MIRACUM-Pipe: An Adaptable Pipeline for Next-Generation Sequencing Analysis, Reporting, and Visualization for Clinical Decision Making.

(1) Background: Next-generation sequencing (NGS) of patients with advanced tumors is becoming an established method in Molecular Tumor Boards. However, somatic variant detection, interpretation, and report generation, require in-depth knowledge of both bioinformatics and oncology. (2) Methods: MIRACUM-Pipe combines many individual tools into a seamless workflow for comprehensive analyses and annotation of NGS data including quality control, alignment, variant calling, copy number variation estimation, evaluation of complex biomarkers, and RNA fusion detection. (3) Results: MIRACUM-Pipe offers an easy-to-use, one-prompt standardized solution to analyze NGS data, including quality control, variant calling, copy number estimation, annotation, visualization, and report generation. (4) Conclusions: MIRACUM-Pipe, a versatile pipeline for NGS, can be customized according to bioinformatics and clinical needs and to support clinical decision-making with visual processing and interactive reporting.

RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells.

RATIONALE: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells. METHODS: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays. RESULTS: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growth Conclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

Automated library preparation for whole genome sequencing by centrifugal microfluidics.

Next generation sequencing is evolving from a research tool into a method applied in diagnostic routine. The complete sequencing workflow includes sample pre-processing, library preparation, sequencing and bioinformatics. High quality in each of these steps is necessary to obtain excellent sequencing results. The tedious and error-prone library preparation poses a significant challenge for smaller laboratories, where high throughput pipetting robots are not cost-effective. Here we present an automated library preparation for whole genome sequencing using centrifugal microfluidics. Two samples can be run per cartridge. Precise metering of reagents allows the required liquid volumes to be reduced by 40% and the amount of sample used by 60%. The functionality of the cartridge is demonstrated with bacteria and DNA extracted from a human FFPE sample. For the bacterial sample, mean sequencing depths from 140 to 183 reads and a coverage of 99.8% of the reference genome were detected. For the human DNA, mean sequencing depths of 4.4-5.7 reads and a coverage of 78.2% of the effective reference genome were observed.

Transitioning the Molecular Tumor Board from Proof of Concept to Clinical Routine: A German Single-Center Analysis.

Molecular precision oncology faces two major challenges: first, to identify relevant and actionable molecular variants in a rapidly changing field and second, to provide access to a broad patient population. Here, we report a four-year experience of the Molecular Tumor Board (MTB) of the Comprehensive Cancer Center Freiburg (Germany) including workflows and process optimizations. This retrospective single-center study includes data on 488 patients enrolled in the MTB from February 2015 through December 2018. Recommendations include individual molecular diagnostics, molecular stratified therapies, assessment of treatment adherence and patient outcomes including overall survival. The majority of MTB patients presented with stage IV oncologic malignancies (90.6%) and underwent an average of 2.1 previous lines of therapy. Individual diagnostic recommendations were given to 487 patients (99.8%). A treatment recommendation was given in 264 of all cases (54.1%) which included a molecularly matched treatment in 212 patients (43.4%). The 264 treatment recommendations were implemented in 76 patients (28.8%). Stable disease was observed in 19 patients (25.0%), 17 had partial response (22.4%) and five showed a complete remission (6.6%). An objective response was achieved in 28.9% of cases with implemented recommendations and for 4.5% of the total population (22 of 488 patients). By optimizing the MTB workflow, case-discussions per session increased significantly while treatment adherence and outcome remained stable over time. Our data demonstrate the feasibility and effectiveness of molecular-guided personalized therapy for cancer patients in a clinical routine setting showing a low but robust and durable disease control rate over time.

Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling.

Cathepsin D (CTSD) is a lysosomal protease and a marker of poor prognosis in breast cancer. However, the cells responsible for this association and the function of CTSD in cancer are still incompletely understood. By using a conditional CTSD knockout mouse crossed to the transgenic MMTV-PyMT breast cancer model we demonstrate that CTSD deficiency in the mammary epithelium, but not in myeloid cells, blocked tumor development in a cell-autonomous manner. We show that lack of CTSD impaired mechanistic Target of Rapamycin Complex 1 (mTORC1) signaling and induced reversible cellular quiescence. In line, CTSD-deficient tumors started to grow with a two-month delay and quiescent Ctsd-/- tumor cells re-started proliferation upon long-term culture. This was accompanied by rewiring of oncogenic gene expression and signaling pathways, while mTORC1 signaling remained permanently disabled in CTSD-deficient cells. Together, these studies reveal a tumor cell-autonomous effect of CTSD deficiency, and establish a pivotal role of this protease in the cellular response to oncogenic stimuli.