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Supplementary figure related to figure 3. Supplementary Fig. 3. Wnt-Met signalling induces YAP-dependent luminal-to-basal transition.

Supplementary material and methods and Supplementary Figure Legends

Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, nuclear YAP interacted with β-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, β-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/β-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers.Significance:These findings show that YAP cooperates with β-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer.

Supplementary figure related to figure 7. Supplementary Fig. 7. YAP is active in human basal-like breast tumours and predicts patient outcome in a subtype-dependent manner.

Supplementary figure related to figure 2. Supplementary Fig. 2. Pharmacological evidence of YAP dependency in Wnt-Met tumours.

Supplementary figure related to figure 1. Supplementary Fig. 1. Met signalling regulates YAP and β-catenin.

Supplementary figure related to figure 6. Supplementary Fig. 6. YAP controls β-catenin activity.

Supplementary figure related to figure 5. Supplementary Fig. 5. Stem cell-enriched spheres recapitulate in vivo cell phenotypes.

Supplementary figure related to figure 4. Supplementary Fig. 4. YAP is activated in CSCs of Wnt-Met-driven mammary gland tumours.

The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALKF1174L (exon 23 position 3522, C>A) and ALKR1275Q (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (n = 496) and a validation set (DKTK MASTER cohort, n = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately reanalyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma., Author summary Only few targeted therapies are currently available to treat high-risk neuroblastoma. To address this issue we characterized the drug response of high risk neuroblastoma cell lines and correlated it with genomic and transcriptomic data. Particularly for MEK inhibition, we saw that our panel could be nicely separated into two groups of resistant and sensitive cell lines. Genomic and transcriptomic markers alone did not help to discriminate between responders and non-responders. We used signalling perturbation data to build cell line specific signalling models. Our models suggest that negative feedbacks within MAPK signalling lead to a stronger reactivation of MEK in MEKi resistant cell lines after MEK inhibition. Model analysis suggested that combining MEK inhibition with IGF1R or RAF inhibition could be an effective treatment and we characterised this combination using phosphoproteomics by mass-spectrometry and growth assays. Our study confirms the importance of quantitative understanding of signalling and may help plan future clinical trials involving MEK inhibition for the treatment of neuroblastoma.

Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, nuclear YAP interacted with β-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, β-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/β-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. Significance: These findings show that YAP cooperates with β-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer.

Targeting cancer stem cells (CSCs) can serve as an effective approach toward limiting resistance to therapies and the development of metastases in many forms of cancer. While basal breast cancers encompass cells with CSC features, rational therapies remain poorly established. Here, we show that receptor tyrosine kinase Met signalling promotes the activity of the Hippo component YAP in basal breast cancer. Further analysis revealed enhanced YAP activity within the CSC population. Using both genetic and pharmaceutical approaches, we show that interfering with YAP activity delays basal cancer formation, prevents luminal to basal trans-differentiation and reduces CSC survival. Gene expression analysis of YAP knock-out mammary glands revealed a strong decrease in β-catenin target genes in basal breast cancer, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, we find that nuclear YAP interacts and overlaps with β-catenin and TEAD4 at common gene regulatory elements. Analysis of proteomic data from primary breast cancer patients identified a significant upregulation of the YAP activity signature in basal compared to other breast cancers, suggesting that YAP activity is limited to basal types. Our findings demonstrate that in basal breast cancers, β-catenin activity is dependent on YAP signalling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/β-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal (triple-negative) breast cancers.

