Use of ColonFlag score for prioritisation of endoscopy in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is the fourth most common cancer in UK. Symptomatic patients are referred via an urgent pathway and although most are investigated with colonoscopy <4% are diagnosed with cancer. There is therefore a need for a suitable triage tool to prioritise investigations. This study retrospectively examined performance of various triage tools in patients awaiting investigation on the urgent lower gastrointestinal cancer pathway DESIGN: All patients over 40 years of age on the urgent pathway awaiting investigation for suspected CRC on 1 May were included. After 6 months, outcomes were evaluated and the performance of the faecal immunochemical test (FIT), faecal haemoglobin concentration, age and sex test (FAST) and the artificial intelligence algorithm ColonFlag were examined. RESULTS: 532 completed investigations and received a diagnosis; 15 had CRC. 388 had a valid FIT result, of whom 11 had CRC; FAST Score ≥4.5 had sensitivity of 72.7%, specificity of 80.6% and would have failed to detect three tumours. Faecal haemoglobin (f-Hb) at cut-off of 10 µg/g and ColonFlag had equal sensitivity of 81.82%, ColonFlag had greater specificity 73.47%, compared with 64.99%. Both tests would have failed to detect two tumours but not in the same patients; when used in combination, sensitivity and specificity were 100% and 49.4%. When ColonFlag was applied to the cohort of 532, an additional four tumours would have been detected in patients without a valid FIT. CONCLUSION: This study showed ColonFlag to have equal sensitivity and greater specificity than f-Hb at a cut-off of 10 µg/g as a triage tool for CRC.

Potential roles of artificial intelligence learning and faecal immunochemical testing for prioritisation of colonoscopy in anaemia.

Iron deficiency anaemia (IDA) is the most common cause of anaemia and a frequent indication for colonoscopy, although the prevalence of colorectal cancer (CRC) in IDA is low. Measurement of faecal haemoglobin by immunochemical techniques (FIT) is used to detect symptomatic patients. We studied FIT in patients with anaemia attending a gastroenterology clinic in Plymouth and looked at an artificial intelligence (AI) learning algorithm (ColonFlag™) in these patients, together with a cohort who had undergone colonoscopy for IDA in London. Of 592 patients referred on the basis of haemoglobin concentration, 21 (3.5%) had CRC. Using ColonFlag™, rather than haemoglobin concentration, in combination with symptoms, would have resulted in prioritisation of 304 patients for urgent referral rather than 592. One CRC would have been missed but might have been detected by FIT, which was not available in this case. In patients aged <55 years in whom the incidence of CRC is low, 15 rather than 109 patients would have been prioritised for urgent referral with no cancers missed. FIT has a high negative predictive value in IDA so its use may enable some patients to avoid investigation and AI learning may be a more useful trigger than haemoglobin concentration for urgent referral for colonoscopy.

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer.

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients.

Can exome scans be expected to be part of real-time decision-making in patients with haematological cancers?

The laboratory aspects of diagnosis of patients with haematological malignancies are forever changing. Microscopic examination of blood and bone marrow smears, cytogenetics, flow cytometry and single-assay molecular diagnostics have been and are still essential tools in cancer diagnostics. Flow cytometry has brought the unprecedented possibility of rapid multiplexing and characterization of complex immunophenotypic patterns. However, the advent of next generation sequencing in the haematology laboratory brings a whole new perspective on multiplexing and potentially lowers the cost per analysis effectively. These informative methods still require skilled technicians and bioinformaticians, evolve at a rapid pace, and call for clinical guidelines and best practice. Here, we discuss the potential and caveats of whole exome sequencing as it moves closer to routine laboratory practice. The question is: Will exome sequencing be performed, real-time, in the standard haemodiagnostic laboratory?

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML.

In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18(hi1727/+) embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

GCS-100, a novel galectin-3 antagonist, modulates MCL-1, NOXA, and cell cycle to induce myeloma cell death.

GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study, the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation, accumulation of cells in sub-G(1) and G(1) phases, and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-X(L) levels also occurred, accompanied by a rapid induction of NOXA protein, whereas BCL-2, BAX, BAK, BIM, BAD, BID, and PUMA remained unchanged. The cell-cycle inhibitor p21(Cip1) was up-regulated by GCS-100, whereas the procycling proteins CYCLIN E2, CYCLIN D2, and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IkappaBalpha, IkappaB kinase, and AKT as well as lack of IkappaBalpha and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1, tumor necrosis factor-alpha). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule, GCS-100, is a potent modifier of myeloma cell biology targeting apoptosis, cell cycle, and intracellular signaling and has potential for myeloma therapy.