Randomised, siteless study to compare systematic atrial fibrillation screening using enrichment by a risk prediction model with standard care in a Swedish population aged ≥ 65 years: CONSIDERING-AF study design.

INTRODUCTION: Atrial fibrillation (AF) is the most common arrhythmia and confers an increased risk of mortality, stroke, heart failure and cognitive decline. There is growing interest in AF screening; however, the most suitable population and device for AF detection remains to be elucidated. Here, we present the design of the CONSIDERING-AF (deteCtiON and Stroke preventIon by moDEl scRreenING for Atrial Fibrillation) study. METHODS AND ANALYSIS: CONSIDERING-AF is a randomised, controlled, siteless, non-blinded diagnostic superiority trial with four parallel groups and a primary endpoint of identifying AF during a 6-month study period set in Region Halland, Sweden. In each group, 740 individuals aged≥65 years will be included. The primary objective is to compare the intervention of AF screening enrichment using a risk prediction model (RPM), followed by 14 days of a continuous ECG patch, with no intervention (standard care). Primary outcome is defined as the incident AF recorded in the Region Halland Information Database after 6 months as compared with standard care. Secondary endpoints include the difference in incident AF between groups enriched or not by the RPM, with and without an invitation to 14 days of continuous ECG recording, and the proportions of oral anticoagulation treatment in the four groups. ETHICS AND DISSEMINATION: This study has ethical approval from the Swedish Ethical Review Authority. Results will be published in peer-reviewed international journals. TRIAL REGISTRATION NUMBER: NCT05838781.

VEGF receptor-2/neuropilin 1 trans-complex formation between endothelial and tumor cells is an independent predictor of pancreatic cancer survival.

Unstable and dysfunctional tumor vasculature promotes cancer progression and spread. Signal transduction by the pro-angiogenic vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) is modulated by VEGFA-dependent complex formation with neuropilin 1 (NRP1). NRP1 expressed on tumor cells can form VEGFR2/NRP1 trans-complexes between tumor cells and endothelial cells which arrests VEGFR2 on the endothelial surface, thus interfering with productive VEGFR2 signaling. In mouse fibrosarcoma, VEGFR2/NRP1 trans-complexes correlated with reduced tumor vessel branching and reduced tumor cell proliferation. Pancreatic ductal adenocarcinoma (PDAC) strongly expressed NRP1 on both tumor cells and endothelial cells, in contrast to other common cancer forms. Using proximity ligation assay, VEGFR2/NRP1 trans-complexes were identified in human PDAC tumor tissue, and its presence was associated with reduced tumor vessel branching, reduced tumor cell proliferation, and improved patient survival after adjusting for other known survival predictors. We conclude that VEGFR2/NRP1 trans-complex formation is an independent predictor of PDAC patient survival. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Tumor vessel up-regulation of INSR revealed by single-cell expression analysis of the tyrosine kinome and phosphatome in human cancers.

The tyrosine kinome and phosphatome harbor oncogenes and tumor suppressor genes and important regulators of angiogenesis and tumor stroma formation. To provide a better understanding of their potential roles in cancer, we analyzed the expression of 85 tyrosine kinases and 42 tyrosine phosphatases by in situ hybridization 48 human normal and 24 tumor tissue specimens. Nine-tenths of the assessed transcripts had tumor cell expression concordant with expression array databases. Further, pan-cancer expression of AATK, PTPRK, and PTPRU and expression of PTPRS in a subset of tumors were observed. To demonstrate tumor subcompartment resolution, we validated the predicted tumor stroma-specific markers HTRA1, HTRA3, MXRA5, MXRA8, and SERPING1 in situ. In addition to known vascular and stromal markers such as PDGFRB, we observed stromal expression of PTK6 and TNS1 and vascular expression of INSR, PTPRF, PTPRG, PTPRU, and TNS1, of which INSR emerged as a tumor-specific vessel marker. This study demonstrates the feasibility of large-scale analyses to chart the transcriptome in situ in human cancers and their ability to identify novel cancer biomarkers.

In situ sequencing identifies TMPRSS2-ERG fusion transcripts, somatic point mutations and gene expression levels in prostate cancers.

Translocations contribute to the genesis and progression of epithelial tumours and in particular to prostate cancer development. To better understand the contribution of fusion transcripts and visualize the clonal composition of multifocal tumours, we have developed a technology for multiplex in situ detection and identification of expressed fusion transcripts. When compared to immunohistochemistry, TMPRSS2-ERG fusion-negative and fusion-positive prostate tumours were correctly classified. The most prevalent TMPRSS2-ERG fusion variants were visualized, identified, and quantitated in human prostate cancer tissues, and the ratio of the variant fusion transcripts could for the first time be directly determined by in situ sequencing. Further, we demonstrate concurrent in situ detection of gene expression, point mutations, and gene fusions of the prostate cancer relevant targets AMACR, AR, TP53, and TMPRSS2-ERG. This unified approach to in situ analyses of somatic mutations can empower studies of intra-tumoural heterogeneity and future tissue-based diagnostics of mutations and translocations.

Local and systemic protumorigenic effects of cancer-associated fibroblast-derived GDF15.

The tumor stroma is vital to tumor development, progression, and metastasis. Cancer-associated fibroblasts (CAF) are among the abundant cell types in the tumor stroma, but the range of their contributions to cancer pathogenicity has yet to be fully understood. Here, we report a critical role for upregulation of the TGFß/BMP family member GDF15 (MIC-1) in tumor stroma. GDF15 was found upregulated in situ and in primary cultures of CAF from prostate cancer. Ectopic expression of GDF15 in fibroblasts produced prominent paracrine effects on prostate cancer cell migration, invasion, and tumor growth. Notably, GDF15-expressing fibroblasts exerted systemic in vivo effects on the outgrowth of distant and otherwise indolent prostate cancer cells. Our findings identify tumor stromal cells as a novel source of GDF15 in human prostate cancer and illustrate a systemic mechanism of cancer progression driven by the tumor microenvironment. Further, they provide a functional basis to understand GDF15 as a biomarker of poor prognosis and a candidate therapeutic target in prostate cancer.

In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics.

Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay.

Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.

STC1 expression by cancer-associated fibroblasts drives metastasis of colorectal cancer.

Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant of cancer-associated fibroblasts (CAF). Elevated expression of PDGF receptors on stromal CAFs is associated with metastasis and poor prognosis, but mechanism(s) that underlie these connections are not understood. Here, we report the identification of the secreted glycoprotein stanniocalcin-1 (STC1) as a mediator of metastasis by PDGF receptor function in the setting of colorectal cancer. PDGF-stimulated fibroblasts increased migration and invasion of cocultured colorectal cancer cells in an STC1-dependent manner. Analyses of human colorectal cancers revealed significant associations between stromal PDGF receptor and STC1 expression. In an orthotopic mouse model of colorectal cancer, tumors formed in the presence of STC1-deficient fibroblasts displayed reduced intravasation of tumor cells along with fewer and smaller distant metastases formed. Our results reveal a mechanistic basis for understanding the contribution of PDGF-activated CAFs to cancer metastasis.

Scalable in situ hybridization on tissue arrays for validation of novel cancer and tissue-specific biomarkers.

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues.

Selective inhibition of proliferation in colorectal carcinoma cell lines expressing mutant APC or activated B-Raf.

Tumor-derived cell lines are indispensable tools for understanding the contribution of activated signaling pathways to the cancer phenotype and for the design and testing of targeted signal therapies. In our study, we characterize 10 colorectal carcinoma cell lines for the presence of mutations in the wnt, Ras/MAPK, PI3K and p53 pathways. The mutational spectrum found in this panel of cell lines is similar to that detected in primary CRC, albeit with higher frequency of mutation in the beta-catenin and B-Raf genes. We have monitored activation of the wnt and Ras/MAPK pathways in these cells and analyzed their sensitivity to selective signaling inhibitors. Using beta-catenin subcellular distribution as a marker, we show that cells harboring APC mutations have low-level activated wnt signaling, which can be blocked by the extracellular wnt inhibitor DKK-1, suggesting autocrine activation of this pathway; proliferation of these cells is also blocked by DKK-1. In contrast, cells with beta-catenin mutations are unresponsive to extracellular wnt inhibition. Constitutive phosphorylation of MAPK is present in the majority of the cell lines and correlates with B-Raf but not K-Ras mutations; correspondingly, the proliferation of cells harboring mutations in B-Raf, but not K-Ras, is exquisitely sensitive inhibition of the MAPK pathway. We find no correlation between PI3K mutation or loss of PTEN expression and increased sensitivity to PI3K inhibitors. Our study discloses clear-cut differences in responsiveness to signaling inhibitors between individual mutations within an activated signaling pathway and suggests likely targets for signal-directed therapy of colorectal carcinomas.