Background Reliable and reproducible interpretation of molecular aberrations constitutes a bottleneck of precision medicine. Evidence-based decision management systems may improve rational therapy recommendations. To cope with an increasing amount of complex molecular data in the clinical care of patients with cancer, we established a workflow for the interpretation of molecular analyses. Methods A specialized physician screened results from molecular analyses for potential biomarkers, irrespective of the diagnostic modality. Best available evidence was retrieved and categorized through establishment of an in-house database and interrogation of publicly available databases. Annotated biomarkers were ranked using predefined evidence levels and subsequently discussed at a molecular tumour board (MTB), which generated treatment recommendations. Subsequent translation into patient treatment and clinical outcomes were followed up. Results One hundred patients were discussed in the MTB between January 2016 and May 2017. Molecular data were obtained for 70 of 100 patients (50 whole exome/RNA sequencing, 18 panel sequencing, 2 immunohistochemistry (IHC)/microsatellite instability analysis). The MTB generated a median of two treatment recommendations each for 63 patients. Thirty-nine patients were treated: 6 partial responses and 12 stable diseases were achieved as best responses. Genetic counselling for germline events was recommended for seven patients. Conclusion The development of an evidence-based workflow allowed for the clinical interpretation of complex molecular data and facilitated the translation of personalized treatment strategies into routine clinical care. The high number of treatment recommendations in patients with comprehensive genomic data and promising responses in patients treated with combination therapy warrant larger clinical studies.

BackgroundSingle cell omics technologies present unique opportunities for biomedical and life sciences from lab to clinic, but the high dimensional nature of such data poses challenges for computational analysis and interpretation. Furthermore, FAIR data management as well as data privacy and security become crucial when working with clinical data, especially in cross-institutional and translational settings. Existing solutions are either bound to the desktop of one researcher or come with dependencies on vendor-specific technology for cloud storage or user authentication.ResultsTo facilitate analysis and interpretation of single-cell data by users without bioinformatics expertise, we present SCelVis, a flexible, interactive and user-friendly app for web-based visualization of pre-processed single-cell data. Users can survey multiple interactive visualizations of their single cell expression data and cell annotation, and download raw or processed data for further offline analysis. SCelVis can be run both on the desktop and cloud systems, accepts input from local and various remote sources using standard and open protocols, and allows for hosting data in the cloud and locally.MethodsSCelVis is implemented in Python using Dash by Plotly. It is available as a standalone application as a Python package, via Conda/Bioconda and as a Docker image. All components are available as open source under the permissive MIT license and are based on open standards and interfaces, enabling further development and integration with third party pipelines and analysis components. The GitHub repository is https://github.com/bihealth/scelvis.

Summary. Managing raw sequencing data and conversion into sequences (demultiplexing) remains a challenging topic for groups running sequencing devices. They face many challenges in such efforts and solutions range from manual management of spreadsheets to very complex and customized LIMS systems handling much more than just sequencing raw data. In this manuscript, we describe the software package DigestiFlow that focuses on the management of Illumina flow cell sample sheets and raw data. Namely, it allows for automated extraction of flow cell raw data information, management of sample sheets, and the automated (and thus reproducible) demultiplexing of Illumina base calls data. Availability and Implementation. The software is available under the MIT license at https://github.com/bihealth/digestiflow-server. The client and demux software components are available via Bioconda.

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.

Immune checkpoint inhibition leads to response in some patients with head and neck squamous cell carcinoma (HNSCC). Robust biomarkers are lacking to date. We analyzed viral status, gene expression signatures, mutational load and mutational signatures in whole exome and RNA-sequencing data of the HNSCC TCGA dataset (N = 496) and a validation set (DKTK MASTER cohort, N = 10). Public single-cell gene expression data from 17 HPV-negative HNSCC were separately re-analyzed. APOBEC3-associated TCW motif mutations but not total single nucleotide variant burden were significantly associated with inflammation. This association was restricted to HPV-negative HNSCC samples. An APOBEC-enriched, HPV-negative subgroup was identified, that showed higher T-cell inflammation and immune checkpoint expression, as well as expression of APOBEC3 genes. Mutations in immune-evasion pathways were also enriched in these tumors. Analysis of single-cell sequencing data identified expression of APOBEC3B and 3C genes in malignant cells. We identified an APOBEC-enriched subgroup of HPV-negative HNSCC with a distinct immunogenic phenotype, potentially mediating response to immunotherapy.

