[Molecular pathological analysis through the ages]. / Molekularpathologische Untersuchungen im Wandel der Zeit.

BACKGROUND: Molecular pathological examinations of tumor samples encompass a wide range of diagnostic analyses. Especially in recent years, numerous new biomarkers have come to the forefront-the analysis of which is crucial for therapy decisions. OBJECTIVES: Within the field of molecular pathology, the demands of next generation sequencing (NGS)-based requirements have experienced massive growth in recent years. To meet this demand, methods are constantly being adapted and further developed. The following sections aim to illuminate how this trend arises and which analyses are gaining importance. METHODS: The article provides an overview of the essential nucleic acid-based analysis techniques in the field of massive parallel sequencing. Terms such as DNA- and RNA-based techniques, as well as the associated analysis methods, are described, particularly with regard to their use in routine molecular pathological diagnostics. RESULTS: The breadth of genomic sequencing has been steadily growing in recent years, particularly due to the increasing relevance of personalized medicine, along with the rising approvals of targeted therapeutics. This necessitates, among other things, the analysis of new biomarkers. The diagnostics as part of interdisciplinary molecular tumor boards (MTB) are now based on large gene panels (> 1 megabase). Furthermore, through the "Modellvorhaben Genomsequenzierung" § 64e, whole exome or whole genome sequencing has been made available for oncological patients. Given these developments, it is evident that future analyses will require the integration of additional omics fields, such as whole transcriptome analysis, epigenomics, and proteomics. CONCLUSION: The challenges of personalized medicine along with the necessity of simultaneously assessing numerous new biomarkers require the implementation and execution of new techniques in molecular pathology whose complexity is steadily increasing.

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.

A Zinc Cluster Transcription Factor Contributes to the Intrinsic Fluconazole Resistance of Candida auris.

The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism.IMPORTANCECandida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast.