Shafi Ahmed

Analysis of colorectal cancers in British Bangladeshi identifies early onset, frequent mucinous histotype and a high prevalence of RBFOX1 deletion.

BackgroundPrevalence of colorectal cancer (CRC) in the British Bangladeshi population (BAN) is low compared to British Caucasians (CAU). Genetic background may influence mutations and disease features.MethodsWe characterized the clinicopathological features of BAN CRCs and interrogated their genomes using mutation profiling and high-density single nucleotide polymorphism (SNP) arrays and compared findings to CAU CRCs.ResultsAge of onset of BAN CRC was significantly lower than for CAU patients (p=3.0 x 10-5) and this difference was not due to Lynch syndrome or the polyposis syndromes. KRAS mutations in BAN microsatellite stable (MSS) CRCs were comparatively rare (5.4%) compared to CAU MSS CRCs (25%; p=0.04), which correlates with the high percentage of mucinous histotype observed (31%) in the BAN samples. No BRAF mutations was seen in our BAN MSS CRCs (CAU CRCs, 12%; p=0.08). Array data revealed similar patterns of gains (chromosome 7 and 8q), losses (8p, 17p and 18q) and LOH (4q, 17p and 18q) in BAN and CAU CRCs. A small deletion on chromosome 16p13.2 involving the alternative splicing factor RBFOX1 only was found in significantly more BAN (50%) than CAU CRCs (15%) cases (p=0.04). Focal deletions targeting the 5’ end of the gene were also identified. Novel RBFOX1 mutations were found in CRC cell lines and tumours; mRNA and protein expression was reduced in tumours.ConclusionsKRAS mutations were rare in BAN MSS CRC and a mucinous histotype common. Loss of RBFOX1 may explain the anomalous splicing activity associated with CRC.

Colorectal cancers with microsatellite instability display mRNA expression signatures characteristic of increased immunogenicity

BackgroundColorectal cancers displaying high-degree microsatellite instability (MSI-H) have an improved prognosis compared to microsatellite stable (MSS) cancers. The observation of pronounced lymphocytic infiltrates suggests that MSI-H cancers are inherently more immunogenic. We aimed to compare the gene expression profiles of MSI-H and MSS cancers to provide evidence for an activated immune response in the former.ResultsWe analysed tissue from 133 colorectal cancer patients with full consent and Local Ethics Committee approval. Genomic DNA was analysed for microsatellite instability in BAT-26. High-quality RNA was used for microarray analysis on the Affymetrix® HG-U133A chip. Data was analysed on GeneSpring software version 6.0. Confirmatory real-time RT-PCR was performed on 28 MSI-H and 26 MSS cancers. A comparison of 29 MSI-H and 104 MSS cancers identified 2070 genes that were differentially expressed between the two groups [P < 0.005]. Significantly, many key immunomodulatory genes were up-regulated in MSI-H cancers. These included antigen chaperone molecules (HSP-70, HSP-110, Calreticulin, gp96), pro-inflammatory cytokines (Interleukin (IL)-18, IL-15, IL-8, IL-24, IL-7) and cytotoxic mediators (Granulysin, Granzyme A). Quantitative RT-PCR confirmed up-regulation of HSP-70 [P = 0.016], HSP-110 [P = 0.002], IL-18 [P = 0.004], IL-8 [0.002] and Granulysin [P < 0.0001].ConclusionsThe upregulation of a large number of genes implicated in immune response supports the theory that MSI-H cancers are immunogenic. The novel observation of Heat Shock Protein up-regulation in MSI-H cancer is highly significant in light of the recognised roles of these proteins in innate and antigen-specific immunogenicity. Increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators also indicate an activated anti-tumour immune response.

Quantification of cytokeratin 20, carcinoembryonic antigen and guanylyl cyclase C mRNA levels in lymph nodes may not predict treatment failure in colorectal cancer patients

Conventional histopathologic staging of primary colorectal cancers does not allow accurate prognostic stratification within a given tumour stage. Therefore, PCR‐based assays are increasingly used to try to predict more accurately the likelihood of disease progression for the individual patient. Real‐time reverse transcription PCR (RT‐PCR) assays were used to detect and quantitate cytokeratin 20 (ck20), carcinoembryonic antigen (CEA) and guanylyl cyclase C (GCC) mRNA in 149 lymph nodes (LN) from 17 patients with benign disease and 302 LN from 42 patients with colorectal cancer who had curative (R0) resections. None of the markers were specific, with ck20, CEA and GCC mRNA detected in 47%, 89% and 13% of 149 LN, respectively, from patients with benign disease. The sensitivity of all 3 markers was very high, with mRNA detected in 93%, 100% and 97% of 30 histologically involved LN, respectively. There was significant overlap in the mRNA levels of all 3 markers between histologically involved and uninvolved LN. There was no association between mRNA levels and distant recurrence (median follow‐up: 3.94 years, range 3.35–5.12). We conclude that the use of molecular techniques to detect occult disease in LN may suffer from the same limitations as conventional methods. Instead, accurate prognostic stratification requires careful assessment of the likely metastatic potential of the primary cancer.

