Genetic Aberrations Associated With Metastasis in Colorectal Cancer: An Insight Into Tumour Heterogeneity

The purpose of this thesis was to obtain some insight into the role of genetic changes in progression of human colorectal cancer. Two approaches were adopted: (i), determination of frequency and nature of mutations in KA-ras and (ii), determination of the nature and extent of genetic aberrations, at chromosomal level, in advanced colorectal cancer. The main theme of the work was the use of paired tumour samples from the same patients in an attempt to measure the extent of genetic heterogeneity and clonal diversity between primary tumours and their synchronous metastases. For Ki-ras gene, I have determined the point mutations occurring in codons 12 and 13 of exon 1 of the Ki-ras gene in 78 patients with colorectal carcinoma (31 Dukes' A and B, 21 Dukes' C, 26 Dukes' D) by allele specific oligonucleotide hybridisation and sequencing. Duplicate samples of invasive primary carcinoma, adjacent normal tissue and available lymph node and liver metastases from the same patients were microdissected from paraffin sections. There were no differences in incidence of Kl-ras mutations between primary carcinomas and secondary deposits: 26 of 78 (33%) primary carcinomas, 10 of 32 (31%) lymph node metastases, and 10 of 26 (38%) liver metastases. Multiple sampling revealed frequent heterogeneity within carcinomas: 9 of 26 primaries with Ki-ras mutations also contained areas of carcinoma with only the wild-type gene, implying that Ki-ras mutation, even when present in a colonic carcinoma, may not have been necessary for establishing the malignant phenotype. Also, 2 of 26 (8%) Dukes' D patients had a mutation in their primary carcinoma but none in liver metastases and 6 of 47 (13%) Dukes' C and D patients had mutations in liver or lymph-node metastases but none in the primary carcinoma. Mutation of codon 12 from GGT (glycine) to GTT (valine) was more prevalent in primary and metastatic deposits of Dukes' C/D carcinomas (p=0.01) than in primary carcinomas from Dukes' A/B patients. Mutations of codon 12 to GAT, AGT, GCT and codon 13 GGC to GAC were also found, but no correlation with carcinoma aggressiveness was apparent. Follow-up of 71/78 patients (up to 12 years) revealed decreased overall survival (P=0.001) in patients with the GGT to GTT transversion in codon 12, even when the analysis was restricted to Dukes' D cases, supporting the suggestion that this mutation may confer a more aggressive phenotype in colorectal carcinoma. I then wanted to determine whether the correlation between valine-12 mutant Ki- RAS and tumour aggressiveness observed in vivo could be reflected in vitro. For this, Rat-1 fibroblasts transfected with valine-12 or aspartate-12 mutant or wild-type Ki-ras gene were assessed in terms of transformation, VEGF production and in vitro invasion. Both mutants demonstrated equal abilities to transform Rat-1 cells and induce VEGF production while cells expressing the wild-type Ki-Ras protein failed to do so. Clones of Rat-1 cells expressing valine-12 mutant Ki-Ras protein demonstrated increased ability to invade matrigel and to stimulate stromelysin-1 production compared to cells expressing aspartate-12 mutant Ki-Ras. A central question in the role of mutant Ras protein in tumour biology is why should one particular mutation be associated with aggressive behaviour while the other is not? To address this question, I have used molecular modelling on the wild type, valine-12 and aspartate-12 Ras molecules and shown there to be a significant conformational differences in the GTP-bound state between the two mutants involving the effector binding domain of Ras. These 'topographic' differences could be responsible for the different biological activities between the two mutants. It is arguable, based on both in vivo and in vitro work, that the biological effects of the two different Ki-ras mutations cannot be assumed to be the same and the use of valine-12 mutant Ki-ras as a clinical marker for worse prognosis should be considered

Comparison of the sensitivity and specificity of urine cytology, urinary nuclear matrix protein-22 and multitarget fluorescence in situ hybridization assay in the detection of bladder cancer

