Leveraging Off-Target Reads in Panel Sequencing for Homologous Recombination Repair Deficiency Screening in Tumor.

Targeted tumor only sequencing has become a standard practice in cancer diagnostics. This study aims to develop an approach for robust copy number variant calling in tumor samples using only off-target region (OTR) reads. We also established a clinical use case for homologous recombination deficiency (HRD) score estimation (HRDest) using the sum of telomeric-allelic imbalance and large-scale state transition scores without the need for loss of heterozygosity information. A strong correlation was found between HRD score and the sum of telomeric-allelic imbalance + large-scale state transition in The Cancer Genome Atlas cohort (ρ = 0.99, P < 2.2 × 10-16) and in a clinical in-house cohort of 34 tumors (ρ = 0.9, P = 5.1 × 10-13) comparing whole-exome sequencing and targeted sequencing data. HRDest scores from 1086 clinical cases were compared with The Cancer Genome Atlas data set. There were no significant differences in HRD score distribution within the analyzed tumor types. As a control, commercially available HRD standards were also sequenced, and the HRDest scores obtained from the OTR reads were well within the HRD reference range provided by the manufacturer. In conclusion, OTR reads of tumor-only panel sequencing can be used to determine genome-wide copy number variant profiles and to approximate HRD scores.

High Concordance of Different Assays in the Determination of Homologous Recombination Deficiency-Associated Genomic Instability in Ovarian Cancer.

PURPOSE: Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promising clinical results in the treatment of ovarian cancer. Analysis of biomarker subgroups consistently revealed higher benefits for patients with homologous recombination deficiency (HRD). The test that is most often used for the detection of HRD in clinical studies is the Myriad myChoice assay. However, other assays can also be used to assess biomarkers, which are indicative of HRD, genomic instability (GI), and BRCA1/2 mutation status. Many of these assays have high potential to be broadly applied in clinical routine diagnostics in a time-effective decentralized manner. Here, we compare the performance of a multitude of alternative assays in comparison with Myriad myChoice in high-grade serous ovarian cancer (HGSOC). METHODS: DNA from HGSOC samples was extracted from formalin-fixed paraffin-embedded tissue blocks of cases previously run with the Myriad myChoice assay, and GI was measured by multiple molecular assays (CytoSNP, AmoyDx, Illumina TSO500 HRD, OncoScan, NOGGO GISv1, QIAseq HRD Panel and whole genome sequencing), applying different bioinformatics algorithms. RESULTS: Application of different assays to assess GI, including Myriad myChoice, revealed high concordance of the generated scores ranging from very substantial to nearly perfect fit, depending on the assay and bioinformatics pipelines applied. Interlaboratory comparison of assays also showed high concordance of GI scores. CONCLUSION: Assays for GI assessment not only show a high concordance with each other but also in correlation with Myriad myChoice. Thus, almost all of the assays included here can be used effectively to assess HRD-associated GI in the clinical setting. This is important as PARPi treatment on the basis of these tests is compliant with European Medicines Agency approvals, which are methodologically not test-bound.

Genomic heterogeneity at baseline is associated with T790M resistance mutations in EGFR-mutated lung cancer treated with the first-/second-generation tyrosine kinase inhibitors.

This study analyzed whether extended molecular profiling can predict the development of epidermal growth factor receptor (EGFR) gene T790M mutation, which is the most frequent resistance alteration in non-small cell lung cancer (NSCLC) after treatment with the first-/second-generation (1G/2G) EGFR inhibitors (tyrosine kinase inhibitors [TKIs]), but only weakly associated with clinical characteristics. Whole exome sequencing (WES) was performed on pretreatment tumor tissue with matched normal samples from NSCLC patients with (n = 25, detected in tissue or blood rebiopsies) or without (n = 14, negative tissue rebiopsies only) subsequent EGFR p.T790M mutation after treatment with 1G/2G EGFR TKI. Several complex genetic biomarkers were assessed using bioinformatic methods. After treatment with first-line afatinib (44%) or erlotinib/gefitinib (56%), median progression-free survival and overall survival were 12.1 and 33.7 months, respectively. Clinical and tumor genetic characteristics, including age (median, 66 years), sex (74% female), smoking (69% never/light smokers), EGFR mutation type (72% exon 19 deletions), and TP53 mutations (41%) were not significantly associated with T790M mutation (p > 0.05). By contrast, complex biomarkers including tumor mutational burden, the clock-like mutation signature SBS1 + 5, tumor ploidy, and markers of subclonality including mutant-allele tumor heterogeneity, subclonal copy number changes, and median tumor-adjusted variant allele frequency were significantly higher at baseline in tumors with subsequent T790M mutation (all p < 0.05). Each marker alone could predict subsequent development of T790M with an area under the curve (AUC) of 0.72-0.77, but the small number of cases did not allow confirmation of better performance for biomarker combinations in leave-one-out cross-validated logistic regression (AUC 0.69, 95% confidence interval: 0.50-0.87). Extended molecular profiling with WES at initial diagnosis reveals several complex biomarkers associated with subsequent development of T790M resistance mutation in NSCLC patients receiving first-/second-generation TKIs as the first-line therapy. Larger prospective studies will be necessary to define a forecasting model.

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.

[Molecular tumor diagnostics as the driving force behind precision oncology]. / Molekulare Tumordiagnostik als Triebfeder der Präzisionsonkologie.

Molecular pathological diagnostics plays a central role in personalized oncology and requires multidisciplinary teamwork. It is just as relevant for the individual patient who is being treated with an approved therapy method or an individual treatment attempt as it is for prospective clinical studies that require the identification of specific therapeutic target structures or complex biomarkers for study inclusion. It is also of crucial importance for the generation of real-world data, which is becoming increasingly important for drug development. Future developments will be significantly shaped by improvements in scalable molecular diagnostics, in which increasingly complex and multi-layered data sets must be quickly converted into clinically useful information. One focus will be on the development of adaptive diagnostic strategies in order to be able to depict the enormous plasticity of a cancer disease over time.

Accurate tumor purity determination is critical for the analysis of homologous recombination deficiency (HRD).

Homologous recombination deficiency (HRD) is a predictive marker for response to poly (ADP-ribose) polymerase inhibitors (PARPi) in ovarian carcinoma. HRD scores have entered routine diagnostics, but the influence of algorithms, parameters and confounders has not been analyzed comprehensively. A series of 100 poorly differentiated ovarian carcinoma samples was analyzed using whole exome sequencing (WES) and genotyping. Tumor purity was determined using conventional pathology, digital pathology, and two bioinformatic methods. HRD scores were calculated from copy number profiles determined by Sequenza and by Sclust either with or without fixed tumor purity. Tumor purity determination by digital pathology combined with a tumory purity informed variant of Sequenza served as reference method for HRD scoring. Seven tumors had deleterious mutations in BRCA1/2, 12 tumors had deleterious mutations in other homologous recombination repair (HRR) genes, 18 tumors had variants of unknown significance (VUS) in BRCA1/2 or other HRR genes, while the remaining 63 tumors had no relevant alterations. Using the reference method for HRD scoring, 68 tumors were HRD-positive. HRDsum determined by WES correlated strongly with HRDsum determined by single nucleotide polymorphism (SNP) arrays (R = 0.85). Conventional pathology systematically overestimated tumor purity by 8% compared to digital pathology. All investigated methods agreed on classifying the deleterious BRCA1/2-mutated tumors as HRD-positive, but discrepancies were observed for some of the remaining tumors. Discordant HRD classification of 11% of the tumors was observed comparing the tumor purity uninformed default of Sequenza and the reference method. In conclusion, tumor purity is a critical factor for the determination of HRD scores. Assistance by digital pathology helps to improve accuracy and imprecision of its estimation.

Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by transcriptome analysis.

TP53 is the most frequently mutated gene in human cancer. While no TP53-targeting drugs have been approved in the USA or Europe so far, preclinical and clinical studies are underway to investigate targeting of specific or all TP53 mutations, for example, by restoration of the functionality of mutated TP53 (TP53mut) or protecting wildtype TP53 (TP53wt) from negative regulation. We performed a comprehensive mRNA expression analysis in 24 cancer types of TCGA to extract (i) a consensus expression signature shared across TP53 mutation types and cancer types, (ii) differential gene expression patterns between tumors harboring different TP53 mutation types such as loss of function, gain of function or dominant-negative mutations, and (iii) cancer-type-specific patterns of gene expression and immune infiltration. Analysis of mutational hotspots revealed both similarities across cancer types and cancer type-specific hotspots. Underlying ubiquitous and cancer type-specific mutational processes with the associated mutational signatures contributed to explaining this observation. Virtually no genes were differentially expressed between tumors harboring different TP53 mutation types, while hundreds of genes were over- and underexpressed in TP53mut compared to TP53wt tumors. A consensus list included 178 genes that were overexpressed and 32 genes that were underexpressed in the TP53mut tumors of at least 16 of the investigated 24 cancer types. In an association analysis of immune infiltration with TP53 mutations in 32 cancer subtypes, decreased immune infiltration was observed in six subtypes, increased infiltration in two subtypes, a mixed pattern of decreased and increased immune cell populations in four subtypes, while immune infiltration was not associated with TP53 status in 20 subtypes. The analysis of a large cohort of human tumors complements results from experimental studies and supports the view that TP53 mutations should be further evaluated as predictive markers for immunotherapy and targeted therapies.

Homologous recombination deficiency is inversely correlated with microsatellite instability and identifies immunologically cold tumors in most cancer types.

Homologous recombination deficiency (HRD) leads to DNA double-strand breaks and can be exploited by the use of poly (ADP-ribose) polymerase (PARP) inhibitors to induce synthetic lethality. Extending the original therapeutic concept, the role of HRD is currently being investigated in clinical trials testing immune checkpoint blockers alone or in combination with PARP inhibitors, but the relationship between HRD and immune cell context in cancer is incompletely understood. We analyzed the association between immune cell composition, gene expression, and HRD in 9,041 tumors of 32 solid cancer types from The Cancer Genome Atlas (TCGA). The numbers of genomic scars were quantified by the HRD sum score (HRDsum) including loss of heterozygosity, large-scale state transitions, and telomeric allelic imbalance. The T-cell inflamed gene expression profile correlated weakly, but significantly positively, with HRDsum across cancer types (ρ = 0.17). Within individual cancer types, a significantly positive correlation was observed only in breast cancer, ovarian cancer, and four other cancer types, but not in the remaining 26 cancer types. HRDsum and tumor mutational burden (TMB) correlated significantly positively across cancer types (ρ = 0.42) and within 18 cancer types. HRDsum and a proliferation metagene correlated significantly positively across cancer types (ρ = 0.52) and within 20 cancer types. Mismatch repair deficiency and HRD as well as proofreading deficiency showed a high level of exclusivity. High HRD scores were associated with an immunologically activated tumor microenvironment only in a minority of cancer types. Our data favor the combination of genetic markers, complex genomic markers (including HRDsum and TMB), and other molecular markers (including proliferation scores) for a precise and comprehensive read-out of the tumor biology and an individually tailored treatment.

NoPeak: k-mer-based motif discovery in ChIP-Seq data without peak calling.

MOTIVATION: The discovery of sequence motifs mediating DNA-protein binding usually implies the determination of binding sites using high-throughput sequencing and peak calling. The determination of peaks, however, depends strongly on data quality and is susceptible to noise. RESULTS: Here, we present a novel approach to reliably identify transcription factor-binding motifs from ChIP-Seq data without peak detection. By evaluating the distributions of sequencing reads around the different k-mers in the genome, we are able to identify binding motifs in ChIP-Seq data that yield no results in traditional pipelines. AVAILABILITY AND IMPLEMENTATION: NoPeak is published under the GNU General Public License and available as a standalone console-based Java application at https://github.com/menzel/nopeak. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RNA-guided retargeting of Sleeping Beauty transposition in human cells.

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

Enhort: a platform for deep analysis of genomic positions.

The rise of high-throughput methods in genomic research greatly expanded our knowledge about the functionality of the genome. At the same time, the amount of available genomic position data increased massively, e.g., through genome-wide profiling of protein binding, virus integration or DNA methylation. However, there is no specialized software to investigate integration site profiles of virus integration or transcription factor binding sites by correlating the sites with the diversity of available genomic annotations. Here we present Enhort, a user-friendly software tool for relating large sets of genomic positions to a variety of annotations. It functions as a statistics based genome browser, not focused on a single locus but analyzing many genomic positions simultaneously. Enhort provides comprehensive yet easy-to-use methods for statistical analysis, visualization, and the adjustment of background models according to experimental conditions and scientific questions. Enhort is publicly available online at enhort.mni.thm.de and published under GNU General Public License.

