The impact of histopathology and NAB2-STAT6 fusion subtype in classification and grading of meningeal solitary fibrous tumor/hemangiopericytoma.

Meningeal solitary fibrous tumor (SFT)/hemangiopericytoma (HPC) is a rare tumor with propensity for recurrence and metastasis. Although multiple classification schemes have been proposed, optimal risk stratification remains unclear, and the prognostic impact of fusion status is uncertain. We compared the 2016 WHO CNS tumor grading scheme (CNS-G), a three-tier system based on histopathologic phenotype and mitotic count, to the 2013 WHO soft-tissue counterpart (ST-G), a two-tier system based on mitotic count alone, in a cohort of 133 patients [59 female, 74 male; mean age 54 years (range 20-87)] with meningeal SFT/HPC. Tumors were pathologically confirmed through review of the first tumor resection (n = 97), local recurrence (n = 35), or distant metastasis (n = 1). A STAT6 immunostain showed nuclear expression in 132 cases. NAB2-STAT6 fusion was detected in 99 of 111 successfully tested tumors (89%) including the single STAT6 immunonegative tumor. Tumors were classified by CNS-G as grade 1 (n = 43), 2 (n = 41), or 3 (n = 49), and by ST-G as SFT (n = 84) or malignant SFT (n = 49). Necrosis was present in 16 cases (12%). On follow-up, 42 patients had at least one subsequent recurrence or metastasis (7 metastasis only, 33 recurrence only, 2 patients had both). Twenty-nine patients died. On univariate analysis, necrosis (p = 0.002), CNS-G (p = 0.01), and ST-G (p = 0.004) were associated with recurrence-free (RFS) but not overall survival (OS). NAB2-STAT6 fusion type was not significantly associated with RFS or OS, but was associated with phenotype. A modified ST-G incorporating necrosis showed higher correlation with RFS (p = 0.0006) and remained significant (p = 0.02) when considering only the primary tumors. From our data, mitotic rate and necrosis appear to stratify this family of tumors most accurately and could be incorporated in a future grading scheme

Utility of different massive parallel sequencing platforms for mutation profiling in clinical samples and identification of pitfalls using FFPE tissue

In the growing field of personalised medicine, the analysis of numerous potential targets is becoming a challenge in terms of work load, tissue availability, as well as costs. The molecular analysis of non-small cell lung cancer (NSCLC) has shifted from the analysis of the epidermal growth factor receptor (EGFR) mutation status to the analysis of different gene regions, including resistance mutations or translocations. Massive parallel sequencing (MPS) allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small formalin-fixed, paraffin-embedded (FFPE) biopsies. In this study, we compared and evaluated currently used MPS platforms for their application in routine pathological diagnostics. We initiated a first round-robin testing of 30 cases diagnosed with NSCLC and a known EGFR gene mutation status. In this study, three pathology institutes from Germany received FFPE tumour sections that had been individually processed. Fragment libraries were prepared by targeted multiplex PCR using institution-specific gene panels. Sequencing was carried out using three MPS systems: MiSeq (TM), GS Junior and PGM Ion Torrent (TM). In two institutes, data analysis was performed with the platform-specific software and the Integrative Genomics Viewer. In one institute, data analysis was carried out using an in-house software system. Of 30 samples, 26 were analysed by all institutes. Concerning the EGFR mutation status, concordance was found in 26 out of 26 samples. The analysis of a few samples failed due to poor DNA quality in alternating institutes. We found 100% concordance when comparing the results of the EGFR mutation status. A total of 38 additional mutations were identified in the 26 samples. In two samples, minor variants were found which could not be confirmed by qPCR. Other characteristic variants were identified as fixation artefacts by reanalyzing the respective sample by Sanger sequencing. Overall, the results of this study demonstrated good concordance in the detection of mutations using different MPS platforms. The failure with samples can be traced back to different DNA extraction systems and DNA quality. Unknown or ambiguous variations (transitions) need verification with another method, such as qPCR or Sanger sequencing

SST gene hypermethylation acts as a pan‐cancer marker for pancreatic ductal adenocarcinoma and multiple other tumors: toward its use for blood‐based diagnosis

Aberrant DNA methylation is often involved in carcinogenesis. Our initial goal was to identify DNA methylation biomarkers associated with pancreatic cancer. A genomewide methylation study was performed on DNA from pancreatic ductal adenocarcinoma (PDAC) and endocrine pancreas tumors. Validation of DNA methylation patterns and concomitant alterations in expression of gene candidates was performed on patient samples and pancreatic cancer cell lines. Furthermore, validation was done on independent data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Finally, droplet digital PCR was employed to detect DNA methylation marks in cell‐free (cf) DNA isolated from plasma samples of PDAC patients and cancer‐free blood donors. Hypermethylation of the SST gene (encoding somatostatin) and concomitant downregulation of its expression were discovered in PDAC and endocrine tumor tissues while not being present in chronic pancreatitis (inflamed) tissues and normal pancreas. Fittingly, treatment with a somatostatin agonist (octreotide) reduced cell proliferation and migration of pancreatic cancer cells. Diagnostic performance of SST methylation in a receiver operating characteristic curve analysis was 100% and 89% for tissue and plasma samples, respectively. A large body of TCGA and GEO data confirmed SST hypermethylation and downregulation in PDAC and showed a similar effect in a broad spectrum of other tumor entities. SST promoter methylation represents a sensitive and promising molecular, pan‐cancer biomarker detectable in tumor tissue, and liquid biopsy samples

Quality Evaluation, Phytochemical Characteristics and Estimation of Beta-Carotene Hydroxylase 2 (Chy2) Alleles of Interspecific Potato Hybrids

Potatoes contain antioxidants such as flavonoids, carotenoids and ascorbic acid. High level consumption worldwide makes potato a valuable source of phytonutrients. Developing new potato varieties with high nutritional value in combination with resistance to pathogens is an important task. In this study, 25 interspecific potato hybrids with resistance to Synchytrium endobioticum, common scab, silver scab, rhizoctonia, nematodes and PVY were evaluated for total phenolics, total flavonoids, total carotenoids, ascorbic acid contents and antioxidant activity. The identification of the dominant allele related with yellow flesh color at the Chy locus was also performed by the specific CAPS marker. Total protein content was detected and ranged from 8.19 ± 0.59 to 30.17 ± 4.56 mg/g dry weight (DW). Total starch and total carbohydrate contents were in the range of 9.0–21.0% and 73.21 ± 20.94–676.36 ± 195.28, respectively. Total phenolic content of hybrids varied from 8.45 to 82.75 mg/100 g, and total flavonoids content—from 0.64 to 9.67 mg/100 g DW. It is possible to distinguish samples with high protein and carbohydrate contents, high level of substances with antioxidant activity and characterized by resistance to pathogens. Quality evaluation has shown that some of the samples have a high potential for processing and chip production in combination with high eating qualities. These samples can be used in breeding programs to develope varieties resistant to pathogens and with high nutritional value

Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing

Objectives: Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in similar to 5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. Materials and methods: We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. Results: The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. Conclusion: Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing. (C) 2014 Elsevier Ireland Ltd. All rights reserved