Semi-automated computational assessment of cancer organoid viability using rapid live-cell microscopy

The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess cellular viability in tissue specimens are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed 'Apex Imaging'. We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.

Serial measurement of cytokines strongly predict COVID-19 outcome

Purpose Cytokines are major mediators of COVID-19 pathogenesis and several of them are already being regarded as predictive markers for the clinical course and outcome of COVID-19 cases. A major pitfall of many COVID-19 cytokine studies is the lack of a benchmark sampling timing. Since cytokines and their relative change during an infectious disease course is quite dynamic, we evaluated the predictive value of serially measured cytokines for COVID-19 cases. Methods In this single-center, prospective study, a broad spectrum of cytokines were determined by multiplex ELISA assay in samples collected at admission and at the third day of hospitalization. Appropriateness of cytokine levels in predicting mortality were assessed by receiver-operating characteristic (ROC) analyses for both sampling times in paralel to conventional biomarkers. Results At both sampling points, higher levels of IL-6, IL-7, IL-10, IL-15, IL-27 IP-10, MCP-1, and GCSF were found to be more predictive for mortality (p<0.05). Some of these cytokines, such as IL-6, IL-10, IL-7 and GCSF, had higher sensitivity and specificity in predicting mortality. AUC values of IL-6, IL-10, IL-7 and GCSF were 0.85 (0.65 to 0.92), 0.88 (0.73 to 0.96), 0.80 (0.63 to 0.91) and 0.86 (0.70 to 0.95), respectively at hospital admission. Compared to hospital admission, on the 3rd day of hospitalization serum levels of IL-6 and, IL-10 decreased significantly in the survivor group, unlike the non-survivor group (IL-6, p = 0.015, and IL-10, p = 0.016). Conclusion Our study results suggest that single-sample-based cytokine analyzes can be misleading and that cytokine levels measured serially at different sampling times provide a more precise and accurate estimate for the outcome of COVID-19 patients.

P-P04 The cost of acute pancreatitis is amylasing!

Background Amylase is the key serum biomarker in the diagnosis of acute pancreatitis, however there is no indication for repeat/serial measurement once the diagnosis is established. It is estimated that £27,000pa is spent unnecessarily on repeat amylase investigations without clinical indication1. Anecdotally, within the department, unnecessary repeats were being routinely performed. Resultantly, we audited in 2019 and 2020 to understand the extent of the issue. Following the first cycle, pre-rotation departmental talks were given to all relevant healthcare staff. Aim(s) Methods Data was collected and analysed retrospectively over 2 audit cycles (C1 & C2) from 79 patient episodes of admissions to the surgical department of a Northern Major Trauma Centre with confirmed diagnoses of acute pancreatitis between 01/05/2019 – 31/07/2019 and 01/08/2020 – 31/12/2020. Resources used included: patient notes, IMPAX and WebICE. Data was collected and analysed by one author in C1 but multiple authors in C2. Results Mean age = 60 years. Male:Female ratio was 8:16 and 24:31, respectively. Initial amylase was diagnostic in &gt; 75% (61/79). 81 unnecessary repeats performed. Most patients underwent imaging (75% and 67%) however, only approximately one-third (30.8% and 32.4%) of scans were performed to confirm the diagnosis. • Despite imaging confirming the diagnosis in 88%, &gt;50% of imaged patients had repeat amylase testing. Conclusions The results demonstrate that our intervention, a pre-rotation departmental talk, has significantly reduced the over-requesting of amylase and current practice is of a good standard. Improvements are still required. Resultantly, we are additionally producing an electronic ‘alert’ into our investigations software that, on requesting a repeat amylase, will prompt clinicians to consider its necessity. Other centres offering acute treatment for similar patients may benefit from performing a similar audit to optimise care while reducing overall clinical costs.

TROPONIN T AFTER CARDIAC SURGERY: WHAT IS NORMAL? A REVIEW OF THE LITERATURE

It is well established that cardiac biomarkers are universally raised post-cardiac surgery. This is due to various preoperative, intraoperative, and postoperative factors. At present, no reliable tool or model considers intraoperative and postoperative factors to predict morbidity and mortality after cardiac surgery. Troponin T as a single or serial measurement to predict postoperative mortality and morbidity, is an attractive diagnostic tool due to the direct relationship to myocardial damage and the availability of testing. Currently, there is no consensus regarding the expected release of troponin T post-cardiac surgery. From a diagnostic and prognostic point of view, troponin T testing could be beneficial in recognising high-risk patients and any imminent compilations early. Therefore, this review aims to assess the expected release profile and prognostic value of troponin T after cardiac surgery. Keywords: troponin T., CABG, MPM, SAPS

