Chronic kidney disease prevalence and clinical outcomes in anterior circulation acute ischemic stroke patients with reperfusion therapy: A meta-analysis.

AIM: Chronic Kidney Disease (CKD) is a common comorbidity among acute ischaemic stroke (AIS) patients undergoing reperfusion therapies, including intravenous thrombolysis (IVT) and endovascular thrombectomy (EVT). Acknowledging CKD's prevalence in this cohort and understanding its influence on outcomes is crucial for prognosis and optimizing care. This study aims to determine the prevalence of CKD among anterior circulation AIS (acAIS) patients undergoing reperfusion therapies and to analyse the role of CKD in mediating outcomes. METHODS: A random-effects meta-analysis was conducted to pool and examine prevalence data. A total of 263 633 patients were included in the meta-analysis. The study assessed CKD's association with functional outcomes, symptomatic intracranial haemorrhage (sICH) and mortality. RESULTS: The overall pooled prevalence of CKD among acAIS ranged from 30% to 56% in IVT-treated patients and 16%-42% for EVT-treated patients. CKD was associated with increased odds of unfavourable functional outcome at 90 days in both IVT (OR 1.837; 95% CI: [1.599; 2.110]; p < .001) and EVT (OR 1.804; 95% CI: [1.525; 2.133]; p < .001) groups. In IVT-treated patients, CKD was associated with increased odds of 30-day mortality (OR 6.211; 95% CI: [1.105; 34.909]; p = .038). CKD in IVT-treated patients exhibited increased odds of sICH, albeit statistically non-significant (OR 1.595; 95% CI: [0.567; 3.275]). CONCLUSIONS: The high prevalence of CKD and its significant impact on outcomes in acAIS patients treated with reperfusion therapies underscore its clinical significance. This insight can guide personalised care strategies and potentially improve the prognosis in the management of acAIS.

Short-chain fatty acid concentrations in the incidence and risk-stratification of colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: The beneficial role of gut microbiota and bacterial metabolites, including short-chain fatty acids (SCFAs), is well recognized, although the available literature around their role in colorectal cancer (CRC) has been inconsistent. METHODS: We performed a systematic review and meta-analysis to examine the associations of fecal SCFA concentrations to the incidence and risk of CRC. Data extraction through Medline, Embase, and Web of Science was carried out from database conception to June 29, 2022. Predefined inclusion/exclusion criteria led to the selection of 17 case-control and six cross-sectional studies for quality assessment and analyses. Studies were categorized for CRC risk or incidence, and RevMan 5.4 was used to perform the meta-analyses. Standardized mean differences (SMD) with 95% confidence intervals (CI) were calculated using a random-effects model. Studies lacking quantitation were included in qualitative analyses. RESULTS: Combined analysis of acetic, propionic, and butyric acid revealed significantly lower concentrations of these SCFAs in individuals with a high-risk of CRC (SMD = 2.02, 95% CI 0.31 to 3.74, P = 0.02). Additionally, CRC incidence was higher in individuals with lower levels of SCFAs (SMD = 0.45, 95% CI 0.19 to 0.72, P = 0.0009), compared to healthy individuals. Qualitative analyses identified 70.4% of studies reporting significantly lower concentrations of fecal acetic, propionic, butyric acid, or total SCFAs in those at higher risk of CRC, while 66.7% reported significantly lower concentrations of fecal acetic and butyric acid in CRC patients compared to healthy controls. CONCLUSIONS: Overall, lower fecal concentrations of the three major SCFAs are associated with higher risk of CRC and incidence of CRC.

Role of diabetes in stroke: Recent advances in pathophysiology and clinical management.

The increasing prevalence of diabetes and stroke is a major global public health concern. Specifically, acute stroke patients, with pre-existing diabetes, pose a clinical challenge. It is established that diabetes is associated with a worse prognosis after acute stroke and the various biological factors that mediate poor recovery profiles in diabetic patients is unknown. The level of association and impact of diabetes, in the setting of reperfusion therapy, is yet to be determined. This article presents a comprehensive overview of the current knowledge of the role of diabetes in stroke, therapeutic strategies for primary and secondary prevention of cardiovascular disease and/or stroke in diabetes, and various therapeutic considerations that may apply during pre-stroke, acute, sub-acute and post-stroke stages. The early diagnosis of diabetes as a comorbidity for stroke, as well as tailored post-stroke management of diabetes, is pivotal to our efforts to limit the burden. Increasing awareness and involvement of neurologists in the management of diabetes and other cardiovascular risk factors is desirable towards improving stroke prevention and efficacy of reperfusion therapy in acute stroke patients with diabetes.

