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Erschienen in: BMC Cancer 1/2016

Open Access 01.12.2016 | Research article

A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing

verfasst von: Elisabeth Mack, Kathleen Stabla, Jorge Riera-Knorrenschild, Roland Moll, Andreas Neubauer, Cornelia Brendel

Erschienen in: BMC Cancer | Ausgabe 1/2016

Abstract

Background

KRAS mutation testing is mandatory in the management of metastatic colorectal cancer prior to treatment with anti-EGFR antibodies as patients whose tumors express mutant KRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved two in-vitro diagnostics kits for determination of KRAS status, there is generally no consensus on the preferred method and new tests are continuously being developed. Most of these techniques focus on the hotspot mutations at codons 12 and 13 of the KRAS gene.

Methods

We describe a two-step approach to KRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using the Therascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified as KRAS wildtype or mutant by HRM. First, we determined KRAS status in a panel of 61 colorectal cancer samples using both methods to compare technical performance and concordance of results. Subsequently, we evaluated practicability and costs of our concept in an independent set of 120 colorectal cancer samples in a routine diagnostic setting.

Results

HRM and pyrosequencing appeared to be equally sensitive, allowing for clear detection of mutant alleles at a mutant allele frequency ≥12.5 %. Pyrosequencing yielded more exploitable results due to lower input requirements and a lower rate of analysis failures. KRAS codon 12/13 status was called concordantly for 98.2 % (56/57) of all samples that could be successfully analysed by both methods and 100 % (19/19) of samples that were identified mutant by HRM. Reviewing the actual effort and expenses for KRAS mutation testing in our laboratory revealed, that the selective use of pyrosequencing for only those samples that could not be analysed by HRM increased the fraction of valid results from 87.5 % for HRM alone to 99.2 % (119/120) while allowing for a net reduction of operational costs of >75 % compared to pyrosequencing alone.

Conclusions

Combination of HRM and pyrosequencing in a two-step diagnostic procedure constitutes a reliable and economic analysis platform for KRAS mutation testing in colorectal cancer in a clinical setting.

Background

The anti EGFR-antibodies cetuximab and panitumumab represent well-established treatments for metastatic colorectal cancer (CRC), the third most prevalent cancer entity and fourth most common cause of cancer-related death around the world [1, 2]. Several studies have shown KRAS status to predict outcome under these anti-EGFR targeting agents, with beneficial effects being seen only in patients whose tumors express wildtype (WT) KRAS [38]. Thus, testing for KRAS mutations, which are found in approximately 40 % of colorectal cancers, has become routine in the management of metastatic CRC (mCRC) prior to cetuximab or panitumumab treatment [9, 10] and is even required by the responsible regulatory agencies. Notably, current standards regarding oncogenic Ras mutation analysis in mCRC issued by the U.S Food and Drug Administration (FDA) require determination of KRAS status by an FDA-approved test, while the European Medical Agency (EMA) just states application of validated methods by an experienced laboratory [1115]. Currently available FDA-approved companion diagnostic devices for cetuximab (Erbitux) and panitumumab (Vectibix) comprise the Cobas KRAS Mutation Test (Roche) and Therascreen KRAS RGQ PCR Kit (Qiagen) [16]. Besides these and other commercially available kits, the spectrum of methods for KRAS mutation testing encompasses multiple PCR-derived and sequencing-based techniques. Of note, most of the previously established assays for KRAS mutation detection focus on the hotspot mutations involving codons 12 and 13, which account for >95 % of Ras mutations in CRC [10]. The advantages and limitations of selected methods have been repeatedly evaluated comparatively [1722], however, beyond the FDA-guideline, there is no consensus on the preferred approach to investigate KRAS status in routine molecular pathological diagnostics [23]. Given the high incidence of CRC resulting in high demand for KRAS mutation testing, an ideal diagnostic assay for this purpose not only needs to be sufficiently sensitive and specific, but, for socio-economic reasons, also should be time- and cost-effective. Therefore, we developed a two-step procedure for KRAS mutation testing including high resolution melting analysis (HRM) followed by pyrosequencing of only those samples that are not clearly identified as KRAS WT or mutant by HRM. HRM is a one-tube qPCR-based technique for DNA-variant detection. The method utilizes alterations in the melting behavior of double-stranded DNA fragments that are conferred by nucleotide exchanges. Melting of qPCR amplicons is monitored in real time using a suitable qPCR instrument capable of time-dense data aquisition and a saturating DNA-intercalating fluorescent dye that does not redistribute during the melting step [24]. Pyrosequencing is a sequencing-by-synthesis approach that involves sequential addition of dNTPs and recording incorporation of a nucleotide based on a light signal that is generated by sulfurylase-catalyzed conversion of the released pyrophosphate to ATP and a subsequent luciferase reaction [25]. Here, we applied a previously described HRM-assay [20] and the Therascreen KRAS Pyro kit (Qiagen) for detection of KRAS codon 12/13 mutations. First we comparatively analysed KRAS status in a panel of 61 colon cancer samples to determine sensitivity, specificity, technical performance and concordance of results of the two methods. Subsequently, we evaluated our two-step approach in the routine setting of our molecular diagnostics laboratory. In summary, we present a reliable, time- and cost-effective operational concept for KRAS mutation testing prior to anti-EGFR antibody treatment in mCRC.

Methods

Tumor samples, control cell lines and DNA isolation

The colorectal cancer samples reported on in this study were obtained from patients with metastatic colorectal cancer (UICC IV) at the University Hospital Marburg, Germany and analysed in a routine diagnostic setting. Tissue samples were fixed, paraffin-embedded, sectioned, hematoxylin-eosin stained and deparaffinated using standard procedures. Tissue sections were reviewed by an experienced pathologist (RM) to establish the diagnosis and to mark regions for microdissections. Microdissection of tumor cells was performed from deparaffinated sections using a scalpel. DNA was isolated from microdissected samples using the QiaAmp DNA Mini kit (Qiagen) as recommended by the manufacturer. KRAS mutant cell lines PL45 (pancreatic adenocarcinoma) and RPMI 8226 (multiple myeloma) were obtained from ATCC and cultured according to standard cell culture methods. Positive control DNA for HRM analyses was isolated from these cell lines using the QiaAmp DNA Mini kit. WT control DNA was extracted from peripheral blood of healthy donors from whom informed consent had been obtained (WT control) with the QiaAmp DNA Mini kit. DNA concentrations were measured using a Nanodrop 1000 spectrophotometer (Peqlab).

High resolution melting analysis

For HRM analysis, a 92 bp amplicon spanning exons 2 and 3 of the KRAS gene was amplified from 60 ng (or less) of sample DNA using the primers KRAS-92_F 5′-ttataaggcctgctgaaaatgactgaa-3′ and KRAS-92_R 5′-tgaattagctgtatcgtcaaggcact-3′ [20], the DNA-intercalating dye SYTO 9 (Thermo) in a final concentration of 5 μM and Platinum Taq polymerase (Thermo). Amplification and melting analysis was performed on a Rotor Gene 6000 instrument (Corbett Life Sciences) under the following temperature conditions: one cycle 95 °C/2 min, 40 cycles 95 °C/15 sec – 67.5 °C/15 sec - 72 °C/15 sec, one cycle 95 °C/1 sec, pre-melt conditioning at 72 °C/90 sec, HRM-ramp from 72 °C to 95 °C rising at 0.2 °C per step/wait 2 sec each step. Controls in each HRM run included a no-template-control, a WT control (gDNA from healthy donor) and two mutation controls (gDNA from the cell lines RPMI 8226, KRAS codon 12 GGT → GCT/heterozygous, corresponding to G12A and PL45, KRAS codon 12 GGT → GAT/heterozygous, corresponding to G12D). All HRM assays were performed in quadruplicate.

