The introduction of next-generation sequencing (NGS) in the molecular diagnostic armamentarium is deeply changing pathology practice and laboratory frameworks. NGS allows for the comprehensive molecular characterization of neoplasms, in order to provide the best treatment to oncologic patients. On the other hand, NGS raises technical issues and poses several challenges in terms of education, infrastructures and costs. The aim of this review is to give an overview of the main NGS sequencing platforms that can be used in current molecular diagnostics and gain insights into the clinical applications of NGS in precision oncology. Hence, we also focus on the preanalytical, analytical and interpretative issues raised by the incorporation of NGS in routine pathology diagnostics.

The introduction of immunotherapy has revolutionized the oncological targeted therapy paradigm. Microsatellite instability (MSI) identifies a subgroup of colorectal cancers (CRCs) which respond to treatment with immune checkpoint inhibitors. Tissue biopsy is currently the gold standard for the assessment of MSI/Mismatch Repair deficiency (MMRd) by means immunohistochemistry or molecular assays. However, the application of liquid biopsy in the clinic may help to overcome several limitations of tissue analysis and may provide great benefit to the diagnostic scenario and therapeutic decision-making process. In the context of MSI/MMRd CRC, the use of liquid biopsy may allow to establish MSI/MMR status if tissue sampling cannot be performed or in case of discordant tissue biopsies. Liquid biopsy may also become a powerful tool to monitor treatment response and the onset resistance to immunotherapy over time and to stratify of MSI/MMRd patients according to their risk of relapse and metastases. The aim of this review is to summarize the main technical aspects and clinical applications, the benefits, and limitations of the use of liquid biopsy in MSI/MMRd colorectal cancer patients.

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3587 Background: The combination of BRAF inhibitors (BRAFi) plus anti-EGFR antibodies is a new standard of care in V600E BRAF mutated (mut) metastatic colorectal cancer (mCRC). Nevertheless, resistance develops during the target therapy (TT). We designed the URBAN study, a translational prospective project, in order to identify possible primary and acquired resistance mechanisms. Methods: Patients (pts) with V600E BRAF mut mCRC treated with BRAFi + anti-EGFR +/- MEK inhibitors at Veneto Institute of Oncology were enrolled. Clinical data and liquid biopsy at baseline and progression were collected. The ctDNA derived from plasma was analyzed by the AVENIO expanded kit, a hybridization capture sequencing-based 77 genes pan-cancer assay contained in NCCN Guidelines. Survival outcomes were calculated using Kaplan–Meier curves, log-rank tests and univariate Cox regression models were also performed. The study is exploratory and no formal hypothesis has been postulated. Results: Forty consecutive V600E BRAF mut mCRC pts were enrolled. Median age was 63 years (42-77), 47.5% of pts were males. Right CRC were 65% and 20% were MSI-H. Only 5% of pts received TT after second line; doublet regimen was administered in 60% of pts while triplet in 40%. According to the mPFS of doublet arm in the BEACON trial (4.2 months, mo), our population was divided in responder (R), 24 (60%), and non-responder (NR), 16 (40%). In R vs NR group, mPFS was 9 vs 3.2 mo while mOS was 21.6 vs 10.7 mo, respectively. The V600E BRAF mut was detected in 85% of the pre-treatment plasma samples without statistically significant differences in the genomic alterations between R and NR groups, but there was a higher frequency of MET and EGFR amplification in NR group. At progression, the mutation of BRAF was lost in 2 cases in R group. After receiving TT, the most common acquired mutations involved RAS genes: 16 pts (40%) acquired at least one activating mutation in KRAS and/or NRAS. Among these, 9 pts showed multiple mutations of the same RAS gene probably due to both intra- and inter-lesional heterogeneities; none of these pts had MSI-H mCRC. We found a higher number of RAS and MAP2K1 acquired mutations in NR and a trend to acquire EGFR amplification in R group. Inactivating mutations in RFN43 gene was observed in 2 cases in R group. These data did not reach statistical significance, probably due to the low number of cases. Interestingly, 37% of NR pts acquired three or more molecular alterations vs 13% in R group. Furthermore, a higher number of genetic alterations was acquired in pts treated with doublet vs triplet regimen. Conclusions: This prospective, observational molecular profiling study provided further evidences to support the use of ctDNA in capturing the dynamic somatic mutational spectrum in V600E BRAF mut mCRC and to identify potential mechanisms of resistance to TT. An expansion of study population is ongoing.