Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples

Herein, we present a simple yet robust approach for the rapid molecular profiling of KRAS mutations in PDAC patients, which combines the amplification refractory mutation system followed by high-resolution melting analyses (ARMS-HRMA), for the detection of two of the most relevant KRAS mutations (G12V and G12D) in these patients. Because these two alterations are single-point missense mutations, we used an allele-specific amplification (ARMS) where the forward primers were designed to favor amplification of the target mutated allelic sequence to the detriment of the wt allele. Even though ARMS-primers provide for the allele specificity of amplification, mismatched priming at 3′ still allows DNA polymerase to yield residual amplification at a much lower efficiency (Supplementary Information S1 and S2). To warrant complete disambiguation of nucleobase call and correctly identify samples containing the mutated allele, HRMA was performed directly on the obtained ARMS products (closed tube). By doing this, a more intense fluorescence signal is retrieved when in the presence of the mutation that provided for correct priming in ARMS reaction, resulting in a higher efficiency of amplification and amplicon yield. Consequently, the ARMS-HRMA approach is based on the difference of the melting profile (melting bands and corresponding intensity) after ARMS reaction, when compared to control genotypes (mutated or wild type).

ARMS-HRMA assay development

First, we used DNA retrieved from cell lines with known genotypes SW480 (homozygous for G12V), LS174T (wt/G12D), and SW48 (wt/wt) to optimize the ARMS-HRMA approach. A critical aspect was the definition of the criteria for allele discrimination, dependent on the melting temperature and fluorescence peak intensity. Following ARMS amplification using the G12V forward primer, the melting profile analysis, HRMA, revealed the presence of a melting peak at approximately the same temperature for both SW480 and SW48 cell lines (80 °C), which indicates that the amplification occurred in both samples (Fig. 2A). However, the melting peaks are clearly distinguishable as they present variations in intensity (fluorescence signal), with the highest peak corresponding to the homozygous G12V cell line, indicating that the degenerated forward primer (allele specific against G12V mutation) is allowing the amplification of DNA containing the G12V allele at a much higher efficiency than the amplification of wt phenotype, yielding more amplification product, which translates to a higher intensity of the melting peak. Overall, this shows a successful identification of samples bearing the KRAS G12V mutation via the proposed ARMS-HRMA approach (Fig. 2A).

Fig. 2figure 2

HRMA derivative plot for KRAS mutation detection. (A1) Derivative plot generated after ARMS amplification for the detection of KRAS G12V and (B1) KRAS G12D mutations. (A2) Average intensity of the melting peak generated after ARMS-HRMA reaction for the detection of G12V and (B2) G12D mutations in G12V/G12V (SW480) (green line), wt/wt (SW48) (gray line), and G12D/wt (LS174T) (yellow line). ****P value < 0.0001 using Mann–Whitney test. Error bars represent the standard error mean of the average result of at least 8 replicates

We then evaluated the potential of ARMS-HRMA to detect the KRAS G12D mutation (GGT > GAT), using a G12D-specific primer. This primer differs from that specific for the G12V mutation in the 3′ nucleotide, which is allele specific. As for the G12V mutation, the detection of G12D was optimized using genomic DNA from cell line SW48 (wt/wt) as wild-type control and LS174T (wt/G12D) as G12D positive control. The derivative plot in Fig. 2B shows two distinct melting peaks, one around 75.5 °C, independent of the sample genotype, and a second around 79.5 °C, exclusive for the G12D mutant DNA (Fig. 2B). Overall, using ARMS-HRMA, it was possible to attain distinct melting profiles that correlate to the distinct genotypes, allowing for correct scoring of the wt/G12D genotype due to the presence of a melting peak at about 79.5 °C.

Our concept relies on a simple and rapid approach to detect the presence of KRAS G12D and/or G12V mutations directly in samples, and not to genotype, i.e., to provide a quick response of “the mutation is present” rather than to characterize the sample as wild-type (wt) homozygous or wt heterozygous. Nevertheless, a wt primer was designed to amplify the wt allele (Figure S2 panel D). SW48 (wt/wt) and LS174T (G12D/wt) cell lines were also used to validate these wt primers. The results show a higher peak intensity of the melting peak (~ 78.5 °C) for the SW48 cell line (wt/wt) (gray line in Figure S2 panel C). Furthermore, for the LS174T cell line (G12D/wt), a melting peak also appears in the same melting temperature but with a much lower intensity (yellow line in Figure S2 panel C), as expected due to the heterozygous state of the cell sample. Finally, the specificity for the wt allele can be observed with the homozygotic mutated cell line, SW480 (G12V/G12V), where the correspondent melting peak (~ 78.5 °C) does not appear (blue line in Figure S2 panel C).

