No	No
			*USP8*^a	5 (39%; 95% CI, 18–65%)	No	No	Yes
22q	Loss	80
			*AP1B1*^c	3 (23%; 95% CI, 8–50%)	No	No	Yes
			NEFH	3 (23%; 95% CI, 8–50%)	No	No	No
			SEC14L4	3 (23%; 95% CI, 8–50%)	No	No	No
			RFPL3	3 (23%; 95% CI, 8–50%)	No	No	No
			*GTSE1*^a	3 (23%; 95% CI, 8–50%)	Yes	No	Yes
			*MAPK8IP2*^a,^b	3 (23%; 95% CI, 8–50%)	Yes	No	Yes
			TUBGCP6	4 (31%; 95% CI, 13–58%)	No	No	No
4q	Gain	4
			PDGFRA	3 (23%; 95% CI, 8–50%)	Yes	Yes	Yes
			*KIT*^a	6 (46%; 95% CI, 23–71%)	Yes	Yes	Yes
5q	Gain	100
			*DIAPH1*^a,^c	3 (23%; 95% CI, 8–50%)	Yes	No	Yes
			FAT2	3 (23%; 95% CI, 8–50%)	Yes	No	Yes
			PCDHB8	3 (23%; 95% CI, 8–50%)	No	No	No
			TCOF1	3 (23%; 95% CI, 8–50%)	No	No	No
			YIPF5	3 (23%; 95% CI, 8–50%)	No	No	No
			C5orf65	4 (31%; 95% CI, 13–58%)	No	No	No
			CDHR2	4 (31%; 95% CI, 13–58%)	No	No	Yes
			F12	4 (31%; 95% CI, 13–58%)	No	No	No
			*FLT4*^a	4 (31%; 95% CI, 13–58%)	Yes	Yes	Yes

↵^aProtein expressions were evaluated immunohistochemically and correlated with risk factors.
↵^bPotential interaction.
↵^cPotential interaction.

Table 4.

Expression of proteins and correlation of overexpression with prognostic parameters

Protein	Number of investigated cases	Primary staining pattern	Positive cases	95% CI	Fletcher risk	Miettinen risk	UICC staging	UICC mitotic rate	Localization	Survival	KIT mutations
RBM5	118	Nucleus	108 (91.5%)	85.1–95.3%	–	–	–	–	–	–	–
RAD54L2	114	Nucleus	11 (9.6%)	5.5–16.5%	Yes	–	Yes	–	–	Shorter OS	–
RB1	106	Nucleus	20 (18.9%)	12.6–27.4%	–	–	–	–	–	–	–
SYNE2	128	Nucleus	23 (18%)	12.3–25.5%	Yes	Yes	–	Yes	Small intestinal	Shorter DFS	–
USP8	120	Cytoplasm	108 (90%)	83.3–94.2%	–	–	–	–	–	–	–
AP1B1	74	Cytoplasm	74 (100%)	95.1–100%	–	–	–	–	–	–	–
GTSE1	119	Cytoplasm	14 (11.8%)	7.1–18.8%	–	–	–	–	Small intestinal	–	–
MAPK8IP2	137	Cytoplasm	69 (50.4%)	42.1–58.6%	–	–	–	–	–	–	–
KIT	145	Cytoplasm	126 (86.9%)	80.4–91.5%	Yes (neg.)	–	Yes (neg.)		–	Longer OS	Yes
DIAPH1	119	Cytoplasm	54 (45.4%)	36.7–54.3%	Yes	Yes	Yes	Yes	–	Shorter DFS
FLT4	75	Cytoplasm	30 (40%)	29.7–51.3%	–	–	–	–	–	–	–

Abbreviation: neg., negative correlation.

Additional Files

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Tables

Supplementary Data

Files in this Data Supplement:

Supplementary Tables 1 and 2 and 5 - PDF file, 95K, Supplemental Table 1: Comparision of copy number variation regions of interest by FISH (n=125) and microarray (n=29). Supplemental Table 2: Antibodies used for detection of genes in regions of recurrent losses or gains Supplemental Table 5: Results of rearward stepwise Cox regression of disease free and overall survival.
Supplementary Tables 3 and 4 - PDF file, 24K, Supplemental Table 3 Variants per sample Supplemental Table 4 Variants unique per normal/cancer pair.