Differential Expression Patterns of the Insulin-Like Growth Factor 2 Gene in Human Colorectal Cancer

Tumour development and metastasis are associated with altered gene expression profiles. The aim of this study was to identify the transcriptional differences in normal, tumour and metastatic tissue. We used oligonucleotide arrays to identify differential expression patterns of insulin-like growth factor 2 (IGF 2) between 139 primary colorectal tumour specimens and 42 tumour-adjacent mucosa specimens from colorectal cancer (CRC) patients. The expression levels of the IGF 2 gene were significantly increased in primary tumours compared with adjacent mucosae. This was concordant with our real-time RT-PCR quantification of 48 matched tumour mucosa samples. IGF 2 expression levels were also measured by RT-PCR quantitative analysis in 18 liver metastases and 10 normal tissues from patients without cancer. The mRNA levels were significantly under-expressed in liver metastases compared with either colorectal tumours or adjacent normal mucosae. The non- malignant normal tissue expressed significantly lower IGF 2 levels than adjacent normal tissue, and this was not due to a field effect originating from the tumour. In addition, our microarray data demonstrated that IGF 2 expression was down-regulated in sporadic microsatellite instability (MSI-H) CRC and parallels under-expression of hMLH1 and IGF 2 receptor genes in these patients. We conclude that IGF 2 plays an important role in CRC development. Also, individuals with loss of genomic imprinting (LOI) causing over-expression of IGF 2 may be at greater risk of developing CRC. However, this LOI may be reversed in MSI-H patients.

Microarray profiling of colorectal cancer in Bangladeshi patients

Objective  We have carried out a retrospective analysis of all cases of colorectal cancer at the Royal London Hospital between April 1998 and March 2002 and determined the differences in presentation and outcome between Bangladeshi and Non‐Bangladeshi patients. DNA microarrays were used to explain any potential genetic differences between these two groups that may explain the different phenotypes.

Ex vivo study of human visceral nociceptors

Objective The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation. Design Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses. Results Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (−20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (−34.9±10.0%, n=7, p<0.05), a TRPV4 antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity. Conclusions Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Relative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer

Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.

The impact of medical student surgical conferences

Recent reports have shown that in the UK there has been a decrease in interest towards pursuing a surgical career, whereas early exposure to surgery may increase interest. We aimed to assess the effect of a 1‐day medical school surgical conference in encouraging attendees to pursue a surgical career.

Performance of a novel optical sensor for intraoperative assessment of intestinal viability - 'proof of principle' study

Introduction Objective assessment of intestinal viability during surgery will allow surgeons to make informed decisions on the safety of intestinal anastomosis and to predict anastomotic related complications.1We investigated the performance of a novel dual sensor incorporating two established methods – photoplethysmography (PPG) and laser Doppler flowmetry (LDF) in a ‘proof of principle’ study. Method Using the new probe, infrared and red PPG amplitude and LDF flux of the bowel surface were recorded by LDF and PPG simultaneously in 30 patients undergoing large bowel resection with or without anastomosis. Each patient had measurements at eight different time points: before and after colonic mobilisation, over the tumour, before and after ligation of major vascular pedicle, immediately before and after anastomosis (+/- 5 min) at distal and proximal limbs. Both laparoscopic and open resectional cases were included. The differences between the mean amplitude and flux between different measurements were analysed. Results Twenty four laparoscopic and six open bowel resection cases had a total of 121 measurements recorded using the new probe. The mean IR AC amplitude in pre-ligation was 433 (± 189) mV and the mean IR AC in post ligation was 301 (± 152) mV. A statistically significant difference (P = 0.047) was observed between pre-ligation and post-ligation for the mean infrared PPG amplitude (student’s t-test). A considerable difference in mean amplitude between pre and post ligation for the red PPG was also observed (the mean amplitude for the red PPG fell by –25.6%). The amplitude of the infrared PPG increased after anastomosis by 38.2% at the proximal site and by 37.8% at the distal site. The amplitude of the red PPG also increased after anastomosis (by 6.3%) at the proximal site although the equivalent increase was not seen at the distal site. The LDF measurements did not follow the same pattern of the changes seen in PPG amplitude. Conclusion The preliminary results show that simultaneous PPG/LDF measurements from a combined sensor is feasible and provide useful information on changes in pulse volume and blood flow. Although the differences in PPG amplitude achieved statistical significance, the LDF measurements produced confounding results, perhaps explained by motion artefacts. Further refinements in design of the probe are required to improve the probe-tissue optical interface and reduce motion artefact. Disclosure of interest None Declared. Reference Urbanavičius L, Pattyn P, Van de Putte D, Venskutonis D. How to assess intestinal viability during surgery: a review of techniques. World J Gastrointest Surg. 2011;3(5):59–69

Idiopathic necrotising fasciitis

An 82-year-old male was admitted to the accident and emergency department complaining of severe pain in his upper right thigh and sudden onset abdominal pain. On examination his temperature was 38.2°C, pulse rate 130 beats/minute and blood pressure 98/65 mm Hg. His right thigh was noted to be extremely tender, swollen and erythematous. In addition, crepitus was notable on palpation. His abdomen was diffusely tender, but was soft with no evidence of peritonism. With regard to his past medical history the patient was known to have insulin dependent …