ARMS-HRMA application to tumor DNA

The successful mutation identification via the ARMS-HRMA scheme, with sensitivity to differentiate between a homozygous wild-type sample from a homozygous or heterozygous mutated sample, was then applied to the analysis of DNA extracted from tumor biopsies and matching plasma samples. ARMS-HRMA was performed in a total of 30 tumor samples from patients diagnosed with PDAC. All the DNA samples with sufficient material were also analyzed by SS and ddPCR. Due to the small amount of patient material available from biopsy, samples P3, P10, P13, P15, P17, and P19 were not assessed by SS (Supplementary Information S3), and samples T9, T10, T13, T24, P1, P10, P15, and P19 were not assessed by ddPCR (Supplementary Information S4).

A first observation was that the fluorescence intensity signals showed some variation between samples, which may be attributed to different efficiencies in amplification and, thus, of the amount of amplified product that is then available for the HRMA step. Therefore, the melting plot of the SW48 cell line (negative for G12V and G12D mutations) was used as reference and, consequently, its melting peak as the threshold. Applying this criterion, the analyzed samples can be divided into two groups: mutated for G12V (G12V positive), which continues to exhibit a melting peak with high fluorescence signal, and not mutated for G12V (G12V negative), showing a melting curve below or equal to the reference line—Fig. 3. To illustrate this issue, a set of four tumor samples (T1–T4) are shown, all analyzed by ARMS-HRMA for presence of the G12V mutation. Figure 3A shows that all samples present a melting temperature consistent with that observed for SW480 or SW48 cell lines, G12V/G12V and wt/wt, respectively. Samples T1 and T3 may be easily scored as mutated for G12V, since a higher melting peak is present, like that of the SW480 cell line, and T2 and T4 samples are not mutated for G12V, due to the low-intensity melting peak (like that of SW48) (Fig. 3B). These results were corroborated by SS, in which the KRAS G12V mutation was present in T1 and T3 and not detected for T2 (G12D mutation) and T4 (wt) samples (Fig. 3C).

Fig. 3figure 3

ARMS-HRMA for the detection of KRAS G12V mutation. A HRMA derivative plot for KRAS G12V mutation analysis for the tumor samples T1 (blue green line), T2 (yellow line), T3 (blue line), and T4 (dark gray line). Cell lines SW480 (turquoise line) and SW48 (gray line) as G12V positive and negative controls, respectively. B Difference plot. The difference plot shows the melting curve of each tumor sample subtracted from the SW48 cell line. C Chromograms for SS results for KRAS codon 12 (wt: GGT). Sequence obtained with forward primer. Arrows indicate the mutations at positions 2 of codon 12 of KRAS. Sanger sequencing showed a G to T transversion at position 2 of codon 2 (G12V: GGT > GTT) in tumors T1 and T3, and a G to A transition (G12D: GGT > GAT) in tumor T2. T4 has a wt KRAS codon 12

Figure 4 shows the output of the ARMS-HRMA approach for G12V scoring in all 30 tumor samples. As shown, all tumor samples positive for G12V mutation by ARMS-HRMA also scored as G12V by ddPCR indicating a concordance of 100%, and only one tumor was not scored as G12V via direct SS (T12 in Fig. 4 and sample 12 in Table 1). It should be mentioned that the described reactions were performed in quadruplicate with the same result, which highlights the high reproducibility of the ARMS-HRMA methodology.

Fig. 4figure 4

ARMS-HRMA for the detection of KRAS G12V mutation in tumor samples. A G12V mutation scoring for the set of 30 tumor samples, based on the intensity of the melting peak at 79.5 °C. The dark blue bars represent tumor samples scored as G12V non-mutated. B Statistical analysis of the average intensity of the melting peak in each sample group. Four asterisks, P value < 0.0001 using Mann–Whitney test. The blue green bar represents tumor samples scored as G12V mutated; the light gray bar represents the SW48 cell line as the control for G12V non-mutated samples; the turquoise bar represents the SW480 cell line as the control for G12V mutated samples. The dagger represents samples scored as G12V mutated with non-concordant result based on SS. Error bars represent the standard error mean of the average result of at least 4 replicates

Table 1 Results obtained for the mutation status of codon 12 from the KRAS gene on tumor samples via ARMS-HRMA, SS, and/or ddPCR

