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BACKGROUND: Colorectal cancer (CRC) is mainly a disease of the elderly in the Western world, but its characteristics are changing globally. Iraq does not have a well established CRC screening program. Understanding trends of CRC incidence, fatality and the clinical features of CRC patients is vital to the design of effective public health measures; public awareness, screening, diagnosis and treatment strategies to meet the future demands. OBJECTIVES: Determine trends in demography, incidence proportion, mortality, topography (primary tumor site) and morphology (histology) over two decades. DESIGN: Registry-based study SETTING: Iraqi National Cancer Registry (INCR) database PATIENTS AND METHODS: We collected and analyzed data from CRC patients obtained from the INCR to calculate incidence and mortality proportion per 100 000 population for the period from 2000 to 2019. In addition to estimation, data were examined by anatomic location and morphological type. MAIN OUTCOME MEASURES: Change in the incidence and mortality proportion, topography and morphology of CRC over 20 years. SAMPLE SIZE: 20 880 CRC patients ranging in age from 14-80 years. RESULTS: The overall (males and females) CRC incidence proportion (CIP) increased from 2.28 to 6.18 per 100 000 population in 2000 and 2019, respectively, with an annual percentage change (APC) of 5.11%. The incidence proportion (IP) of CRC in patients from 20 to CONCLUSIONS: CRC in Iraq is still a disease of the elderly and is rising in incidence and mortality in all age groups. This necessitates reconsidering health policy regarding CRC; public awareness, screening and management strategies to accommodate for these alarming changes. LIMITATIONS: Data about stages, grades and molecular characterisations are not available in the INCR. CONFLICT OF INTEREST: None.

Heavy metal poisoning is a worldwide problem that is caused by different human industrial activities such as battery and painting manufacturing and occupational exposure of those working at petrol stations. Wastewater is known to contain higher amounts of heavy metals such as lead (Pd) and cadmium (Cd) and might be sources of exposure for workers at the wastewater treatment plant. However, to our best knowledge, no studies were done to evaluate the level of cadmium and lead in blood of workers at wastewater treatment plants and evaluate the subsequent effect of lead and cadmium on delta-aminolevulinic acid dehydratase (δ-ALAD), urinary delta-aminolevulinic acid (Uδ-ALA), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) as markers of lead and cadmium toxicity. In this case-control study, 79 workers at the Al-Rustumiya wastewater plant in Baghdad, Iraq, and 40 control subjects were included. The levels of lead and cadmium were measured in blood of the study subjects using the atomic absorption spectroscopy (AAS) method. 8-OHdG was analysed using enzyme-linked immunosorbent assay (ELISA) technique. δ-ALAD and Uδ-ALA were estimated using spectrophotometry-based methods. Our work showed that workers had a significantly higher level of lead and cadmium when compared with the control group (P<0.05), yet, still within the World Health Organization permissible limit. The level of both metals was positively associated with duration of work at the plant (P<0.01). The activity of δ-ALAD was inversely associated with the lead level, while both Uδ-ALA and 8-OHdG were positively correlated with the lead level (P<0.05). These three markers lacked any statistically significant association with the cadmium level (P>0.05). To sum up, working at the wastewater treatment plant was associated with a higher blood level of lead and cadmium and their possible health hazard. Health and occupational safety authorities are required to set up tighter regulations and protocols to minimize these hazards and ensure a safe working environment.

Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and β-catenin (through gene knock-down and dominant-negative TCF4 expression) in nine CRC cell lines. β-catenin inhibition did not affect STAT3 levels, whereas STAT3 knock-down resulted in reduced β-catenin mRNA and protein levels. The reduction in β-catenin protein was not prevented by proteasome inhibition, and IL6-induced STAT3 activation resulted in increased β-catenin mRNA. This suggests that STAT3 positively regulates β-catenin (at a transcriptional level) and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear β-catenin (P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and β-catenin individually and in combination. Knock-down of β-catenin and STAT3 individually inhibited cell proliferation (P

Fadhil W, Ibrahem S, Seth R, AbuAli G, Ragunath K, Kaye P & Ilyas M (2012) Histopathology The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer Aims: If stratified medicine is to be applied in the neoadjuvant setting, predictive testing will have to be undertaken on preoperative diagnostic biopsy specimens. The aim of this study was to evaluate whether a diagnostic biopsy was adequately representative of the main tumour in colorectal cancer. Methods and results: Thirty cases of paired biopsy and subsequent resection specimens were randomly selected. Samples were screened for mutation in KRAS (codons 12/13, 61, and 146), BRAF (codon 600 and exon 11), PIK3CA (exons 1, 9, and 20), TP53 (exons 5–8), and microsatellite instability, using the quick multiplex consensus or standard polymerase chain reaction (PCR) protocols followed by high-resolution melting analysis. A total of 570 paired PCR tests were performed for mutation detection, and identical results were obtained in both biopsy and resection specimens in 569 tests (>99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens. Conclusions: This is the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer.

Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.

KRAS and BRAF mutations occur in colorectal cancers (CRCs) and are considered mutually exclusive methods of activating the RAS/RAF/MEK/ERK pathway. This pathway is a therapeutic target and KRAS mutation may predict tumour responsiveness. The purpose of this study was to investigate the relationship between KRAS and BRAF mutations in 24 CRC cell lines and 29 advanced CRCs.KRAS and BRAF mutations were detected using high resolution melting and sequencing. Expression of mutations was confirmed by reverse transcription- PCR (RT-PCR) and sequencing. CpG island methylator phenotype (CIMP) was tested by methylation-specific PCR.KRAS or BRAF mutation occurred in 79% of cell lines and 59% of CRCs. In the cell lines, KRAS mutations occurred in 54% of cases (with 62% in codons 12/13 and 38% in other codons). Four cell lines had a homozygous mutation. Only heterozygous BRAF mutations were detected in 29% cell lines. The V600E mutation occurred most commonly and was associated with CIMP+ status (p = 0.005). Mutations at codons 529 and 581 were also found and, in one case, BRAF and KRAS mutation co-occurred. Unexpectedly, BRAF splice variants (with a predicted kinase-dead protein) were found in 5/24 (21%) cell lines. In advanced CRCs, KRAS mutations occurred in 48% of cases (64% codons 12/13, 36% other codons) and BRAF mutations in 10% (66% V600E, 33% exon 11). A compound KRAS/BRAF mutation was not seen.Disrupted Ras/Raf signalling is common in CRC. Homozygous KRAS mutations and concomitant KRAS/BRAF mutations may be indicative of a gene dosage effect. The significance of BRAF splice variants is uncertain but may represent another layer of complexity. Finally, if KRAS mutation is to be used for predictive testing, then the whole gene may need to be screened as mutations occur outside codons 12/13.

If stratified medicine is to be applied in the neoadjuvant setting, predictive testing will have to be undertaken on preoperative diagnostic biopsy specimens. The aim of this study was to evaluate whether a diagnostic biopsy was adequately representative of the main tumour in colorectal cancer.Thirty cases of paired biopsy and subsequent resection specimens were randomly selected. Samples were screened for mutation in KRAS (codons 12/13, 61, and 146), BRAF (codon 600 and exon 11), PIK3CA (exons 1, 9, and 20), TP53 (exons 5-8), and microsatellite instability, using the quick multiplex consensus or standard polymerase chain reaction (PCR) protocols followed by high-resolution melting analysis. A total of 570 paired PCR tests were performed for mutation detection, and identical results were obtained in both biopsy and resection specimens in 569 tests (99% concordance). Four cases (13%) showed microsatellite instability, and, in all four cases, instability was seen at identical mononucleotide markers in both biopsy and matched resection specimens.This is the first study to show that diagnostic biopsy specimens, even though they are a tiny sample of the tumour, are sufficiently representative for use in predictive testing for early driver mutations in colorectal cancer.

Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.

Background Mutation detection in tumours will become increasingly important in pathological diagnosis as ‘predictive’ mutations are identified. A cheap and reliable test that works on formalin-fixed paraffin-embedded (FFPE) tissue is required. Methods The quick-multiplex-consensus (QMC)-PCR protocol was developed to be used with high-resolution melting (HRM) analysis. The assay was compared with Sanger sequencing. Robustness of the assay was tested in DNA from FFPE tissue. Results QMC-PCR with HRM could detect a minimum of 2.5% of mutant alleles (compared with 20% detectable for Sanger sequencing). Ten mutation hotspots in KRAS , BRAF , PIK3CA and CDC4 were screened in 29 cell lines with 100% sensitivity and specificity. Forty-three FFPE colorectal tumours were sequenced for hotspots in KRAS and PIK3CA and then screened by QMC-PCR. There was 100% sensitivity, although, of 21 mutations detected by QMC-PCR, 16 were confirmed by sequencing (71% specificity, positive predictive value 76%). All 43 samples were then screened for mutations in all 10 hotspots. Of 430 tests, 43 (10%) showed aberrant melting and 36 were confirmed mutant (positive predictive value 84%). As our technique is more sensitive than direct sequencing, the remaining seven tests are probably sequencing false-negatives. Precision tests showed that there was little intra-assay and interassay variation. Conclusions QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues.