Project description:Colorectal cancer remains the commonest cancer among men, and third commonest among women in Saudi Arabia . Presentation with metastatic disease occurs in almost one third of patients , with 5-year survival decreasing significantly from 90% in stage 1 to 14% once the disease is metastatic . There is enthusiasm in the potential for liquid biopsies to provide easily accessible genetic biomarkers for mutational cancer characterization . Epidermal growth factor receptor (EGFR) monoclonal antibodies are widely used in the treatment of advanced colorectal cancer that do not harbor RAS mutations (RAS wild type). Hence genotyping of oncogenic RAS mutations is essential prior to the initiation of systemic therapy for such patients as the presence of these mutations predict resistance to EGFR targeted antibodies such as Cetuximab and Panitumumab . Detection of such mutations has been done on tissue biopsies with the disadvantage of this being an invasive procedure, and data suggesting that such testing may not be reflective of the true mutational burden of the disease since a single fragment of tissue may be inadequate to reflect the intratumoral heterogeneity. There is increasing evidence suggesting that liquid biopsies or blood based mutational profiling can provide a more comprehensive molecular profile of the disease, and carries the advantage of being minimally invasive. Serial liquid biopsies can act as a tool to identify spatial and temporal heterogeneity predicting response or resistance to targeted agents, and can shed light into the emergence (or disappearance) of specific mutations that may potentially be targeted with newer anti cancer agents . Circulating cell free DNA (cfDNA) consists of small nucleic acid fragments liberated from cells by rupture, necrosis or apoptosis, and is now increasingly being used to detect RAS (and other) mutations in patients with advanced colorectal cancers. KRAS has remained an "undruggable" target for decades until the most recent evidence that showed a new anticancer drug that targets KRAS G12C mutation. The investigators aim to perform cfDNA testing on patients with advanced colorectal cancers who have no RAS mutations (and hence start on EGFR inhibitors) as baseline, compare the results with mutational analysis on fresh tumor tissue, and perform cfDNA at first progression to determine what mutations have emerged, and specifically look for KRAS G12C mutation, which can be targeted with a new novel anti cancer drug . These patients will be collected over a 12 month period (with the aim of performing this on at least 100 patients), and followed from diagnosis (with baseline cfDNA) and until progression on EGFR inhibitors (where another cfDNA sample will be taken). A detailed proposal delineating this process will follow once accepted. This project is unique as it examines mechanisms of resistance to anti-EGFR inhibitors in our patients with advanced colorectal cancers, determines the prevalence of a specific mutation using liquid biopsies and examining cfDNA use, and may have therapeutic implications in facilitating obtaining KRAS G12C inhibitors for such patients.

Project description:Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high intra-tumoral heterogeneity, frequently resistant to treatment and no known targeted therapy available to improve patient outcomes. It has been hypothesized that the genomic architecture of a TNBC tumour evolves over time, both before, and during therapy, leading to therapy resistance and a high propensity to relapse. Whether this is an inherent property of the tumour or acquired over time is not well characterized. Despite this important clinical implication, limited studies have been carried out to unravel temporal evolution of TNBC over time. Herein, we report an OMICS based analysis of three TNBC patients who were longitudinally sampled during their treatment at different times of relapse. We recruited three TNBC patients at the time of their first relapse who were then followed-up through the course of their treatment. We obtained retrospective samples (tumour samples) from patient tumours at diagnosis (before neo-adjuvant chemotherapy - NACT) at surgery (post NACT) and prospectively sampled them at each subsequent relapse (tumour, blood plasma, and buffy coat) as determined by RECIST criteria. Tumor and buffy coat DNA were subjected to whole exome sequencing (WES) at 200x, and SNP arrays for copy number variation (CNV) analysis. RNA from tumour samples at relapse was subjected to whole transcriptome sequencing. Pathogenic germline BRCA1 variants identified in WES were validated using Sanger sequencing. 1084 somatic mutations identified in whole exome sequencing of all tumour tissues (n=13) from three patients, were subjected to a custom amplicon ultra-deep sequencing assay at 30,000X in their germline DNA (n=3), tumour DNA (n=10), and cfDNA from plasma samples at relapse (n=8). Copy number corrected allele frequencies, tumour ploidy, tumour purity, and ultra-deep sequencing assay derived variant allele frequencies were used to infer clonal and phylogenetic architecture of each patient as it evolved under selective pressure of therapy over time. Clonality analysis incorporating allele fractions from ultra-deep sequencing identified clones comprising of mutations that are present throughout the course of therapy which we term as founding clones and stem mutations respectively. Such founding clones comprising of stem mutations in all 3 patients were present throughout the course of treatment, irrespective of change in treatment modalities. These stem clones included well characterized cancer related genes like PDGFRB & ARID2 (Patient 02), TP53, BRAF & CSF3R (Patient 04) and ESR1, APC, EZH2 & TP53 (Patient 07). Such branching evolution is seen in all three patients wherein the dominant clone (stem clone) acquires additional mutations to form sub-clones, while persisting over time. These sub-clones may be chemo and radio resistant, while also providing for organ specific metastatic potential. Allele fractions of expressed variants inferred from RNA-Seq data co-related with allele fractions from WES data indicating that all somatic.

Dataset Information

Evaluation of RAS mutations in cell free deoxyribonucleic acid (cfDNA) in response to cetuximab anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer.

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