[Precision oncology and molecular tumor boards]. / Präzisionsonkologie und molekulare Tumorboards.

Precision oncology is a field of personalized medicine in which tumor biology forms the basis for tailored treatments. The preferred approach currently applied in clinical practice is based on the concept of malignant tumors as genetic diseases that are caused by mutations in oncogenes and tumor suppressors. On the one hand, these can be targeted by molecular drugs, while on the other hand, next-generation sequencing allows for comprehensive analysis of all relevant aberrations, thus enabling the matching of appropriate treatments across entities based on molecular information. Rational molecular therapies are developed and annotated with supporting evidence by molecular tumor boards, which have been established at various academic centers in recent years. Advancing precision oncology to a new standard of care requires improved applicability of personalized molecular therapies and thorough scientific evaluation of precision oncology programs.

Measuring Progress in Precision Oncology.

SUMMARY: In this issue of Cancer Discovery, Suehnholz and colleagues describe their efforts to quantify the gradual yet steady progress of precision oncology by surveying the regulatory approvals of targeted cancer therapies, and thus the actionability of corresponding molecular alterations in clinical practice, over more than 20 years. Their work also suggests a relationship between the discovery of candidate therapeutic targets through comprehensive tumor profiling and molecularly guided cancer drug development. See related article by Suehnholz et al., p. 49 (5).

High power Raman second stokes generation in a methane filled hollow core fiber.

We demonstrate a multi-watt, picosecond pulse duration laser source by exploiting a cascaded Raman process to the second Stokes signal at a wavelength of 2.58 µm in a methane-filled Nested Anti-Resonant Nodeless fiber from a 1 µm disk laser source. A maximum average power of 2.89 W (14.45 µJ) is produced in a 160 cm length of custom-designed and in-house fabricated fiber filled with methane at a pressure of 2 bar. The impact of gas pressure and propagation distance on the second Stokes signal power are investigated experimentally. The experimental results are simulated by solving the Generalized Nonlinear Schrodinger Equation with the experiment carefully modelled by accounting for the impacts of pressure dependent gas-light interactions along the pressure gradient of the fiber. This work offers a laser source for a variety of applications as well as expanding the modelling space to methane filled fibers including pressure gradients, and nonlinear optical activity in the presence of infrared gas absorption.

Multicentric pilot study to standardize clinical whole exome sequencing (WES) for cancer patients.

A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79-1, TMB: 0.97-0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

NCT/DKFZ MASTER handbook of interpreting whole-genome, transcriptome, and methylome data for precision oncology.

Analysis of selected cancer genes has become an important tool in precision oncology but cannot fully capture the molecular features and, most importantly, vulnerabilities of individual tumors. Observational and interventional studies have shown that decision-making based on comprehensive molecular characterization adds significant clinical value. However, the complexity and heterogeneity of the resulting data are major challenges for disciplines involved in interpretation and recommendations for individualized care, and limited information exists on how to approach multilayered tumor profiles in clinical routine. We report our experience with the practical use of data from whole-genome or exome and RNA sequencing and DNA methylation profiling within the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) program of the National Center for Tumor Diseases (NCT) Heidelberg and Dresden and the German Cancer Research Center (DKFZ). We cover all relevant steps of an end-to-end precision oncology workflow, from sample collection, molecular analysis, and variant prioritization to assigning treatment recommendations and discussion in the molecular tumor board. To provide insight into our approach to multidimensional tumor profiles and guidance on interpreting their biological impact and diagnostic and therapeutic implications, we present case studies from the NCT/DKFZ molecular tumor board that illustrate our daily practice. This manual is intended to be useful for physicians, biologists, and bioinformaticians involved in the clinical interpretation of genome-wide molecular information.

Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity.

Targeted therapies are effective in treating cancer, but success depends on identifying cancer vulnerabilities. In our study, we utilize small RNA sequencing to examine the impact of pathway activation on microRNA (miRNA) expression patterns. Interestingly, we discover that miRNAs capable of inhibiting key members of activated pathways are frequently diminished. Building on this observation, we develop an approach that integrates a low-miRNA-expression signature to identify druggable target genes in cancer. We train and validate our approach in colorectal cancer cells and extend it to diverse cancer models using patient-derived in vitro and in vivo systems. Finally, we demonstrate its additional value to support genomic and transcriptomic-based drug prediction strategies in a pan-cancer patient cohort from the National Center for Tumor Diseases (NCT)/German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) precision oncology trial. In conclusion, our strategy can predict cancer vulnerabilities with high sensitivity and accuracy and might be suitable for future therapy recommendations in a variety of cancer subtypes.

Convolutional neural networks for mode on-demand high finesse optical resonator design.

We demonstrate the use of machine learning through convolutional neural networks to solve inverse design problems of optical resonator engineering. The neural network finds a harmonic modulation of a spherical mirror to generate a resonator mode with a given target topology ("mode on-demand"). The procedure allows us to optimize the shape of mirrors to achieve a significantly enhanced coupling strength and cooperativity between a resonator photon and a quantum emitter located at the center of the resonator. In a second example, a double-peak mode is designed which would enhance the interaction between two quantum emitters, e.g., for quantum information processing.

BRAFΔß3-αC in-frame deletion mutants differ in their dimerization propensity, HSP90 dependence, and druggability.

In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAFΔß3-αC oncoproteins usually lack five amino acids in the ß3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAFΔß3-αC oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAFΔLNVTAP>F and two novel mutants, BRAFdelinsFS and BRAFΔLNVT>F, and compare them with other BRAFΔß3-αC oncoproteins. We show that BRAFΔß3-αC oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAFΔß3-αC oncoproteins, e.g., BRAFΔLNVTAP>F, increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAFΔß3-αC mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.

Tumour mutational burden and survival with molecularly matched therapy.

BACKGROUND: The impact of tumour mutational burden (TMB) on outcome with molecularly matched therapy is unknown. Higher TMB could predict resistance to molecularly matched therapy through co-occurring driver mutations. METHODS: One hundred and four patients with advanced cancers underwent molecular profiling in the DKTK-MASTER program. Fifty-five patients received systemic therapy excluding immunotherapy. Patients with molecularly matched (n = 35) or non-molecularly informed therapy (n = 20) were analysed for TMB and survival. Results were validated in an independent cohort of patients receiving molecularly matched (n = 68) or non-molecularly informed therapy (n = 40). Co-occurring driver mutations and TMB were analysed in the exploratory cohort and The Cancer Genome Atlas (TCGA) datasets. RESULTS: Patients were stratified by the median TMB of 1.67 mutations per Megabase (mut/Mb) of 35 patients receiving molecularly matched therapy into TMB-high or TMB-low groups. Median overall survival (4 months [95% CI, 3.3-7.6] versus 12.8 months [95% CI, 10-not reached], p < 0.001) and progression-free survival (1.8 months [95% CI, 1.1-3.7] versus 7.9 months [95% CI, 2.8-17.0], p = 0.003) were significantly shorter in the TMB-high group compared to the TMB-low group. In the validation cohort, shorter OS and PFS were identified in the TMB-high group (TMB cut-off of 4 mut/Mb) treated with molecularly matched therapy. No differences were observed in patients receiving non-molecularly informed systemic therapy. A significant correlation between co-occurring driver mutations and TMB (n = 104, r = 0.78 [95% CI, 0.68-0.85], p < 0.001) was found in the exploratory cohort as well as the majority (24/33) of TCGA studies. CONCLUSION: A high TMB was associated with unfavourable outcome in patients receiving molecularly matched therapy, indicating untargeted resistance pathways. Therefore, TMB should be further investigated as a predictive biomarker in precision oncology programs.

Bi-frequency operation in a membrane external-cavity surface-emitting laser.