This study aimed to compare the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of urine cytology, BladderChek nuclear matrix protein-22 (NMP22) and UroVysion fluorescence in situ hybridization (FISH) tests in patients with newly diagnosed bladder cancer, those with recurrent bladder cancer, and those with bladder cancer but in remission during surveillance. Material and methods. Voided urine samples obtained from 178 patients with suspected or known bladder cancer about to undergo diagnostic or surveillance cystoscopy and 25 control subjects without the disease were divided into four and used for urine culture and cytology, NMP22 BladderChek and UroVysion FISH tests

Prevalence and Correlation of Human Papilloma Virus and its Types with Prognostic Markers in Patients with Invasive Ductal Carcinoma of the Breast in Kuwait

Objectives: This study aimed to document the association of human papilloma virus (HPV) and its types in breast carcinoma tissues in Kuwaiti women, and correlate this with known prognostic markers. Methods: The clinicopathological data of archived tissue from 144 cases of invasive ductal breast carcinoma were studied (age, histological grade, size of tumour, lymph node metastases, oestrogen/progesterone receptors and human epidermal growth factor receptor 2 status). HPV frequency was documented using immunohistochemistry (IHC) and chromogenic in-situ hybridisation (CISH). HPV types were documented by CISH using HPV probes. CISH and IHC techniques were compared and HPV correlated with prognostic parameters. Results: The HPV prevalence as determined by CISH and IHC was 51 (35.4%) and 24 (16.7%) cases, respectively. The sensitivity of HPV by IHC was 37.3% and specificity was 94.6%. The sensitivity and specificity of HPV-CISH compared to HPVIHC was statistically significant (P <0.001). HPV-CISH was seen in 51 cases. A combination of HPV 6 and 11, and 16 and 18 was seen in 2 (3.9%) cases, and a combination of HPV 6, 11, 31 and 33 was seen in 7 (13.7%) cases. All three HPV probes: 6 and 11, 16 and 18, as well as 31 and 33 were present in 2 (3.9%) cases. The prevalence of HPVCISH in the Kuwaiti and non-Kuwaiti populations was 27 (52.9%) and 19 (37.2%), respectively. No correlation was observed with the prognostic parameters. Conclusion: The frequency of HPV in breast carcinoma cases in Kuwait was 35.4% (CISH). Of those, 52.9% were Kuwaitis in whom both low- and high-risk HPV types were detected

Correlation Profile of Suppression of Tumorigenicity 2 and/or Interleukin-33 with Biomarkers in the Adipose Tissue of Individuals with Different Metabolic States

Purpose: The suppression of tumorigenicity 2 (ST2) has two main splice variants including a membrane bound (ST2) form, which activates the myeloid differentiation primary response 88 (MyD88)/nuclear factor-kappa B (NF-kappa B) signaling pathway, and a secreted soluble form (sST2), which acts as a decoy receptor for ST2 ligand, interleukin (IL)-33. The IL-33/ST2 axis is protective against obesity, insulin resistance, and type 2 diabetes (T2D). In humans, adipose tissue IL-33 displays distinct correlation profiles with glycated hemoglobin, ST2, and other immunometabolic mediators, depending on the glycemic health of the individuals. We determined whether adipose tissue ST2 displays distinct correlation profiles with immunometabolic mediators and whether ST2 and/or IL-33 are correlated with intracellular signaling molecules. Patients and Methods: A total of 91 adults with normal glycemia, prediabetes, and T2D were included. After measuring their anthropometric and biochemical parameters, subcutaneous adipose tissues were isolated and mRNA expression of biomarkers was measured. Results: In individuals with normal glycemia, adipose tissue ST2 was directly correlated with chemokine (C-C motif) ligand (CCL)-2, CCL5, IL-12, fibrinogen-like protein 2 (FGL2) and interferon regulatory factor (IRF)-4, but inversely correlated with cytochrome C oxidase subunit 7A1. IL-33 and ST2 were directly correlated with tumor necrosis factor receptorassociated factor 6 (TRAF6), NF-kappa B, and nuclear factor of activated T-cells 5 (NFAT5). In individuals with prediabetes, ST2 was inversely correlated with IL-5, whereas IL-33 but not ST2 was directly correlated with MyD88 and NF-kappa B. In individuals with T2D, ST2 was directly correlated with CCL2, IL-1 beta, and IRF5. IL-33 and ST2 were directly correlated with MyD88, TRAF6, and NF-kappa B. Conclusion: Adipose tissue ST2 and IL-33 show different correlation profiles with various immunometabolic biomarkers depending on the metabolic state of the individuals. Therefore, targeting the IL-33/ST2 axis might form the basis for novel therapies to combat metabolic disorders.Peer reviewe

