Computer-Assisted Annotation of Digital H&E/SOX10 Dual Stains Generates High-Performing Convolutional Neural Network for Calculating Tumor Burden in H&E-Stained Cutaneous Melanoma

Deep learning for the analysis of H&E stains requires a large annotated training set. This may form a labor-intensive task involving highly skilled pathologists. We aimed to optimize and evaluate computer-assisted annotation based on digital dual stains of the same tissue section. H&E stains of primary and metastatic melanoma (N = 77) were digitized, re-stained with SOX10, and re-scanned. Because images were aligned, annotations of SOX10 image analysis were directly transferred to H&E stains of the training set. Based on 1,221,367 annotated nuclei, a convolutional neural network for calculating tumor burden (CNN(TB)) was developed. For primary melanomas, precision of annotation was 100% (95%CI, 99% to 100%) for tumor cells and 99% (95%CI, 98% to 100%) for normal cells. Due to low or missing tumor-cell SOX10 positivity, precision for normal cells was markedly reduced in lymph-node and organ metastases compared with primary melanomas (p < 0.001). Compared with stereological counts within skin lesions, mean difference in tumor burden was 6% (95%CI, −1% to 13%, p = 0.10) for CNN(TB) and 16% (95%CI, 4% to 28%, p = 0.02) for pathologists. Conclusively, the technique produced a large annotated H&E training set with high quality within a reasonable timeframe for primary melanomas and subcutaneous metastases. For these lesion types, the training set generated a high-performing CNN(TB), which was superior to the routine assessments of pathologists

Fueling the flames of colon cancer – does CRP play a direct pro-inflammatory role?

Background: Systemic inflammation, diagnostically ascribed by measuring serum levels of the acute phase reactant C-reactive protein (CRP), has consistently been correlated with poor outcomes across cancer types. CRP exists in two structurally and functionally distinct isoforms, circulating pentameric CRP (pCRP) and the highly pro-inflammatory monomeric isoform (mCRP). The aim of this pilot study was to map the pattern of mCRP distribution in a previously immunologically well-defined colon cancer (CC) cohort and explore possible functional roles of mCRP within the tumor microenvironment (TME). Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue samples from 43 stage II and III CC patients, including 20 patients with serum CRP 0-1 mg/L and 23 patients with serum CRP >30 mg/L were immunohistochemically (IHC) stained with a conformation-specific mCRP antibody and selected immune and stromal markers. A digital analysis algorithm was developed for evaluating mCRP distribution within the primary tumors and adjacent normal colon mucosa. Results: mCRP was abundantly present within tumors from patients with high serum CRP (>30 mg/L) diagnostically interpreted as being systemically inflamed, whereas patients with CRP 0-1 mg/L exhibited only modest mCRP positivity (median mCRP per area 5.07‰ (95%CI:1.32-6.85) vs. 0.02‰ (95%CI:0.01-0.04), p<0.001). Similarly, tissue-expressed mCRP correlated strongly with circulating pCRP (Spearman correlation 0.81, p<0.001). Importantly, mCRP was detected exclusively within tumors, whereas adjacent normal colon mucosa showed no mCRP expression. Double IHC staining revealed colocalization of mCRP with endothelial cells and neutrophils. Intriguingly, some tumor cells also colocalized with mCRP, suggesting a direct interaction or mCRP expression by the tumor itself. Conclusion: Our data show that the pro-inflammatory mCRP isoform is expressed in the TME of CC, primarily in patients with high systemic pCRP values. This strengthens the hypothesis that CRP might not only be an inflammatory marker but also an active mediator within tumors.publishedVersio

Prognostic significance of T-cell–inflamed gene expression profile and PD-L1 expression in patients with esophageal cancer

PURPOSE: The ability of the T‐cell–inflamed gene expression profile (GEP) to predict clinical outcome in esophageal cancer (EC) is unknown. This retrospective observational study assessed the prognostic value of GEP and programmed death ligand 1 (PD‐L1) expression in patients with EC treated in routine clinical practice. METHODS: Tumor samples of 294 patients from three centers in Denmark, South Korea, and the United States, collected between 2005 and 2017, were included. T‐cell–inflamed GEP score was defined as non‐low or low using a cutoff of −1.54. A combined positive score (CPS) ≥10 was defined as PD‐L1 expression positivity. Associations between overall survival (OS) and GEP status and PD‐L1 expression were explored by Cox proportional hazards models adjusting for age, sex, histology, stage, and performance status. RESULTS: Median age was 65 years; 63% of patients had adenocarcinoma (AC) and 37% had squamous cell carcinoma (SCC). Thirty‐six percent of tumors were GEP non‐low, with higher prevalence in AC (46%) than SCC (18%). Twenty‐one percent were PD‐L1–positive: 32% in South Korean samples versus 16% in non‐Asian samples and 26% in SCC versus 18% in AC. GEP scores and PD‐L1 CPS were weakly correlated (Spearman’s R = 0.363). OS was not significantly associated with GEP status (non‐low vs low; adjusted hazard ratio, 0.91 [95% CI, 0.69–1.19]) or PD‐L1 expression status. CONCLUSION: Neither GEP nor PD‐L1 expression was a prognostic marker in Asian and non‐Asian patients with EC

PD-L1 Expression and Survival among Patients with Advanced Non–Small Cell Lung Cancer Treated with Chemotherapy