Considerations for the selection of tests for SARS-CoV-2 molecular diagnostics.

During the course of 2020, the outbreak of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spread rapidly across the world. Clinical diagnostic testing for SARS-Cov-2 infection has relied on the real-time Reverse Transcriptase Polymerase Chain Reaction and is considered the gold standard assay. Commercial vendors and laboratories quickly mobilised to develop diagnostic tests to detect the novel coronavirus, which was fundamentally important in the pandemic response. These SARS-Cov-2 assays were developed in line with the Food Drug Administration-Emergency Use Authorization guidance. Although new tests are continuously being developed, information about SARS-CoV-2 diagnostic molecular test accuracy has been limited and at times controversial. Therefore, the analytical and clinical performance of SARS-CoV-2 test kits should be carefully considered by the appropriate regulatory authorities and evaluated by independent laboratory validation. This would provide improved end-user confidence in selecting the most reliable and accurate diagnostic test. Moreover, it is unclear whether some of these rapidly developed tests have been subjected to rigorous quality control and assurance required under good manufacturing practice. Variable target gene regions selected for currently available tests, potential mutation in target gene regions, non-standardized pre-analytic phase, a lack of manufacturer independent validation data all create difficulties in selecting tests appropriate for different countries and laboratories. Here we provide information on test criteria which are important in the assessment and selection of SARS-CoV-2 molecular diagnostic tests and outline the potential issues associated with a proportion of the tests on the market.

Neutrophil-lymphocyte ratio in acute ischemic stroke: Immunopathology, management, and prognosis.

There is an ongoing need for accurate prognostic biomarkers in the milieu of acute ischemic stroke (AIS) receiving reperfusion therapy. Neutrophil-lymphocyte ratio (NLR) has been implicated in emergency medicine and acute stroke setting as an important biomarker in the prognosis of patients. However, there are ongoing questions around its accuracy and translation into clinical practice given suboptimal sensitivity and specificity results, as well as varying thresholds and lack of clarity around which NLR time points are most clinically indicative. This article provides a comprehensive overview of the role of NLR in AIS patients receiving reperfusion therapy and perspectives on areas of future research. NLR may be an important biomarker in risk stratifying patients in AIS to identify and select those who are more likely to benefit from reperfusion therapy. Appropriate clinical decision-making tools and models are required to harness the predictive value of NLR, which could be useful in identifying and monitoring high-risk patients to guide early treatment and achieve improved outcomes. Our understanding of the role of NLR in the immunopathogenesis of AIS is also suboptimal, which hinders the ability to translate this into clinical practice.

Rate of dissemination and prognosis in early and advanced stage colorectal cancer based on microsatellite instability status: systematic review and meta-analysis.

INTRODUCTION: For the past two decades, microsatellite instability (MSI) has been reported as a robust clinical biomarker associated with survival advantage attributed to its immunogenicity. However, MSI is also associated with high-risk adverse pathological features (poorly differentiated, mucinous, signet cell, higher grade) and exhibits a double-edged sword phenomenon. We performed a systematic review and meta-analysis to evaluate the rate of dissemination and the prognosis of early and advanced stage colorectal cancer based on MSI status. METHODS: A systematic literature search of original studies was performed on Ovid searching MEDLINE, Embase, Cochrane Database of Systematic Reviews, American College of Physicians ACP Journal Club, Database of Abstracts of Reviews of Effects DARE, Clinical Trials databases from inception of database to June 2019. Colorectal cancer, microsatellite instability, genomic instability and DNA mismatch repair were used as key words or MeSH terms. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline was followed. Data were pooled using a random-effects model with odds ratio (OR) as the effect size. Statistical analysis was performed using RevMan ver 5.3 Cochrane Collaboration. RESULTS: From 5288 studies, 136 met the inclusion criteria (n = 92,035; MSI-H 11,746 (13%)). Overall, MSI-H was associated with improved OS (OR, 0.81; 95% CI 0.73-0.90), DFS (OR, 0.73; 95% CI 0.66-0.81) and DSS (OR, 0.69; 95% CI 0.52-0.90). Importantly, MSI-H had a protective effect against dissemination with a significantly lower rate of lymph node and distant metastases. By stage, the protective effect of MSI-H in terms of OS and DFS was observed clearly in stage II and stage III. Survival in stage I CRC was excellent irrespective of MSI status. In stage IV CRC, without immunotherapy, MSI-H was not associated with any survival benefit. CONCLUSIONS: MSI-H CRC was associated with an overall survival benefit with a lower rate of dissemination. Survival benefit was clearly evident in both stage II and III CRC, but MSI-H was neither a robust prognostic marker in stage I nor stage IV CRC without immunotherapy.