[Fe19 ] "Super-Lindqvist" Aggregate and Large 3D Interpenetrating Coordination Polymer from Solvothermal Reactions of [Fe2 (OtBu)6 ] with Ethanol.

The syntheses, crystal structures, and physical properties of [HFe19 O14 (OEt)30 ] and {Fe11 (OEt)24 }∞ are reported. [HFe19 O14 (OEt)30 ] has an octahedral shape. Its core with a central Fe metal ion surrounded by six µ6 -oxo ligands is arranged in the rock salt structure. {Fe11 (OEt)24 }∞ is a mixed-valence coordination polymer in which Fe(III) metal ions form three 3D interpenetrating (10,3)-b nets. The arrangement of the Fe(III) ions can also be compared to that of Si ions in α-ThSi2 . Thus, the described structures are at the interface between molecular and solid-state chemistry.

Evaluation of the PotoClean(®) decontamination technology for reprocessing of water supply lines in dental units during routine work.

BACKGROUND: A frequent problem in dental units is the microbial contamination of water and biofilm formation in the water supply lines. After random identification of a bacterial contaminated dental unit (310 cfu/ml) in a practise with 3 dental units we implemented the present study to evaluate the efficacy of the PotoClean(®) technology, based on anodic oxidation. METHOD: The efficacy of a regular low concentrated permanent decontamination (1 mg Cl/L) with an additional intensive decontamination by PotoClean(®) (three times 20 mg Cl/ml for 2 h) on three dental units was tested over 7 months. Microbial contamination, total chlorine concentration and redox potential have been analyzed. Dental unit A and B was 15 years old, unit C 5 years. RESULTS: After 3 intensive decontaminations, in dental unit A and B the number of bacteria and moulds could be reduced less than 7 d. Thereafter the bacteria counts increased again during the subsequent 7 month period and the amount of moulds was with some exceptions 300 cfu/ml, although PotoClean(®) was constantly added in the system (1 mg Cl/L). After further 7.5 month only with low concentrated permanent disinfection (1 mg Cl/L) both units were successful decontaminated. Dental unit C represented an object which was easier to decontaminate because of the advanced construction (prevention of water stagnation) and the shorter useful life. At the beginning of the decontamination it was no bacterial contamination, but moulds were contained (300 cfu/ml). Already after the first intensive decontamination, no further bacteria and moulds could be detected. DISCUSSION: An important factor for the efficacy of PotoClean(®) was the age of the units and their construction. For a new generation of dental units PotoClean(®) was effective during the whole period of monitoring. For two old types of dental unit with massive biofilm development the successful decontamination needed more than 7 month. CONCLUSION: The PotoClean(®) technology has resulted in even old-type turbines with intensive biofilm formation to complete decontamination. In a recent turbine design already after the first intensive decontamination with PotoClean(®) and its continuous use (1 mg Cl/L) no more contamination by bacteria and moulds were detectable.

Online gas chromatography combustion/pyrolysis-isotope ratio mass spectrometry (HRGC-C/P-IRMS) of (+/-)-Dihydroactinidiolide from tea ( Camellia sinensis ) and rooibos tea ( Aspalathus linearis ).

Online capillary gas chromatography-isotope ratio mass spectrometry in both the combustion and the pyrolysis modes (HRGC-C/P-IRMS) was employed to perform authentication studies of the flavoring agent (+/-)-dihydroactinidiolide. Thus, the delta(13)C(V-PDB) and delta(2)H(V-SMOW) values of synthetic (ex synthetic beta-ionone and natural beta-carotene) as well as enzymatically (ex synthetic and natural beta-carotene) produced references were studied in comparison with those of the natural substance isolated from black (n = 17) and green teas (n = 6) ( Camellia sinensis ) as well as Rooibos tea ( Aspalathus linearis ) (n = 7). The isotope values determined for both the synthetic and enzymatically produced samples of (+/-)-dihydroactinidiolide reflected the influence of the origin of their educts. Hence, in cases when synthetic educts were used, the delta(13)C(V-PDB) and delta(2)H(V-SMOW) values ranged from -27.0 to -28.4 per thousand and from -28 to -169 per thousand, respectively, whereas the use of natural educts led to ranges from -30.3 to -31.6 per thousand and from -154 to -228 per thousand, respectively. As to the tea samples, delta(13)C(V-PDB) and delta(2)H(V-SMOW) values ranging from -29.0 to -34.1 per thousand and from -153 to -274 per thousand, respectively, were recorded for (+/-)-dihydroactinidiolide from black and green teas, whereas that from Rooibos tea showed (2)H/(1)H ratios ranging from -189 to -210 per thousand as well as slightly enriched values in the (13)C/(12)C ratios ranging from -24.4 to -27.1 per thousand.

