RAS mutation is the most frequent oncogenic alteration in human cancers. KRAS is the most frequently mutated followed by NRAS. The emblematic KRAS mutant cancers are pancreatic, colorectal, lung ...adenocarcinomas and urogenital cancers. KRAS mutation frequencies are relatively stable worldwide in various cancer types with the one exception of lung adenocarcinoma. The frequencies of KRAS variant alleles appears cancer type specific, reflecting the various carcinogenic processes. In addition to point mutation KRAS, allelic imbalances are also frequent in human cancers leading to the predominance of a mutant allele. KRAS mutant cancers are characterized by typical, cancer-type-specific co-occurring mutations and distinct gene expression signatures. The heterogeneity of KRAS mutant primary cancers is significant, affecting the variant allele frequency, which could lead to unpredictable branching development in metastases. Selection of minute mutant subclones in the primary tumors or metastases during target therapies can also occur frequently in lung or colorectal cancers leading to acquired resistance. Ultrahigh sensitivity techniques are now routinely available for diagnostic purposes, but the proper determination of mutant allele frequency of KRAS in the primary or metastatic tissues may have larger clinical significance.
Fusions involving NTRK1, NTRK2, and NTRK3 are oncogenic drivers occurring in a spectrum of mesenchymal neoplasms ranging from benign to highly malignant tumors. To gain further insights into the ...staining profile with the pan-TRK assay, we analyzed a large number of soft tissue sarcomas and correlated our findings with molecular testing. Additionally, we expand the spectrum of NTRK-fusion tumors by reporting a mesenchymal lesion in the lung as well as a mesenchymal skin lesion in the spectrum of benign fibrous histiocytoma with NTRK—fusion. We retrospectively reviewed soft tissue sarcomas diagnosed at the Diagnostic and Research Institute of Pathology, Medical University of Graz, between 1999 and 2019, and cases from the consultation files of one of the authors (BLA). In total, 494 cases were analyzed immunohistochemically with pan-TRK antibody (clone EPR17341, RTU, Roche/Ventana) and positive cases (defined as any cytoplasmic/nuclear staining in more than 1% of tumor cells) underwent next-generation sequencing (NGS). Immunohistochemical staining was observed in 16 (3.2%) cases. Eleven cases with focal weak and moderate cytoplasmic/membranous or focal moderate to strong nuclear staining did not harbor an NTRK-fusion (three synovial sarcomas, three leiomyosarcomas, two extraskeletal myxoid chondrosarcomas, and one each: dedifferentiated liposarcoma, pleomorphic liposarcoma, and myxofibrosarcoma). Four cases showed strong diffuse nuclear and/or cytoplasmatic staining, and one case showed diffuse, but weak cytoplasmic staining. All these cases demonstrated an NTRK-fusion (LMNA-NTRK1, IRF2BP2-NTRK1, TMB3-NTRK1, ETV6-NTRK3, RBPMS-NTRK3). Pan-TRK assay (clone EPR17341, RTU, Roche, Ventana) immunohistochemistry serves as a reliable diagnostic marker that can also be expressed in non-NTRK-rearranged mesenchymal neoplasms. It can be used as a surrogate marker for identification of NTRK fusion, nevertheless, an RNA-based NGS for detection of the specific fusion should be performed to confirm the rearrangement, if patients are undergoing targeted therapy. Additionally, we identified NTRK-fusion-positive, primary mesenchymal tumors of the lung and the skin.
This study evaluates the frequency and type of TP53 gene mutations and HPV status in 72 consecutively diagnosed primary invasive vulvar squamous cell carcinomas (SCC) during the past 5years.
DNA of ...formalin-fixed and paraffin embedded tumour tissue was analysed for 32 HPV subtypes and the full coding sequence of the TP53 gene, and correlated with results of p53 immunohistochemistry.
13/72 (18%) cancers were HPV-induced squamous cell carcinomas, of which 1/13 (8%) carcinoma harboured a somatic TP53 mutation. Among the 59/72 (82%) HPV-negative cancers, 59/72 (82%) SCC were HPV-negative with wild-type gene in 14/59 (24%) SCC and somatic TP53 mutations in 45/59 (76%) SCC. 28/45 (62%) SCC carried one (n=20) or two (n=8) missense mutations. 11/45 (24%) carcinomas showed a single disruptive mutation (3× frame shift, 7× stop codon, 1× deletion), 3/45 SCC a splice site mutation. 3/45 (7%) carcinomas had 2 or 3 different mutations. 18 different “hot spot” mutations were observed in 22/45 cancers (49%; 5× R273, 3× R282; 2× each Y220, R278, R248). Immunohistochemical p53 over expression was identified in most SCC with missense mutations, but not in SCC with disruptive TP53 mutations or TP53 wild-type. 14/45 (31%) patients with TP53 mutated SCC died of disease within 12months (range 2–24months) versus 0/13 patients with HPV-induced carcinomas and 0/14 patients with HPV-negative, TP53 wild-type carcinomas.
80% of primary invasive vulvar SCC were HPV-negative carcinomas with a high frequency of disruptive mutations and “hot spot” TP53 gene mutations, which have been linked to chemo- and radioresistance. The death rate of patients with p53 mutated vulvar cancers was 31%. Immunohistochemical p53 over expression could not reliably identify SCC with TP53 gene mutation. Pharmacological therapies targeting mutant p53 will be promising strategies for personalized therapy in patients with TP53 mutated vulvar cancers.
•In a 5-year consecutive analysis of invasive vulvar SCC 80% were HPV-negative.•75% of HPV-negative SCC harboured disruptive and “hot spot” TP53 gene mutations.•Patients with TP53 gene mutated SCC had poorer survival.
Highlights • Antibiotic treatment of adult mice impairs object recognition but not spatial memory. • Gut microbiota disruption alters the metabolic profiles in colon and plasma. • The cerebral ...neuropeptide Y system is severely disturbed in gut dysbiosis.
Analysis of RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathological diagnostics. We have performed a comprehensive and systematic ...investigation of the impact of factors in the pre-analytical workflow, such as different fixatives, fixation time, RNA extraction method and storage of tissues in paraffin blocks, on several downstream reactions including complementary DNA (cDNA) synthesis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray hybridization. We compared the effects of routine formalin fixation with the non-crosslinking, alcohol-based Tissue Tek Xpress Molecular Fixative (TTXMF, Sakura Finetek), and cryopreservation as gold standard for molecular analyses. Formalin fixation introduced major changes into microarray gene expression data and led to marked gene-to-gene variations in delta-ct values of qRT-PCR. We found that qRT-PCR efficiency and gene-to-gene variations were mainly attributed to differences in the efficiency of cDNA synthesis as the most sensitive step. These differences could not be reliably detected by quality assessment of total RNA isolated from formalin-fixed tissues by electrophoresis or spectrophotometry. Although RNA from TTXMF fixed samples was as fragmented as RNA from formalin fixed samples, much higher cDNA yield and lower ct-values were obtained in qRT-PCR underlining the negative impact of crosslinking by formalin. In order to better estimate the impact of pre-analytical procedures such as fixation on the reliability of downstream analysis, we applied a qRT-PCR-based assay using amplicons of different length and an assay measuring the efficiency of cDNA generation. Together these two assays allowed better quality assessment of RNA extracted from fixed and paraffin-embedded tissues and should be used to supplement quality scores derived from automated electrophoresis. A better standardization of the pre-analytical workflow, application of additional quality controls and detailed sample information would markedly improve the comparability and reliability of molecular studies based on formalin-fixed and paraffin-embedded tissue samples.
Objectives
Anorexia nervosa (AN) is a heterogeneous eating disorder associated with alterations of body structure and the gut microbiome. We aimed to investigate the gut microbiota composition of a ...large female cohort including different BMI groups and activity levels along with body composition parameters.
Method
106 female participants were included in this cross‐sectional study: AN patients (n = 18), athletes (n = 20), normal weight (n = 26), overweight (n = 22), and obese women (n = 20). DNA was extracted from stool samples and subjected to 16S rRNA gene analysis. The software Quantitative Insights Into Microbial Ecology (QIIME) was used to analyze data. Additionally, we performed anthropometric assessments, ultrasound measurements of subcutaneous adipose tissue thickness, bioimpedance analysis, administered depression inventories, and ascertained laboratory parameters and dietary intakes.
Results
Alpha diversity was particularly lower in AN patients and obese participants compared to other groups, while athletes showed highest alpha diversity. Several categories significantly associated with community structure were identified: body fat parameters, serum lipids, CRP, depression scales and smoking. Comparative analysis revealed Coriobacteriaceae as the only enriched phylotype in AN compared to other entities (LDA score >3.5).
Discussion
This study provides further evidence of intestinal dysbiosis in AN and sheds light on characteristics of the gut microbiome in different BMI and physical activity groups. These insights point to new modulation possibilities of the gut microbiota which could improve the standard therapy of AN.
Monitoring the T cell receptor (TCR) repertoire in health and disease can provide key insights into adaptive immune responses, but the accuracy of current TCR sequencing (TCRseq) methods is unclear. ...In this study, we systematically compared the results of nine commercial and academic TCRseq methods, including six rapid amplification of complementary DNA ends (RACE)-polymerase chain reaction (PCR) and three multiplex-PCR approaches, when applied to the same T cell sample. We found marked differences in accuracy and intra- and inter-method reproducibility for T cell receptor α (TRA) and T cell receptor β (TRB) TCR chains. Most methods showed a lower ability to capture TRA than TRB diversity. Low RNA input generated non-representative repertoires. Results from the 5' RACE-PCR methods were consistent among themselves but differed from the RNA-based multiplex-PCR results. Using an in silico meta-repertoire generated from 108 replicates, we found that one genomic DNA-based method and two non-unique molecular identifier (UMI) RNA-based methods were more sensitive than UMI methods in detecting rare clonotypes, despite the better clonotype quantification accuracy of the latter.
Although particle therapy with protons has proven to be beneficial in the treatment of chondrosarcoma compared to photon-based (X-ray) radiation therapy, the cellular and molecular mechanisms have ...not yet been sufficiently investigated. Cell viability and colony forming ability were analyzed after X-ray and proton irradiation (IR). Cell cycle was analyzed using flow cytometry and corresponding regulator genes and key players of the DNA repair mechanisms were measured using next generation sequencing, protein expression and immunofluorescence staining. Changes in metabolic phenotypes were determined with nuclear magnetic resonance spectroscopy. Both X-ray and proton IR resulted in reduced cell survival and a G2/M phase arrest of the cell cycle. Especially 1 h after IR, a significant dose-dependent increase of phosphorylated γH2AX foci was observed. This was accompanied with a reprogramming in cellular metabolism. Interestingly, within 24 h the majority of clearly visible DNA damages were repaired and the metabolic phenotype restored. Involved DNA repair mechanisms are, besides the homology directed repair (HDR) and the non-homologous end-joining (NHEJ), especially the mismatch mediated repair (MMR) pathway with the key players EXO1, MSH3, and PCNA. Chondrosarcoma cells regenerates the majority of DNA damages within 24 h. These molecular mechanisms represent an important basis for an improved therapy.
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