Clonal Evolution in Inflammatory Bowel Disease: "Are some Bowels Born to Be Bad"?

PhD Doctoral Theses Bart's.Patients with longstanding, extensive colonic inflammation from inflammatory bowel disease (IBD) have a significantly increased risk of developing colorectal cancer (CRC). As a result, these patients are entered into endoscopic surveillance programmes with the aim of detecting and treating precursor dysplastic lesions and early cancers. However, surveillance is complicated by high cost, patient discomfort and missed pathology. The majority of patients with dysplastic lesions do not develop cancer, and many IBD patients with cancer do not have a preceding dysplasia diagnosis. Moreover, the histological grading of IBD-associated dysplasia suffers from significant interobserver variability, and the most appropriate clinical management strategy for patients with evidence of dysplasia remains uncertain. Accurate CRC risk stratification in IBD represents an unmet clinical need; these clinical challenges reflect our poor understanding of the molecular processes underpinning IBD-mediated CRC. We now recognise that clonal evolution underpins IBD-mediated carcinogenesis, whereby epithelial cells acquire heritable genomic changes that provide them with a survival advantage in an environment of relapsing-remitting inflammation. In this thesis, I begin with an assessment of long-term patient outcomes following the endoscopic resection of neoplastic lesions in patient with IBD. Next, I analysed the pattern of clonal evolution over time and space in colonic mucosa by measuring the burden of genomic alterations accumulating during transition from histologically normal epithelium through to dysplasia and cancer, with a particular focus on the role of chromosomal copy number alterations. The results generated from these studies are leveraged to compare the pattern of genomic alterations in epithelial tissue samples (both dysplastic and non-neoplastic) from patients who subsequently progress to CRC, against samples from patients who never progress. In particular, I highlight the translational potential of the generated findings in the development of a novel clinically applicable “evolutionary biomarker” of cancer risk in patients with IBD

The mutational signatures of formalin fixation on the human genome

Clinical archives of patient material near-exclusively consist of formalin-fixed and paraffin-embedded (FFPE) blocks. The ability to precisely characterise mutational signatures from FFPE-derived DNA has tremendous translational potential. However, sequencing of DNA derived from FFPE material is known to be riddled with artefacts. Here we derive genome-wide mutational signatures caused by formalin fixation. We show that the FFPE-signature is highly similar to signature 30 (the signature of Base Excision Repair deficiency due to NTHL1 mutations), and chemical repair of DNA lesions leads to a signature highly similar to signature 1 (clock-like signature due to spontaneous deamination of methylcytosine). We demonstrate that using uncorrected mutational catalogues of FFPE samples leads to major mis-assignment of signature activities. To correct for this, we introduce FFPEsig, a computational algorithm to rectify the formalin-induced artefacts in the mutational catalogue. We demonstrate that FFPEsig enables accurate mutational signature analysis both in simulated and whole-genome sequenced FFPE cancer samples. FFPEsig thus provides an opportunity to unlock additional clinical potential of archival patient tissues.Peer reviewe

Comparative evaluation of PCR success with universal primers of maturase K (matK) and ribulose-1, 5-bisphosphate carboxylase oxygenase large subunit (rbcL) for barcoding of some arid plants

Abstract DNA barcoding is the use of short DNA sequences (~650 bp) of the standard segment of the genome for large scale species identification. The Consortium for the Barcode of Life (CBOL) plant-working group recommended the 2-locus combination of rbcL and matK as the standard plant barcode. These two regions of chloroplast DNA were chosen due to efficient recovery of quality sequences and high levels of species discrimination. We evaluated the success rates of universal primers for amplification of matK and rbcL loci in 26 different plant species (covering 14 families) from Saudi Arabia. Success rate in PCR was higher for rbcL (88%) compared with matK (69%). The universal primers of both matK and rbcL failed to amplify the DNA form 3 plant species belonging to the family Asteraceae (Anthemis deserti, Pulicaria undulate, and Sonchus oleraceus). Two plant species Malva parviflora (Malvaceae) and Salsola imbricate (Chenopodiaceae) indicated different primer binding site (matK) as the amplified PCR products were of lower size than expected for these species. These findings indicate that although currently used universal primers of rbcL and matK are able to amplify many of the plant species they may fail in certain cases due to primer mismatch at the annealing site. Further studies are therefore needed for protocol development, particularly designing of novel universal primers, to extend the barcoding for a broader coverage of plant species