Pyrosequencing

Pyrosequencing of the KRAS codon 12/13 region was performed using the Therascreen KRAS Pyro Kit (Qiagen) as recommended by the manufacturer. 2 ng of DNA were used per analysis. PCR amplification of the target region was performed on a T-100 thermocycler (Biorad). For the pyrosequencing reaction on the PyroMark Q24 platform (Qiagen), amplicons were immobilized to the wells of a PyroMark Q24 plate using streptavidin high performance beads (GE Healthcare). Pyrosequencing results were analysed using the PyroMark Q24 software version 2.0 with the Therascreen KRAS Pyro-plugin report, which already incorporated the thresholds for mutation calls (detection limit for the mutation (LOD) + 3 %).

Statistical analysis

HRM and pyrosequencing results were compared by contingency table analysis test using GraphPad Prism 5 software (GraphPad). Technical performance (1st run success vs. 1st run failure) was evaluated by two-sided Fisher’s exact test at a significance level of 5 %. The agreement between HRM and pyrosequencing results was quantified by kappa using the appropriate Graphpad Prism online calculator (http://​graphpad.​com/​quickcalcs/​kappa2).

Results

Sensitivity of HRM and pyrosequencing

In order to test whether pyrosequencing allows for KRAS mutation detection with at least equal sensitivity compared to HRM, we analysed serial dilutions of DNA from a KRAS mutant cell line (PL45, codon 12 GGT → GAT heterozygous mutation) in WT DNA by both HRM and pyrosequencing. For HRM, we found, that the presence of KRAS mutant DNA in the sample was clearly reflected by a shifted or skewed melting curve for a fraction of PL45-DNA exceeding 25 %, which corresponded to a mutant allele frequency of 12.5 % (Fig. 1). Similarly, pyrosequencing definitely yielded a mutation if the sample contained ≥25 % PL45-DNA. On the other hand, samples with 5–10 % cell line DNA were indicated to exhibit a potential low level mutation as the mutant allele frequency was quantified below the threshold for accurate WT/mutant discrimination for the G12D mutation (LOD + 3 %; LOD = 2,2 %) for both the 5 and 10 % samples (Fig. 2). Thus, HRM and pyrosequencing appeared to be equally sensitive methods for the detection of KRAS codon 12/13 mutations.

Technical reliability of HRM and pyrosequencing

To further assess the suitability of pyrosequencing to serve as a backup-assay allowing for accurate diagnosis of KRAS mutation status in case of failed HRM analysis, we investigated KRAS status of 61 colorectal cancer samples by both HRM and pyrosequencing and compared the two methods with regard to their technical performance and concordance of results. In a first run of HRM analysis, 11/61 samples (18.0 %) could not be analysed due to PCR-failures or ambiguous melting curves (Table 1). Repetition of the assay for seven samples, which most likely had been compromised technically, allowed for assigning KRAS mutation status in all cases. The remaining four samples were directly subjected to pyrosequencing without a second round of HRM analysis. Indeed, KRAS status could each be determined, although one sample yielded a potential low level mutation. Of the 57 samples that could be definitely classified as KRAS WT or mutant by HRM, 26 WT samples (45.6 % of all samples/68.4 % of WT samples) yielded skewed HRM curves, which, however, did not prevent establishment of a diagnosis (Tables 1 and 2). Moreover, we noted that low DNA content of the samples below the detection limit of the Nanodrop spectrophotometer not necessarily prevented successful HRM analysis. In contrast to HRM, the pyrosequencing assay had to be repeated for only one sample (Table 1). Thus, the failure rate of a first analysis run as a consequence of technical and/or sample-issues was significantly higher for HRM analysis than for pyrosequencing (p = 0.0042). Together, pyrosequencing is technically more reliable than HRM due to lower input requirements and a lower incidence of invalid results.
Table 1
KRAS codon 12/13 status by HRM and pyrosequencing in 61 CRC samples
  
