br II trial aimed at testing
II trial aimed at testing trastuzumab and lapatinib in patients with
ERBB2 amplified CRC, was performed with very encouraging results.26
In addition, molecular alterations leading to the constitutive activation of other receptor tyrosine kinases also play a pivotal role
in colorectal tumorigenesis and drive primary resistance to EGFR targeted monoclonal antibodies. For example, fusions involving ALK, RET, ROS1, and NTRK family genes occur in 0.2% to 1% of the cases27 and represent valuable therapeutic targets for highly selected patients with mCRC.28,29
As previously mentioned, the CRC mutational landscape has also prognostic value. Mismatch repair proficient CRCs comprise 85% of the total cases and often arise in the left colon.30 Mismatch repair deficient (MMRd) cancers that carry defects in the DNA repair machinery, and preferentially arise in the right colon, account for the remaining 15% of cases. MMR deficiency causes insertions and de-letions in regions of repetitive DNA sequences called microsatellites. For this reason, MMRd often leads to the onset of a phenotype called “microsatellite instability,”31,32 which, importantly, is associated with favorable prognosis.33,34 Accordingly, the fraction of MMRd cases decreases to 5% to 7% in the metastatic setting. In addition to molecular analyses performed on tissue samples, profiling circulating tumor DNA (ctDNA) offers unprecedented opportunities for genotyping, tracking minimal residual disease, and monitoring the emergence of drug resistance in CRC and other tumor types.35 In light of its predictive role in response to EGFR blockade, the analysis of RAS mutational status in ctDNA from patients with mCRC has been recommended by the European Society of Digestive Oncology and the European Society for Medical Oncology when tumor tissue is not readily available.36 Liquid biopsies also allow tracking of clonal Conessine during treatment, for example, by detecting acquired alterations before disease progression is clinically manifest.23 As compared with ana-lyses performed on tissue, detection of mutant tumor DNA in blood is more challenging and requires dedicated methodologies.35
For the reasons discussed above, defining the complex genomic landscape of CRCs and identification of genetically distinct CRC subtypes involve deep molecular characterization of individual pa-tients. This is necessary for diagnostic purposes and to properly tailor treatments. In this work, we describe multiple DNA next generation sequencing (NGS) approaches that, coupled with computational and bioinformatic algorithms, allow determination of clinically relevant parameters in this clinical setting.
Materials and Methods
Patients with mCRC were treated with a dual HER2 blockade by trastuzumab and lapatinib within the HERACLES multicenter, open-label, phase II trial performed at 4 academic cancer centers in Italy, as previously described.26,37 The study was conducted ac-cording to the provisions of the Declaration of Helsinki and the International Conference on Harmonization and Good Clinical Practice guidelines. All patients provided written informed consent for participation to the study and associated procedures, including the molecular analyses described in this work.
NGS: Target Enrichment and Custom Panel Design
We carried out and optimized specific workflows for both DNA extraction and the initial steps of library preparation (see Supplemental Material section in the online version) based on DNA-specific features (Figure 1A, B).
Independently from these initial steps, all libraries proceeded through enrichment of target regions. For whole exome sequencing (WES, 30 Mb), we used Nextera Rapid Capture Exome kit, or the latest equivalent TruSeq Rapid Exome Enrichment kit (Illumina Inc). For the IRCC-TARGET and FUSION custom panels, we designed capture probes exploiting the DesignStudio tool available online (https://designstudio.illumina.com). In particular, for the IRCC-TARGET panel, we identified a target covering all coding regions of 224 genes known to be involved in CRC tumorigenesis, progres-sion, oncogenic signaling and sensitivity, or resistance to targeted therapy, for a total of 603 kb (see Supplemental Table 1 in the online