Table 1.
Patient characteristics and results from a droplet digital PCR experiment on NRAS p.Q61R mutation
| Patient number (diagnosis) | Age (years) / sex | Affected organs | Complications | Sample material (sample numbers in Fig. 3b) | Target gene | Concentration (copies/μL)a | Accepted dropletsb | Fractional abundance (%)c | Poisson fractional abundance (max)d | Poisson fractional abundance (min)e |
|---|---|---|---|---|---|---|---|---|---|---|
| P1 (KLA) | 21/ M | Chest wall and mediastinum | Hemorrhagic PE, gastrointestinal hemorrhage and coagulation disorder | Plasma (P1–1) | NRAS Q61R | 0.41 | 40,029 | 0.18 | 0.28 | 0.08 |
| NRAS WT | 227.07 | 40,029 | – | – | – | |||||
| PE (P1–2) | NRAS Q61R | 0.41 | 31,317 | 0.44 | 0.70 | 0.18 | ||||
| NRAS WT | 93.64 | 31,317 | – | – | – | |||||
| P2 (KLA) | 12/ M | Bone, thoracic and mediastinal | Hemorrhagic PE and coagulation disorder | Plasma (P2–1) | NRAS Q61R | 0.20 | 41,269 | 0.07 | 0.13 | 0.02 |
| NRAS WT | 275.93 | 41,269 | – | – | – | |||||
| PE (P2–2) | NRAS Q61R | 0.03 | 39,120 | 0.16 | 0.55 | 0.02 | ||||
| NRAS WT | 18.28 | 39,120 | – | – | – | |||||
| FFPE tissuef (P2–3) | NRAS Q61R | 0.14 | 33,434 | 0.2 | 0.4 | 0.00 | ||||
| NRAS WT | 70.7 | 33,434 | – | |||||||
| P3 (KLA) | 4/ F | Thoracic and mediastinal | Hemorrhagic PE and coagulation disorder | Plasma (P3–1) | NRAS Q61R | 0.06 | 37,013 | 0.02 | 0.06 | 0.00 |
| NRAS WT | 280.55 | 37,013 | – | – | – | |||||
| PE (P3–2) | NRAS Q61R | 0.03 | 38,740 | 0.01 | 0.04 | 0.00 | ||||
| NRAS WT | 229.55 | 38,740 | – | – | – | |||||
| P4 (KLA) | 12/ M | Bone, thoracic and mediastinal | Scoliosis, hemorrhagic PE and coagulation disorder | Plasma (P4) | NRAS Q61R | 0.06 | 41,040 | 0.02 | 0.06 | 0.00 |
| NRAS WT | 243.08 | 41,040 | – | – | – | |||||
| P5 (KLA) | 20/ M | Bone, thoracic and mediastinal | PE and coagulation disorder | Plasma (P5) | NRAS Q61R | 0.00 | 37,721 | 0.00 | 0.00 | 0.00 |
| NRAS WT | 27.52 | 37,721 | – | – | – | |||||
| P6 (GLA) | 18/ M | Bone, thoracic and mediastinal | PE and coagulation disorder | Plasma (P6) | NRAS Q61R | 0.06 | 39,832 | 0.03 | 0.07 | 0.00 |
| NRAS WT | 226.91 | 39,832 | – | – | – | |||||
| P7 (GLA) | 35/ F | Abdominal cavity and skin | Lymphorrhea, pain, and cellulitis | Plasma (P7) | NRAS Q61R | 0.00 | 36,076 | 0.00 | 0.00 | 0.00 |
| NRAS WT | 39.09 | 36,076 | – | – | – | |||||
| P8 (GLA) | 32/ F | Abdominal cavity and skin | Ascites, coagulation disorder, and lymphorrhea | Plasma (P8) | NRAS Q61R | 0.00 | 42,470 | 0.00 | 0.00 | 0.00 |
| NRAS WT | 49.65 | 42,470 | – | – | – | |||||
| Positive control | Plasma (PC) | NRAS Q61R | 0.00 | 13,194 | 0.00 | 0.00 | 0.00 | |||
| NRAS WT | 352.38 | 13,194 | – | – | – | |||||
| Negative control | (NC) | NRAS Q61R | 0.00 | 12,603 | 0.00 | 0.00 | 0.00 | |||
| NRAS WT | 0.00 | 12,603 | – | – | – | |||||
| Human skin melanoma cell lines (SK-MEL-2)g | NRAS Q61R | 106.16 | 34,258 | 69.15 | 70.52 | 67.77 | ||||
| NRAS WT | 47.37 | 34,258 | – | – | – | |||||
Characteristics (diagnosis, age, sex, affected organs, and complications) of the patients with KLA (P1–5) and GLA (P6–8) are shown in the table. The information of the samples (sample materials) and results (concentration of target gene, accepted droplets, fractional abundance and percentage of Poisson fractional abundance) from a droplet digital PCR experiment on NRAS p.Q61R mutations are shown. Positive controls included wild-type DNA. Negative controls did not include DNA. Human melanoma cell line with NRAS p.Q61R mutation was used as a positive control for NRAS mutation
KLA kaposiform lymphangiomatosis, GLA generalized lymphatic anomaly, P patient, PC positive control, NC negative control, M male, F female, PE pleural effusion, WT wild type, FFPE formalin-fixed, paraffin-embedded
aTarget gene copy number per microliter
bNumber of droplets measured—i.e., the sum of three measured wells
cPercentage of mutant droplets relative to the sum of wild-type droplets. It was statistically calculated from the measurements of three wells
dPercentage of Poisson fractional abundance (upper limit of the 95% confidence interval)
ePercentage of Poisson fractional abundance (lower limit of the 95% confidence interval)
fFFPE tissue of the affected lesion in P2
gNRAS Q61R mutated sample was the positive control