Involvement of Krüppel-like factor 6 (KLF6) mutation in the development of nonpolypoid colorectal carcinoma

Table 1 PCR primer sets

Priming region		Primer sequence
Loss of heterozygosity analysis
	p53 (TP53)	5′-AGGGATACTATTCAGCCCGAGGTG-3′
		5′-ACTGCCACTCCTTGCCCCATTC-3′
	KLF6 (D10S591)	5′-ACCTCGAAGGTCTGTTCTCC-3′
		5′-GGCTTTATGGATCATATTAATCCAC-3′
	KLF6 (D10S594)	5′-GGGCAGCGTTGCTGAGA-3′
		5′-GCACCCAGATAGGCATAGAGA-3′
Mutation analysis
	K-ras exon 1:	5′-GGCCTGCTGAAAATGACTGA-3′
		5′-GGTGCAGGACCATTCTTTGAT-3′
	B-raf exon 11:	5′-AAACACTTGGTAGACGGGAC-3′
		5′-AATGTGGTGACATTGTGACAAGT-3′
	B-raf exon 15:	5′-CTTCATGAAGACCTCACAGT-3′
		5′-TCCACTGATTAAATTTTTGGCC-3′
KLF6	exon 1:	5′-GCCTCGCCGCCCTCGC-3′
		5′-AACTCCAAACAGCCGACCC-3′
	exon 2:	5′-CAATCACGTGCCTTCTCTGG-3′
		5′-GAGAAAGTGAGGATTTGTCTG-3′
	exon 3	5′-GTCTGCGGGTCAGTGAAGTC-3′
		5′-GTCATCACATTCCCAAGGCC-3′

Table 2 Summary of clinicopathologic features of 55 colorectal tumors

Characteristics	Number (%)
Sex
Male	39 (71)
Female	16 (29)
Tumor location1
Right-sided	20 (36)
Left-sided	35 (64)
Tumor histology2
Well-differentiated adenocarcinoma	46 (84)
Moderately differentiated adenocarcinoma	9 (16)
Tumor depth
Mucosal	10 (18)
Submucosal	45 (82)

¹Right-sided: from the cecum to the transverse colon; Left-sided: from the splenic flexure to the rectum.

²According to the Japanese Classification of Colorectal Carcinoma.

Table 3 Individual data for genetic analysis of 55 nonpolypoid colorectal tumors

	KLF6		p53		K-ras	B-raf
Case	LOH1	Mutation	IHC2	LOH4	Mutation	Mutation
Intramucosal cancer
16	NI3	Wild	(-)	NI	GGC to GCC [13, Gly to Ala (1/4)]	Wild
17	(-)		(-)		Wild	Wild
29	LOH (3/3)	Wild	(-)		Wild	Wild
30	LOH (2/4)	Wild	(-)	(-)	Wild	Wild
31		Wild	(-)	(-)	Wild	Wild
32		Wild	(++)	LOH (3/3)	Wild	Wild
33	(-)		(+)	LOH (1/3)	Wild	AAA to CAA [560, Lys to Gln (1/3)]
34	(-)		(+++)		Wild	Wild
35		Wild	(-)	LOH (1/3)	Wild	Wild
36	(-)		(-)	LOH (1/4)	Wild	Wild
Submucosal cancer
1	LOH (1/4)	Wild	(-)		Wild	Wild
2		Wild	(-)	(-)	Wild	Wild
3	NI	Wild	(+)		GGC to GCC [13, Gly to Ala (1/4)]	CAT to CTT [396, His to Leu (1/4)]
4	(-)		(+++)	LOH (1/5)	Wild	Wild
5		Wild	(+++)	(-)	Wild	Wild
6	NI	Wild	(-)	LOH (2/3)	Wild	Wild
7	NI		(+++)	(-)	Wild	Wild
8	LOH (2/3)	Wild	(++)	(-)	Wild	Wild
9	NI	Wild	(+++)		Wild	Wild
10	NI	Wild	(-)	LOH (2/3)	Wild	Wild
11	NI		(+++)	(-)	Wild	Wild
12	NI		(+++)	LOH (2/4)	Wild	Wild
13	LOH (3/7)	Wild	(+++)	(-)	Wild	Wild
14	NI	Wild	(-)	(-)	Wild	Wild
15	LOH (3/4)	Wild	(+++)		Wild	Wild
18	(-)		(+)		Wild	Wild
19	NI		(-)		Wild	Wild
20		Wild	(-)		GGC to GCC [13, Gly to Ala (1/4)]	CAG to CCG [608, Glu to Pro (1/4)]
21	NI	Wild	(++)	(-)	Wild	GTG to ATG [378, Val to Met (1/3)]
22	LOH (2/4)	CTG to GCT [735, Leu to Ala (2/4)]	(++)		Wild	Wild
23	NI	CTG to GCT [735, Leu to Ala (2/4)]	(+)	(-)	Wild	Wild
24	NI	Wild	(-)	(-)	GGC to GCC [13, Gly to Ala (1/5)]	Wild
25			(+)		Wild	AAA to CAA [560, Lys to Gln (1/5)]
26	(-)	Wild	(-)		Wild	AAA to CAA [560, Lys to Gln (2/6)]
27	(-)		(-)	NI	Wild	Wild
28		Wild	(-)		Wild	Wild
37	(-)		(-)	LOH (4/4)	Wild	Wild
38	(-)		(+++)	LOH (4/4)	Wild	Wild
39	(-)		(-)		Wild	Wild
40	(-)		(++)	(-)	Wild	Wild
41		Wild	(-)		Wild	Wild
42			(-)		Wild	Wild
43	(-)		(++)		Wild	Wild
44	LOH (3/3)	Wild	(-)	NI	Wild	Wild
45	LOH (1/5)	Wild	(+++)	LOH (5/5)	Wild	Wild
46	(-)		(+++)	(-)	Wild	Wild
47	LOH (3/3)	Wild	(-)	(-)	Wild	Wild
48	LOH (2/4)	Wild	(-)		Wild	Wild
49		Wild	(-)	(-)	GGC to GCC [13, Gly to Ala (1/4)]	Wild
50		Wild	(-)	LOH (3/5)	Wild	Wild
51	(-)		(-)	(-)	Wild	Wild
52	LOH (3/4)	Wild	(-)		Wild	Wild
53			(-)	LOH (1/4)	Wild	Wild
54	LOH (1/5)	Wild	(-)		Wild	Wild
55	LOH (1/4)	Wild	(+++)	LOH (3/4)	Wild	Wild

¹LOH: loss of heterozygosity;

²IHC: immunohistochemistry;

³NI: not informative; blank space, not examined;

⁴Number of LOH or mutation-positive foci per number of microdissected tumor foci is indicated in parenthesis.

Table 4 Summary for genetic analysis of 55 nonpolypoid colorectal tumors

Genetic alteration	Frequency
	Mucosal cancer	Submucosal cancer	Total
KLF6, LOH1 (+)	2/6 (33.3%)	12/23 (52.2%)	14/29 (48.3%)
KLF6, mutation	0/6 (0%)	2/28 (7.1%)	2/34 (5.9%)
p53, LOH (+)	4/8 (50.0%)	10/23 (43.5%)	14/31 (45.2%)
p53, overexpression	2/10 (20.0%)	17/45 (37.8%)	19/55 (34.5%)
K-ras, mutation	1/10 (10.0%)	4/45 (8.9%)	5/45 (9.1%)
B-raf, mutation	1/10 (10.0%)	5/45 (11.1%)	6/55 (10.9%)

¹LOH, Loss of heterozygosity.