Background
Methods
Tumor samples, control cell lines and DNA isolation
High resolution melting analysis
Pyrosequencing
Statistical analysis
Results
Sensitivity of HRM and pyrosequencing
Technical reliability of HRM and pyrosequencing
HRM | Pyrosequencing | |||||||
---|---|---|---|---|---|---|---|---|
Run 1 | Run 2 | |||||||
Sample | cDNA [ng/μl] | Run 1 | Run 2 | Result | % mut. Alleles | Result | % mut. Alleles | Final result |
1 | 11 | failed | WT a | WT | WT | |||
2 | 31 | WT a | WT | WT | ||||
3 | 10 | WT a | G12C | 13.4 | mut | |||
4 | 93 | WT a | WT | WT | ||||
5 | 26 | WT a | WT | WT | ||||
6 | 27 | WT a | WT | WT | ||||
7 | 10 | WT a | WT | WT | ||||
8 | 43 | mut | G13D | 73.9 | mut | |||
9 | 85 | failed | WT | WT | WT | |||
10 | N/A | mut | G13D | 44.3 | mut | |||
11 | N/A | failed | G12V | 2.6 | WT b | |||
12 | N/A | WT | WT | WT | ||||
13 | N/A | mut | G12V | 41.9 | mut | |||
14 | 126 | mut | failed | G12D | 65.7 | mut | ||
15 | 133 | mut | G12D | 74.1 | mut | |||
16 | 97 | mut | G13D | 52.8 | mut | |||
17 | 47 | failed | WT | WT | WT | |||
18 | 14 | failed | WT | WT | WT | |||
19 | 44 | WT a | WT | WT | ||||
20 | 20 | failed | WT a | WT | WT | |||
21 | N/A | failed | WT | WT | ||||
22 | 138 | mut | G12V | 12.9 | mut | |||
23 | 325 | mut | G12D | 56.1 | mut | |||
24 | 140 | WT a | WT | WT | ||||
25 | 7 | mut | G12C | 61.2 | mut | |||
26 | 4 | WT a | WT | WT | ||||
27 | 27 | WT a | WT | WT | ||||
28 | 13 | WT a | WT | WT | ||||
29 | 207 | mut | G12D | 54.5 | mut | |||
30 | 10 | failed | WT | G12V | 1.5 | WT b | ||
31 | 54 | WT a | WT | WT | ||||
32 | 120 | mut | G12V | 29.2 | mut | |||
33 | 33 | failed | mut | G12C | 33.5 | mut | ||
34 | N/A | mut | G12C | 71.4 | mut | |||
35 | N/A | WT | WT | WT | ||||
36 | N/A | failed | WT | WT | ||||
37 | 67 | WT a | WT | WT | ||||
38 | 137 | mut | G12C | 76.7 | mut | |||
39 | 63 | mut | G12A | 56 | mut | |||
40 | 13 | WT a | WT | WT | ||||
41 | 113 | WT a | WT | WT | ||||
42 | 82 | mut | G12C | 75.4 | mut | |||
43 | 39 | WT | WT | WT | ||||
44 | 7 | WT a | G12S | 2 | WT b | |||
45 | 23 | WT a | WT | WT | ||||
46 | 24 | faileda | G13D | 3.5 | WT b | |||
47 | 9 | WT a | WT | WT | ||||
48 | 34 | WT a | G12S | 2 | WT b | |||
49 | 25 | WT a | WT | WT | ||||
50 | 17 | WT | WT | WT | ||||
51 | 5 | WT a | WT | WT | ||||
52 | 29 | WT a | WT | WT | ||||
53 | 31 | mut | G12D | 71.3 | mut | |||
54 | 68 | WT | WT | WT | ||||
55 | 93 | mut | G12D | 74.2 | mut | |||
56 | 221 | WT | WT | WT | ||||
57 | 83 | WT | WT | WT | ||||
58 | N/A | WT a | G12V | 1.2 | WT b | |||
59 | 35 | mut | G12D | 83.3 | mut | |||
60 | N/A | WT | WT | WT | ||||
61 | 37 | WT a | WT | WT |
Run 1 | Run 2 | Summary | ||||
---|---|---|---|---|---|---|
Summary of Results | ||||||
HRM | n | % | n | % | n | % |
Number of samples | 61 | 100.0 | 7 | 100.0 (11.5) | 61 | 100.0 |
Analysis passed | 50 | 82.0 | 7 | 100.0 | 57 | 93.4 |
WT (total) | 32 | 64.0 | 6 | 85.7 | 38 | 66.7 |
WT (skewed HRM curve) | 24 | 75.0 | 2 | 33.3 | 26 | 68.4 |
Mutant (total) | 18 | 36.0 | 1 | 14.3 | 19 | 33.3 |
Mutant (skewed HRM curve) | 0 | 0 | 0 | |||
Analysis failed | 11 | 18.0 | 0 | |||