Obeticholic Acid Improves Aminotransferases Early during Treatment in Patients with Primary Biliary Cholangitis Not Responding to Ursodeoxycholic Acid: A Study in Clinical Practice

Obeticholic acid (OCA) improves cholestasis and is generally well tolerated in patients with primary biliary cholangitis (PBC) not responding, or intolerant, to ursodeoxycholic acid (UDCA). As PBC is mainly a cholestatic disorder, less attention is paid to aminotransferase behavior in the course of treatment. In this study we evaluated, in clinical practice, the efficacy of OCA treatment on both alkaline phosphatase (ALP) and alanine aminotransferase (ALT) using updated healthy ranges for aminotransferases. Fifteen PBC patients, non-responders to UDCA, were evaluated at baseline and during OCA treatment with serial measurement of cholestasis indexes and ALT, that were also assessed using updated normal ranges (<30 IU/L in males, <19 IU/L in females). Median ALP and ALT decreased from 2.16 to 1.27 × upper limit of normal (p = 0.003) and from 0.93 to 0.78 × upper limit of normal (p = 0.008), respectively, in the course of OCA treatment. At treatment day-15, median ALT decreased by 29.7% and ALP by 8.8%. Bilirubin and albumin were unmodified throughout treatment. Using updated normal ranges, ALT levels were normal in 6.7% of patients at baseline and in 33.3% of patients at 18 months of treatment. OCA treatment improves cholestasis and, also, indexes of hepatocyte necrosis, with a decline in necro-inflammatory activity even predating the improvement in cholestasis. Use of recalibrated healthy ranges for aminotransferases might be a useful tool to assess hepatic histological activity and its improvement with OCA treatment.

Clinical Relevance of the Serial Measurement of Krebs von den Lungen-6 Levels in Patients with Systemic Sclerosis-Associated Interstitial Lung Disease

Krebs von den Lungen-6 (KL-6) levels measured at baseline have been reported as a circulating biomarker useful for the detection, evaluation of severity and assessment of risk of the progression of interstitial lung disease (ILD) in patients with systemic sclerosis (SSc). In this retrospective study, longitudinal changes in serum KL-6 levels over 2 years were examined in 110 patients with SSc using prospectively collected cohort data. Serum KL-6 levels fluctuated in a significant proportion of the patients but remained stable in the remaining patients. A wide range of variability of longitudinal KL-6 levels was associated with the presence of ILD, diffuse cutaneous SSc, positive anti-topoisomerase I antibodies, negative anticentromere antibodies, increased ILD extent on high-resolution computed tomography, extensive disease, low pulmonary function parameters, high KL-6 levels at baseline and immunomodulatory treatment. Extensive disease was consistently identified as an independent factor associated with variability in KL-6 levels in different models of multiple regression analysis. We failed to demonstrate correlations between trends for KL-6 level changes during the 6 months after SSc diagnosis and ILD progression over 2 years in patients with SSc-ILD. Serum KL-6 levels fluctuate in SSc patients with ILD, especially in those with extensive disease, but the clinical utility of a serial KL-6 level measurement remains uncertain.

EP.TU.582Impact of enteral prophylactic antibiotic course on the trends of inflammatory markers following laparoscopic colorectal cancer surgery

Introduction The study aimed to assess the effect of oral prophylactic antibiotic (OAB) with mechanical bowel preparation (MBP) on the serial measurement of postoperative inflammatory markers and clinical outcomes of the patients undergoing laparoscopic colorectal cancer resection surgery. Methods A retrospective data collection was carried out from January 2019 to March 2020 for the patients undergoing laparoscopic colorectal cancer resection. Daily measurements of inflammatory markers were obtained upto 7 days following surgery. The measurements of inflammatory markers were compared between patients who received a 1 week course of OAB along with MBP to those who only received MBP. Results There were a total of 110 patients that were divided into 2 groups: patients who received OAB and MBP (n = 44, 40%) and those who had MBP only (n = 66, 60%). There was no significant difference between the patient characteristics and preoperative staging of the cancer between the 2 groups. The overall length of stay was significantly lower in the patients who received OAB (9.09 days [SD 7.94] vs. 6.63 days [SD 4.96], P 0.02). The patients with OAB and MAP had persistently and significantly low levels of white blood cell count, CRP, and neutrophil count throughout the postoperative period as compared to those who only had MBP. Conclusion The study demonstrated reduction in serial measurement of inflammatory markers throughout postoperative stay for the patients receiving preoperative OAB. The use of OAB helps in physiological recovery of the patient by reducing the inflammatory process postoperatively.