Frequent mutations of the CA simple sequence repeat in intron 1 of EGFR in mismatch repair-deficient colorectal cancers

Table 1 Allelic and genotype distribution of EGFR CA-SSR I polymorphism in normal tissues of patients with colorectal tumors n (%)

	CA-SSR I status of the tumora
Genotypes	CA-SSR I MSI	CA-SSR I MSS	Total
in normal colon	(n = 36)	(n = 50)	(n = 86)
14/20	1 (2.8)	0 (0)	1 (1.2)
15/20	1 (2.8)	0 (0)	1 (1.2)
16/16	10 (27.8)	14 (28)	24 (27.9)
16/17	2 (5.6)	2 (4.0)	4 (4.7)
16/18	4 (11.1)	11 (22.0)	15 (17.4)
16/19	0 (0)	1 (2.0)	1 (1.2)
16/20	6 (16.7)	9 (18)	15 (17.4)
16/21	0 (0)	2 (4.0)	2 (2.3)
17/20	0 (0)	1 (2.0)	1 (1.2)
18/18	2 (5.6)	2 (4.0)	4 (4.7)
18/19	1 (2.8)	0 (0)	1 (1.2)
18/20	2 (5.6)	3 (6.0)	5 (5.8)
18/21	1 (2.8)	1 (2.0)	2 (2.3)
19/21	1 (2.8)	0 (0)	1 (1.2)
20/20	4 (11.1)	3 (6.0)	7 (8.1)
20/21	1 (2.8)	0 (0)	1 (1.2)
21/21	0 (0)	1 (2.0)	1 (1.2)

^aP = 0.673, CA-SSR I MSI vs CA-SSR I MSS according to the allele lengths (patients with at least one allele ≥ 20 CA (17/19) vs both alleles < 20 CA (19/23), respectively) (χ² test).

Table 2 Status of EGFR CA-SSR I in patients with MSI-H and MSS colorectal tumors n/n (%)

	CA-SSR I MSIb	CA-SSR I MSSb	CA-SSR I MSS1
			AI+	AI-
MSI-H (n = 42)	36/42 (85.7)	6/42 (14.3)	0/1 (0)	1/1 (100)
MSS (n = 44)	0/44 (0)	44/44 (100)	5/30 (16.7)	25/30 (83.3)

¹Tumors from informative patients for AI score determination, i.e. heterozygous for CA-SSR I.

^bP < 0.001, regarding the comparison of CA-SSR I MSI vs CA-SSR I MSS distribution according to the MSI status (χ² test).

Table 3 Relationship between EGFR CA-SSR I genotypes and EGFR expression levels in colorectal tumors

	CA-SSR genotypes1		CA-SSR I status2 of the tumor	EGFR expression
	Normal colon	Tumor		QRT-PCR3	IHC4
1	16/16	16/20	MSI	0.87	++ m
2	16/16	16/16	MSS	0.70	++ m
3	16/18	16/18	MSS	2.68	++ m
4	16/16	14/16/20/(21/22/23)	MSI	5.10	+++ m, c
5	20/20	16/25/26/27	MSI	0.27	++ m, c
6	20/20	20/20	MSS	0.76	++ m
7	16/20	16/20	MSS (AI +)	1.11	+++ m
8	20/20	20/20	MSS	0.48	++ m
9	16/20	16/20	MSS	3.68	++ m
10	16/16	16/16	MSS	1.32	++ m
11	16/18	16/18	MSS (AI +)	0.47	+ m
12	19/21	19/21/(22/23)	MSI	1,57	+++ m, c
13	16/18	14/16/18	MSI	ND	++ m, c
14	14/20	12/14/20/(22/23)	MSI	ND	++ m, c
15	16/18	16/18/(23/24)	MSI	ND	+++ m, c
16	16/20	16/20	MSS	ND	-
17	16/16	16/16	MSS	ND	-
18	18/18	18/18	MSS	ND	++ m

¹CA-SSR I alleles determined in colorectal tumors and corresponding normal tissues (germline alleles). All observed alleles are indicated, with minor alleles in brackets;

²MSI, presence of CA-SSR I instability; MSS, absence of CA-SSR I instability; AI +, presence of allelic imbalance;

³Relative expression of EGFR mRNA levels (tumor relative to matching normal tissues), as assessed by QRT-PCR and calculated using the 2^-ΔΔCt formula. Values corresponding to a fold change > 2 or < 0.5 are indicated in bold. ND: not determined;

⁴Expression of EGFR peptide determined by immunohistochemistry. Score 0, 0% of positive cells with membranous staining; 1+, > 0%-1%; 2+, 2%-50%; 3+, > 50%. m: Membranous staining; c: Cytoplasmic staining.