Digital image analysis improves precision of PD-L1 scoring in cutaneous melanoma.

AIMS: Immune checkpoint inhibitors have become a successful treatment in metastatic melanoma. The high response rates in a subset of patients suggest that a sensitive companion diagnostic test is required. The predictive value of programmed death ligand 1 (PD-L1) staining in melanoma has been questioned due to inconsistent correlation with clinical outcome. Whether this is due to predictive irrelevance of PD-L1 expression or inaccurate assessment techniques remains unclear. The aim of this study was to develop a standardised digital protocol for the assessment of PD-L1 staining in melanoma and to compare the output data and reproducibility to conventional assessment by expert pathologists. METHODS AND RESULTS: In two cohorts with a total of 69 cutaneous melanomas, a highly significant correlation was found between pathologist-based consensus reading and automated PD-L1 analysis (r = 0.97, P < 0.0001). Digital scoring captured the full diagnostic spectrum of PD-L1 expression at single cell resolution. An average of 150 472 melanoma cells (median 38 668 cells; range = 733-1 078 965) were scored per lesion. Machine learning was used to control for heterogeneity introduced by PD-L1-positive inflammatory cells in the tumour microenvironment. The PD-L1 image analysis protocol showed excellent reproducibility (r = 1.0, P < 0.0001) when carried out on independent workstations and reduced variability in PD-L1 scoring of human observers. When melanomas were grouped by PD-L1 expression status, we found a clear correlation of PD-L1 positivity with CD8-positive T cell infiltration, but not with tumour stage, metastasis or driver mutation status. CONCLUSION: Digital evaluation of PD-L1 reduces scoring variability and may facilitate patient stratification in clinical practice.

COVID-19 Autopsies Reveal Underreporting of SARS-CoV-2 Infection and Scarcity of Co-infections.

Coronavirus disease 2019 (COVID-19) mortality can be estimated based on reliable mortality data. Variable testing procedures and heterogeneous disease course suggest that a substantial number of COVID-19 deaths is undetected. To address this question, we screened an unselected autopsy cohort for the presence of SARS-CoV-2 and a panel of common respiratory pathogens. Lung tissues from 62 consecutive autopsies, conducted during the first and second COVID-19 pandemic waves in Switzerland, were analyzed for bacterial, viral and fungal respiratory pathogens including SARS-CoV-2. SARS-CoV-2 was detected in 28 lungs of 62 deceased patients (45%), although only 18 patients (29%) were reported to have COVID-19 at the time of death. In 23 patients (37% of all), the clinical cause of death and/or autopsy findings together with the presence of SARS-CoV-2 suggested death due to COVID-19. Our autopsy results reveal a 16% higher SARS-CoV-2 infection rate and an 8% higher SARS-CoV-2 related mortality rate than reported by clinicians before death. The majority of SARS-CoV-2 infected patients (75%) did not suffer from respiratory co-infections, as long as they were treated with antibiotics. In the lungs of 5 patients (8% of all), SARS-CoV-2 was found, yet without typical clinical and/or autopsy findings. Our findings suggest that underreporting of COVID-19 contributes substantially to excess mortality. The small percentage of co-infections in SARS-CoV-2 positive patients who died with typical COVID-19 symptoms strongly suggests that the majority of SARS-CoV-2 infected patients died from and not with the virus.

Integrated Analysis Of Immunotherapy Treated Clear Cell Renal Cell Carcinomas: An Exploratory Study.

Molecular or immunological differences between responders and nonresponders to immune checkpoint inhibitors (ICIs) of clear cell renal cell carcinomas (ccRCCs) remain incompletely understood. To address this question, we performed next-generation sequencing, methylation analysis, genome wide copy number analysis, targeted RNA sequencing and T-cell receptor sequencing, and we studied frequencies of tumor-infiltrating CD8+ T cells, presence of tertiary lymphoid structures (TLS) and PD-L1 expression in 8 treatment-naive ccRCC patients subsequently treated with ICI (3 responders, 5 nonresponders). Unexpectedly, we identified decreased frequencies of CD8+ tumor-infiltrating T cells and TLS, and a decreased expression of PD-L1 in ICI responders when compared with nonresponders. However, neither tumor-specific genetic alterations nor gene expression profiles correlated with response to ICI or the observed immune features. Our results underline the challenge to stratify ccRCC patients for immunotherapy based on routinely available pathologic primary tumor material, even with advanced technologies. Our findings emphasize the analysis of pretreated metastatic tissue in line with recent observations describing treatment effects on the tumor microenvironment. In addition, our data call for further investigation of additional parameters in a larger ccRCC cohort to understand the mechanistic implications of the observed differences in tumor-infiltrating CD8+ T cells, TLS, and PD-L1 expression.

Combined Simplified Molecular Classification of Gastric Adenocarcinoma, Enhanced by Lymph Node Status: An Integrative Approach.

