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.

Independent Tissue-Based Biomarkers in Endometrioid Endometrial Cancer: Tumor Budding in Microsatellite Instability and WHO Grading in Copy-Number-Low Patients.

The molecular characterization of endometrial endometrioid adenocarcinomas has provided major advances in its prognostic stratification. However, risk assessment of microsatellite instability (MSI) and copy-number (CN)-low cases remains a challenge. Thus, we aimed to identify tissue-based morphologic biomarkers that might help in the prognostic stratification of these cases. Histomorphologic parameters (WHO grading, tumor budding (TB), tumor-stroma ratio (as a quantitative description of stromal desmoplasia), tumor-infiltrating lymphocytes (TIL), "microcystic, elongated, fragmented" (MELF) pattern) were analyzed in resection specimens of the TCGA-UCEC cohort (n = 228). For each quantitative parameter, a two-tiered system was developed utilizing systematically determined cutoffs. Associations with survival outcomes were calculated in univariate and multivariate analysis and validated in two independent cohorts. In MSI tumors, only TB remained an independent prognostic factor. TB (≥3 buds/high-power field) was associated with inferior outcomes and with lymph node metastases. The prognostic significance of TB was confirmed in two validation cohorts. For CN-low tumors, established grading defined by the WHO was independently prognostic with inferior outcomes for high-grade tumors. The evaluation of TB might help in identifying MSI-patients with unfavorable prognosis who, e.g., could benefit from lymphadenectomy. WHO-based grading facilitates independent prognostic stratification of CN-low endometrioid adenocarcinomas. Therefore, we propose the utilization of TB and WHO-based grading, two tissue-based and easy-to-assess biomarkers, in MSI/CN-low endometrial carcinomas for improved clinical management.

Tumour budding-based grading as independent prognostic biomarker in HPV-positive and HPV-negative head and neck cancer.

BACKGROUND: The prognostic significance of tumour budding (TB) and minimal cell nest size (MCNS) was shown in human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC). However, the optimisation of cutpoints, the prognostic impact in HPV-positive HNSCC, and the comparison with other histopathological grading systems are insufficiently investigated. METHODS: TB and MCNS were analysed digitally in 1 and 10 high-power fields (HPF) of 331 HPV-positive and HPV-negative cases from TCGA. Optimising the cutpoints a new cellular dissociation grading (CDG) system was defined and compared to the WHO grading and the Brandwein-Gensler (BG) risk model. RESULTS: The two-tiered CDG system based solely on TB yielded optimal prognostic stratification with shortened overall survival for CDG-high cases. Optimal cut-offs were two buds (1 HPF) and six buds (10 HPF), respectively. Analysing MCNS did not add prognostic significance to quantifying TB. CDG was a significant prognostic marker in HPV-negative and HPV-positive tumours and prognostically superior to the WHO and BG systems. High CDG was associated with clinically occult lymph-node metastases. CONCLUSIONS: The most comprehensive study of TB in HNSCC so far confirmed its prognostic impact in HPV-negative tumours and for the first time in HPV-positive tumours. Further studies are warranted to evaluate its applicability for therapy guidance in HNSCC.

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.

Cyanobacteria of Greece: an annotated checklist.

BACKGROUND: The checklist of Greek Cyanobacteria was created in the framework of the Greek Taxon Information System (GTIS), an initiative of the LifeWatchGreece Research Infrastructure (ESFRI) that has resumed efforts to compile a complete checklist of species reported from Greece. This list was created from exhaustive search of the scientific literature of the last 60 years. All records of taxa known to occur in Greece were taxonomically updated. NEW INFORMATION: The checklist of Greek Cyanobacteria comprises 543 species, classified in 130 genera, 41 families, and 8 orders. The orders Synechococcales and Oscillatoriales have the highest number of species (158 and 153 species, respectively), whereas these two orders along with Nostocales and Chroococcales cover 93% of the known Greek cyanobacteria species. It is worth mentioning that 18 species have been initially described from Greek habitats. The marine epilithic Ammatoidea aegea described from Saronikos Gulf is considered endemic to this area. Our bibliographic review shows that Greece hosts a high diversity of cyanobacteria, suggesting that the Mediterranean area is also a hot spot for microbes.