Identification of 42 genes linked to stage II colorectal cancer metastatic relapse

Colorectal cancer (CRC) is one of the leading causes of cancer mortality. Metastasis remains the primary cause of CRC death. Predicting the possibility of metastatic relapse in early-stage CRC is of paramount importance to target therapy for patients who really need it and spare those with low-potential of metastasis. Ninety-six stage II CRC cases were stratified using high-resolution array comparative genomic hybridization (aCGH) data based on a predictive survival algorithm and supervised clustering. All genes included within the resultant copy number aberrations were each interrogated independently at mRNA level using CRC expression datasets available from public repositories, which included 1820 colon cancers, and 167 normal colon tissues. Reduced mRNA expression driven by copy number losses and increased expression driven by copy number gains revealed 42 altered transcripts (29 reduced and 13 increased transcripts) associated with metastatic relapse, short disease-free or overall survival, and/or epithelial to mesenchymal transition (EMT). Resultant genes were classified based on gene ontology (GO), which identified four functional enrichment groups involved in growth regulation, genomic integrity, metabolism, and signal transduction pathways. The identified 42 genes may be useful for predicting metastatic relapse in stage II CRC. Further studies are necessary to validate these findings

A Perception on Genome-Wide Genetic Analysis of Metabolic Traits in Arab Populations

Despite dedicated nation-wide efforts to raise awareness against the harmful effects of fast-food consumption and sedentary lifestyle, the Arab population continues to struggle with an increased risk for metabolic disorders. Unlike the European population, the Arab population lacks well-established genetic risk determinants for metabolic disorders, and the transferability of established risk loci to this population has not been satisfactorily demonstrated. The most recent findings have identified over 240 genetic risk loci (with similar to 400 independent association signals) for type 2 diabetes, but thus far only 25 risk loci (ADAMTS9, ALX4, BCL11A, CDKAL1, CDKN2A/B, COL8A1, DUSP9, FTO, GCK, GNPDA2, HMG20A, HNF1A, HNF1B, HNF4A, IGF2BP2, JAZF1, KCNJ11 , KCNQ1, MC4R, PPAR gamma, SLC30A8, TCF7L2, TFAP2B, TP53INP1, and WFS1) have been replicated in Arab populations. To our knowledge, large-scale population- or family-based association studies are non-existent in this region. Recently, we conducted genome-wide association studies on Arab individuals from Kuwait to delineate the genetic determinants for quantitative traits associated with anthropometry, lipid profile, insulin resistance, and blood pressure levels. Although these studies led to the identification of novel recessive variants, they failed to reproduce the established loci. However, they provided insights into the genetic architecture of the population, the applicability of genetic models based on recessive mode of inheritance, the presence of genetic signatures of inbreeding due to the practice of consanguinity, and the pleiotropic effects of rare disorders on complex metabolic disorders. This perspective presents analysis strategies and study designs for identifying genetic risk variants associated with diabetes and related traits in Arab populations.Peer reviewe

Urocortin 3 overexpression reduces ER stress and heat shock response in 3T3-L1 adipocytes

The neuropeptide urocortin 3 (UCN3) has a beneficial effect on metabolic disorders, such as obesity, diabetes, and cardiovascular disease. It has been reported that UCN3 regulates insulin secretion and is dysregulated with increasing severity of obesity and diabetes. However, its function in the adipose tissue is unclear. We investigated the overexpression of UCN3 in 3T3-L1 preadipocytes and differentiated adipocytes and its effects on heat shock response, ER stress, inflammatory markers, and glucose uptake in the presence of stress-inducing concentrations of palmitic acid (PA). UCN3 overexpression significantly downregulated heat shock proteins (HSP60, HSP72 and HSP90) and ER stress response markers (GRP78, PERK, ATF6, and IRE1 alpha) and attenuated inflammation (TNF alpha) and apoptosis (CHOP). Moreover, enhanced glucose uptake was observed in both preadipocytes and mature adipocytes, which is associated with upregulated phosphorylation of AKT and ERK but reduced p-JNK. Moderate effects of UCN3 overexpression were also observed in the presence of 400 mu M of PA, and macrophage conditioned medium dramatically decreased the UCN3 mRNA levels in differentiated 3T3-L1 cells. In conclusion, the beneficial effects of UCN3 in adipocytes are reflected, at least partially, by the improvement in cellular stress response and glucose uptake and attenuation of inflammation and apoptosis.Peer reviewe

