Research Article: Integrated routine workflow using next-generation sequencing and a fully-automated platform for the detection of KRAS, NRAS and BRAF mutations in formalin-fixed paraffin embedded samples with poor DNA quality in patients with colorectal carcinoma

Date Published: February 27, 2019

Publisher: Public Library of Science

Author(s): Claire Franczak, Ludovic Dubouis, Pauline Gilson, Marie Husson, Marie Rouyer, Jessica Demange, Agnès Leroux, Jean-Louis Merlin, Alexandre Harlé, Giancarlo Troncone.


KRAS and NRAS mutations are identified resistance mutations to anti-epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancer. BRAF status is also routinely assessed for its poor prognosis value. In our institute, next-generation sequencing (NGS) is routinely used for gene-panel mutations detection including KRAS, NRAS and BRAF, but DNA quality is sometimes not sufficient for sequencing. In our routine practice, Idylla platform is used for the analysis of samples that don’t reach sufficient quality criteria for NGS assay.

In this study, data from mCRC samples analyzed from May 2017 to 2018 were retrospectively collected. All samples with a poor DNA quality for sequencing have been assessed using Idylla platform. First, KRAS Idylla assay cartridge has been used for the determination of KRAS mutational status. All KRAS wild-type samples have then been analyzed using NRAS-BRAF assay. Among 669 samples, 67 samples failed the DNA quality control and have been assessed on Idylla KRAS mutation test.

Among 67 samples, 50 (75%) samples had a valid result with Idylla KRAS mutation test including 22 carrying a KRAS mutation. For 28 samples, NRAS and BRAF mutational statuses have been assessed using Idylla NRAS-BRAF mutation test. Among 28 samples, 27 (96%) had a valid result including 2 samples bearing a NRAS mutation and 3 samples bearing a BRAF mutation.

Our study shows that an integrated workflow using NGS and Idylla platform allows the determination of KRAS, NRAS and BRAF mutational statuses of 651/669 (97.3%) samples and retrieve 49/67 (73.1%)samples that don’t reach DNA quality requirements for NGS.

Partial Text

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide [1]. Despite current early detection strategies for CRC, 20% of CRC are diagnosed at a metastatic stage [2,3].

Among 669 samples analyzed in routine from May 2017 to May 2018, 67 samples failed to reach the quality requirement (ΔQC > 6, n=53) or reached the quality requirement (ΔQC ≤ 6, n=14) but did not allow library preparation or gave invalid results after DNA sequencing.

In this study, we retrospectively collected data from 669 samples from patients with mCRC for KRAS, NRAS and BRAF mutational status assessment. DNA was of too poor quality for a NGS analysis for 10% of samples. The most common cause of DNA degradation in FFPE tissues is an inappropriate length of tissue fixation. In the large majority of the cases, the same poor DNA quality is found in a second block from the same lesion, because of the pre-analytical steps were similar. Moreover, asking a second block to the pathologist can improve delays for RAS and BRAF testing. In our experience, analyzing poor quality DNA (ΔQC > 6) using amplicon-based NGS assay leads to no library amplification or to results with a very high background noise which is not compatible with a good interpretation for samples with variant allele frequency (VAF) under 10% or more. The risk of false positive or false negative is too high when sequencing poor quality DNA, thus we chose a second assay which is less stringent on DNA quality to avoid a second biopsy to the patient. These samples have been analyzed with the Idylla platform and 75% of the samples have been retrieved. We assume that Idylla assay amplifies shorter amplicons than the library preparation kit we use and is then less influenced by DNA fragmentation than NGS assay. We tested this hypothesis by analyzing DNA fragments sizes from samples with a range of ΔQC and the results confirmed that a high ΔQC is associated with shorter DNA fragments (S1 Fig). Fourteen samples were found with a ΔQC ≤ 6 but did not allow library preparation or gave non interpretable results after sequencing. We assume that for these samples, ΔQC close to 6 combined with low DNA yield led to a library preparation failure and or high background noise. Using shorter amplicons for library preparation or using capture-based sequencing may address this issue.




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