Preservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of... Show morePreservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of translocations in preserved samples.In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens. Show less
Background: CDC4/FBXW7, encoding a ubiquitin ligase, maps to 4q32 and has been implicated as a tumor suppressor gene and therapeutic target in many tumor types. Mutations in colonic adenomas, and... Show moreBackground: CDC4/FBXW7, encoding a ubiquitin ligase, maps to 4q32 and has been implicated as a tumor suppressor gene and therapeutic target in many tumor types. Mutations in colonic adenomas, and the frequent losses on 4q described in gastric cancer prompt speculation about the role of CDC4/FBXW7 in gastric carcinogenesis. Methods: We assessed the role of CDC4/FBXW7 in gastric cancer, through loss of heterozygosity (LOH) and multiplex ligation-dependent probe amplification (MLPA) on 47 flow-sorted gastric carcinomas including early-onset gastric cancers (EOGC) and xenografted conventional gastric carcinomas. Ploidy analysis was carried out on 39 EOGCs and immunohistochemistry of CDC4/FBXW7 and its substrates c-myc, c-jun, Notch and cyclin E was performed on 204 gastric carcinomas using tissue microarrays (TMAs). Sequence analysis of CDC4/FBXW7 was carried out on gastric carcinoma cell lines and xenografts. Results: Loss of heterozygosity of CDC4/FBXW7 occurred in 32% of EOGCs, and correlated with loss of expression in 26%. Loss of expression was frequent in both EOGC and conventional gastric cancers. No CDC4/FBXW7 mutations were found and loss of CDC4/FBXW7 did not correlate with ploidy status. There was a significant correlation between loss of CDC4/FBXW7 expression and upregulation of c-myc. Conclusion: Loss of CDC4/FBXW7 appears to play a role in both EOGC and conventional gastric carcinogenesis, and c-myc overexpression is likely to be an important oncogenic consequence of CDC4/FBXW7 loss. Show less