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
aSkewed HRM curve
bLOD/threshold for potential low level mutation (cf. Therascreen KRAS Pyro Kit handbook version 1, July 2011): G12D 2.2 %/5.2 %, G12V 1.0 %/4.0 %, G12C 2.1 %/5.1 %, G12S 1.9 %/4.9 %, G13D 1.9 %/4.9 %
Table 2
Comparison of HRM and pyrosequencing results in 61 CRC samples
 
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

In order to evaluate the diagnostic validity of HRM analysis as a basic test for KRAS mutation detection, we compared the results from this assay to pyrosequencing in the 57 samples that could be successfully analysed by both methods. KRAS status was assigned concordantly for 56 samples (98.2 %; kappa = 0.961), while the result for one sample with a low mutant allele frequency of 13.4 % (#3, Table 1) was inconsistent between HRM and pyrosequencing (Tables 1 and 2). Importantly, pyrosequencing indicated the presence of potential low level mutations (mutant allele frequency < 4.0–5.2 %, cf. Table 1) in four samples that were called WT by HRM. Given that this output is generated due to low signal strength for the potential mutation near the technical detection limit of the pyrosequencing method we finally classified these samples as WT. Conversely, all 19 samples that were clearly identified as mutant by HRM were classified identically by pyrosequencing. Therefore, defining pyrosequencing as the reference method, the specificity of HRM for detection of mutant KRAS alleles was 100 %. On the other hand, the specificity for the detection of WT alleles was slightly reduced (97.4 %) due to erroneous interpretation of the HRM curve for the one sample mentioned (#3, Table 1) with a mutant allele frequency only slightly above the sensitivity threshold of the method. When we applied a different HRM assay for the detection of NRAS codon 61 mutations on an independent set of 19 CRC samples, we found a 100 % concordance of results with reports from a reference laboratory (Additional file 1: Table S1). Of note, sensitivity of the NRAS HRM assay was comparable to the KRAS assay and allowed for reliable identification of mutations at a mutant sample fraction of 20 % (Additional file 2: Figure S1). Taken together, these findings indicate that HRM represents a very reliable basic method for KRAS mutation testing.

Two-step KRAS mutation testing in routine diagnostics

To evaluate the actual effectiveness of our two-step analysis platform (Fig. 3) in a routine diagnostic setting, we reviewed effort and outcome of KRAS codon 12/13 mutation testing in 120 independent colorectal cancer samples that were examined consecutively in our laboratory according to this concept (Table 3). We found, that KRAS status could be determined for 87.5 % of samples by HRM and for 99.2 % of samples in total, when pyrosequencing was applied to samples that could not be successfully analysed by HRM (Table 4). However, for both HRM and pyrosequencing, the failure rate was slightly higher than anticipated based on the observations from our initial 61 sample set (Tables 2 and 4). Also of note, the number of samples that were subjected to pyrosequencing in routine diagnostics exceeded the previously estimated need of this analysis (19/120 = 15.5 % vs. 4/61 = 6.6 %) because 15 samples were directly analysed by pyrosequencing after the first failed HRM run in order to utilize otherwise wasted capacities. Yet, in summary, these data strongly support the rationale of our two-step approach to KRAS codon 12/13 mutation analysis, confirming the accuracy of our diagnostic platform.
Table 3
Detailed results of KRAS codon 12/13 mutation testing in 120 CRC samples
  
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
aSkewed HRM curve
Table 4
Operational analysis of two-step KRAS mutation testing of 120 CRC samples
 
