Digital Quantification of Tumor PD-L1 Predicts Outcome of PD-1-Based Immune Checkpoint Therapy in Metastatic Melanoma

BackgroundPD-1-based immune checkpoint blockade (ICB) is a highly effective therapy in metastatic melanoma. However, 40-60% of patients are primarily resistant, with valid predictive biomarkers currently missing. This study investigated the digitally quantified tumor PD-L1 expression for ICB therapy outcome prediction.Patients and MethodsTumor tissues taken prior to PD-1-based ICB for unresectable metastatic disease were collected within the prospective multicenter Tissue Registry in Melanoma (TRIM). PD-L1 expression (clone 28-8; cut-off=5%) was determined by digital and physician quantification, and correlated with therapy outcome (best overall response, BOR; progression-free survival, PFS; overall survival, OS).ResultsTissue samples from 156 patients were analyzed (anti-PD-1, n=115; anti-CTLA-4+anti-PD-1, n=41). Patients with PD-L1-positive tumors showed an improved response compared to patients with PD-L1-negative tumors, by digital (BOR 50.5% versus 32.2%; p=0.026) and physician (BOR 54.2% versus 36.6%; p=0.032) quantification. Tumor PD-L1 positivity was associated with a prolonged PFS and OS by either digital (PFS, 9.9 versus 4.6 months, p=0.021; OS, not reached versus 13.0 months, p=0.001) or physician (PFS, 10.6 versus 5.6 months, p=0.051; OS, not reached versus 15.6 months, p=0.011) quantification. Multivariable Cox regression revealed digital (PFS, HR=0.57, p=0.007; OS, HR=0.44, p=0.001) and physician (OS, HR=0.54, p=0.016) PD-L1 quantification as independent predictors of survival upon PD-1-based ICB. The combination of both methods identified a patient subgroup with particularly favorable therapy outcome (PFS, HR=0.53, p=0.011; OS, HR=0.47, p=0.008).ConclusionPre-treatment tumor PD-L1 positivity predicted a favorable outcome of PD-1-based ICB in melanoma. Herein, digital quantification was not inferior to physician quantification, and should be further validated for clinical use.

CD10 and Das1: A Biomarker Study Using Immunohistochemistry to Subtype Gastric Intestinal Metaplasia

Background: Intestinal metaplasia (IM) is considered a key pivot point in the Correa model of gastric cancer (GC). It is histologically subtyped into the complete and incomplete subtypes, the latter being associated with a greater risk of progression. However, the clinical utility of IM subtyping remains unclear, partially due to the absence of reliable defining biomarkers.Methods: Based on gene expression data and existing literature, we selected CD10 and Das1 as candidate biomarkers to distinguish complete and incomplete IM glands in tissues from patients without GC (IM-GC) and patients with GC (IM+GC). Immunohistochemical staining of individually subtyped IM glands was scored after blinding by two researchers using tissue belonging to both IM-GC and IM+GC patients. Whole tissue Das1 staining was further assessed using digital image quantification (cellSens Dimension, Olympus).Results: Across both cohorts CD10 stained the IM brush border and was shown to have a high sensitivity (87.5% and 94.9% in IM-GC and IM+GC patients respectively) and specificity (100.0% and 96.7% respectively) with an overall AUROC of 0.944 for complete IM glands. By contrast Das1 stained mainly goblet cells and the apical membrane of epithelial cells, mostly of incomplete IM glands with a low sensitivity (28.6% and 29.3% in IM-GC and IM+GC patients respectively) but high specificity (98.3% and 85.1% respectively) and an overall AUROC of 0.603 for incomplete IM glands. A combined logistic regression model showed a significant increase in AUROC for detecting complete IM glands (0.955 vs 0.970). Whole tissue digital quantification of Das1 staining showed a significant association with incomplete IM compared to complete IM, both in IM-GC and in IM+GC patients (p=0.016 and p=0.009 respectively, Mann-Whitney test and unpaired t test used). Additionally, complete IM in IM+GC patients exhibited significantly more Das1 staining than in IM-GC patients (p=0.019, Mann-Whitney test). Conclusions: These findings suggest that CD10 is an outstanding biomarker for complete IM and Das1 may be useful as a secondary biomarker for IM glands at greater risk of progression irrespective of IM subtype. Overall, the clinical use of these biomarkers could lead to improved patient stratification and targeted surveillance.

Digital Quantification of Occlusal Contacts: A Methodological Study

Objective: To compare the reliability of digital occlusal contacts quantification and the validity of digital occlusal contacts quantification with traditional methods used for occlusal contact determination. Subjects and Methods: Thirty participants, all of whom were students at the Sahlgrenska Academy, University of Gothenburg in Gothenburg, Sweden, were included in the study. Three different methods were used to evaluate occlusal contacts: (I) a digital examination of the patients’ casts, using the Ortho 3D Models (O3DM) software and measuring the total occlusal contact area in square millimeters (DE); (II) an examination involving the measurement of the total number of occlusal contacts on stone casts mounted in an articulator (AE); and (III) a clinical examination with the measurement of the total number of occlusal contacts with 8 μm-thick articulating foil (CE). Results: The repeated digital measurements (same casts scanned multiple times) showed a significant correlation of 0.85 (p < 0.01), which shows a diagnostic consistency. Furthermore, there was a significant correlation between the results obtained with the DE method and the AE of 0.41 (p < 0.05), and between those acquired with the AE method and the CE of 0.37 (p < 0.05). However, no significant correlation was found between the DE method and the CE method with a correlation coefficient of 0.10 (p > 0.05). Conclusions: Digital casts can be used for quantification of the total occlusal contact area (in mm2) owing to the high reliability of repeated measurements and the strong validity of the method compared to traditionally employed stone cast measurements.

Rotational scan digital LAMP for accurate quantitation of nucleic acids

Droplets generated by centrifugation can be packed as a thin layer by flipping a micro-centrifugal tube with a plastic insert. RS-dLAMP applies line-scan to enable high-speed and contamination-free digital quantification of nucleic acids.

Multiplexed Droplet Loop-mediated Isothermal Amplification with Scorpion-shaped Probes and Fluorescence Microscopic Counting for Digital Quantification of Virus RNAs

Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplex droplet loop-mediated isothermal amplification (multiplexed...