Search

Single‐cell transcriptomics have revolutionized our understanding of the cell composition of tumors and allowed us to identify new subtypes of cells. Despite rapid technological advancements, single‐cell analysis remains resource‐intense hampering the scalability that is required to profile a sufficient number of samples for clinical associations. Therefore, more scalable approaches are needed to understand the contribution of individual cell types to the development and treatment response of solid tumors such as esophageal adenocarcinoma where comprehensive genomic studies have only led to a small number of targeted therapies. Due to the limited treatment options and late diagnosis, esophageal adenocarcinoma has a poor prognosis. Understanding the interaction between and dysfunction of individual cell populations provides an opportunity for the development of new interventions. In an attempt to address the technological and clinical needs, we developed a protocol for the separation of esophageal carcinoma tissue into leukocytes (CD45+), epithelial cells (EpCAM+), and fibroblasts (two out of PDGFRα, CD90, anti‐fibroblast) by fluorescence‐activated cell sorting and subsequent RNA sequencing. We confirm successful separation of the three cell populations by mapping their transcriptomic profiles to reference cell lineage expression data. Gene‐level analysis further supports the isolation of individual cell populations with high expression of CD3, CD4, CD8, CD19, and CD20 for leukocytes, CDH1 and MUC1 for epithelial cells, and FAP, SMA, COL1A1, and COL3A1 for fibroblasts. As a proof of concept, we profiled tumor samples of nine patients and explored expression differences in the three cell populations between tumor and normal tissue. Interestingly, we found that angiogenesis‐related genes were upregulated in fibroblasts isolated from tumors compared with normal tissue. Overall, we suggest our protocol as a complementary and more scalable approach compared with single‐cell RNA sequencing to investigate associations between clinical parameters and transcriptomic alterations of specific cell populations in esophageal adenocarcinoma.

Abstract The prevalence of germ line mutations in non‐BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95%CI: 2.67–4.94), CDH1 (OR: 17.04, 95%CI: 3.54–82), CHEK2 (OR: 2.93, 95%CI: 2.29–3.75), PALB2 (OR: 9.53, 95%CI: 6.25–14.51), and TP53 (OR: 7.30, 95%CI: 1.22–43.68). NBN germ line mutations were not significantly associated with BC risk (OR:1.39, 95%CI: 0.73–2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple‐negative breast cancer (TNBC) and estrogen receptor (ER)‐negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)‐positive tumors.

Abstract Background Serrated or Hyperplastic Polyposis Syndrome (SPS, HPS) is a yet poorly defined colorectal cancer (CRC) predisposition characterised by the occurrence of multiple and/or large serrated polyps throughout the colon. A serrated polyp-CRC sequence (serrated pathway) of CRC formation has been postulated, however, to date only few molecular signatures of serrated neoplasia (BRAF, KRAS, RNF43 mutations, CpG Island Methylation, MSI) have been described in a subset of SPS patients and neither the etiology of the syndrome nor the distinct genetic alterations during tumorigenesis have been identified. Methods To identify somatic point mutations in potential novel candidate genes of SPS-associated lesions and the involved pathways we performed exome sequencing of eleven early serrated polyps obtained from a 41 year-old female patient with clinically confirmed SPS. For data filtering and analysis, standard pipelines were used. Somatic mutations were identified by comparison with leukocyte DNA and were validated by Sanger sequencing. Results The BRAF p.V600E or KRAS p.G12D mutation was identified in six polyps (~50%) and not found in polyps from the distal colon. In addition, we found seven unique rare somatic alterations of seven different genes in four serrated tumours, all of which are missense variants. The variant in ABI3BP and CATSPERB are predicted to be deleterious. No established cancer gene or candidate genes related to serrated tumorigenesis were affected. Conclusions Somatic mutations seem to be rare events in early hyperplastic and serrated lesions of SPS patients. Neither frequently affected genes nor enrichment of specific pathways were observed. Thus, other alterations such as non-coding variants or epigenetic changes might be the major driving force of tumour progression in SPS.

Olfactory receptors (ORs) are known to be expressed in a variety of human tissues and act on different physiological processes, such as cell migration, proliferation, or secretion and have been found to function as biomarkers for carcinoma tissues of prostate, lung, and small intestine. In this study, we analyzed the OR expression profiles of several different carcinoma tissues, with a focus on breast cancer. The expression of OR2B6 was detectable in breast carcinoma tissues; here, transcripts of OR2B6 were detected in 73% of all breast carcinoma cell lines and in over 80% of all of the breast carcinoma tissues analyzed. Interestingly, there was no expression of OR2B6 observed in healthy tissues. Immunohistochemical staining of OR2B6 in breast carcinoma tissues revealed a distinct staining pattern of carcinoma cells. Furthermore, we detected a fusion transcript containing part of the coding exon of OR2B6 as a part of a splice variant of the histone HIST1H2BO transcript. In addition, in cancer tissues and cell lines derived from lung, pancreas, and brain, OR expression patterns were compared to that of corresponding healthy tissues. The number of ORs detected in lung carcinoma tissues was significantly reduced in comparison to the surrounding healthy tissues. In pancreatic carcinoma tissues, OR4C6 was considerably more highly expressed in comparison to the respective healthy tissues. We detected OR2B6 as a potential biomarker for breast carcinoma tissues.

Abstract Activation of MAPK signaling via BRAF mutations may limit the activity of EGFR inhibitors in EGFR-mutant lung cancer patients. However, the impact of BRAF mutations on the selection and fitness of emerging resistant clones during anti-EGFR therapy remains elusive. We tracked the evolution of subclonal mutations by whole-exome sequencing and performed clonal analyses of individual metastases during therapy. Complementary functional analyses of polyclonal EGFR-mutant cell pools showed a dose-dependent enrichment of BRAF V600E and a loss of EGFR inhibitor susceptibility. The clones remain stable and become vulnerable to combined EGFR, RAF, and MEK inhibition. Moreover, only osimertinib/trametinib combination treatment, but not monotherapy with either of these drugs, leads to robust tumor shrinkage in EGFR-driven xenograft models harboring BRAF V600E mutations. These data provide insights into the dynamics of clonal evolution of EGFR-mutant tumors and the therapeutic implications of BRAF co-mutations that may facilitate the development of treatment strategies to improve the prognosis of these patients.

Abstract Background The role of the BARD1 gene in breast cancer (BC) and ovarian cancer (OC) predisposition remains elusive, as published case-control investigations have revealed controversial results. We aimed to assess the role of deleterious BARD1 germline variants in BC/OC predisposition in a sample of 4920 BRCA1/2-negative female BC/OC index patients of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC). Methods A total of 4469 female index patients with BC, 451 index patients with OC, and 2767 geographically matched female control individuals were screened for loss-of-function (LoF) mutations and potentially damaging rare missense variants in BARD1. All patients met the inclusion criteria of the GC-HBOC for germline testing and reported at least one relative with BC or OC. Additional control datasets (Exome Aggregation Consortium, ExAC; Fabulous Ladies Over Seventy, FLOSSIES) were included for the calculation of odds ratios (ORs). Results We identified LoF variants in 23 of 4469 BC index patients (0.51%) and in 36 of 37,265 control individuals (0.10%), resulting in an OR of 5.35 (95% confidence interval [CI] = 3.17–9.04; P

Abstract Background Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. Methods To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. Results BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02–36.57, P