Correlation of Circulating ANGPTL5 Levels With Obesity, High Sensitivity C-Reactive Protein and Oxidized Low Density Lipoprotein in Adolescents

Angiopoietin-like proteins (ANGPTL) is a family of eight members known to play an important role in metabolic diseases. Of these, ANGPTL5 is suggested to regulate triglyceride metabolism and is increased in obesity and diabetes. However, its role in metabolic diseases in adolescents is not well-studied. In this study, we tested the hypothesis of a positive association between plasma ANGPTL5, and obesity, high sensitivity C-reactive protein (HsCRP) and oxidized low-density lipoprotein (Ox-LDL) in adolescents. Adolescents (N = 431; age 11–14 years) were randomly selected from middle schools in Kuwait. Obesity was classified by the BMI-for-age based on the WHO growth charts. Plasma ANGPTL5, HsCRP, and Ox-LDL were measured using ELISA. The prevalence of overweight and obesity was 20.65% and 33.18%, respectively. Mean (SD) plasma ANGPTL5 levels were significantly higher in obese, compared with overweight and normal-weight adolescents (23.05 (8.79) vs 18.39 (7.08) ng/mL, and 18.26 (6.95) ng/ml, respectively). ANGPTL5 was positively associated with both HsCRP (ρ=0.27, p \u3c 0.001) and Ox-LDL (ρ = 0.24, p \u3c 0.001). In Conclusion, ANGPTL5 levels are elevated in obese adolescents and are associated with cardiovascular disease risk factors, HsCRP and Ox-LDL. The use of ANGPTL5 as a powerful diagnostic and prognostic tool in obesity and metabolic diseases needs to be further evaluated

Metabolically Healthy and Unhealthy Obese Phenotypes among Arabs and South Asians:Prevalence and Relationship with Cardiometabolic Indicators

Obesity is a public health crisis in Kuwait. However, not all obese individuals are metabolically unhealthy (MuHO) given the link between obesity and future cardiovascular events. We assessed the prevalence of the metabolically healthy obese (MHO) phenotype and its relationship with high sensitivity C-reactive protein (hs-CRP), serum alanine aminotransferase (ALT), and insulin resistance (HOMA-IR) in Arab and South Asian ethnic groups in Kuwait. The national cross-sectional survey of diabetes and obesity in Kuwait adults aged 18–60 years were analysed. The harmonised definition of metabolic syndrome was used to classify metabolic health. Multinomial logistic regression analysis was used to model the relationship between the MHO and MuHO phenotypes and hs-CRP, ALT and HOMA-IR levels. Overall, the prevalence of MHO for body mass index (BMI)- and waist circumference (WC)-defined obesity was 30.8% and 56.0%, respectively; it was greater in women (60.4% and 61.8%, respectively) than men (39.6% and 38.2%, respectively). Prevalence rates were also lower for South Asians than for Arabs. The MHO phenotype had hs-CRP values above 3 µg/mL for each age group category. Men compared to women, and South Asians compared to Arabs had a lower relative risk for the MHO group relative to the MuHO group. This study shows there is high prevalence of MHO in Kuwait

A novel PKD1 variant demonstrates a disease-modifying role in trans with a truncating PKD1 mutation in patients with Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common form of Polycystic Kidney Disease (PKD) and occurs at a frequency of 1/800 to 1/1000 affecting all ethnic groups worldwide. ADPKD shows significant intrafamilial phenotypic variability in the rate of disease progression and extra-renal manifestations, which suggests the involvement of heritable modifier genes. Here we show that the PKD1 gene can act as a disease causing and a disease modifier gene in ADPKD patients