Evaluation of epigenetic methylation biomarkers for the detection of colorectal cancer using droplet digital PCR.

Colorectal cancer (CRC) is the third most common cancer worldwide. Screening programs allow early diagnosis and have improved the clinical management of this disease. Aberrant DNA methylation is increasingly being explored as potential biomarkers for many types of cancers. In this study we investigate the methylation of ten target genes in 105 CRC and paired normal adjacent colonic tissue samples using a MethylLight droplet digital PCR (ML-ddPCR) assay. Receiver operator characteristic (ROC) curves were used to determine the diagnostic performance of all target genes individually and in combination. All 515 different combinations of genes showed significantly higher levels of methylation in CRC tissue. The combination of multiple target genes into a single test generally resulted in greater diagnostic accuracy when compared to single target genes. Our data confirms that ML-ddPCR is able to reliably detect significant differences in DNA methylation between CRC tissue and normal adjacent colonic tissue in a specific selection of target genes.

CD36 polymorphisms and the age of disease onset in patients with pathogenic variants within the mutation cluster region of APC.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant condition that predisposes patients to colorectal cancer. FAP is the result of a loss of APC function due to germline pathogenic variants disrupting gene expression. Genotype-phenotype correlations are described for FAP. For example attenuated forms of the disease are associated with pathogenic variants at the 5' and 3' ends of APC whilst severe forms of the disease appear to be linked to variants occurring in the mutation cluster region (MCR) of the gene. Variants occurring in the MCR are phenotypically associated with hundreds to thousands of adenomas carpeting the colon and rectum and patients harbouring changes in this region have a high propensity to develop colorectal cancer. Not all patients who carry pathogenic variants in this region have severe disease which may be a result of environmental factors. Alternatively, phenotypic variation observed in these patients could be due to modifier genes that either promote or inhibit disease expression. Mouse models of FAP have provided several plausible candidate modifier genes, but very few of these have survived scrutiny. One such genetic modifier that appears to be associated with disease expression is CD36. We previously reported a weak association between a polymorphism in CD36 and a later age of disease onset on a relatively small FAP patient cohort. METHODS: In the current study, we enlarged the FAP cohort. 395 patients all carrying pathogenic variants in APC were tested against three CD36 Single Nucleotide Polymorphisms (SNP)s (rs1049673, rs1761667 rs1984112), to determine if any of them were associated with differences in the age of disease expression. RESULTS: Overall, there appeared to be a statistically significant difference in the age of disease onset between carriers of the variant rs1984112 and wildtype. Furthermore, test equality of survivor functions for each SNP and mutation group suggested an interaction in the Log Rank, Wilcoxon, and Tarone-Ware methods for rs1049673, rs1761667, and rs1984112, thereby supporting the notion that CD36 modifies disease expression. CONCLUSIONS: This study supports and strengthens our previous findings concerning CD36 and an association with disease onset in FAP, AFAP and FAP-MCR affected individuals. Knowledge about the role CD36 in adenoma development may provide greater insight into the development of colorectal cancer.

C-reactive protein trajectory to predict colorectal anastomotic leak: PREDICT Study.

BACKGROUND: Anastomotic leak is a common complication after colorectal surgery, associated with increased morbidity and mortality, and poorer long-term survival after oncological resections. Early diagnosis improves short-term outcomes, and may translate into reduced cancer recurrence. Multiple studies have attempted to identify biomarkers to enable earlier diagnosis of anastomotic leak. One study demonstrated that the trajectory of C-reactive protein (CRP) levels was highly predictive of anastomotic leak requiring intervention, with an area under the curve of 0·961. The aim of the present study was to validate this finding externally. METHODS: This was a prospective international multicentre observational study of adults undergoing elective colorectal resection with an anastomosis. CRP levels were measured before operation and for 5 days afterwards, or until day of discharge if earlier than this. The primary outcome was anastomotic leak requiring operative or radiological intervention. RESULTS: Between March 2017 and July 2018, 933 patients were recruited from 20 hospitals across Australia, New Zealand, England and Scotland. Some 833 patients had complete CRP data and were included in the primary analysis, of whom 41 (4·9 per cent) developed an anastomotic leak. A change in CRP level exceeding 50 mg/l between any two postoperative days had a sensitivity of 0·85 for detecting a leak, and a high negative predictive value of 0·99 for ruling it out. A change in CRP concentration of more than 50 mg/l between either days 3 and 4 or days 4 and 5 after surgery had a high specificity of 0·96-0·97, with positive likelihood ratios of 4·99-6·44 for a leak requiring intervention. CONCLUSION: This study confirmed the value of CRP trajectory in accurately ruling out an anastomotic leak after colorectal resection.

