Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics

Abstract

Background: Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.

Methodology/principal findings: Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect<or=1% mutant cells, provided that samples yielded cycle thresholds (Ct)<29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).

Conclusions/significance: Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. FFPE DNA fragmentation and real time PCR method performance.

A: A typical series of diagnostic samples tested with the multiplex PCR assay for DNA fragmentation and KRAS exon 2 (intron spanning amplicon). Samples positive for products ≥300 bp are considered as good quality samples yielding the 340 bp product for exon 2 KRAS sequencing. In lanes 1, 3 and 4, faint KRAS product bands could be obtained from samples with very fragmented DNA but the corresponding capillary electropherograms were usually non-informative. In B, real time PCR efficiency largely depends on DNA fragmentation. DNA control Ct values reflect the amplification efficiency of FFPE DNA. With both TMGB and DxS-KRAS assays, Ct values from good quality samples were substantially lower than Ct values from very fragmented samples. The majority of FFPE DNA samples yielded DNA control Ct values between 29–33 (dotted lines). In C, typical results from diagnostic samples with DxS-KRAS. DNA control Cts for the standards contained in the kit are below 29 (arrow), but corresponding values for the diagnostic samples are >29.

Figure 2. Performance of a typical good quality FFPE-DNA diagnostic sample with the three methods applied for KRAS mutation assessment.

A and B: Corresponding tissue section with areas marked for macrodissection containing ∼70% tumor cells. Some necrotic areas can not be avoided but in this analogy these do not interfere with DNA extraction. The estimated number of sectioned neoplastic cells in B is ∼4000. For good quality samples, KRAS mutation assessment is reliable with any method, as shown in C (sequencing, c. 34 G>A corresponding to the G12S change), in D (TMGB-KRAS, G12S mutation positive) and in E (DxS-KRAS, G12S mutation positive). DNA control Ct values <29 (arrow) were yielded with both real time PCR methods (red curves in D and E).

Figure 3. KRAS-TMGB validation on good quality FFPE DNA samples.

A. The method can provide reliable results with a broad range of DNA input, as shown here for a G12V mutant sample that was serially diluted up to 1.5 ng/reaction. dCts in all samples are kept close to 0 (safe mutation calling with this method). Red curves, DNA control; Green curves, G12V targeting assay. B. TMGB-KRAS was sensitive to detect <1% tumor cells in good quality FFPE-DNA samples. Seven different mutant FFPE samples containing ∼70% tumor cells (arrows) were diluted at 1∶10 and 1∶100 with different wild-type FFPE-DNA samples. Amplification curve Cts of the diluted mutant DNA showed some degree of linear increase in Ct values for the mutant target, as expected (diagonal lines).

Figure 4. Evaluation of KRAS-TMGB profiles.

As shown in A, mutant samples have significantly lower dCT values than non-mutant ones. The mutation status of KRAS codons 12 and 13 is evaluated based on the lowest value of the 7-dCt-profile for the corresponding mutant targets, provided that the lowest value falls below the cut-off in each case. In some cases, dCts for the non-mutant alleles can be very low, falling within the range of mutation calling, due to cross-reactivity (labeled as OTHER MUT). Such cross-reactions can be troublesome if evaluating each assay separately. By contrast, if evaluating the profile of dCt values for each sample, as shown in the two examples in B with TMGB, the mutant allele can readily be recognized. Here, on the left a sample with G12A, on the right a sample with G13D (duplicates). The dCt profiles of all samples analyzed in this study are shown in the colormap in C.

Figure 5. Typical example of a diagnostic case with very low content in neoplastic cells.

If macrodissection is avoided in such cases, erroneous results are likely to be obtained. A, whole section of the CRC metastatic site (M) surrounded by normal liver (L). Circled areas are marked for macrodissection. In B, the metastatic site is largely composed of necrotic (N) and calcified (Ca++) elements within a loose stroma (s), while neoplastic cells (asterisks) correspond to <<1% in the whole section (A) and to ∼10% in the macrodissected areas (B). Two DNA samples were extracted from this specimen, one upon macrodissection and one from the whole section. As shown in C, although both DNA samples were of the same unfavorable quality (DNA control Ct∼34.5), it was possible to identify the G12D mutation in the macrodissected sample, while the sample obtained from the whole section appeared as wild type. Arrow in C, Ct = 29.