Immune escape is one of the hallmarks of cancer and several new treatment approaches attempt to modulate and restore the immune system’s capability to target cancer cells. At the heart of the immune recognition process lies antigen presentation from somatic mutations. These neo-epitopes are emerging as attractive targets for cancer immunotherapy and new strategies for rapid identification of relevant candidates have become a priority. We carefully screen TCGA data sets for recurrent somatic amino acid exchanges and apply MHC class I binding predictions. We propose a method for in silico selection and prioritization of candidates which have a high potential for neo-antigen generation and are likely to appear in multiple patients. While the percentage of patients carrying a specific neo-epitope and HLA-type combination is relatively small, the sheer number of new patients leads to surprisingly high reoccurence numbers. We identify 769 epitopes which are expected to occur in 77629 patients per year. While our candidate list will definitely contain false positives, the results provide an objective order for wet-lab testing of reusable neo-epitopes. Thus recurrent neo-epitopes may be suitable to supplement existing personalized T cell treatment approaches with precision treatment options.

e17577 Background: Salivary gland tumors (SGT) represent a rare and heterogeneous group of malignancies. No standard treatment exists in the advanced situation and the prognosis is poor. We here report characteristics and clinical outcomes of patients with SGT discussed at the Charité molecular tumor board (MTB). Methods: Patients with advanced cancer and no curative treatment option were discussed at the Charité MTB. Eligible patients underwent fresh tissue sampling and subsequent whole exome (WES) and RNA sequencing (RNA-seq) and immunohistochemical analyses (EGFR, HER2, AR as well as validation tests) or panel sequencing. Results from molecular testing were discussed at the MTB and patients were followed-up after recommendations were made. Results: 24 patients (median age 56 years, 13 male, 11 female) with advanced SGT were presented at the MTB between 2016 and 2019 (9 adenoidcystic carcinomas, 5 adenocarcinomas, 3 mucoepidermoid, 2 carcinosarcoma, 5 miscellaneous). WES/RNA sequencing was performed on tumor tissue from 16 patients. 2 patients were not included in the sequencing program and WES/RNA-Seq was ongoing for another 4 patients at the time of analysis. For another 2 patients, panel sequencing and IHC analysis, respectively was done. Results from analyses were discussed and a median of 2 recommendations, ranked by priority according to prespecified evidence levels, were made for 17 patients, each. Most commonly proposed treatment options by the MTB were FGFR inhibitors in 6 patients, mTOR or PARP inhibitors in 5 each, EGFR, HDAC inhibitors or antiandrogen therapy in 4, each. Treatments following MTB recommendations were initiated in 8 patients, 1 of which received a second recommended therapy after progression (antiandrogen therapy in 4, EGFR inhibitor in 2, a PDGFR, mTOR and PARP inhibitor in 1, each). A clinical benefit (CR = 1; Mixed Response = 1, SD = 3) was achieved in 5 patients, including a complete response in a patient with a metastatic adenocarcinoma of the parotid gland, treated with antiandrogen therapy. Conclusions: Precision oncology represents a feasible treatment strategy in patients with advanced SGT and shows early evidence of activity in a subset of patients. These results suggest further exploration of personalized therapy in these hard-to-treat tumors.