ANTECEDENTES: La fuga anastomótica es una complicación frecuente después de la cirugía colorrectal que se asocia con morbilidad y mortalidad, con una peor supervivencia a largo plazo tras resecciones oncológicas. El diagnóstico precoz mejora los resultados a corto plazo y puede traducirse en una reducción de la recidiva del cáncer. Múltiples estudios han tratado de identificar biomarcadores para lograr un diagnóstico precoz de la fuga anastomótica. Un estudio demostró que la evolución de la proteína C reactiva (PCR) era altamente predictiva de una fuga anastomótica que requería intervención, con un área bajo la curva de 0,961. Nuestro estudio tuvo como objetivo validar externamente este hallazgo. MÉTODOS: Se llevó a cabo un estudio internacional prospectivo observacional y multicéntrico de pacientes adultos sometidos a resección colorrectal electiva con anastomosis. Los niveles de PCR se midieron antes de la operación y diariamente hasta el día 5 después de la cirugía, o hasta el día del alta si fue anterior. El criterio de valoración principal fue la fuga anastomótica que requirió intervención quirúrgica o radiológica. RESULTADOS: Entre marzo de 2017 y julio de 2018, se reclutaron 933 pacientes de 20 hospitales de Australia, Nueva Zelanda, Inglaterra y Escocia. Se obtuvieron datos completos de PCR en 833 pacientes y se incluyeron en el análisis primario, de los cuales 41 (4,9%) presentaron una fuga anastomótica. Un aumento de la PCR > 50 mg/L entre dos días del postoperatorio fue sensible para detectar una fuga (0,85) y tuvo un alto valor predictivo negativo para descartarla (0,99). El porcentaje de cambio de PCR > 50 mg/L por día entre los días 3-4 o 4-5 después de la operación fue altamente específico (0,96) con un cociente de probabilidad positivo > 5,0 para las fugas que requirieron una intervención. CONCLUSIÓN: Este estudio confirma la utilidad de la evolución de la PCR para descartar con precisión una fuga anastomótica después de una resección colorrectal.

Randomized clinical trial of short-term outcomes following purse-string versus conventional closure of ileostomy wounds.

BACKGROUND: Ileostomy closure is an operation with an underappreciated morbidity, including surgical-site infection, small bowel obstruction and anastomotic leakage. Surgical-site infections, in particular, are a frequent occurrence following closure of contaminated wounds. This randomized controlled trial compared a purse-string closure technique with conventional linear closure. METHODS: Sixty-one patients were randomized to conventional or purse-string closure of ileostomy wounds. The primary endpoint was the incidence of surgical-site infection, including infections requiring hospital or community treatment. RESULTS: Purse-string closure resulted in fewer surgical-site infections than conventional closure: two of 30 versus 12 of 31 respectively (P = 0.005). CONCLUSION: The purse-string method results in a clinically relevant reduction in surgical-site infections after ileostomy closure. REGISTRATION NUMBER: ACTRN12609000021279 (Australian New Zealand Clinical Trials Registry: http://www.anzctr.org.au/).

Recognition of skin malignancy by general practitioners: observational study using data from a population-based randomised controlled trial.

Skin malignancy is an important cause of mortality in the United Kingdom and is rising in incidence every year. Most skin cancer presents in primary care, and an important determinant of outcome is initial recognition and management of the lesion. Here we present an observational study of interobserver agreement using data from a population-based randomised controlled trial of minor surgery. Trial participants comprised patients presenting in primary care and needing minor surgery in whom recruiting doctors felt to be able to offer treatment themselves or to be able to refer to a colleague in primary care. They are thus relatively unselected. The skin procedures undertaken in the randomised controlled trial generated 491 lesions with a traceable histology report: 36 lesions (7%) from 33 individuals were malignant or pre-malignant. Chance-corrected agreement (kappa) between general practitioner (GP) diagnosis of malignancy and histology was 0.45 (0.36-0.54) for lesions and 0.41 (0.32-0.51) for individuals affected with malignancy. Sensitivity of GPs for the detection of malignant lesions was 66.7% (95% confidence interval (CI), 50.3-79.8) for lesions and 63.6% (95% CI, 46.7-77.8) for individuals affected with malignancy. The safety of patients is of paramount importance and it is unsafe to leave the diagnosis and treatment of potential skin malignancy in the hands of doctors who have limited training and experience. However, the capacity to undertake all of the minor surgical demand works demanded in hospitals does not exist. If the capacity to undertake it is present in primary care, then the increased costs associated with enhanced training for general medical practitioners (GPs) must be borne.

