Tumor irradiation may facilitate the detection of tumor-specific mutations in plasma

Table 1 Patients, tumors and treatment characteristics

Patient ID	Gender	Age	сТ	сN	Stage	CRM	EMV	Tumor location	RAS/RAFstatus	Total RT dose, Gy	Chemosensibilization1	RECIST	Surgery	MRI TRG	Mandard TRG	ypT	уN	Progression2	Follow-up, mo	ctDNA positive at baseline
																		Yes/no
ArAS	M	44	3	1	3	No	No	L	KRAS G12S	50	Yes	SD	Yes	III	NA	3	0	No	6.90	No
GaZM	F	66	3	2	3	Yes	No	U-M	KRAS G13D	46	Yes	PR	Yes	IV	4	4	1	No	4.03	No
DaKS	M	73	4	1	3	Yes	No	L	KRAS G12A	25 (short course)	No	PD	No	IV	NA	NA	NA	Yes	4.73	Yes
ArTP	F	81	4	1	3	Yes	Yes	L	KRAS Q61L	50	No	SD	Yes	III	3	3	0	Nd	5.80	Yes
MaLI	F	78	3	1	3	No	No	L	NRAS G12D	25(short course)	No	PD	No	IV	NA	NA	NA	Yes	3.97	Yes
MaNK	F	48	3	2	3	No	Yes	M	KRAS G12D	50	Yes	SD	Yes	III	2	3	2	No	6.57	Yes
ZuNM	F	63	3	2	3	No	No	L-M	BRAF V600E	44	Yes	PR	Yes	II	2	3	0	No	5.13	No
MiMF	F	74	3	1	3	No	Yes	M	NRAS G12D	25(short course)	No	PR	No	III	NA	NA	NA	No	3.13	No
SaVV	M	65	3	1	3	Yes	No	L-M	NRAS Q61R	50	Yes	SD	No	III	NA	NA	NA	Nd	1.13	No

¹Capecitabine 825 mg/m² twice daily was delivered on the days of RT (D1-5, D8-12, D 15-19, D22-26, D29-33).

²Disease status at March 1,.2021; Nd – lost from follow-up.

Tumor localization: U: Upper rectum, M: Middle rectum, L: Lower rectum; EMV: Extramural venous invasion; CRM: Circumferential resection margin; TRG: Tumor regression grade; CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease; NA: Not evaluated; ctDNA: Circulating tumor DNA.

Table 2 Changes in circulating tumor DNA content during radiotherapy for locally advanced rectal cancer

Patient ID	Mutation	Baseline mutated ctDNA	ctDNA analysis	-1 h	0	1 h	3 h	6 h	12 h	24 h	36 h	48 h	72 h	96 h	Radiation dose per day / number of fractions / total dose accumulated for the blood collection time
ArAS	KRAS G12S	Neg	C (mut)1	0	0	0	3	0	0	0	0	14	12	nd	2.0 Gy/4 fr/8 Gy + capecitabine3
			C (mut + wt)2	642	1006	1337	1963	633	906	829	762	4298	3088	nd
			VAF, %	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.3	0.4	nd
GaZM	KRAS G13D	Neg	C (mut)	0	0	0	26	37	59	40	50	46	5	80	2.0 Gy/5 fr/10 Gy + capecitabine3
			C (mut + wt)	6	7	8	866	808	1450	604	441	506	112	1219
			VAF, %	0.0	0.0	0.0	3.0	4.6	4.1	6.6	11.3	9.1	4.5	6.6
DaKS	KRAS G12A	Pos	C(mut)	22	22	52	134	42	33	32	102	27	65	36	5.0 Gy/5fr/25 Gy
			C (mut + wt)	295	118	655	1802	588	292	170	549	359	391	331
			VAF, %	7.5	18.6	7.9	7.4	7.1	11.3	18.8	18.6	7.5	16.6	10.9
ArTP	KRAS Q61L	Pos	C (mut)	39	0	0	26	10	0	39	nd	42	107	nd	2.0 Gy/4 fr/8 Gy
			C (mut + wt)	7606	9430	4036	5028	3187	3033	12574	nd	10622	14443	nd
			VAF, %	0.5	0.0	0.0	0.5	0.3	0.0	0.3	nd	0.4	0.7	nd
MaLI	NRAS G12D	Pos	C (mut)	23	15	0	0	0	5	13	15	10	21	15	5.0 Gy/5 fr/25 Gy
			C (mut + wt)	981	1011	1073	1303	1309	1160	1010	724	815	673	1088
			VAF, %	2.3	1.5	0.0	0.0	0.0	0.0	1.3	2.1	1.2	3.1	1.4
MaNK	KRAS G12D	Pos	C (mut)	244	257	nd	nd	nd	Nd	254	335	415	418	387	2.0 Gy/5 fr/10 Gy + capecitabine3
			C (mut + wt)	897	1091	nd	nd	nd	Nd	1424	1832	2241	1719	1955
			VAF, %	27.2	23.6	nd	nd	nd	Nd	17.8	18.0	18.5	24.3	19.8
ZuNM	BRAF V600E	Neg	C (mut)	0	0	0	0	0	0	0	0	0	0	0	2.0 Gy/5 fr/10 Gy + capecitabine3
			C (mut + wt)	1652	372	522	522	522	522	671	837	1004	791	2805
			VAF, %	0	0	0	0	0	0	0	0	0	0	0
MiMF	NRAS G12D	Neg	C (mut)	0	0	0	0	0	0	0	0	0	0	0	5.0 Gy/5 fr/25 Gy
			C (mut + wt)	594	500	740	2294	804	681	681	386	644	2891	1536
			VAF, %	0	0	0	0	0	0	0	0	0	0	0
SaVV	NRAS Q61R	Neg	C (mut)	0	0	0	0	0	0	0	0	0	0	0	2.0 Gy/5 fr/10 Gy + capecitabine3
			C (mut + wt)	196	237	541	840	621	748	353	372	272	261	249
			VAF, %	0	0	0	0	0	0	0	0	0	0	0

¹C (mut): Number of mutated copies per 1 mL of plasma measured by droplet digital PCR.

²C (mut + wt): Total number of target fragment (both wt and mut) copies per 1 mL of plasma measured by droplet digital PCR.

³Capecitabine 825 mg/m² twice daily was delivered on the 1-5 d of RT.

ctDNA: Circulating tumor DNA; VAF: Variant allele frequency.