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

In order to estimate the economic benefits of our two-step approach to KRAS mutation testing, we compared analysis costs in our routine setting to a pyrosequencing-only platform. Based on current list prices for reagents and consumables, we estimated the assay costs for HRM analysis and pyrosequencing at approximately € 7.50 and € 100, respectively (Table 5). The costs for the essential technical devices for both methods have not been converted to per-sample costs because operation expenses are highly dependent on sample throughput, including not only the KRAS mutation assay but also other applications. Moreover, investments for technical equipment are in the same range for pyrosequencing and HRM. Considering the failure rates of each assay in our set of 120 routine samples (23.6 % for HRM and 9.1 % for pyrosequencing), leading to repeated testing of some samples, our two-step approach allows for net reduction of operational costs of approximately 75 % compared to pyrosequencing alone. Moreover, according to our experience, hands-on time for processing the maximum number of samples for one HRM-run (14 + 4 controls) is only half of the time required to prepare and perform a pyrosequencing run at full capacity (22 + 2 controls) (Table 5). Therefore, our concept to maintain two sequential assays for KRAS codon 12/13 mutation testing represents cost- and time-effective approach for routine diagnostics.
Table 5
Per-sample costs and hands-on time for HRM and pyrosequencing analyses
 
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
Costs for the controls were estimated based on the maximum number of samples that can be processed in one HRM- or pyrosequencing run, respectively. Costs for HRM controls also include DNA isolation from KRAS WT and mutant cell lines. Hands-on time is indicated for full capacity runs

Discussion

Here we present a two-step approach to KRAS codon 12/13 mutation testing for mCRC employing HRM analysis and pyrosequencing using the Therascreeen KRAS Pyro Kit. Comparing the performance of the two methods in a panel of 61 samples, we observed a 98.2 % concordance of results with a 100 % specificity of HRM for the detection of mutant alleles. Thus, HRM analysis needs methodically independent confirmation of results by pyrosequencing only in exceptional cases and therefore can serve as a filter assay to exclude clearly WT or mutant samples from the more expensive and more laborious pyrosequencing analysis. Specifically, based on our observations reported here, this approach can reduce throughput of the pyrosequencing assay by >85 %, resulting in a >75 % cost reduction compared to using pyrosequencing only. We emphasize, that our comparison of the two methods in the first place aimed on diagnostic accuracy for sequential application in order to establish a reliable and economized platform for KRAS mutation testing. Of note, we reached this goal in spite we were able to detect mutant KRAS alleles only at a frequency >12.5 % instead of 5 % as reported in the literature [20, 26].
With respect to technical performance, although we successfully applied HRM to very low input samples, we state a clear advantage for the pyrosequencing assay due to lower input requirements and an apparently relatively high susceptibility of HRM to artifacts. More precisely, previous authors have pointed out, that especially WT HRM curves show a certain degree of variation resulting from poor quality of FFPE-derived template DNA, differing salt- or inhibitor concentrations or unspecific amplification [20, 27], that may complicate correct determination of KRAS status. Consistent with this notion, 6 of the 7 samples in our 61-sample validation set that were subjected to a second round of HRM analysis due to poor interpretability of first round results were eventually called WT by this method. Conversely, we did not obtain false positive results by HRM, i.e., none of our samples that had been identified as mutant by HRM was found to be WT according to pyrosequencing. Yet, we state that the mutation frequency of KRAS codon 12/13 observed in our study was slightly lower than reported in the literature [9, 10], which may be explained by our homogenous patient population from a single center (Marburg, Germany).
Concerning diagnostic value of results from our sequential KRAS mutation analysis procedure, it is important to point out that pyrosequencing results include information on the site, type and frequency of the nucleotide exchange, while HRM only allows for categorical discrimination of WT and mutant tumors. According to current standards, such a dual output is actually sufficient to establish the indication for anti-EGFR treatment, although certain authors have suggested that not all KRAS mutations are equal regarding outcome in mCRC patients treated with cetuximab [3]. Consequently, as clinical routine testing at present in principle does not require sequence-based analysis, the more differentiated output of the pyrosequencing assay does not warrant the higher costs for this analysis. Therefore, the two-step procedure for KRAS mutation testing presented here represents a reasonable diagnostic approach not only from a technical-practical and economical, but also from a clinical perspective. More specifically, using our diagnostic platform focused on KRAS codon 12/13 mutation testing, even small diagnostic laboratories can provide accurate and clinically meaningful results within a short processing time for the most relevant genetic alteration that determines a treatment decision for mCRC patients. Consequently, only a small fraction of patient samples has to be sent to an external reference laboratory for further molecular studies in accordance with the current EMA standards and recommendations by the American Society of Clinical Oncology, which state that Ras mutation testing prior to initiation of treatment with cetuximab and panitumumab has to include analysis of both KRAS and NRAS exons 2, 3 and 4 (codons 12, 13, 59, 61, 117 and 146). Also of note, besides KRAS and NRAS mutations, alterations in several other genes such as BRAF and PIK3CA have been proposed to predict outcome with EGFR antibody treatment [2830]. Thus, identification of patients eligible for cetuximab or panitumumab treatment in fact requires either a broad panel of single mutation tests or a multiplex approach. Optimized methods for DNA melting analysis of short PCR amplicons have been suggested to allow for comprehensive hot spot mutation testing in a clinical setting as they require only standard qPCR equipment. However, each assay requires careful optimization, implying considerable efforts for a diagnostic laboratory to set up all tests on site [31]. Alternatively, next generation sequencing (NGS) with a targeted resequencing approach appears to be a suitable technology for extensive clinically relevant mutation testing in the future, which has already been evaluated for the molecular diagnostics of colorectal cancer [32, 33]. Given the high frequency of KRAS codon 12/13 mutations compared to other KRAS- or NRAS mutations and the fact that these mutations occur mutually exclusive [10], it still seems reasonable, to filter the samples that actually need advanced testing method as proposed here. Thus, a two-step approach including HRM analysis of KRAS codon 12/13 mutations followed by next generation targeted resequencing might be the most attractive implementation for routine KRAS mutation diagnostics in the future.