We report on the achievement of continuous wave bi-frequency operation in a membrane external-cavity surface-emitting laser (MECSEL), which is optically pumped with up to 4 W of 808 nm pump light. The presence of spatially specific loss of the intra-cavity high reflectivity mirror allows loss to be controlled on certain transverse cavity modes. The regions of spatially specific loss are defined through the removal of Bragg layers from the surface of the cavity high reflectivity mirror in the form of crosshair patterns with undamaged central regions, which are created using a laser ablation system incorporating a digital micromirror device (DMD). By aligning the laser cavity mode with the geometric centre of the loss patterns, the laser simultaneously operated on two Hermite-Gaussian spatial modes: the fundamental HG00 and the higher order HG11 mode. We demonstrate bi-frequency operation over a range of pump powers and sizes of spatial loss features, with a wavelength separation of approximately 5 nm centred at 1005 nm.

Hollow core fiber Fabry-Perot interferometers with reduced sensitivity to temperature: erratum.

In the original publication of our research article "Hollow core fiber Fabry-Perot interferometers with reduced sensitivity to temperature" [Opt. Lett.47, 2510 (2022)10.1364/OL.456589OPLEDP0146-9592], we identified an error that requires correction. The authors sincerely apologize for any confusion that may have arisen from this error. The correction does not affect the overall conclusions of the paper.

CIViCdb 2022: evolution of an open-access cancer variant interpretation knowledgebase.

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC's functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications.

Comprehensive molecular profiling of sarcomas in adolescent and young adult patients: Results of the EORTC SPECTA-AYA international proof-of-concept study.

BACKGROUND: Adolescent and young adult (AYA) patients with cancer are poorly recruited to molecularly targeted trials and have not witnessed the advances in cancer treatment and survival seen in other age groups. We report here a pan-European proof-of-concept study to identify actionable alterations in some of the worst prognosis AYA cancers: bone and soft tissue sarcomas. DESIGN: Patients aged 12-29 years with newly diagnosed or recurrent, intermediate or high-grade bone and soft tissue sarcomas were recruited from six European countries. Pathological diagnoses were centrally reviewed. Formalin-fixed tissues were analysed by whole exome sequencing, methylation profiling and RNA sequencing and were discussed in a multidisciplinary, international molecular tumour board. RESULTS: Of 71 patients recruited, 48 (median 20 years, range 12-28) met eligibility criteria. Central pathological review confirmed, modified and re-classified the diagnosis in 41, 3, and 4 cases, respectively. Median turnaround time to discussion at molecular tumour board was 8.4 weeks. whole exome sequencing (n = 48), methylation profiling (n = 44, 85%) and RNA sequencing (n = 24, 50%) led to therapeutic recommendations for 81% patients, including 4 with germ line alterations. The most common were for agents targeted towards tyrosine kinases (n = 20 recommendations), DNA repair (n = 18) and the PI3K/mTOR/AKT pathway (n = 15). Recommendations were generally based on weak evidence such as activity in a different tumour type (n = 68, 61%), reflecting the dearth of relevant molecular clinical trial data in the same tumour type. CONCLUSIONS: We demonstrate here that comprehensive molecular profiling of AYA patients' samples is feasible and deliverable in a European programme.

Out-of-plane beam shaping with UV-written tilted Bragg gratings for beam delivery on quantum chips.

We theoretically and numerically investigate the performance of tilted Bragg gratings in planar waveguides, fabricated by direct UV writing in photosensitive silica, to couple light out of a chip. An analytic expression is derived for the coupling efficiency and validated numerically by finite element simulations. Using the analytic result, we can design gratings to generate output beams in free space of any specific shape and calculate their overall power coupling efficiency. Our simulations indicate that for currently achievable grating index contrasts devices of millimeter length are most suitable for this technology.

Sub-ppm gas phase Raman spectroscopy in an anti-resonant hollow core fiber.