Micro RNA Expression after Ingestion of Fucoidan; A Clinical Study.

Fucoidans are a class of fucose-rich sulfated polysaccharides derived from brown macroalgae that exert a range of biological activities in vitro and in vivo. To generate an unbiased assessment of pathways and processes affected by fucoidan, a placebo-controlled double-blind pilot study was performed in healthy volunteers. Blood samples were taken immediately before and 24 h after ingestion of a single dose of 1 g of Undaria pinnatifida fucoidan (UPF) or placebo. Levels of isolated miRNAs were analyzed using Taqman Open Array Human MicroRNA panels. Out of 754 miRNAs screened, UPF affected a total of 53 miRNAs. Pathway analysis using the TALOS data analysis tool predicted 29 different pathways and processes that were largely grouped into cell surface receptor signaling, cancer-related pathways, the majority of which were previously associated with fucoidans. However, this analysis also identified nine pathways and processes that have not been associated with fucoidans before. Overall, this study illustrates that even a single dose of fucoidans has the potential to affect the expression of genes related to fundamental cellular processes. Moreover, it confirms previous data that fucoidans influence immunity, cancer cells, inflammation, and neurological function.

Essential developmental, genomic stability, and tumour suppressor functions of the mouse orthologue of hSSB1/NABP2.

Single-stranded DNA binding proteins (SSBs) regulate multiple DNA transactions, including replication, transcription, and repair. We recently identified SSB1 as a novel protein critical for the initiation of ATM signaling and DNA double-strand break repair by homologous recombination. Here we report that germline Ssb1(-/-) embryos die at birth from respiratory failure due to severe rib cage malformation and impaired alveolar development, coupled with additional skeletal defects. Unexpectedly, Ssb1(-/-) fibroblasts did not exhibit defects in Atm signaling or γ-H2ax focus kinetics in response to ionizing radiation (IR), and B-cell specific deletion of Ssb1 did not affect class-switch recombination in vitro. However, conditional deletion of Ssb1 in adult mice led to increased cancer susceptibility with broad tumour spectrum, impaired male fertility with testicular degeneration, and increased radiosensitivity and IR-induced chromosome breaks in vivo. Collectively, these results demonstrate essential roles of Ssb1 in embryogenesis, spermatogenesis, and genome stability in vivo.

New frontiers in circulating tumor cell analysis: A reference guide for biomolecular profiling toward translational clinical use.

Circulating tumor cells (CTCs) are now routinely isolated from blood, and measurement of CTC concentrations appears to correlate well with survival in patients with cancer. Interrogation of the molecular profile of CTCs for expression of protein biomarkers, genetic variants and gene expression provides opportunities to use this information to guide personalized treatment, monitor therapy and detect emerging resistance. However, successful application of profiling techniques requires analyses that deliver a reliable and clinically relevant representation of a patient's cancer as it changes with time. Here, we comprehensively review the current knowledge of therapeutically relevant biomarkers in isolated CTCs obtained by fluorescence imaging and genomic profiling approaches. The reviewed data support the notion that molecular profiling of CTCs will provide a reliable representation or surrogate index of tumor burden. Large-scale translational trials, many currently in progress, will provide critical data to progress CTC analysis toward wider clinical use in personalized treatment.

