Supplementary

Patient number / Age / Sex / uT / uN / M / ypT / ypN / M1 / TRG
P1 / 52 / m / 4 / 1 / 1 / 3c / 0 (0/10) / 1a(hep) / 2
P2 / 74 / f / 2 / 1 / 0 / 3a / 2 (4/20) / 0 / n/a
P3 / 68 / m / 3 / 1 / 0 / 2 / 0 (0/26) / 0 / 2
P4 / 60 / m / 4 / 1 / 0 / 0 / 0 (0/15) / 0 / 4
P5 / 46 / f / 3 / 1 / 0 / 3a / 0 (0/11) / 0 / 3
P6 / 65 / m / 3 / 0 / 0 / 3 / 2 (4/16) / 0 / n/a
P7 / 60 / f / 3 / 0 / 0 / 2 / 1 (1/24) / 0 / 2
P8 / 73 / f / 3 / 0 / 0 / 2 / 0 (0/22) / 0 / 2
P9 / 57 / m / 3 / 1 / 0 / 2 / 0 (0/48) / 0 / 1
P10 / 74 / m / 3 / 0 / 0 / 0 / 0 (0/25) / 0 / 4
P11 / 75 / m / 3 / 1 / 0 / 3c / 1 (3/31) / 0 / 1
P12 / 66 / f / 3 / 1 / 0 / 2 / 0 (0/39) / 0 / 3
P13 / 74 / m / 3 / 1 / 0 / 0 / 0 (0/19) / 0 / 4
P14 / 43 / f / 3 / 1 / 0 / 3b / 2 (4/32) / 0 / 2
P15 / 83 / f / 3 / 1 / 0 / 1 / 2 (4/21) / 0 / 3
P16 / 64 / m / 3 / 1 / 0 / 1 / 0 (0/16) / 0 / 2
P17 / 79 / f / 3 / 0 / 0 / 2 / 0 (0/21) / 0 / 3
P18 / 80 / f / 3 / 1 / 0 / 3a / 0 (0/36) / 0 / 2
P19 / 61 / f / 3 / 1 / 0 / 2 / 0 (0/30) / 0 / 2
P20 / 85 / m / 3 / 1 / 0 / 3a / 0 (0/19) / 0 / 2
P21 / 59 / m / 3 / 1 / 0 / 2 / 0 (0/19) / 0 / 3
P22 / 58 / m / 4 / 1 / 0 / 3c / 2 (4/21) / 0 / 1
P23 / 72 / m / 2 / 1 / 0 / 3b / 2 (10/17) / 0 / 2
P24 / 62 / m / 3 / 1 / 0 / 2 / 0 (0/27) / 0 / 3
P25 / 21 / f / 4 / 1 / 0 / 4b / 2 (5/29) / 0 / 2
P26 / 71 / f / 3 / 1 / 0 / 3a / 0 (0/17) / 0 / 3
P27 / 70 / m / 3 / 1 / 0 / 3c / 2 (16/16) / 1a(hep) / 0
P28 / 56 / m / 3 / 0 / 0 / 3b / 2 (13/24) / 0 / 2
P29 / 69 / f / 3 / 1 / 1 / 3b / 1 (1/16) / 1a(hep) / 2
P30 / 71 / m / 3 / 1 / 0 / 3c / 0 (0/14) / 0 / 1
P31 / 71 / m / 3 / 1 / 0 / 3c / 0 (0/16) / 1a(hep) / 1
P32 / 55 / f / 3 / 1 / 0 / 2 / 0 (0/27) / 0 / 2
P33 / 59 / f / 3 / 1 / 0 / 2 / 0 (0/25) / 0 / 2
P34 / 39 / f / 3 / 1 / 0 / 0 / 0 (0/10) / 0 / n/a
P35 / 69 / m / 3 / 1 / 0 / 0 / 0 (0/19) / 0 / 4
P36 / 62 / m / 3 / 1 / 0 / 2 / 0 (0/15) / 0 / 2
P37 / 56 / m / 3 / 1 / 0 / 0 / 0 (0/12) / 0 / 4
P38 / 69 / m / 3 / 1 / 0 / 2 / 0 (0/20) / 0 / 2
P39 / 66 / m / 3 / 1 / 0 / 2 / 0 (0/21) / 0 / 2
P40 / 79 / f / 3 / 1 / 0 / 3c / 0 (0/15) / 0 / 2
P41 / 63 / m / 3 / 1 / 0 / 2 / 0 (0/24) / 0 / 3
P42 / 71 / m / 2 / 1 / 0 / 0 / 0 (0/25) / 0 / 4
Supplementary Table 1: Clinical data of each patient. m = male, f = female, M1 represents the postoperative status whether a patient has any distant metastasis or not; hep hepatic metastasis. Total of 42 patients including 25 male and 17 female. TRG = tumor regression grade according to Dworak.

Supplementary Figure 1:Treatment of locally advanced rectal cancer. It is based on chemoradiotherapy (CRT), surgical resection with total mesorectal excision (TME) and adjuvant chemotherapy (CTx). For this study, blood was taken during CRT at three different time points: before starting CRT (time point 1), during CRT, precisely after the first cycle of CRT (time point 2) and after completion of CRT but before surgery (time point 3). Preoperative CRT consisted of radiotherapy accompanied by either 5-FU (n = 25) or 5-FU and Oxaliplatin followed by three applications of a shortened FOLFOX regimen (n = 17). Four weeks after completion of CRT TME-surgery was performed.

Supplementary Figure 2:Normalized ct values per miRNA (miR-17, miR-18b, miR-20a, miR-31 and miR-193-3p) and added C. elegans miRNA mimics as spike-ins (Cel_miR-39, Cel_miR-54, Cel_miR-238) are shown. The spike-ins hardly vary, which demonstrates a high-quality miRNA isolation.

Supplementary Figure 3:Development of microRNA expression levels in plasma during CRT, from time point 1 (TP1) to time point 2 (TP2), and from time point 2 to time point 3. Number of patients (n) is given for those who showed increased and decreased expression levels, respectively. On the right the development of microRNA expression levels is showed between TP1 and TP3. Time points are explained in supplementary figure 1.