scholarly journals High-throughput proteomic analysis of FFPE tissue samples facilitates tumor stratification

2019 ◽  
Author(s):  
Yi Zhu ◽  
Tobias Weiss ◽  
Qiushi Zhang ◽  
Rui Sun ◽  
Bo Wang ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Temporal Stability ◽  
B Cell Lymphoma ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Pattern Similarity ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Peroxidase Enzyme ◽  
High Degree

AbstractFormalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

scholarly journals High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Y. Cheng ◽  
Lauren E. Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael T. Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

AbstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

Accelerated Lysis and Proteolytic Digestion of Biopsy-Level Fresh-Frozen and FFPE Tissue Samples Using Pressure Cycling Technology

2020 ◽  
Vol 19 (5) ◽  
pp. 1982-1990 ◽  
Author(s):  
Huanhuan Gao ◽  
Fangfei Zhang ◽  
Shuang Liang ◽  
Qiushi Zhang ◽  
Mengge Lyu ◽  
...  
Keyword(s):  
Ffpe Tissue ◽  
Proteolytic Digestion ◽  
Tissue Samples ◽  
Pressure Cycling ◽  
Fresh Frozen ◽  
Pressure Cycling Technology

scholarly journals High Sensitivity Sanger Sequencing Detection of BRAF Mutations in Metastatic Melanoma FFPE Tissue Specimens

2020 ◽  
Author(s):  
Lauren Cheng ◽  
Lauren Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

Abstract Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTraceTM BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.26% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

scholarly journals Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples

2019 ◽  
Author(s):  
Aditya Vijay Bhagwate ◽  
Yuanhang Liu ◽  
Stacey J. Winham ◽  
Samantha J. McDonough ◽  
Melody L. Stallings-Mann ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Genetic Variants ◽  
Variant Calling ◽  
Allelic Frequency ◽  
Tissue Samples ◽  
Mutational Signature ◽  
Ffpe Samples ◽  
Fresh Frozen ◽  
Wet Lab ◽  
Control Samples

Abstract Background Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. Results As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different library preparations. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (<5%), and collectively shared a “C>T|G>A” mutational signature known to be representative of FFPE artifacts resulting fromcytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. Conclusions Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Measurement of gene expression biomarkers by qNPA from archived NSCLC FFPE: Prognosis of 5-year survival

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 11098-11098
Author(s):  
M. M. Joshi ◽  
B. Seligmann ◽  
C. Sabalos ◽  
D. H. Harpole
Keyword(s):  
Gene Expression ◽  
B Cell Lymphoma ◽  
Base Hydrolysis ◽  
Data Set ◽  
S1 Nuclease ◽  
Tissue Samples ◽  
Frozen Tissue ◽  
Fresh Frozen ◽  
Cox Analysis ◽  
Stage 1

11098 Background: There are vast archives of formalin fixed paraffin-embedded (FFPE) tissue samples that are clinically annotated offering great research potential. However, the technology currently available to assess gene expression is limited to fresh, frozen, tissue. Recently, a method for measuring gene expression from FFPE using the quantitative Nuclease Protection Assay (qNPA) has been published in a model of diffuse large B-cell lymphoma. It was demonstrated that identical results were obtained from small amounts of FFPE as from matched frozen tissue, or from freshly fixed versus 18 year archived FFPE. Methods: This study used the qNPA assay to measure gene expression in archived FFPE primary tumor samples of patients with stage 1 NSCLC for whom the survival outcomes are known (n=86). HTG lysis buffer is added to the sample; nuclease protection probes that are complementary to the mRNA of interest are then added to the solution. The probes hybridize to all RNA, soluble and cross-linked. After hybridization, S1 nuclease was added and destroys all nonspecific, single strand nucleic acid, producing a stoichiometric amount of target-mRNA to probe duplexes. Base hydrolysis releases the probe from these duplexes. Probes were transferred to a programmed ArrayPlate, detection linker added, and both probes and detection linkers were captured onto the array. The ArrayPlate was washed, HRP-labeled detection probe added, incubated, washed, and chemiluminescent substrate was added. Finally, the ArrayPlate was imaged, to measure the expression of each gene in all the wells. Results: Mantel-Cox analysis indicates that the detection of increased expression of G-CSF (p = 0.07; H.R. =1.904; 95% CI=0.9003–4.028) and Leptin (p=0.09; H.R. =1.910; 95% CI=0.9299–3.924) individually suggest an improved survival. Age, gender and T size were found to not be significant in this data set. Conclusions: These results suggest an improved survival advantage in patients with an elevated native GCSF level in stage 1 NSCLC that is consistent with the survival benefits associated with the prophylactic treatment of GCSF for chemosensitivity in stage III or IV NSCLC patients. These results are currently being assessed using a larger cohort. [Table: see text]

scholarly journals Accelerated Protein Biomarker Discovery from FFPE Tissue Samples Using Single-Shot, Short Gradient Microflow SWATH MS

2020 ◽  
Vol 19 (7) ◽  
pp. 2732-2741 ◽  
Author(s):  
Rui Sun ◽  
Christie Hunter ◽  
Chen Chen ◽  
Weigang Ge ◽  
Nick Morrice ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Ffpe Tissue ◽  
Single Shot ◽  
Protein Biomarker ◽  
Tissue Samples

scholarly journals Whole mitochondrial genome sequencing highlights mitochondrial impact in gastric cancer

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Giovanna Chaves Cavalcante ◽  
Anderson N. R. Marinho ◽  
Ana Karyssa Anaissi ◽  
Tatiana Vinasco-Sandoval ◽  
André Ribeiro-dos-Santos ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Native American ◽  
Mitochondrial Genome ◽  
Internal Control ◽  
Ffpe Tissue ◽  
Cancer Development ◽  
External Control ◽  
Tissue Samples ◽  
Significant Difference ◽  
Ffpe Samples

Abstract Mitochondria are organelles that perform major roles in cellular operation. Thus, alterations in mitochondrial genome (mtGenome) may lead to mitochondrial dysfunction and cellular deregulation, influencing carcinogenesis. Gastric cancer (GC) is one of the most incident and mortal types of cancer in Brazil, particularly in the Amazon region. Here, we sequenced and compared the whole mtGenome extracted from FFPE tissue samples of GC patients (tumor and internal control – IC) and cancer-free individuals (external control – EC) from this region. We found 3-fold more variants and up to 9-fold more heteroplasmic regions in tumor when compared to paired IC samples. Moreover, tumor presented more heteroplasmic variants when compared to EC, while IC and EC showed no significant difference when compared to each other. Tumor also presented substantially more variants in the following regions: MT-RNR1, MT-ND5, MT-ND4, MT-ND2, MT-DLOOP1 and MT-CO1. In addition, our haplogroup results indicate an association of Native American ancestry (particularly haplogroup C) to gastric cancer development. To the best of our knowledge, this is the first study to sequence the whole mtGenome from FFPE samples and to apply mtGenome analysis in association to GC in Brazil.

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