Chilodonella uncinata is not pathogenic to Culex pipiens mosquitoes.

Chilodonella uncinata was previously reported to be pathogenic to Culex and other mosquitoes based on the observation of high densities of ciliates inside of dead mosquito larvae. Here, we tested whether co-culturing Cx. pipiens with Ch. uncinata resulted in decreased survival of the mosquito larvae. We find no evidence that Ch. uncinata is pathogenic to Cx. pipiens. We suggest that Ch. uncinata may instead feed on already deceased larvae.

Circulating Tumour Cell Associated MicroRNA Profiles Change during Chemoradiation and Are Predictive of Response in Locally Advanced Rectal Cancer.

Locally advanced rectal cancer (LARC) has traditionally been treated with trimodality therapy consisting of neoadjuvant radiation +/- chemotherapy, surgery, and adjuvant chemotherapy. There is currently a clinical need for biomarkers to predict treatment response and outcomes, especially during neoadjuvant therapy. Liquid biopsies in the form of circulating tumour cells (CTCs) and circulating nucleic acids in particular microRNAs (miRNA) are novel, the latter also being highly stable and clinically relevant regulators of disease. We studied a prospective cohort of 52 patients with LARC, and obtained samples at baseline, during treatment, and post-treatment. We enumerated CTCs during chemoradiation at these three time-points, using the IsofluxTM (Fluxion Biosciences Inc., Alameda, CA, USA) CTC Isolation and detection platform. We then subjected the isolated CTCs to miRNA expression analyses, using a panel of 106 miRNA candidates. We identified CTCs in 73% of patients at baseline; numbers fell and miRNA expression profiles also changed during treatment. Between baseline and during treatment (week 3) time-points, three microRNAs (hsa-miR-95, hsa-miR-10a, and hsa-miR-16-1*) were highly differentially expressed. Importantly, hsa-miR-19b-3p and hsa-miR-483-5p were found to correlate with good response to treatment. The latter (hsa-miR-483-5p) was also found to be differentially expressed between good responders and poor responders. These miRNAs represent potential predictive biomarkers, and thus a potential miRNA-based treatment strategy. In this study, we demonstrate that CTCs are present and can be isolated in the non-metastatic early-stage cancer setting, and their associated miRNA profiles can potentially be utilized to predict treatment response.

Immunohistochemical testing of conventional adenomas for loss of expression of mismatch repair proteins in Lynch syndrome mutation carriers: a case series from the Australasian site of the colon cancer family registry.

Debate continues as to the usefulness of assessing adenomas for loss of mismatch repair protein expression to identify individuals with suspected Lynch syndrome. We tested 109 polyps from 69 proven mutation carriers (35 females and 34 males) belonging to 49 Lynch syndrome families. All polyps were tested by immunohistochemistry for four mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. Detailed pathology review was performed by specialist gastrointestinal pathologists. The majority of polyps (86%) were conventional adenomas (n=94), with 65 tubular and 28 tubulovillous adenomas and a single villous adenoma. The remaining 15 lesions (14%) were serrated polyps. Overall, loss of mismatch repair expression was noted for 78/109 (72%) of polyps. Loss of mismatch repair expression was seen in 74 of 94 (79%) conventional adenomas, and 4 of 15 (27%) serrated polyps from mismatch repair gene mutation carriers. In all instances, loss of expression was consistent with the underlying germline mutation. Mismatch repair protein expression was lost in 27 of 29 adenomas with a villous component compared with 47 of 65 adenomas without this feature (93 vs 73%; P=0.028). A strong trend was observed for high-grade dysplasia. Mismatch repair deficiency was observed in 12 of 12 conventional adenomas with high-grade dysplasia compared with 60 of 79 with low-grade dysplasia (100 vs 76%; P=0.065). We were unable to demonstrate a significant association between conventional adenoma size or site and mismatch repair deficiency. All (4/4 or 100%) of the serrated polyps demonstrating mismatch repair deficiency were traditional serrated adenomas from a single family. Diagnostic testing of adenomas in suspected Lynch syndrome families is a useful alternative in cases where cancers are unavailable. The overwhelming majority of conventional adenomas from mutation carriers show loss of mismatch repair protein expression concordant with the underlying germline mutation.