Pyrosequencing | n | % | n | % | n | % |
Number of samples | 61 | 100.0 | 1 | 100.0 (1.6) | 61 | 100.0 |
Analysis passed | 60 | 98.4 | 1 | 100.0 | 61 | 100.0 |
WT (total) | 41 | 68.3 | 0 | 41 | 67.2 | |
WT (call: WT) | 35 | 58.3 | 35 | |||
WT (call: potential low level mutation) | 6 | 10.0 | 0 | 6 | ||
Mutant | 19 | 31.7 | 1 | 100.0 | 20 | 32.8 |
Analysis failed | 1 | 1.6 | 0 | |||
Concordance of Results | HRM | Pyrosequencing | ||||
n | % | n | % | |||
Number of samples | 57 | 100 | 57 | 100 | ||
WT (total) | 38 | 66.7 | 37 | 64.9 | ||
WT (call: WT) | 33 | 57.9 | ||||
WT (call: potential low level mutation) | 4 | 7.0 | ||||
Mutant | 19 | 33.3 | 20 | 35.1 | ||
Concordant | 56 | 98.2 | ||||
Discordant | 1 | 1.8 | ||||
Correctly classified WT | 37 | 97.4 | ||||
Incorrectly classified WT | 1 | 2.6 | ||||
Correctly classified mutant | 19 | 100 | ||||
Incorrectly classified mutant | 0 | 0 |
Concordance of HRM and pyrosequencing results
Two-step KRAS mutation testing in routine diagnostics
HRM | Pyrosequencing | |||||||
---|---|---|---|---|---|---|---|---|
Run 1 | Run 2 | |||||||
Sample | cDNA [ng/μl] | Run 1 | Run 2 | Result | % mut. Alleles | Result | % mut. Alleles | Final result |
62 | 541 | WT a | WT | WT | ||||
63 | 29 | WT a | WT | WT | ||||
64 | 378 | WT | WT | |||||
65 | 342 | WT a | WT | WT | ||||
66 | 176 | failed | WT a | G12V | 3.2 | G12V | 5.2 | WT |
67 | 139 | mut | mut | |||||
68 | 520 | WT | WT | |||||
69 | 42 | WT | WT | |||||
70 | 202 | WT | WT | |||||
71 | 157 | mut | mut | |||||
72 | 259 | mut | mut | |||||
73 | 145 | WT | WT | |||||
74 | 21 | failed | failed | WT | WT | |||
75 | 22 | failed | WT | WT | ||||
76 | 66 | mut | mut | |||||
77 | 55 | mut | mut | |||||
78 | 199 | mut | mut | |||||
79 | 197 | WT | WT | |||||
80 | 171 | WT | WT | |||||
81 | 55 | failed | G12V | 41.1 | mut | |||
82 | 231 | failed | WT | WT | ||||
83 | 250 | failed | WT | WT | ||||
84 | 57 | failed | WT a | WT | ||||
85 | 21 | mut | mut | |||||
86 | 248 | WT | WT | |||||
87 | 258 | WT | WT | |||||
88 | 83 | failed | failed | failed | N/A | |||
89 | 38 | failed | WT | WT | ||||
90 | 279 | WT | WT | |||||
91 | 122 | failed | WT | WT | ||||
92 | 129 | failed | Low Mut. | 5.2 | Low Mut. | 8.2 | mut | |
93 | 58 | WT | WT | |||||
94 | 129 | WT | WT | |||||
95 | 254 | WT | WT | |||||
96 | 373 | WT | WT | |||||
97 | 158 | WT | WT | |||||
98 | 96 | mut | mut | |||||
99 | 22 | WT | WT | |||||
100 | 30 | mut | mut | |||||
101 | 26 | muta | muta | G13D | 7.3 | mut | ||
102 | 49 | WT | WT | |||||
103 | 47 | WT | WT | |||||
104 | 43 | WT | WT | |||||
105 | 54 | WT | WT | |||||
106 | 363 | failed | mut | mut | ||||
107 | 521 | failed | WT | WT | ||||
108 | 199 | mut | mut | |||||
109 | 260 | WT | WT | |||||
110 | 67 | WT | WT | |||||
111 | 103 | WT | WT | |||||
112 | 24 | mut | mut | |||||
113 | 150 | WT | WT | |||||
114 | 5 | WT | WT | |||||
115 | 25 | WT | WT | |||||
116 | 33 | WT | WT | |||||
117 | 26 | mut | mut | |||||
118 | 72 | WT | WT | |||||
119 | 16 | failed | G12V | 12.8 | mut | |||
120 | 33 | failed | WT | WT | ||||
121 | 48 | WT | WT | |||||
122 | 74 | WT | WT | |||||
123 | 474 | mut | mut | |||||