A Simple Method for DNA Extraction from Mature Date Palm Leaves: Impact of Sand Grinding and Composition of Lysis Buffer

Molecular marker techniques have been widely used for cultivar identification of inbred date palms (Phoenix dactylifera L.; Arecaceae) and biodiversity conservation. Isolation of highly pure DNA is the prerequisite for PCR amplification and subsequent use such as DNA fingerprinting and sequencing of genes that have recently been developed for barcoding. To avoid problems related to the preservation and use of liquid nitrogen, we examined sterile sand for grinding the date palm leaves. Individual and combined effects of sodium chloride (NaCl), polyvinylpyrrolidone (PVP) and lithium chloride (LiCl) with the cetyltrimethylammonium bromide (CTAB) method for a DNA yield of sufficient purity and PCR amplification were evaluated in this study. Presence of LiCl and PVP alone or together in the lysis buffer did not significantly improve the DNA yield and purity compared with the addition of NaCl. Our study suggested that grinding of date palm leaf with sterile sand and inclusion of NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and LiCl, provides a DNA yield of sufficient purity, suitable for PCR amplification

The ligational behavior of a phenolic quinolyl hydrazone towards copper(II)- ions

<p>Abstract</p> <p>Background</p> <p>The heterocyclic hydrazones constitute an important class of biologically active drug molecules. The hydrazones have also been used as herbicides, insecticides, nematocides, redenticides, and plant growth regulators as well as plasticizers and stabilizers for polymers. The importance of the phenolic quinolyl hydrazones arises from incorporating the quinoline ring with the phenolic compound; 2,4-dihydroxy benzaldehyde. Quinoline ring has therapeutic and biological activities whereas, phenols have antiseptic and disinfectants activities and are used in the preparation of dyes, bakelite and drugs. The present study is planned to check the effect of the counter anions on the type and geometry of the isolated copper(II)- complexes as well as the ligational behavior of the phenolic hydrazone; 4-[(2-(4,8-dimethylquinolin-2-yl)hydrazono)methyl] benzene-1,3-diol; (H₂L).</p> <p>Results</p> <p>A phenolic quinolyl hydrazone (H₂L) was allowed to react with various copper(II)- salts (Cl‾, Br‾, NO₃‾, ClO₄‾, AcO‾, SO₄^2-). The reactions afforded dimeric complexes (ClO₄‾, AcO‾ ), a binuclear complex (NO₃‾ ) and mononuclear complexes (the others; Cl‾, Br‾, SO₄^2-). The isolated copper(II)- complexes have octahedral, square pyramid and square planar geometries. Also, they reflect the strong coordinating ability of NO₃‾, Cl‾, Br‾, AcO‾ and SO₄^2-anions. Depending on the type of the anion, the ligand showed three different modes of bonding <it>viz</it>. (NN)⁰for the mononuclear complexes (<b>3, 4, 6</b>), (NO)^-with O- bridging for the dimeric complexes (<b>1, 5</b>) and a mixed mode [(NN)⁰+ (NO)^-with O- bridging] for the binuclear nitrato- complex (<b>2</b>).</p> <p>Conclusion</p> <p>The ligational behavior of the phenolic hydrazone (H₂L) is highly affected by the type of the anion. The isolated copper(II)- complexes reflect the strong coordinating power of the SO₄^2-, AcO‾, Br‾, Cl‾ and NO₃‾ anions. Also, they reflect the structural diversity (octahedral, square pyramid and square planar) depending on the type of the counter anion.</p