Role of Neutrophil-Lymphocyte Ratio in the Prognosis of Acute Ischaemic Stroke After Reperfusion Therapy: A Systematic Review and Meta-analysis.

Background: Inflammation may mediate response to acute reperfusion therapy (RT) in acute cerebral ischaemia. Neutrophil-lymphocyte ratio (NLR), an inflammatory biomarker, may play an important role in acute ischaemic stroke (AIS) prognostication. Objective: This meta-analysis sought to examine the effect of NLR on functional outcomes, mortality and adverse outcomes in AIS patients receiving RT. Methods: Individual studies were retrieved from PubMed/Medline, EMBASE and Cochrane databases. Data were extracted using a standardised data sheet and meta-analysis on association of admission (pre-RT) or delayed (post-RT) NLR with clinical/safety outcomes after RT was conducted. Results: Thirty-five studies (n = 10 308) were identified for the systematic review with 27 (n = 8537) included in the meta-analyses. Lower admission NLR was associated with good functional outcomes (GFOs), defined as 3-month modified Rankin scale (mRS) 0-2 (SMD = -.46; 95% CI = -.62 to -.29; P < .0001), mRS 0-1 (SMD = -.44; 95% CI = -.66 to -.22; P < .0001) and early neurological improvement (ENI) (SMD = -.55; 95 %CI = -.84 to -.25; P < .0001). Lower delayed admission NLR was also associated with GFOs (SMD = -.80; 95%CI = -.91 to -.68; P < .0001). Higher admission NLR was significantly associated with mortality (SMD = .49; 95%CI = .12 to .85; P = .009), intracerebral haemorrhage (ICH) (SMD = .34; 95% CI = .09 to .59; P = .007), symptomatic ICH (sICH) (SMD = .48; 95% CI = .07 to .90; P = .022) and stroke-associated infection or pneumonia (SMD = .85; 95% CI = .50, 1.19; P < .0001). Higher delayed NLR was significantly associated with sICH (SMD = 1.40; 95% CI = .60 to 2.19; P = .001), ICH (SMD = .94; 95% CI = .41 to 1.46; P < .0001) and mortality (SMD = 1.12; 95% CI = .57 to 1.67; P < .0001). There were variations in outcomes across RT groups. Conclusion: Higher admission or delayed NLR is significantly associated with worse morbidity, mortality and safety outcomes in AIS patients receiving RT.

Association of Lesion Topography with Functional Outcomes in Acute Ischemic Stroke Patients Considered for, or Receiving, Reperfusion Therapy: A Meta-Analysis.

Background: The impact of lesion topography (LT), characterised by the Alberta Stroke Programme Early CT Score (ASPECTS), on outcomes after reperfusion therapy in acute ischemic stroke (AIS) is poorly elucidated. We investigated the prognostic accuracy of ASPECTS-based LT assessment and its association with clinical outcomes in AIS patients considered for reperfusion therapy or receiving intravenous thrombolysis (IVT), endovascular thrombectomy (EVT), or none or both. Methods: Studies were identified from PubMed with additional studies added from Google Scholar. The prevalence of individual ASPECTS regions will also be determined. The association of individual ASPECTS regions with the functional outcome at 90 days will be assessed using random-effects modelling for various cut-offs, such as 6, 7 and 8. The association of continuous ASPECTS with the functional outcome at 90 days will also be undertaken. Forest plots of odds ratios (ORs) will be generated. Results: A total of 25 studies have been included in the final analysis, encompassing 11,404 patients. Pooled estimates indicate that the highest prevalence rates were in cases involving the insula and lentiform nucleus. Subgroup analysis for ASPECTS < 6 (OR 6.10; 95% CI 2.50−14.90; p < 0.0001), ASPECTS < 7 (OR 4.58; 95% CI 1.18−17.86; p < 0.0001) and ASPECTS < 8 (OR 2.26; 95% CI 1.32−3.89; p < 0.0001) revealed a significant association with poor functional outcome at 90 days. Decreasing ASPECTS significantly increased the odds of poor functional outcomes at 90 days (SMD −1.15; 95% CI −1.77−−0.52; p < 0.0001). Conclusions: Our meta-analysis demonstrates that decreasing ASPECTS is significantly associated with poor functional outcomes. Individual ASPECTS regions associated with the highest odds of poor functional outcomes were identified. Future studies on the association of LT and clinical outcomes specific to EVT are required.