Carbon and hydrogen stable isotope ratios of carotenoids and beta-carotene-based dietary supplements.

Considering the increasing nutritional and commercial importance of carotenoids, there is an interest in developing a reliable method for authenticity assessment of these compounds. Applying isotope ratio mass spectrometry using elemental analysis in the "combustion" (C) and "pyrolysis" (P) modes (EA-C/P-IRMS), the delta (13)C V-PDB and delta (2)H V-SMOW values of selected carotenoids and alpha/beta-carotene-based commercial dietary supplements were determined in comparison to those of synthetic and "natural" references. The delta (13)C V-PDB and delta (2)H V-SMOW values of synthetic beta-carotene samples ( n = 4), ranging from -25.3 per thousand to -26.4 per thousand and from -144 per thousand to -155 per thousand, respectively, differed clearly from the data determined for carotenoids from various natural sources, including C 3 plant material ( n = 9; delta (13)C V-PDB ranging from -28.5 per thousand to -32.8 per thousand and delta (2)H V-SMOW from -180 per thousand to -275 per thousand) and microalgae Dunaliella salina ( n = 1; delta (13)C V-PDB value of -15.6 per thousand and delta (2)H V-SMOW value of -191 per thousand). From five commercial dietary supplements under study, two revealed delta (13)C V-PDB and delta (2)H V-SMOW values in areas as found for synthetic references, and the other three had values near those of biotechnological beta-carotene produced by D. salina. The delta (13)C V-PDB and delta (2)H V-SMOW values recorded for natural lycopene ( n = 4) and lutein ( n = 5) ranged from -31.1 per thousand to -31.8 per thousand and from -180 to -201 per thousand, as well as from -28.8 per thousand to -32.2 per thousand and from -186 per thousand to -245 per thousand, respectively. Synthetic canthaxanthin ( n = 3) exhibited delta (13)C V-PDB and delta (2)H V-SMOW values ranging from -25.0 per thousand to -28.6 per thousand and from -133 per thousand to -153 per thousand, respectively. The EA-C/P-IRMS application of this study showed that the natural stable isotopic composition of carotenoids is a powerful tool for determining their origin.

The solid-state architecture of a metallosupramolecular polyelectrolyte.

Self-assembly of Fe(II) and the ditopic ligand 1,4-bis(2,2':6',2''-terpyridine-4'-yl)benzene results in equilibrium structures in solutions, so-called metallosupramolecular coordination polyelectrolytes (MEPEs). It is exceedingly difficult to characterize such macromolecular assemblies, because of the dynamic nature. Therefore, hardly any structural information is available for this type of material. Here, we show that from dilute solutions, where small aggregates predominate, it is possible to grow nanoscopic crystals at an interface. A near atomic resolution structure of MEPE is obtained by investigating the nanoscopic crystals with electron diffraction in combination with molecular modeling. The analysis reveals a primitive monoclinic unit cell (P2(1)/c space group, a = 10.4 A, b = 10.7 A, c = 34.0 A, alpha = gamma = 90 degrees , beta = 95 degrees , rho = 1.26 g/cm(3), and Z = 4). The MEPE forms linear rods, which are organized into sheets. Four sheets intersect the unit cell, while adjacent sheets are rotated by 90 degrees with respect to each other. The pseudooctahedral coordination geometry of the Fe(II) centers is confirmed by Mössbauer spectroscopy. The combination of diffraction and molecular modeling presented here may be of general utility to address problems in structural materials science.