title: Genetic profiling of circulating tumour cells and DNAs in patients with colorectal carcinoma
Creators: name: Hamid, Faysal-Bin
Publisher: name: Griffith University
Year: 2021
Subjects: subject: blood-based biopsy
subject: liquid biopsy
subject: circulating tumour cell
subject: circulating tumour DNA

description: Over the decades, it has been considered that blood-based biopsy or liquid biopsy could be an alternative approach of tumour biopsy. To date, blood-based biomarkers such as circulating tumour cell (CTC) and circulating tumour DNA (ctDNA) have been widely studied and found their significance in patients with several carcinomas including colorectal carcinoma (CRC). Biologically, both CTC and cell-free DNA (cfDNA) have been released from the tumour and circulate freely in the blood. Although they are present in trivial quantity due to minute scale of discharge and rapid clearance from the blood, the status of the circulating biomarkers at a given time-point can be “snapshot” of the entire tumour landscape. With the advancement of detection techniques, trace quantity of biomarkers can even be detected in the blood while the existing diagnostic biomarkers, e.g., CEA often fails to detect the disease at the early stages of CRC. In addition, an elevation of CTC and ctDNA levels have been observed in patients with advanced stages of CRC suggested the possible role in CRC diagnosis. Molecular analyses of the biomarkers revealed that of CTC and ctDNA have distinct biological, genetic and genomic signatures compared to the primary tumours which can highlight on the critical insights of their lifecycle and pinpoint the novel therapeutic targets. In the thesis, we have presented the isolation techniques, novel biological and molecular characteristics and clinical relevance of CTC and ctDNA in patients with CRC. For CTC detection, we used two techniques- immunomagnetic negative selection and filtration method. Ten millilitres (ml) of the whole blood were collected from the healthy individuals and patients with CRC. To validate the techniques, three colon cancer cell lines SW 48, SW 480 and HCT 116 were spiked in the blood of healthy persons and recovered using the two techniques. After validation, we isolated CTCs from patients with CRC and detected using epithelial cell adhesion molecule (EPCAM) and cytokeratin 18 (CK 18) based immunofluorescence experiments. We have also studied novel morphological characteristics of CTCs from their sizes and phenotypical characteristics of CTCs from different expressions of two proteins in CTCs. In addition, genetic heterogeneities of the CTCs were studied using mRNA expression profiling of a novel multigene panel and compared to the primary tumours. Next, the precise role of the individual CTCs likely could be obscured due to the other contaminated blood cells. Therefore, we have isolated 28 single CTCs from eight patients with CRC to study the genetic heterogeneity of CTCs in single-cell resolution. We have initially validated our single-cell-analysis platform using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in six single cells from colon cancer cell lines. Then we used a panel of 19 genes to investigate the single CTCs (n=28), primary carcinomas (n=8), and colon cancer cell lines (n=6) using real-time qPCR (RT-qPCR). In addition, we compared the number of CTCs and gene expression patterns of CTCs with pathological stages to assess the correlation with pathological stages of CRC. Moreover, we aimed to study the novel biological characteristics of cell-free DNAs (cfDNA) such as the concentration and DNA integrity in the plasma of patients with CRC. The cfDNA samples were extracted from the plasma samples of patients with CRC and healthy donors. The measurement of cfDNA concentration was performed using two approaches: Alu-based qPCR and Qubit techniques. In the Alu-based qPCR approach, the ratio of the long (Alu 247) and short (Alu 115 and Alu 81) amplicons represented cfDNA integrity (cfDI). To compare the efficiencies of these methods, ROC (Receiver-Operative Characteristic) curve was analysed. Furthermore, only a few of the DNA fragments called ctDNA carry mutations found in the cfDNA pool. Hence, we sought to investigate the genomic status of ctDNA by detecting a KRAS mutation (G12C) using duplex digital PCR (dPCR) in the plasma of patients with CRC. Given that screening KRAS exon 2 and 3 mutations are frequently used to diagnose CRC in clinical practise and G12C mutation is barely (approximately 3%) found in patients with CRC. The rational of KRAS G12C selection was to evaluate the sensitivity and specificity of dPCR technique in KRAS mutation testing in clinical settings. Followed by a validating step with commercially DNA fragment harbouring KRAS G12C mutation, we used the duplex dPCR to detect KRAS wild-type (WT) and G12C mutation from the plasma of patients with CRC. Finally, we compared the concentration, DNA integrity and frequency of KRAS G12C allele of ctDNAs in the plasma with pathological stages to investigate the association with pathological stages of CRC. We have detected CTCs from 61.3% and 69.3% of patients with CRC using negative selection and filtration methods, respectively. The total number and diameter of CTCs were significantly higher (p&lt;0.0001) in the advanced stages of CRC. In addition, the number of the novel EPCAM+CK18 and E-Cadherin+MMP9 phenotypes of CTCs detected by both techniques were significantly higher in patients with advanced pathological stages of CRC. Gene expression profiling of CTCs unravelled three unique CTC subtypes expressing epithelial, epithelium-mesenchymal transition (EMT) and stemness features, which were divergent from the primary tumour specimens. Also, significant alterations in expressions of EPCAM, HRAS, BRAF, TP53, SLUG, TWIST1, CD44 and MMP9 genes of CTCs were observed compared to the primary tumours in patients with CRC. Moreover, gene expression profiling of the single CTCs displayed an extensive heterogeneity of the selected genes among the CTCs. Hierarchical clustering analyses highlighted diverse variations between CTCs and the primary tumours. In addition, the genetic heterogeneities were observed in an individual patient as well as different patients. Our results showed that AKT1 expression in CTCs was significantly (p= 0.0129) higher in advanced pathological stages compared to early stages of CRC. We have also measured the concentration of cfDNA using ALU-sequences and found relatively higher Alu 247 amplicon in patients with CRC compared to the healthy donors. Similarly, fluorometry-based Qubit 2.0 was also showed higher cfDNA concentration in patients with CRC. For the first time, we showed cfDI 2 was significantly higher and had more diagnostic accuracy than cfDI 1. We observed significantly higher concentration of Alu 247 and Alu 115 as well as cfDI 1 and cfDI 2 in patients with advanced pathological stages than early stages of CRC. Furthermore, we have detected G12C allele harbouring ctDNA from 48.53% (33 out of 68) patients with CRC. Our dPCR platform could detect efficiently as low as 0.1% ctDNAs in the validation step. MAF of G12C alleles was observed significantly higher (p= 0.0308) in patients with advanced stages than the early stages of CRC. In conclusion, this study has found that both CTC and ctDNA have the potential to predict and diagnosis of CRC. For CTC isolation, the filtration method is better than immunomagnetic-based negative selection. Also, CTC analyses revealed that CTCs could be heterogeneous in different ways-morphologically, phenotypically, genetically in an individual patient as well as different patients of CRC. Nevertheless, the biological features such as concentration and fragmentation patterns of cfDNA fragments are also distinct in the plasma of patients with CRC. Assessment of dPCR-based KRAS mutation harbouring ctDNA from plasma can be an alternative approach for the early detection of CRC. Therefore, sensitive and accurate detection of CTC and ctDNA will assist in the early screening and progression of CRC. Molecular analyses of CTC and ctDNA can offer critical insights of the drug resistance and offers improved therapeutic targets.