Unsupervised hierarchical clustering based on our 2188-gene signature applied to external data sets.

<p>a) Clustering based on GSE4554 and b) Clustering based on the four largest batches of the TCGA RNAseqv2 data sets. MMR proficient tumors (green), microsatellite-low tumors (blue) and MMR deficient tumors (red) along the x-axis.</p

Clinical characteristics of the colorectal cancer subsets.

<p>Clinical characteristics of the colorectal cancer subsets.</p

Up-regulated signaling pathways in FCCTX and Lynch syndrome tumors.

<p>Up-regulated signaling pathways in FCCTX and Lynch syndrome tumors.</p

qRT-PCR analysis of 5 target genes in the four different colorectal cancer subsets.

<p>Differential expression of <i>MYC, NDUFA9</i>, <i>H2AFZ</i>, <i>AXIN2</i>, and <i>DNA2</i> was done for 12 representative samples (3 from each group) and qRT-PCR ratios were normalized to rRNA18S and median centered.</p

Table_1_Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.xlsx

IntroductionLynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers.MethodsNinety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively.ResultsAmong the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays.ConclusionOur results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.</p

Additional file 1 of Colorectal cancer incidences in Lynch syndrome: a comparison of results from the prospective lynch syndrome database and the international mismatch repair consortium

Additional file 1