Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

Table 1 Patient characteristics

		Colorectal cancer patients, n = 70
		Right colon, n = 28	Left colon, n = 34	Rectum, n = 8
Sex	Female	13	13	3
	Male	15	21	5
Age (mean)		76.89	72.26	70.63
pT	T1	3	4	2
	T2	6	9	2
	T3	16	18	3
	T4	3	3	1
pN	N0	20	22	8
	N+	8	12	-
Stage	I	9	10	4
	IIa	9	10	3
	IIb	1	1	1
	IIIa	1	3	-
	IIIb	4	5	-
	IIIc	2	2	-
	IV	2	3	-
KRAS status	Mutant	11	15	6
	Wild-type	17	19	2

Table 2 KRAS mutational status by pyrosequencing

	KRAS wild-type	KRAS mutations
		Codon 12				Codon 13		Others
		G12D	G12V	G12R	G12S	G13D	G13R	A146V	A146T	A59T	Q61R	Q61H	Q61L
Right colon	17	2	0	1	0	3	1	1	1	0	1	1	0
Left colon	19	6	5	0	0	1	0	1	1	1	0	0	0
Rectum	2	2	0	0	1	1	0	0	0	0	0	1	1
		10	5	1	1	5	1	2	2	1	1	2	1
Total	38 (54.29%)	32 (45.71%)

Table 3 KRAS G12D mutation levels in DNA from stool samples of wild-type control patients

				G12D								Wild-type
				Single				Merged				Single					Merged
Patient	Tumor location	Tumor stage	Positive events		Copies/μL	Copies/20 μL reaction	Positive events		Copies/μL	Copies/20 μL reaction	Copies/ng DNA		Positive events	Copies/μL	Copies/20 μL reaction	Positiveevents		Copies/μL	Copies/20 μL reaction	Copies/ng DNA
53	Left	I	1		0.1	2	3		0.07	1.40	0.011		94	9	180	469		10.50	210.00	1.615
			2		0.17	3.4							12	10.8	216
			0		0	0							116	9.9	198
			0		0	0							136	12.1	242
56	Left	IIA	1		0.08	1.6	1		0.02	0.40	0.003		17	1.4	28	76		1.56	31.20	0.240
			0		0	0							25	2.1	42
			0		0	0							16	1.3	26
			0		0	0							18	1.4	28
63	Right	IIA	1		0.08	1.6	1		0.02	0.42	0.003		258	20.4	408	957		20.30	406.00	3.123
			0		0	0							222	22.1	442
			0		0	0							217	19	380
			0		0	0							260	19.9	398
71	Right	IIIB	1		0.08	1.6	3		0.06	1.20	0.009		89	6.7	134	377		7.20	144.00	1.108
			2		0.15	3							104	7.9	158
			0		0	0							93	6.9	138
			0		0	0							91	7.3	146
96	Left	IV	1		0.09	1.8	3		0.06	1.20	0.009		32	3	60	133		2.84	56.80	0.437
			1		0.08	1.6							33	2.6	52
			1		0.09	1.8							34	3	60
			0		0	0							34	2.9	58

Table 4 KRAS G12D mutation levels in DNA from stool samples of patients with KRAS G12D mutated tumors

			G12D								Wild-type
			Single			Merged					single			Merged
Patient	Tumor location	Tumor stage	Positive events	Copies/μL	Copies/20 μL reaction		Positive events	Copies/μL	Copies/20 μL reaction	Copies/ng DNA	Positive events	Copies/μL	Copies/20 μL reaction	Positive events	Copies/μL	Copies/20 μL reaction	Copies/ng DNA
12	Rectum	I	4	0.32	6.4		14	0.37	7.40	0.051	155	12.6	252	493	13.10	262.00	1.819
			5	0.39	7.8						158	12.5	250
			5	0.39	7.8						180	14.1	282
17	Left	I	1147	95	1900		4849	97.20	1944.00	41.362	4684	446	8920	19664	456.00	9120.00	194.043
			1243	98	1960						4996	458	9160
			1269	99	1980						5094	460	9200
			1190	97	1940						4890	461	9220
29	Left	IIIB	5	0.41	8.2		11	0.30	6.00	0.024	94	7.7	154	257	7.10	142.00	0.577
			3	0.24	4.8						90	7.4	148
			3	0.25	5						73	6.2	124
30	Right	I	6	0.57	11.4		18	0.54	10.80	0.017	238	22.8	456	757	23.10	462.00	0.728
			7	0.64	12.8						268	24.8	496
			5	0.43	8.6						251	21.8	436
43	Right	IIIC	6	0.59	11.8		19	0.59	11.80	0.035	418	42	840	1393	44.10	882.00	2.602
			8	0.7	14						492	45.5	910
			5	0.46	9.2						483	44.8	896
461	Left	IIC	1	0.09	1.8		3	0.09	1.80	0.014	81	7	140	265	7.80	156.00	1.238
			1	0.09	1.8						97	8.3	166
			1	0.09	1.8						87	8.2	164
51	Rectum	I	13	1	20		47	0.90	18.00	0.295	1292	103	2060	5110	102.60	2052.00	33.639
			12	0.9	18						1291	102	2040
			9	0.68	13.6						1297	102	2040
			13	1	20						1230	103	2060
641	Left	I	0	0	0		0	0.00	0.00	0.000	141	12	240	653	12.20	244.00	1.877
			0	0	0						163	12.1	242
			0	0	0						154	11.4	228
			0	0	0						195	13.3	266
70	Left	I	11	1.1	22		45	1.37	27.40	0.036	2515	282	5640	8516	294.00	5880.00	7.656
			24	1.9	38						3313	305	6100
			10	1	20						2688	291	5820
75	Left	IIA	0	0	0		11	0.33	6.60	0.032	304	27.2	544	964	29.00	580.00	2.788
			2	0.18	3.6						326	29.8	596
			9	0.8	16						334	30.1	602

¹Patients 46 and 64 were considered negative because they had values less than or equal to the positivity threshold.