High-throughput automated scoring of Ki67 in breast cancer tissue microarrays from the Breast Cancer Association Consortium (BCAC)

Automated methods are needed to facilitate high-throughput and reproducible scoring of Ki67 and other markers in breast cancer tissue microarrays (TMAs) in large-scale studies. To address this need, we developed an automated protocol for Ki67 scoring and evaluated its performanc

Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium

Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large-scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65–70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose-response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96–98%), but yielded many false positives (positive predictive value = 30–32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large-scale, quantitative scoring of immunohistochemically stained tissue microarrays available in consortia. However, continued optimization, rigorous marker-specific quality control measures and standardization of tissue microarray designs, staining and scoring protocols is needed to enhance results.Peer reviewe

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily

YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co(2+), Mg(2+), Zn(2+) and Mn(2+), the Nudix hydrolase activity is Mn(2+)-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity

Research data supporting "Crowdsourcing the General Public for Large Scale Molecular Pathology Studies in Cancer"

Complete data set for the manuscript "Crowdsourcing the general public for large scale molecular pathology studies in cancer". Published at: http://dx.doi.org/10.1016/j.ebiom.2015.05.009This work was supported by the Cancer Research UK [C490/A10124, C490/A16561

Association of p16 expression with prognosis varies across ovarian carcinoma histotypes: an Ovarian Tumor Tissue Analysis consortium study.

We aimed to validate the prognostic association of p16 expression in ovarian high-grade serous carcinomas (HGSC) and to explore it in other ovarian carcinoma histotypes. p16 protein expression was assessed by clinical-grade immunohistochemistry in 6525 ovarian carcinomas including 4334 HGSC using tissue microarrays from 24 studies participating in the Ovarian Tumor Tissue Analysis consortium. p16 expression patterns were interpreted as abnormal (either overexpression referred to as block expression or absence) or normal (heterogeneous). CDKN2A (which encodes p16) mRNA expression was also analyzed in a subset (n = 2280) mostly representing HGSC (n = 2010). Association of p16 expression with overall survival (OS) was determined within histotypes as was CDKN2A expression for HGSC only. p16 block expression was most frequent in HGSC (56%) but neither protein nor mRNA expression was associated with OS. However, relative to heterogeneous expression, block expression was associated with shorter OS in endometriosis-associated carcinomas, clear cell [hazard ratio (HR): 2.02, 95% confidence (CI) 1.47-2.77, p < 0.001] and endometrioid (HR: 1.88, 95% CI 1.30-2.75, p = 0.004), while absence was associated with shorter OS in low-grade serous carcinomas (HR: 2.95, 95% CI 1.61-5.38, p = 0.001). Absence was most frequent in mucinous carcinoma (50%), and was not associated with OS in this histotype. The prognostic value of p16 expression is histotype-specific and pattern dependent. We provide definitive evidence against an association of p16 expression with survival in ovarian HGSC as previously suggested. Block expression of p16 in clear cell and endometrioid carcinoma should be further validated as a prognostic marker, and absence in low-grade serous carcinoma justifies CDK4 inhibition