Analysis of tumor samples by ARMS-HRMA for G12D mutation showed similar performance to that of G12V. Figure 5 depicts the typical ARMS-HRMA output from tumor samples for G12D mutation detection using 4 different tumor samples (T2 to T5) (Fig. 5A). Since the melting peak around 79.5 °C is exclusive for G12D mutated samples, there is no need to perform a difference plot for G12D scoring. The analysis of the melting profile allowed scoring T2 and T5 as mutated for G12D (due to the presence of a similar melting peak to the LS174T cell line; Table 1), whereas T1, T3, and T4 were scored as not mutated for G12D. All these mutation profiles were corroborated by direct SS results (Table 1). As for G12V, the same set of 30 tumor samples was screened for the presence of the G12D mutation by ARMS-HRMA (Fig. 4B and Supplementary Information S5, S6, and S7; Table 1).

Fig. 5figure 5

ARMS-HRMA for KRAS G12D mutation detection. A Derivative plot generated after ARMS amplification for the detection of KRAS G12D mutation in LS174T (wt/G12D) (yellow line) and SW48 (wt/wt) (gray line) cell lines and four tumor samples (T2 (dark yellow line), T3 (blue line), T4 (dark blue line), and T5 (brown line)). B G12D mutation scoring for the set of 30 tumor samples, based on the intensity of the melting peak at 79.5 °C; the dagger represents samples scored as G12D mutated with non-concordant result based on SS, and the asterisk represents samples with non-concordant results by both ddPCR and SS. C Statistical analysis of the average intensity of the melting peak in each sample group. Four asterisks, P value < 0.0001 using Mann–Whitney test. The dark gray bar represents tumor samples scored as G12D non-mutated; the dark yellow bar represents tumor samples scored as G12D mutated; the light gray SW48 cell line as control for G12D non-mutated samples; the light yellow bar represents the LS174T cell line as the control for G12D mutated samples. Error bars represent the standard error mean of the average result of at least 4 replicates

Interestingly, two samples positive for G12D by ARMS-HRMA (T6 and T7) were not scored as mutated by SS (Table 1 and Fig. 5), which might be attributed to the higher sensitivity of the combined techniques [53]. However, ddPCR on these samples showed that T6 was indeed G12D mutated but T7 did not harbor this mutation. The ARMS-HRMA result for T7 might be considered as a false positive or a higher sensitivity of the technique or even the result of genetic heterogeneity of the tumor sample. This is a common aspect when assessing biopsies where the proportion of normal/mutant cells is very high. Indeed, the limit of detection of both PCR-based techniques (ARMS-HRMA and ddPCR) is often much lower (single cell detection) than that of direct SS (requiring at least 10% of mutated cells) [54, 55].

In summary, apart from T7, all KRAS G12V or G12D mutated tumor samples identified by ARMS-HRMA were confirmed by ddPCR, and all these KRAS G12V or G12D mutated tumor samples, except for T6 and T7, also scored mutated for SS (which disagreed with the scoring for T6 against ddPCR). These results demonstrate that different techniques have different sensitivities, which is of relevance when assessing liquid biopsies where the amount of available template and the level of genetic heterogeneity might lead to disparate results. A 17% increase in the detection of mutant alleles in colon cancer tissue samples by a stand-alone ARMS-based approach in comparison to SS has been already reported [43]. Indeed, several studies have conveyed that SS is less sensitive when analyzing tumor samples with less than 30% of neoplastic cells, where ARMS-based approaches increased the mutant detection rate by 12% [56, 57].

ARMS-HRMA for mutation detection in liquid biopsies

We then assessed the performance of the proposed ARMS-HRMA approach in the context of liquid biopsies. ARMS-HRMA was performed on DNA extracted from plasma samples collected from the same set of 30 patients whose tumor samples had also been characterized in Table 1 (Supplementary Information S5 and S6 and Table 2). It should be noted that for some plasma samples, some techniques could not be performed due to the low amount of genetic material (DNA). Foremost, obtaining informative SS results of DNA extracted from plasma samples proved to be a challenge, most likely due to the low amount of genetic material retrieved from each extraction. In fact, it was only possible to obtain enough genetic material from 23 plasma samples (out of the 30 plasma samples). From these, it was not possible to obtain a readable SS for 10 of them; for three other samples, sequencing was only possible with one of the primers. This is in line with what has been observed for liquid biopsy characterization of patients with solid tumors and highlights the need for the development of improved approaches for the recovery of ctDNA from these samples and for mutation detection [56, 57].