Gastric adenocarcinoma (GAC) is a heterogeneous disease and at least two major studies have recently provided a molecular classification for this tumor: The Cancer Genome Atlas (TCGA) and the Asian Cancer Research Group (ARCG). Both classifications quote four molecular subtypes, but these subtypes only partially overlap. In addition, the classifications are based on complex and cost-intensive technologies, which are hardly feasible for everyday practice. Therefore, simplified approaches using immunohistochemistry (IHC), in situ hybridization (ISH) as well as commercially available next generation sequencing (NGS) have been considered for routine use. In the present study, we screened 115 GAC by IHC for p53, MutL Homolog 1 (MLH1) and E-cadherin and performed ISH for Epstein-Barr virus (EBV). In addition, sequencing by NGS for TP53 and tumor associated genes was performed. With this approach, we were able to define five subtypes of GAC: (1) Microsatellite Instable (MSI), (2) EBV-associated, (3) Epithelial Mesenchymal Transition (EMT)-like, (4) p53 aberrant tumors surrogating for chromosomal instability and (5) p53 proficient tumors surrogating for genomics stable cancers. Furthermore, by considering lymph node metastasis in the p53 aberrant GAC, a better prognostic stratification was achieved which finally allowed us to separate the GAC highly significant in a group with poor and good-to-intermediate prognosis, respectively. Our data show that molecular classification of GAC can be achieved by using commercially available assays including IHC, ISH and NGS. Furthermore, we present an integrative workflow, which has the potential to overcome the uncertainty resulting from discrepancies from existing classification schemes.

Single-Center Experience with a Targeted Next Generation Sequencing Assay for Assessment of Relevant Somatic Alterations in Solid Tumors.

Companion diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. Here we report the clinical application and validation of the Oncomine Focus Assay (OFA), an integrated, commercially available next-generation sequencing (NGS) assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance. Two independent patient cohorts were investigated to define the workflow, turnaround times, feasibility, and reliability of OFA targeted sequencing in clinical application and using archival material. Cohort I consisted of 59 diagnostic clinical samples from the daily routine submitted for molecular testing over a 4-month time period. Cohort II consisted of 39 archival melanoma samples that were up to 15years old. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent PGM sequencer. Sequencing datasets were analyzed using the Ion Reporter software. Genomic alterations were identified and validated by orthogonal conventional assays including pyrosequencing and immunohistochemistry. Sequencing results of both cohorts, including archival formalin-fixed, paraffin-embedded material stored up to 15years, were consistent with published variant frequencies. A concordance of 100% between established assays and OFA targeted NGS was observed. The OFA workflow enabled a turnaround of 3½ days. Taken together, OFA was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics. Thus, OFA has strong potential to become an important asset for precision oncology.

Interleukin-33 Expression Indicates a Favorable Prognosis in Malignant Salivary Gland Tumors.

The cytokine interleukin-33 (IL-33) is abundantly expressed in epithelial barrier tissues such as salivary glands. Here, we characterized nuclear IL-33 protein expression by immunohistochemistry in benign and malignant salivary gland tumors and associated it with disease outcome. Most benign salivary gland tumors expressed IL-33, and all Warthin's tumors showed strong and consistent IL-33 expression in the basally oriented cells of their bilayered epithelium. In the malignant group of neoplasms, nuclear IL-33 expression was limited to specific tumor entities-for example, to epithelial-myopepithelial carcinomas (n = 9/11), acinic cell carcinomas (n = 13/27), and oncocytic carcinomas (n = 2/2). IL-33 expression in the combined group of malignant salivary gland neoplasms was significantly associated with favorable histological parameters, lack of metastasis, and longer overall survival, compared with IL-33-negative tumors. We conclude that IL-33 expression is a novel prognostic marker for malignant salivary gland tumors with potential use in clinical diagnostics.

Granulomas are a source of interleukin-33 expression in pulmonary and extrapulmonary sarcoidosis.

Sarcoidosis is a chronic inflammatory disease characterized by noncaseating epithelioid granulomas. These granulomas consist of highly differentiated mononuclear phagocytes--epithelioid cells and multinucleated giant cells (MNGCs)--surrounded by a proinflammatory infiltrate. Interleukin-33 (IL-33) is an inflammatory cytokine that is constitutively expressed in barrier tissues such as skin and lung and up-regulated in inflammation. Because sarcoidosis occurs most frequently in lung and skin, we studied the expression of this cytokine by immunohistochemistry in these tissues from patients with sarcoidosis, with foreign body granulomas, with other granulomatous diseases, and in corresponding normal tissues. We identified nuclear IL-33 staining of epithelioid cells and MNGCs in biopsies of skin (18/25 patients, 72%) and lung (10/19 patients, 53%) sarcoidosis. In contrast, sarcoidal granulomas in lymph nodes did not show IL-33 expression. Other granulomatous diseases showed only occasional and weak IL-33 expression. In sarcoidosis, we found a strong correlation between IL-33 expression and systemic disease, presence of MNGCs, and an M2-like macrophage phenotype as assessed by CD163 staining. Therefore, we propose that IL-33 plays a critical role in pathogenesis and disease progression of sarcoidosis. Because IL-33 is less commonly and only weakly expressed in other granulomatous diseases, the detection of IL-33 might serve as an adjunctive diagnostic marker. IL-33 expression in sarcoidosis seems to be dependent on the specific tissue microenvironment of sarcoidal granulomas and represents a novel biomarker for systemic involvement.