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

Tumor Ulceration, Reduced Infiltration of CD8-Lymphocytes, High Neutrophil-to-CD8-Lymphocyte Ratio and Absence of MC Virus are Negative Prognostic Markers for Patients with Merkel Cell Carcinoma

(1) Background: Merkel cell carcinoma (MCC) is caused by the Merkel cell polyomavirus and UV radiation. Understanding of the underlying biology is limited, but identification of prognostic markers may lead to better prognostic stratification for the patients. (2) Methods: Ninety patients diagnosed with MCC (1996&ndash;2012) were included. Virus status was estimated by polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Ulceration status, PD-L1, cd66b neutrophils, cd8 lymphocytes and biomarkers of vascularization (cd34 endothelial cells) and migration (e-cadherin) were estimated by IHC and analyzed with digital pathology. (3) Results: Virus was present in 47% of patient samples and correlated with lower E-cadherin expression (p = 0.0005), lower neutrophil-to-CD8 lymphocyte ratio (N:CD8 ratio) (p = 0.02) and increased PD-L1 expression (p = 0.03). Ulceration was associated with absence of virus (p = 0.03), increased neutrophil infiltration (p &lt; 0.0001) and reduced CD8 lymphocyte infiltration (p = 0.04). In multivariate analysis, presence of virus (p = 0.01), ulceration (p = 0.05) and increased CD8 lymphocyte infiltration (p = 0.001) showed independent prognostic impacts on MCC-specific survival. (3) Conclusions: In this study, we found that a high N:CD8 ratio, ulceration, virus-negative status and absence of CD8 lymphocytes are negative prognostic markers. Accurate prognostic stratification of the patients may be important in the clinical setting for determination of adjuvant treatment

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

Occurrence of sinonasal intestinal‐type adenocarcinoma and non‐intestinal‐type adenocarcinoma in two countries with different patterns of wood dust exposure

Sinonasal intestinal-type adenocarcinoma is strongly associated with hardwood dust exposure. Non-intestinal-type adenocarcinoma is a rarer and less well-known subtype considered not to be related with wood dust exposure. We determined the relative numbers of these two tumor types in 56 sinonasal adenocarcinoma patients in France and Finland, relating them with carefully assessed wood dust exposure histories. Diagnostic workup including immunohistochemistry for the intestinal markers CDX2 and CK20 indicated that the proportions of the two tumors differed significantly between France and Finland. In Finnish samples non-intestinal adenocarcinomas were more common than intestinal-type adenocarcinomas (12 non-intestinal vs. nine intestinal), while in the French samples the reverse was true (six non-intestinal vs. 29 intestinal). Such remarkably dissimilar occurrence of these tumors in France and Finland presumably reflects different pathogenetic circumstances in the two countries, and perhaps their different patterns of wood dust exposure. In France the main source of wood dust is from hardwoods. In Finland it is derived from softwoods. This is the first systematic comparison of the occurrence of intestinal-type adenocarcinoma and non-intestinal-type adenocarcinoma in two countries with different wood usage. It appears to be the first systematic study on differences in wood dust exposure between intestinal-type adenocarcinoma and non-intestinal-type adenocarcinoma.</p