Harnessing Liquid Biopsies to Guide Immune Checkpoint Inhibitor Therapy.

Immunotherapy (IO), involving the use of immune checkpoint inhibition, achieves improved response-rates and significant disease-free survival for some cancer patients. Despite these beneficial effects, there is poor predictability of response and substantial rates of innate or acquired resistance, resulting in heterogeneous responses among patients. In addition, patients can develop life-threatening adverse events, and while these generally occur in patients that also show a tumor response, these outcomes are not always congruent. Therefore, predicting a response to IO is of paramount importance. Traditionally, tumor tissue analysis has been used for this purpose. However, minimally invasive liquid biopsies that monitor changes in blood or other bodily fluid markers are emerging as a promising cost-effective alternative. Traditional biomarkers have limitations mainly due to difficulty in repeatedly obtaining tumor tissue confounded also by the spatial and temporal heterogeneity of tumours. Liquid biopsy has the potential to circumvent tumor heterogeneity and to help identifying patients who may respond to IO, to monitor the treatment dynamically, as well as to unravel the mechanisms of relapse. We present here a review of the current status of molecular markers for the prediction and monitoring of IO response, focusing on the detection of these markers in liquid biopsies. With the emerging improvements in the field of liquid biopsy, this approach has the capacity to identify IO-eligible patients and provide clinically relevant information to assist with their ongoing disease management.

Cytotoxic CD8+ T cells and tissue resident memory cells in colorectal cancer based on microsatellite instability and BRAF status.

BACKGROUND: Recent studies in non-colorectal malignancy have associated T resident memory (TRM) cells with improved patient survival. It is unknown if TRM plays a role in colorectal cancer (CRC). AIM: To examine the potential role of TRM cells in providing immunogenicity in CRC stratified by microsatellite instability (MSI) and BRAF status. METHODS: Patients with known MSI and BRAF mutation status were eligible for inclusion in this study. CRC tumour sections stained with haematoxylin and eosin were microscopically reviewed and the images scanned prior to assessment for location of invading edge and core of tumour. Sequential sections were prepared for quantitative multiplex immunohistochemistry (IHC) staining. Opal Multiplex IHC staining was performed with appropriate positive and negative controls and imaged using a standard fluorescent microscope fitted with a spectral scanning camera (Mantra) in conjunction with Mantra snap software. Images were unmixed and annotated in inForm 2.2.0. Statistical analysis was performed using Graphpad Prism Version 7 and Stata Version 15. RESULTS: Seventy-two patients with known MSI and BRAF status were included in the study. All patients were assessed for MSI by IHC and high resolution capillary electrophoresis testing and 44 of these patients successfully underwent quantitative multiplex IHC staining. Overall, there was a statistically significant increase in CD8+ TRM cells in the MSI (BRAF mutant and wild type) group over the microsatellite stable (MSS) group. There was a statistically significant difference in CD8+ TRM between high level MSI (MSI-H):BRAF mutant [22.57, 95% confidence interval (CI): 14.31-30.84] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0076 andMSI-H:BRAF wild type [16.18 (95%CI: 10.44-21.93)] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0279. There was no statistically significant difference in CD8 T cells (both CD8+CD103- and CD8+CD103+TRM) between MSI-H: BRAF mutant and wild type CRC. CONCLUSION: This study has shown that CD8+ TRM are found in greater abundance in MSI-H CRC, both BRAF mutant and MSI-H:BRAF wild type, when compared with their MSS counterpart. CD8+ TRM may play a role in the immunogenicity in MSI-H CRC (BRAF mutant and BRAF wild type). Further studies should focus on the potential immunogenic qualities of TRM cells and investigate potential immunotherapeutic approaches to improve treatment and survival associated with CRC.

A Simple, Accurate and Cost-Effective Capillary Electrophoresis Test with Computational Methods to Aid in Universal Microsatellite Instability Testing.