We demonstrate recent progress in the development of a Raman gas sensor using a single cladding ring anti-resonant hollow core micro-structured optical fiber (HC-ARF) and a low power pump source. The HC-ARF was designed specifically for low attenuation and wide bandwidth in the visible spectral region and provided low loss at both the pump wavelength (532 nm) and Stokes wavelengths up to a Raman shift of 5000 cm-1. A novel selective core pressurization scheme was also implemented to further reduce the confinement loss, improving the Raman signal enhancement by a factor of 1.9 compared to a standard fiber filling scheme. By exploiting longer lengths of fiber, direct detection of both methane and hydrogen at concentrations of 5 and 10 ppm respectively is demonstrated and a noise equivalent limit-of-detection of 0.15 ppm is calculated for methane.

Current Developments in Cellular Therapy for Castration Resistant Prostate Cancer: A Systematic Review of Clinical Studies.

Recently, the development of immunotherapies such as cellular therapy, monoclonal antibodies, vaccines and immunomodulators has revolutionized the treatment of various cancer entities. In order to close the existing gaps in knowledge about cellular immunotherapy, specifically focusing on the chimeric antigen receptors (CAR) T-cells, their benefits and application in clinical settings, we conducted a comprehensive systematic review. Two co-authors independently searched the literature and characterized the results. Out of 183 records, 26 were considered eligible. This review provides an overview of the cellular immunotherapy landscape in treating prostate cancer, honing in on the challenges of employing CAR T-cell therapy. CAR T-cell therapy is a promising avenue for research due to the presence of an array of different tumor specific antigens. In prostate cancer, the complex microenvironment of the tumor vastly contributes to the success or failure of immunotherapies.

Feasibility and outcome of reproducible clinical interpretation of high-dimensional molecular data: a comparison of two molecular tumor boards.

BACKGROUND: Structured and harmonized implementation of molecular tumor boards (MTB) for the clinical interpretation of molecular data presents a current challenge for precision oncology. Heterogeneity in the interpretation of molecular data was shown for patients even with a limited number of molecular alterations. Integration of high-dimensional molecular data, including RNA- (RNA-Seq) and whole-exome sequencing (WES), is expected to further complicate clinical application. To analyze challenges for MTB harmonization based on complex molecular datasets, we retrospectively compared clinical interpretation of WES and RNA-Seq data by two independent molecular tumor boards. METHODS: High-dimensional molecular cancer profiling including WES and RNA-Seq was performed for patients with advanced solid tumors, no available standard therapy, ECOG performance status of 0-1, and available fresh-frozen tissue within the DKTK-MASTER Program from 2016 to 2018. Identical molecular profiling data of 40 patients were independently discussed by two molecular tumor boards (MTB) after prior annotation by specialized physicians, following independent, but similar workflows. Identified biomarkers and resulting treatment options were compared between the MTBs and patients were followed up clinically. RESULTS: A median of 309 molecular aberrations from WES and RNA-Seq (n = 38) and 82 molecular aberrations from WES only (n = 3) were considered for clinical interpretation for 40 patients (one patient sequenced twice). A median of 3 and 2 targeted treatment options were identified per patient, respectively. Most treatment options were identified for receptor tyrosine kinase, PARP, and mTOR inhibitors, as well as immunotherapy. The mean overlap coefficient between both MTB was 66%. Highest agreement rates were observed with the interpretation of single nucleotide variants, clinical evidence levels 1 and 2, and monotherapy whereas the interpretation of gene expression changes, preclinical evidence levels 3 and 4, and combination therapy yielded lower agreement rates. Patients receiving treatment following concordant MTB recommendations had significantly longer overall survival than patients receiving treatment following discrepant recommendations or physician's choice. CONCLUSIONS: Reproducible clinical interpretation of high-dimensional molecular data is feasible and agreement rates are encouraging, when compared to previous reports. The interpretation of molecular aberrations beyond single nucleotide variants and preclinically validated biomarkers as well as combination therapies were identified as additional difficulties for ongoing harmonization efforts.

Bcl-xL as prognostic marker and potential therapeutic target in cholangiocarcinoma.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-xL and Mcl-1. Expression of Bcl-xL , Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-xL and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl-xL (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-xL and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-xL as a key protein in cell death resistance of CCA and may pave the way for clinical application.