Abstract Administration of targeted therapies provides a promising treatment strategy for rare cancers such as urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC), however, the selection of appropriate drugs remains difficult. Therefore, in the present study, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. Next-generation sequencing using a 161 cancer driver gene panel was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered by four publicly available databases. Therapeutic interpretation has been performed by in silico evaluation of drug-gene interactions using an evidence-based decision support tool. After data processing, 45/54 samples (33 UrC and 12 PBAC) passed the quality control. The sequencing analyses revealed a total of 191 pathogenic SNVs in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), MYC (15%), EGFR (9%) and ERBB2 (9%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways in both tumour types were related to cell cycle regulation, DNA damage control and the MAPK/RAS pathway. Actionable mutations with at least one available, regulatory agency-approved drug could be identified for 31/33 (94%) of UrC and 8/12 (67%) of PBAC patients. In this study, we used a commercially available assay and developed a data processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, including EGFR, BRCA, CCND1/2/3, ERBB2, METex14 suggesting a potentially feasible strategy for both UrC and PBAC treatment.
This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose - hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.
Abstract Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants by the gold standard Sanger sequencing has been well established, however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging. To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes implicated in tumorigenesis (e.g. TP53, KRAS and NRAS). We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations in Sanger sequencing studies. To demonstrate the workflow of these panels with MVF, we analyzed DNA from lung cancer FFPE samples. We initially determined variants of TP53 and KRAS in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing (NGS). We confirmed the identity and variant allele frequency of these variants by Sanger sequencing coupled with MVF. Furthermore, we were able to confirm these results in 1 ng, 0.5 ng or 0.1 ng of DNA from these samples. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD). We show that this workflow can detect as little as 3% of a minor variant in an FFPE sample. Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available. For Research Use only - Not for use in diagnostic procedures. Citation Format: Arpad Gerstner, Edgar Schreiber, Stephen Jackson, Kamini Varma. Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3645.
Abstract Formalin-Fixed Paraffin Embedded (FFPE) tissue samples are the standard for clinical oncology tissue sample archiving worldwide. It is estimated that there are approximately a billion FFPE tissue cancer samples archived in hospitals and tissue banks around the world, and many of these samples have full clinical annotation including long term follow-up data. Current methods of analyzing gene expression in these samples involve laborious and expensive sample preparation protocols and long turnaround times (2 days or longer). We have developed a novel, rapid (3 hours), sample-to-answer technology to analyze gene expression from clinical FFPE tissues without the need for RNA extraction. The technique is highly sensitive and requires only 2 mm x 5 mm x 10 microns of FFPE material scraped from a pathology slide. The gene expression data obtained directly from FFPE without sample preparation correlates highly with data obtained on RNA extracted from the same FFPE sample using conventional methods. Importantly, the gene expression data obtained from prostate and breast FFPE samples correlate highly with microarray data, and with data from matching mirrored FF (Fresh Frozen) samples. The assay can be developed for detection using Luminex instrument or Capillary Electrophoresis instrument (Caliper GX or ABI 3500). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3204. doi:1538-7445.AM2012-3204