Evolutionary history of human colitis-associated colorectal cancer

Objective: IBD confers an increased lifetime risk of developing colorectal cancer (CRC), and colitis-associated CRC (CA-CRC) is molecularly distinct from sporadic CRC (S-CRC). Here we have dissected the evolutionary history of CA-CRC using multiregion sequencing. Design: Exome sequencing was performed on fresh-frozen multiple regions of carcinoma, adjacent non-cancerous mucosa and blood from 12 patients with CA-CRC (n=55 exomes), and key variants were validated with orthogonal methods. Genome-wide copy number profiling was performed using single nucleotide polymorphism arrays and low-pass whole genome sequencing on archival non-dysplastic mucosa (n=9), low-grade dysplasia (LGD; n=30), high-grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=19). Phylogenetic trees were reconstructed, and evolutionary analysis used to reveal the temporal sequence of events leading to CA-CRC. Results: 10/12 tumours were microsatellite stable with a median mutation burden of 3.0 single nucleotide alterations (SNA) per Mb, ~20% higher than S-CRC (2.5 SNAs/Mb), and consistent with elevated ageing-associated mutational processes. Non-dysplastic mucosa had considerable mutation burden (median 47 SNAs), including mutations shared with the neighbouring CA-CRC, indicating a precancer mutational field. CA-CRCs were often near triploid (40%) or near tetraploid (20%) and phylogenetic analysis revealed that copy number alterations (CNAs) began to accrue in non-dysplastic bowel, but the LGD/HGD transition often involved a punctuated ‘catastrophic’ CNA increase. Conclusions: Evolutionary genomic analysis revealed precancer clones bearing extensive SNAs and CNAs, with progression to cancer involving a dramatic accrual of CNAs at HGD. Detection of the cancerised field is an encouraging prospect for surveillance, but punctuated evolution may limit the window for early detection

A Brief Review of Molecular Techniques to Assess Plant Diversity

Massive loss of valuable plant species in the past centuries and its adverse impact on environmental and socioeconomic values has triggered the conservation of plant resources. Appropriate identification and characterization of plant materials is essential for the successful conservation of plant resources and to ensure their sustainable use. Molecular tools developed in the past few years provide easy, less laborious means for assigning known and unknown plant taxa. These techniques answer many new evolutionary and taxonomic questions, which were not previously possible with only phenotypic methods. Molecular techniques such as DNA barcoding, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), microsatellites and single nucleotide polymorphisms (SNP) have recently been used for plant diversity studies. Each technique has its own advantages and limitations. These techniques differ in their resolving power to detect genetic differences, type of data they generate and their applicability to particular taxonomic levels. This review presents a basic description of different molecular techniques that can be utilized for DNA fingerprinting and molecular diversity analysis of plant species

Evolutionary history of human colitis-associated colorectal cancer

Objective IBD confers an increased lifetime risk of developing colorectal cancer (CRC), and colitis-associated CRC (CA-CRC) is molecularly distinct from sporadic CRC (S-CRC). Here we have dissected the evolutionary history of CA-CRC using multiregion sequencing. Design Exome sequencing was performed on fresh-frozen multiple regions of carcinoma, adjacent non-cancerous mucosa and blood from 12 patients with CA-CRC (n=55 exomes), and key variants were validated with orthogonal methods. Genome-wide copy number profiling was performed using single nucleotide polymorphism arrays and low-pass whole genome sequencing on archival non-dysplastic mucosa (n=9), low-grade dysplasia (LGD; n=30), high-grade dysplasia (HGD; n=13), mixed LGD/HGD (n=7) and CA-CRC (n=19). Phylogenetic trees were reconstructed, and evolutionary analysis used to reveal the temporal sequence of events leading to CA-CRC. Results 10/12 tumours were microsatellite stable with a median mutation burden of 3.0 single nucleotide alterations (SNA) per Mb, ~20% higher than S-CRC (2.5 SNAs/Mb), and consistent with elevated ageing-associated mutational processes. Non-dysplastic mucosa had considerable mutation burden (median 47 SNAs), including mutations shared with the neighbouring CA-CRC, indicating a precancer mutational field. CA-CRCs were often near triploid (40%) or near tetraploid (20%) and phylogenetic analysis revealed that copy number alterations (CNAs) began to accrue in non-dysplastic bowel, but the LGD/HGD transition often involved a punctuated ‘catastrophic’ CNA increase. Conclusions Evolutionary genomic analysis revealed precancer clones bearing extensive SNAs and CNAs, with progression to cancer involving a dramatic accrual of CNAs at HGD. Detection of the cancerised field is an encouraging prospect for surveillance, but punctuated evolution may limit the window for early detection