124 | 431 | mut | mut | |||||
125 | 66 | mut | mut | |||||
126 | 143 | mut | mut | |||||
127 | 49 | failed | WT | WT | ||||
128 | 143 | WT | WT | |||||
129 | 122 | mut | mut | |||||
130 | 139 | WT | WT | |||||
131 | 21 | failed | WT | WT | ||||
132 | 39 | failed | WT | WT | ||||
133 | 128 | failed | G12D | 26.7 | mut | |||
134 | 60 | mut | mut | |||||
135 | 330 | failed | mut | mut | ||||
136 | 165 | mut | mut | |||||
137 | 213 | mut | mut | |||||
138 | 31 | failed | mut | mut | ||||
139 | 156 | mut | mut | |||||
140 | 59 | WT | WT | |||||
141 | 68 | WT | WT | |||||
142 | 164 | WT | WT | |||||
143 | 238 | mut | mut | |||||
144 | 12 | mut | mut | |||||
145 | 33 | failed | WT | WT | ||||
146 | 81 | WT | WT | |||||
147 | 11 | WT | WT | |||||
148 | 13 | mut | mut | |||||
149 | 71 | WT | WT | |||||
150 | 11 | mut | mut | |||||
151 | 40 | failed | WT | WT | ||||
152 | 50 | WT | WT | |||||
153 | 128 | mut | mut | |||||
154 | 146 | WT | WT | |||||
155 | 69 | WT | WT | |||||
156 | 182 | WT | WT | |||||
157 | 32 | failed | WT | WT | ||||
158 | 142 | WT | WT | |||||
159 | 53 | WT | WT | |||||
160 | 91 | failed | WT | WT | ||||
161 | 334 | WT | WT | |||||
162 | 86 | failed | WT | WT | ||||
163 | 61 | WT | WT | |||||
164 | 64 | WT | WT | |||||
165 | 141 | WT | WT | |||||
166 | 271 | WT | WT | |||||
167 | 40 | WT | WT | |||||
168 | 34 | failed | WT | WT | ||||
169 | 29 | failed | failed | WT | WT | |||
170 | 354 | WT | WT | |||||
171 | 66 | WT | WT | |||||
172 | 43 | failed | WT | WT | ||||
173 | 114 | WT | WT | |||||
174 | 268 | WT | WT | |||||
175 | 107 | WT | WT | |||||
176 | 170 | mut | mut | |||||
177 | 65 | failed | mut | mut | ||||
178 | 31 | failed | mut | mut | ||||
179 | 61 | WT | WT | |||||
180 | 659 | mut | mut | |||||
181 | 34 | WT | WT |
Run 1 | Run 2 | Summary | ||||
---|---|---|---|---|---|---|
HRM | n | % | n | % | n | % |
Number of samples | 120 | 100.0 | 20 | 100.0 (16.7) | 120 | 100.0 |
Analysis passed | 89 | 74.2 | 18 | 90.0 | 105 | 87.5 |
WT (total) | 60 | 67.4 | 12 | 66.7 | 71 | 67.6 |
WT (skewed HRM curve) | 3 | 5.0 | 2 | 16.7 | 4 | 5.6 |
Mutant (total) | 29 | 32.6 | 6 | 33.3 | 34 | 32.4 |
Mutant (skewed HRM curve) | 1 | 3.4 | 1 | 16.7 | 1 | 2.9 |
Analysis failed | 31 | 25.8 | 2 | 10.0 | ||
Pyrosequencing | n | % | n | % | n | % |
Number of samples | 19 | 100.0 (15.8) | 3 | 100.0 (2.5) | 19 | 100.0 |
Analysis passed | 18 | 94.7 | 2 | 66.7 | 18 | 94.7 |
WT | 12 | 66.7 | 0 | 0.0 | 13 | 72.2 |
Potential low level mutation | 2 | 11.1 | 1 | 50.0 | 0 | 0.0 |
Mutant | 4 | 22.2 | 1 | 50.0 | 5 | 27.8 |
Analysis failed | 1 | 5.3 | 1 | 33.3 | 1 | |
Combined HRM + Pyrosequencing | n | % | ||||
Number of samples | 120 | 100.0 | ||||
Number of HRM runs | 140 | 116.7 | ||||
Number of pyrosequencing runs | 22 | 18.3 | ||||
Analysis passed | 119 | 99.2 | ||||
WT | 81 | 68.1 | ||||
Mutant | 38 | 31.9 | ||||
Analysis failed | 1 | 0.8 |
Assay costs
HRM | Pyrosequencing | |
---|---|---|
Costs (Euro) | ||
Reagents | 3.40 | 90.00 |
Consumables | 2.40 | 3.50 |
Controls | 1.70 | 8.50 |
Total | 7.50 | 102.00 |
Time (minutes) | ||
14 samples + 4 controls | 60 | |
22 samples + 2 controls | 120 |