A prospective randomised comparison of minor surgery in primary and secondary care. The MiSTIC trial.

OBJECTIVE: To determine whether there is equivalence in the competence of GPs and hospital doctors to perform a range of elective minor surgical procedures, in terms of the safety, quality and cost of care. DESIGN: A prospective randomised controlled equivalence trial was undertaken in consenting patients presenting at general practices and needing minor surgery. SETTING: The study was conducted in the south of England. PARTICIPANTS: Consenting patients presenting at general practices who needed minor surgery in specified categories for whom the recruiting doctor felt able to offer treatment or to be able to refer to a colleague in primary care. INTERVENTIONS: On presentation to their GP, patients were randomised to either treatment within primary care or treatment at their local hospital. Evaluation was by assessment of clinical quality and safety of outcome, supplemented by examination of patient satisfaction and cost-effectiveness. MAIN OUTCOME MEASURES: Two independent observers assessed surgical quality by blinded assessment of wound appearance, between 6 and 8 weeks postsurgery, from photographs of wounds. Other measures included satisfaction with care, safety of surgery in terms of recognition of and appropriate treatment of skin malignancies, and resource use and implications. RESULTS: The 568 patients recruited (284 primary care, 284 hospital) were randomised by 82 GPs. In total, 637 skin procedures plus 17 ingrowing toenail procedures were performed (313 primary care, 341 hospital) by 65 GPs and 60 hospital doctors. Surgical quality was assessed for 273 (87%) primary care and 316 (93%) hospital lesions. Mean visual analogue scale score in hospital was significantly higher than that in primary care [mean difference=5.46 on 100-point scale; 95% confidence interval (CI) 0.925 to 9.99], but the clinical importance of the difference was uncertain. Hospital doctors were better at achieving complete excision of malignancies, with a difference that approached statistical significance [7/16 GP (44%) versus 15/20 hospital (75%), chi(2)=3.65, p=0.056]. The proportion of patients with post-operative complications was similar in both groups. The mean cost for hospital-based minor surgery was 1222.24 pounds and for primary care 449.74 pounds. Using postoperative complications as an outcome, both effectiveness and costs of the alternative interventions are uncertain. Using completeness of excision of malignancy as an outcome, hospital minor surgery becomes more cost-effective. The 705 skin procedures undertaken in this trial generated 491 lesions with a traceable histology report: 36 lesions (7%) from 33 individuals were malignant or premalignant. Chance-corrected agreement (kappa) between GP diagnosis of malignancy and histology was 0.45 (95% CI 0.36 to 0.54) for lesions and 0.41 (95% CI 0.32 to 0.51) for individuals affected by malignancy. Sensitivity of GPs for detection of malignant lesions was 66.7% (95% CI 50.3 to 79.8) for lesions and 63.6% (95% CI 46.7 to 77.8) for individuals affected by malignancy. CONCLUSIONS: The quality of minor surgery carried out in general practice is not as high as that carried out in hospital, using surgical quality as the primary outcome, although the difference is not large. Patients are more satisfied if their procedure is performed in primary care, largely because of convenience. However, there are clear deficiencies in GPs' ability to recognise malignant lesions, and there may be differences in completeness of excision when compared with hospital doctors. The safety of patients is of paramount importance and this study does not demonstrate that minor surgery carried out in primary care is safe as it is currently practised. There are several alternative models of minor surgery provision worthy of consideration, including ones based in primary care that require all excised tissue to be sent for histological examination, or that require further training of GPs to undertake the necessary work. The results of this study suggest that a hospital-based service is more cost-effective. It must be concluded that it is unsafe to leave minor surgery in the hands of doctors who have never been trained to do it. Further work is required to determine GPs' management of a range of skin conditions (including potentially life-threatening malignancies), rather than just their recognition of them. Further economic modelling work is required to look at the potential costs of training sufficient numbers of GPs and GPs with special interests to meet the demand for minor surgery safely in primary care, and of the alternative of transferring minor surgery large-scale to the hospital sector. Different models of provision need thorough testing before widespread introduction.