Table 2 Results obtained for the mutation status of codon 12 from the KRAS gene on plasma samples via ARMS-HRMA, SS, and ddPCR

Regarding G12V mutation, from the 10 tumor samples previously characterized as mutated for G12V (T1, T3, T8, T12, T13, T18, T19, T21, T27, T29), it was only possible to obtain SS data for 4 plasma samples (P1, P9, P18, and P27), from which ARMS-HRMA and ddPCR identified three as G12V positive (P8, P9, and P18) (see Fig. 6), whereas SS only pointed P9 as G12V mutated. Regarding the two other samples, one was scored as wt (P18) by SS and the other one without a readable sequence (P8). These results underline the higher sensitivity of ARMS-HRMA in comparison to SS, since the proposed approach attained accurate mutation discrimination for samples with the concentration of genetic material below SS working range. Furthermore, samples P8, P9, and P18 belong to patients with G12V-positive tumors detected by SS (T8, T9, and T18), ARMS-HRMA (T8 and T18), and ddPCR (T8 and T18) (Tables 1 and 2). Sample T9 genetic material (DNA) only allowed performing SS genotyping, and the lack of additional tumor material did not allow its genotyping by ARMS-HRMA or ddPCR.

Fig. 6figure 6

ARMS-HRMA detection of KRAS G12V mutation in plasma samples. A G12V mutation scoring for the set of 30 plasma samples, based on the intensity of the melting peak at 79.5 °C. The dark blue bars represent tumor samples scored as G12V non-mutated. B Statistical analysis of the average intensity of the melting peak in each sample group. Four asterisks, P value < 0.0001 using Mann–Whitney test. The dark blue bar represents plasma samples scored as G12V mutated; the dark gray bar represents the SW48 cell line as the control for G12V non-mutated samples; the blue bar represents the SW480 cell line as the control for G12V mutated samples. The dagger represents samples scored as G12V mutated with non-concordant result based on SS. Error bars represent the standard error mean of the average result of at least 4 replicates

Regarding KRAS G12D, the plasma samples correspondent to the 12 patients whose tumor samples were determined as G12D mutated by ARMS-HRMA (P2, P5, P6, P7, P11, P17, P20, P22, P23, P24, P28, and P30) were analyzed. Only sample P7 was scored as positive by ARMS-HRMA (Fig. 7), while SS and ddPCR did not score any plasma sample as G12D mutated (Table 2). Interestingly, P7 plasma corresponds to the tumor sample (T7) that was also scored as positive for G12D by ARMS-HRMA but not by SS and ddPCR (see discussion above). The fact that ARMS-HRMA detected the presence of the G12D mutation in both the plasma and tumor of this patient supports the high sensitivity of the ARMS-HRMA detection method, where, again, the disparate scoring might be due to sample condition/integrity or tumor heterogeneity or even be a false positive. Interestingly, this patient presented a high level of metastases.

Fig. 7figure 7

ARMS-HRMA for KRAS G12D mutation detection on plasma samples. A Derivative plot generated after ARMS amplification for the detection of KRAS G12D mutation in plasma sample P7. B Statistical analysis of the average intensity of the melting peak in each sample group. Four asterisks, P value < 0.0001 using Mann–Whitney test. C Mutation scoring for the set of 24 plasma samples, based on the average intensity of the melting peak at 79.5 °C/80 °C. The dark gray bar represents plasma samples scored as G12D non-mutated; the dark yellow bar represents plasma samples scored as G12D mutated; the light gray bar represents the SW48 cell line as the control for G12D non-mutated samples; the yellow bar represents the LS174T cell line as the control for G12D mutated samples. The asterisk represents samples scored as G12D mutated with non-concordant result based on SS and ddPCR

In summary, the KRAS G12D mutation was detected in one plasma sample by ARMS-HRMA (P7), but not detected by SS or ddPCR, in either plasma or tumor samples of patient 7. However, since both tumor and plasma samples revealed the presence of the mutated allele by ARMS-HRMA, the KRAS G12D genotype may be inferred as a true-positive result. In fact, the presence of ctDNA fragments with mutated KRAS in the blood circulation is by itself a validating factor for the presence of mutated cells in a tumor [58].

Altogether, data show that ARMS-HRMA detected all G12V and G12D mutant alleles also assessed and detected by ddPCR in tumor and plasma samples. When compared to SS, the proposed ARMS-HRMA seems to have a higher sensitivity.

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