BACKGROUND: Microsatellite instability (MSI) testing is important for the classification of Lynch syndrome, as a prognostic marker and as a guide for adjuvant chemotherapy in colorectal cancer (CRC). The gold standard for determining MSI status has traditionally been fluorescent multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE). However, its use in the clinical setting has diminished and has been replaced by immunohistochemical (IHC) detection of loss of mismatch repair protein expression due to practicability and cost. The aim of this study was to develop a simple, cost-effective and accurate MSI assay based on CGE. METHOD: After amplification of microsatellites by polymerase chain reaction (PCR) using the National Cancer Institute (NCI) panel (BAT 25, BAT26, D5S346, D2S123, D17S250) of MSI markers, parallel CGE was utilized to classify colorectal cancers as MSI-H, MSI-L and MSS using the 5200 Fragment Analyzer System. Cell lines and patient cancer specimens were tested. DNA from 56 formalin-fixed paraffin-embedded cancer specimens and matched normal tissue were extracted and CGE was performed. An automated computational algorithm for MSI status determination was also developed. RESULTS: Using the fragment analyser, MSI status was found to be 100% concordant with the known MSI status of cell lines and was 86% and 87% concordant with immunohistochemistry (IHC) from patient cancer specimens using traditional assessment and our MSI scoring system, respectively, for MSI determination. The misclassification rate was mainly attributed to IHC, with only one (1.8%) sampling error attributed to CGE testing. CGE was also able to distinguish MSI-L from MSI-H and MSS, which is not possible with IHC. An MSI score based on total allelic variability that can accurately determine MSI status was also successfully developed. A significant reduction in cost compared with traditional fluorescent multiplex PCR and CGE was achieved with this technique. CONCLUSIONS: A simple, cost-effective and reliable method of determining MSI status and an MSI scoring system based on an automatic computational algorithm to determine MSI status, as well as degree of allelic instability in colorectal cancer, has been developed using the 5200 Fragment Analyzer System.

Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation.

BACKGROUND: Thrombospondin-4 (THBS4) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of THBS4 expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter. METHODS: Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of THBS4 transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon. RESULTS: THBS4 expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and THBS4 methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high THBS4 methylation, but the correlation was not significant. THBS4 methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon. CONCLUSIONS: THBS4 shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence THBS4 expression. THBS4 may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation in vitro. THBS4 methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.

Association Between Microsatellite Instability Status and Peri-Operative Release of Circulating Tumour Cells in Colorectal Cancer.

Microsatellite instability (MSI) in colorectal cancer (CRC) is a marker of immunogenicity and is associated with an increased abundance of tumour infiltrating lymphocytes (TILs). In this subgroup of colorectal cancer, it is unknown if these characteristics translate into a measurable difference in circulating tumour cell (CTC) release into peripheral circulation. This is the first study to compare MSI status with the prevalence of circulating CTCs in the peri-operative colorectal surgery setting. For this purpose, 20 patients who underwent CRC surgery with curative intent were enrolled in the study, and peripheral venous blood was collected at pre- (t1), intra- (t2), immediately post-operative (t3), and 14-16 h post-operative (t4) time points. Of these, one patient was excluded due to insufficient blood sample. CTCs were isolated from 19 patients using the IsofluxTM system, and the data were analysed using the STATA statistical package. CTC number was presented as the mean values, and comparisons were made using the Student t-test. There was a trend toward increased CTC presence in the MSI-high (H) CRC group, but this was not statistically significant. In addition, a Poisson regression was performed adjusting for stage (I-IV). This demonstrated no significant difference between the two MSI groups for pre-operative time point t1. However, time points t2, t3, and t4 were associated with increased CTC presence for MSI-H CRCs. In conclusion, there was a trend toward increased CTC release pre-, intra-, and post-operatively in MSI-H CRCs, but this was only statistically significant intra-operatively. When adjusting for stage, MSI-H was associated with an increase in CTC numbers intra-operatively and post-operatively, but not pre-operatively.

Bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer development.

Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well-described functions in bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down-regulated as assessed by real-time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hypermethylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aberrant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01), the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001). Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re-introduction of BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tumor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early and frequent event in colorectal tumors progressing via the serrated and traditional pathways.