Summary We propose the simple method HLA-MA for consistency checking in pipelines operating on human HTS data. The method is based on the HLA typing result of the state-of-the-art method OptiType. Provided that there is sufficient coverage of the HLA loci, comparing HLA types allows for simple, fast and robust matching of samples from whole genome, exome and RNA-seq data. Our approach uses information from small but genetically highly variable regions and thus complements approaches that rely on genome or exon-wide variant profiles. Availability and Implementation The software is implemented In Python 3 and freely available under the MIT license at https://github.com/bihealth/hlama and via Bioconda. Supplementary information Supplementary data are available at Bioinformatics online.

e24164Background: Precision Oncology programs hold promise to improve the treatment of cancer patients. The interpretation and prioritization of complex molecular data from comprehensive analyses h...

e14613 Background: Immune checkpoints are an emerging treatment target in head and neck squamous cell carcinoma (HNSCC). Expression of PD-L1 and an IFNG immune signature have been shown as biomarkers of response. In this study we analyzed mutational signatures to identify recurrent mutational patterns associated with inflammation. Methods: HNSCC mutation and gene expression data were downloaded (n = 496, TCGA). Inflamed tumors were identified through upregulation of a predefined 6-gene IFNG immune signature. Mutational load, base exchange motifs and a BRCAness signature were analyzed in inflamed and non-inflamed tumors. Results were validated in independent datasets of cervical, urothelial and lung cancer (TCGA). Results: Total mutational load did not correlate with tumor inflammation (r = -0.03, p = 0.5). An APOBEC-associated tCw (thymine – cytosine – adenine/thymine) mutational signature was significantly associated with inflammation (r = 0.26, p = 2.923e-09). The tCw signature was furthermore correlated with the expression of PD-L1 and other immune checkpoints (r = 0.19, p = 1.723e-05). The TCW mutational signature correlated with the expression of APOBEC3 genes. Expression of APOBEC3 genes also correlated with tumor inflammation (r = 0.62, p < 2.2e-16). As a second mutational signature, indels and an independent BRCAness signature were significantly enriched in non-inflamed tumors (p = 6.464e-14). Enrichment of an APOBEC mutational signature in inflamed tumors could be validated in cervical and urothelial carcinoma but not NSCLC. Conclusions: In HNSCC, overall mutational load is not associated with inflammation. BRCAness is significantly associated with immune-desertion whereas expression of APOBEC genes and an APOBEC mutational signature significantly correlates with the expression of PD-L1 and a T-cell inflamed signature. These findings suggest that a specific mutational pattern might serve as a biomarker for immune checkpoint inhibition in cancer.

3082 Background: KEAP1 and STK11 mutations are associated with resistance to immune checkpoint inhibition (ICI) in non-small cell lung cancer (NSCLC). Mechanisms are currently unknown. Methods: We examined mutation, methylation, copy number and gene expression data from the cancer genome atlas (TCGA) lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LSCC), head and neck squamous cell carcinoma (HNSCC) and cervical carcinoma (CESC) data sets as well as public single cell gene expression data from a HNSCC cohort. Pathway annotations were performed using gene set enrichment analysis. A previously published cohort of NSCLC patients treated with ICI was analyzed for the predictive value of NFE2L2 mutations on PFS. Results: Annotation of STK11 and KEAP1 mutant LUAD revealed identical gene set enrichment for mitochondrial metabolism and downregulation of the STING-pathway, immune checkpoints, and interferon signaling. A STK11/KEAP1-mutation derived gene expression signature was established in LUAD and found to be driven by NFE2L2-regulated genes. This gene expression signature was independently predictive of immune desertion in LSCC, CESC and HNSCC and associated with STING-pathway downregulation in single cell sequencing analyses in HNSCC. KEAP1 and STK11 mutations were less frequent in LSCC, CESC and HNSCC but NFE2L2 mutations were identified in 15, 6 and 5%, respectively. NFE2L2 mutant SCC exhibited upregulation of the 15-gene- signature as well as immune desertion. In NSCLC, NFE2L2 mutations were associated with significantly worse PFS with ICI. Conclusions: Alterations of KEAP1, STK11, NFE2L2 and other related genes are linked to NFE2L2 target gene upregulation and immune desertion in LUAD, CESC, LSCC and HNSCC alike. The NFE2L2 pathway should be investigated clinically as a putative negative predictive biomarker for ICI and a potential therapeutic target.