Table 1. Characteristics of next generation sequencing outcomes of resection specimens, tumor biopsies and cfDNA per point in time.
| T | Moment of sample | Analysis | DNA concentration (ng/μL) | Total DNA (ng) | Allele | Allele Mol. Freq. | Mol. Cov. | Allele Mol. Cov. | Allele Read Freq. | Coverage | Allele Read Cov. | Remarks/allele call |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| t=0 (baseline) | Baseline, tissue | NGS with diagV5 panel | 1.57 | 4.7 | c.524G>A; p.R175H | – | – | – | 71% | n/a | n/a | – |
| c.646G>A; p.V216M | – | – | – | – | – | – | – | |||||
| c.659A>G; p.Y220C | – | – | – | – | – | – | – | |||||
| NGS with Oncomine | c.524G>A; p.R175H | 52.24% | 1,003 | 524 | 54.00% | 62,022 | 33,492 | Heterozygous | ||||
| cfDNA colon panel | c.646G>A; p.V216M* | 0.00%* | 876* | 0* | 0* | 74,997* | 0* | Absent | ||||
| c.659A>G; p.Y220C* | 0.00%* | 876* | 0* | 0* | 74,997* | 0* | Absent | |||||
| t=0 (baseline) | Baseline, cfDNA | NGS with Oncomine | 2.14 | 20 | c.524G>A; p.R175H* | 0.03%* | 2,968* | 1* | 0.05%* | 43,333* | 20* | No call |
| cfDNA Colon panel | c.646G>A; p.V216M | 0.37% | 3,006 | 11 | 0.30% | 54,264 | 162 | Heterozygous | ||||
| c.659A>G; p.Y220C* | 0.00%* | 3,006* | 0* | 0.00%* | 54,264* | 0* | Absent | |||||
| t=2 (baseline + 14 w) | CRE-I | NGS with Oncomine | 2.07 | 21 | c.524G>A; p.R175H* | 0.04%* | 5,091* | 2* | 0.03%* | 71,538* | 22* | No call |
| cfDNA Colon panel | c.646G>A; p.V216M | 0.34% | 5,040 | 17 | 0.27% | 91,216 | 249 | Heterozygous | ||||
| c.659A>G; p.Y220C* | 0.02%* | 5,040* | 1* | 0.01%* | 91,216* | 10* | Absent | |||||
| t=3 (baseline + 21 w) | CRE-II | NGS with Oncomine | 2.58 | 21 | c.524G>A; p.R175H* | 0.00%* | 4,433* | 0* | 0.00%* | 68,079* | 0* | Absent |
| cfDNA colon panel | c.646G>A; p.V216M | 0.32% | 4,354 | 14 | 0.30% | 83,850 | 253 | Heterozygous | ||||
| c.659A>G; p.Y220C* | 0.00%* | 4,354* | 0* | 0.00%* | 83,850* | 0* | Absent | |||||
| t=4 (baseline + 22 w) | Resection, specimen | n/a | – | – | c.524G>A; p.R175H | – | – | – | – | – | – | – |
| c.646G>A; p.V216M | – | – | – | – | – | – | – | |||||
| c.659A>G; p.Y220C | – | – | – | – | – | – | – | |||||
| t=5 (baseline + 21 m) | Biopsy, metastasis | NGS with diagV5 panel | 0.2 | 0.6 | c.524G>A; p.R175H | – | – | – | 26% | n/a | n/a | – |
| c.646G>A; p.V216M | – | – | – | – | – | – | – | |||||
| c.659A>G; p.Y220C | – | – | – | – | – | – | – | |||||
| t=7 (baseline + 26 m) | Resection, specimen, metastasis | NGS with diagV5 panel | 1.2 | 3.6 | c.524G>A; p.R175H | – | – | – | 59% | n/a | n/a | – |
| c.646G>A; p.V216M | – | – | – | – | – | – | – | |||||
| c.659A>G; p.Y220C | – | – | – | – | – | – | – | |||||
| NGS with Oncomine | c.524G>A; p.R175H | 39.52% | 2,715 | 1,073 | 39.38% | 59,754 | 23,533 | Heterozygous | ||||
| cfDNA Colon panel | c.646G>A; p.V216M* | 0.06%* | 3,260* | 2* | 0.08%* | 92,215* | 77* | Absent | ||||
| c.659A>G; p.Y220C* | 0.00%* | 3,260* | 0* | 0* | 92,215* | 0* | Absent | |||||
| t=8 (baseline + 27 m) | FU I | NGS with Oncomine | 1.76 | 35.2 | c.524G>A; p.R175H* | 0.04%* | 5,217* | 2* | 0.05%* | 64,998* | 29* | No call |
| cfDNA Colon panel | c.646G>A; p.V216M | 0.59% | 4,935 | 29 | 0.50% | 73,632 | 367 | Heterozygous | ||||
| c.659A>G; p.Y220C* | 0.00%* | 4,935* | 0* | 0.00%* | 72,632* | 0* | Absent | |||||
| t=9 (baseline + 30 m) | FU II | NGS with Oncomine | 2.76 | 55.2 | c.524G>A; p.R175H | 0.23% | 4,325 | 10 | 0.22% | 48,465 | 105 | Heterozygous |
| cfDNA Colon panel | c.646G>A; p.V216M | 0.86% | 4,169 | 36 | 0.79% | 57,420 | 454 | Heterozygous | ||||
| c.659A>G; p.Y220C* | 0.05%* | 4,169* | 2* | 0.07%* | 57,420* | 39* | No call | |||||
| WBCs-FU II# | NGS with Oncomine# | 3.82# | 359.08# | c.524G>A; p.R175H* | 0.00%* | 6,483* | 0* | 0.00%* | 48,676* | 0* | Absent | |
| cfDNA colon panel# | c.646G>A; p.V216M# | 0.77%# | 5,980# | 46# | 0.71%# | 51,804# | 367# | Heterozygous | ||||
| c.659A>G; p.Y220C# | 0.05%# | 5,980# | 3# | 0.04%# | 51,804# | 22# | Heterozygous | |||||
| t=10 (baseline + 33 m) | FU III | NGS with Oncomine | 1.16 | 15.66 | c.524G>A; p.R175H | 0.19% | 2,710 | 5 | 0.09% | 33,774 | 31 | Heterozygous |
| cfDNA Colon panel | c.646G>A; p.V216M | 0.83% | 2,166 | 18 | 1.02% | 32,718 | 333 | Heterozygous | ||||
| c.659A>G; p.Y220C* | 0.05%* | 2,166* | 1* | 0.05%* | 32,718* | 15* | No call | |||||
| WBCs-FU III# | NGS with Oncomine# | 105.6# | 1425.6# | c.524G>A; p.R175H* | 0.00%* | 3,513* | 0* | 0.00%* | 35,466* | 0* | Absent | |
| cfDNA colon panel# | c.646G>A; p.V216M# | 0.48%# | 3,108# | 15# | 0.49%# | 38,982# | 191# | Heterozygous | ||||
| c.659A>G; p.Y220C# | 0.10%# | 3,108# | 3# | 0.09%# | 38,982# | 36# | Heterozygous |
Reference sequence TP53 (NM_000546) was used for DNA variant annotation. *, measurements below quality control threshold (VAF <0.1%; Allele Mol. Cov. <3); #, analysis of white blood cells. VAF, variant allele frequency (percentage based on the number of variant alleles covered divided by the total amount of molecules covered); Mol. Cov., molecular coverage (total number of independent molecules sequenced); Allele Mol. Cov., allele molecular coverage: number of independent molecules with a variant; allele read freq., allele read frequency (percentage of variant allele reads in total coverage); Allele Read Cov., allele read coverage; Cov., coverage (number of unique reads); NGS, next generation sequencing; cfDNA, cell-free DNA; CRE, clinical response evaluation; WBC, white blood cells; FU, follow-up.