Table 1.

Non-exhaustive list of published trials with personalized methods to identify MRD through ctDNA detection.

Reference	Number of Patients Included (n)	Tumor Type and Indication	Methodology	Conclusions
Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patientswith urothelial bladder carcinoma [91]	68	Muscle invasive bladder cancer treated with neoadjuvant chemotherapy before cystectomy	Tumor sequencing: WES Plasma sequencing: 16 mutations/patient by multiplex PCR.	A total of 76% of ctDNA-positive patients post cystectomy had recurrence (median 96 days before). A total of 0% of ctDNA-negative had recurrence.
Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer [86]	55	Early breast cancer patients receiving neoadjuvant chemotherapy	Tumor sequencing: NGS on panel with 14 known breast cancer driver genes (26). Plasma sequencing: 1 (or more) mutation(s) was (were) followed using ddPCR.	ctDNA was detected in the single post-operative blood test in 19% (7 of 37) of patients. ctDNA detection was predictive of early relapse (median 6.5 months).
Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer [89]	33	Stage I to Stage III breast cancer	Tumor sequencing: WES Plasma sequencing: Using TARDIS (combinaison of NGS + PCR + UMIs): 6 to 115 mutations per patient.	Before treatment, ctDNA detected in 32 of 32 patients at tumor fractions of 0.002% to 1.06%. Plasma samples after completion of NAT were analyzed in 22 patients. ctDNA+ in 17 out of 22 patients, including 12 out of 13 patients with invasive or in situ residual disease and 5 out of 9 patients with pathological CR. In patients who achieved pathological CR, the median decrease in ctDNA was 96%, whereas in patients with residual disease observed at surgery, the median decrease was 77%.
Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer [92]	94	Resectable colon cancers with plasma available	Tumor sequencing: NGS on custom targeted panel of 29 genes. Plasma sequencing: personalized ddPCR assays for each somatic mutation identified in the tissue.	ctDNA was detected in 63.8% at baseline. ctDNA was detected at 6–8 weeks post-surgery, before starting adjuvant chemotherapy, in 20.3% (14 of 69) patients with plasma available at this time. In ctDNA-positive post-op: 57.1% (8 of 14 patients) experienced reccurence. The presence of ctDNA immediately after surgery was associated with poorer DFS.
Circulating tumor DNA analyses as markers of recurrence risk and benefit of adjuvant therapy for Stage III colon cancer [90]	96	Stage III colon cancer	Tumor sequencing: NGS on 15 genes recurrently mutated in colorectal cancer. Plasma sequencing: 1 mutation/patient with Safe-Seq (NGS + UMIs).	A tumor-specific mutation was detected (ctDNA-positive finding) in the post-surgical plasma sample of 20 of 96 patients (21%). ctDNA was detectable in 15 of 88 (17%) post-chemotherapy samples. Post-surgical ctDNA was detectable in 10 of 24 patients (42%) with recurrence.
Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response andsurvival [88]	84	High-risk earlybreast cancer patients with NAT (I-SPY2 Trial)	Tumor sequencing: WES Plasma sequencing: 16 mutations/patient by multiplex PCR	After NAC, all patients who achieved pCR were ctDNA-negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had significantly increased risk of metastatic recurrence (HR 10.4; 95% CI, 2.3–46.6). Patients who did not achieve pCR but were ctDNA negative (86%) had a similar outcome to those who achieved pCR.
Circulating Tumor DNA predicts pathologic and clinical outcomes following neoadjuvantchemoradiation and surgery for patients with locally advanced rectal cancer [93]	29	Locally advanced rectal cancer	Tumor sequencing: WES Plasma sequencing: personalized ddPCR assays for each somatic mutation identified in the tissue.	Patients with detectable postoperative ctDNA experienced poorer RFS (hazard ratio, 11.56; p = 0.007). All patients (4 out of 4) with detectable postoperative ctDNA recurred (positive predictive value = 100%), whereas only 2 out of 15 patients with undetectable ctDNA recurred (negative predictive value = 87%).
Galaxy Study: Preoperative ctDNA levels are detectable in the majority of patients with resectable colorectal cancer [94]	808	Resectable CRC	Tumor sequencing: WES Plasma sequencing: personalized ddPCR assays for each somatic mutation identified in the tissue.	Longitudinal ctDNA positivity at postoperative weeks 4, 12, and 24 was significantly associated with inferior disease-free survival (DFS) with a hazard ratio (HR) of 46.8. Sensitivity of relapse detection was 93.1%. Positivity at postoperative week 4 was significantly associated with inferior DFS with HR 19.5 overall, and HR 24.4 in pathologic Stage I–III, indicating it is a suitable time point for ctDNA-based adjuvant study.
Dynamics of cell-free tumour DNA correlate with treatment response of head and neck cancer patients receiving radiochemotherapy [87]	20	Non-resecable locally advanced head and neck squamous cell carcinoma	Tumor sequencing: NGS with 327 genes panel. Plasma sequencing: 127 driver mutations + E7 NGS panel	Baseline: ctDNA-positive: 17/20 patients Post RCT ctDNA-positive-: 2/16 patients Eight patients relapsed: 2ctDNA-positive Eight patients without relapse: 8ctDNA-negative PPV 100%, Sn 25%

WES: whole exome sequencing; PCR: polymerase chain reaction; ddPCR: droplet digital polymerase chain reaction; NGS: next generation sequencing, UMI: unique molecular identifier; NAT: neoadjuvant treatment; CR: complete response; NAC: neoadjuvant chemotherapy; pCR: pathological complete response; DFS: disease-free survival; RFS: relapse-free survival; HR: hazard ratio; RCT: radio-chemotherapy; PPV: positive predictive value; Sn: sensitivity; ctDNA: circulating tumor DNA.