Conclusion

We present a diagnostically reliable and cost-effective two-step approach to KRAS codon 12/13 mutation testing of CRC samples prior to initiation of treatment with anti-EGFR antibodies. The platform appears to be especially attractive for small to medium diagnostic laboratories that don’t have the capacities to maintain an extensive spectrum of rare mutation tests according to regulatory standards for diagnostic laboratories [34] or to adopt NGS-technology with its complex associated infrastructure including bioinformatics.

Abbreviations

CRC, colorectal cancer; EMA. European medical agency; FDA, U.S. food and drug administration; HRM, high resolution melting analysis; LOD, limit of detection; mCRC, metastatic colorectal cancer; NGS, next generation sequencing; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; WT, wildtype; UICC, Union internationale contre le cancer.

Acknowledgements

We like to thank Viktoria Wischmann, Department of Pathology, University Hospital Marburg for microdissection of CRC specimens. Petra Ross, Lisa-Marie Koch and the staff of the Molecular Biology Laboratory, Department of Hematology and Oncology are acknowledged for excellent technical assistance and helpful discussion.

Funding

This work was supported by DFG Klinische Forschergruppe KFO210 (AN, CB) and the José Carreras Leukemia Foundation (AH06-01, to AN).

Availability of data and materials

The dataset supporting the conclusions of this article is included within the article and its supplementary material.

Authors’ contributions

CB, EM and RM conceived and designed the study. RM reviewed tissue sections. KS established the method and performed KRAS mutation analysis. KS, CB and EM analysed data. AN and JRK contributed to the analysis and interpretation of the data. EM wrote the manuscript, which was critically revised by CB, AN, RM and JRK. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.
Not applicable.
This study was conducted in a routine diagnostic setting for internal quality control purposes and did not require formal ethics approval according to the guidelines of the local ethics comitee. Verbal informed consent to perform routine pathological examinations on their samples as needed was obtained from all patients.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://​creativecommons.​org/​licenses/​by/​4.​0/​), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.
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Metadaten
Titel
A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing
verfasst von
Elisabeth Mack
Kathleen Stabla
Jorge Riera-Knorrenschild
Roland Moll
Andreas Neubauer
Cornelia Brendel
Publikationsdatum
01.12.2016
Verlag
BioMed Central
Erschienen in
BMC Cancer / Ausgabe 1/2016
Elektronische ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-016-2589-2

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