BACKGROUND: Recent clinical trial results have suggested that programmed cell death ligand 1 (PD-L1) expression measured by immunohistochemistry may predict response to anti–programmed cell death 1 (PD-1) therapy. Results on the association between PD-L1 expression and survival among patients with advanced non–small cell lung cancer (NSCLC) treated with chemotherapy are inconsistent. MATERIAL AND METHODS: We evaluated the relationship between PD-L1 expression and overall survival (OS) among 204 patients with advanced NSCLC treated at Aarhus University Hospital, Aarhus, Denmark, from 2007 to 2012. PD-L1 expression was measured using a prototype immunohistochemistry assay with the anti–PD-L1 22C3 antibody (Merck). PD-L1 strong positivity and weak positivity were defined to be traceable to the clinical trial version of the assay. RESULTS: Twenty-five percent of patients had PD-L1 strong-positive tumors, and 50% had PD-L1 weak-positive tumors. No statistically significant association was found between PD-L1 expression and survival; adjusted hazard ratio of 1.34 (95% confidence interval, 0.88-2.03; median OS, 9.0 months) for the PD-L1 strong-positive group and 1.07 (0.74-1.55; median OS, 9.8 months) for the PD-L1 weak-positive group compared with the PD-L1–negative group (median OS, 7.5 months). No association was seen between PD-L1 expression and OS when PD-L1 expression levels were stratified by median or tertiles. CONCLUSIONS: In concordance with previous studies, we found PD-L1 measured by immunohistochemistry to be frequently expressed in patients with advanced NSCLC. However, PD-L1 expression is not a strong prognostic marker in patients with advanced NSCLC treated with chemotherapy

DataSheet_1_Molecular epidemiology study of programmed death ligand 1 and ligand 2 protein expression assessed by immunohistochemistry in extensive-stage small-cell lung cancer.pdf

ObjectivesPrevalence of tumor PD-L1 expression in extensive-stage small-cell lung cancer (ES-SCLC) is variable, and data on PD-L2 expression are limited. The prognostic values of these biomarkers are not well understood. The current study was conducted to address these data gaps.MethodsA retrospective cohort study of Danish patients with histologically confirmed ES-SCLC and evaluable tumor samples who were receiving usual care before the introduction of immunotherapy was conducted. Protein expression of PD-L1 and PD-L2 was determined by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx assay and a PD-L2 IHC assay using a propriety mouse monoclonal antibody. A combined positive score (CPS) of ≥1 was used to define biomarker positivity. Kaplan-Meier plots and Cox proportional hazard models were employed to assess the relationship between PD-L1 and PD-L2 protein expression and OS.ResultsAmong 80 patients, 31% (n=25) and 36% (n=29) had disease positive for PD-L1 and PD-L2, respectively. Overall, 85% (n=68) of patients had concordant PD-L1/PD-L2 status; 26% (n=21) had double positive disease (both PD-L1 and PD-L2 CPS ≥1) and 59% (n=47) had double negative disease (both PD-L1 and PD-L2 CPS ConclusionPD-L1 and PD-L2 positivity were observed in approximately one-third of assessed ES-SCLC tumor samples and were highly congruent. Patients with PD-L1 and PD-L2 positivity, alone or combined, were associated with longer OS, independent of other prognostic factors.</p

Molecular analysis and favorable clinical outcomes in real-world patients with metastatic renal cell carcinoma

Prior biomarker studies have mainly been restricted to advanced RCC patients treated in clinical trials or have had limited integration of immunotherapy features such as programmed death ligand (PD-L)-1 with gene expression signatures intended to capture other canonical pathways to confirm their prognostic value. PD-L1 and PD-L2 by immunohistochemistry (IHC), PD-L2 messenger RNA (mRNA), and 10 gene expression profile (GEP) signatures targeting immune, angiogenesis and canonical pathways were analyzed in nephrectomy specimens from 227 advanced clear cell RCC (ccRCC) and 42 non-clear cell RCC (nccRCC) patients treated with targeted therapies including VEGF and mTOR inhibitors. Biomarker association with best overall response (BOR), progression-free survival (PFS), and overall survival (OS) were evaluated using multivariable modeling. Except for PD-L1 IHC and angiogenesis, tested with a nominal p-value of .05, multiplicity control was applied with a 0.1 significance level given limited experience in this setting. The strongest biomarker correlations were observed for hypoxia inducible factor (HIF)-2a and angiogenesis signatures (rho = 0.860 [ccRCC], 0.819 [nccRCC]); hypoxia and glycolysis signatures (rho = 0.943 [ccRCC], 0.973 [nccRCC]); PD-L2 mRNA and T-cell-inflamed GEP signatures (rho = 0.764 [ccRCC], 0.897 [nccRCC]); and PD-L2 mRNA and monocytic myeloid-derived suppressor cell signature (rho = 0.787 [ccRCC], 0.815 [nccRCC]). For ccRCC, higher angiogenesis expression was associated with improved BOR (OR:2.85 [95%CI:1.37, 5.93]), longer PFS (HR:0.61 [95%CI:0.45, 0.82]) and OS (HR:0.74 [95%CI:0.54, 1.00]); higher PD-L1 expression with shorter OS (HR:1.44 [95%CI:1.01, 2.07]). For nccRCC, there was more than a two-fold increased risk with longer OS associated with lower angiogenesis (HR:2.43 [95%CI:1.04, 5.68]), glycolysis (HR:7.03 [95%CI:1.51, 32.76]) and hypoxia (HR:8.83 [95%CI:1.69, 46.05]) gene signature expression. Data pointed at PD-L1 IHC and angiogenesis expression in ccRCC and hypoxia, glycolysis, and angiogenesis expression in nccRCC as potential prognostic factors. These findings may have implications for the design and interpretation of advanced RCC trials and to identify potential targets for combination therapy strategies.</p