Appropriate patient selection in the management of common bile duct stones:when not to do ERCP

Background. Magnetic resonance cholangiopancreatography (MRCP) is noninvasive and accurate for diagnosing intra common bile duct stones (ICSs). However, given limited access, routine utilisation for investigating all patients with gallstone disease is neither practical nor cost-effective. Conversely, many individuals proceed directly to endoscopic retrograde cholangiopancreatography (ERCP), an invasive test with appreciable complications. Aim. Identify factors associated with ICS in order to improve risk-stratification for MRCP/ERCP. Methods. All patients having undergone cholecystectomy between November 2007 and October 2008 were reviewed. High-risk features for ICS were predefined, and their true presence confirmed by ERCP or intraoperative cholangiogram. Multivariate logistic regression was performed on candidate risk features. Results. Of 231 patients, 10.4% had ICS. Defining a high-risk group with “both” biochemical and ultrasound risk factors predicted ICS with 92% specificity and also bore strong association (OR 8.88). However, isolated hyperbilirubinaemia, ultrasound impression of CBD stones, and clinical risk factors did not (OR 1.10, 0.97, and 1.26). Normal liver biochemistry and normal ultrasound had a NPV of 99.5% for ICS. Conclusions. Ultrasound impression of CBD calculi without ductal dilatation is not predictive of ICS. Patients with normal liver biochemistry and normal CBD diameter on ultrasound are unlikely to have ICS and should not proceed to ERCP

Multicentre derivation and validation of a colitis-associated colorectal cancer risk prediction web tool

Objective Patients with ulcerative colitis (UC) diagnosed with low-grade dysplasia (LGD) have increased risk of developing advanced neoplasia (AN: high-grade dysplasia or colorectal cancer). We aimed to develop and validate a predictor of AN risk in patients with UC with LGD and create a visual web tool to effectively communicate the risk. Design In our retrospective multicentre validated cohort study, adult patients with UC with an index diagnosis of LGD, identified from four UK centres between 2001 and 2019, were followed until progression to AN. In the discovery cohort (n=246), a multivariate risk prediction model was derived from clinicopathological features using Cox regression. Validation used data from three external centres (n=198). The validated model was embedded in a web tool to calculate patient-specific risk. Results Four clinicopathological variables were significantly associated with AN progression in the discovery cohort: endoscopically visible LGD >1 cm (HR 2.7; 95% CI 1.2 to 5.9), unresectable or incomplete endoscopic resection (HR 3.4; 95% CI 1.6 to 7.4), moderate/severe histological inflammation within 5 years of LGD diagnosis (HR 3.1; 95% CI 1.5 to 6.7) and multifocality (HR 2.9; 95% CI 1.3 to 6.2). In the validation cohort, this four-variable model accurately predicted future AN cases with overall calibration Observed/Expected=1.01 (95% CI 0.64 to 1.52), and achieved 100% specificity for the lowest risk group over 13 years of available follow-up. Conclusion Multicohort validation confirms that patients with large, unresected, multifocal LGD and recent moderate/severe inflammation are at highest risk of developing AN. Personalised risk prediction provided via the Ulcerative Colitis-Cancer Risk Estimator ( www.UC-CaRE.uk ) can support treatment decision-making