Dataset Information

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

ABSTRACT: Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2?×?5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r?=?0.963). We also achieved area under the curve (AUC) at 0.9998?±?0.0002 (p?

SUBMITTER: Yu G

PROVIDER: S-EPMC7385113 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

Yu Guohua G Zhang Wenfeng W Zhang Yunyun Y Lv Jiahong J Wu Shishou S Sui Xiaolong X Zhang Jiandi J Tang Fangrong F

Scientific reports 20200727 1

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE s ...[more]

PMID: 32719400

Similar Datasets

Project description:The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Project description:Grapevine red blotch is a recently identified viral disease that was first recognized in the Napa Valley of California. Infected plants showed foliar symptoms similar to leafroll, another grapevine viral disease, on vines testing negative for known grapevine leafroll-associated virus. Later, the Grapevine red blotch virus (GRBV) was independently discovered in the US states of California and New York and was demonstrated to be the causal agent of red blotch disease. Due to its wide occurrence in the United States, vector transmission, and impacts on grape industry, this virus has the potential to cause serious economic losses. Despite numerous attempts, it has yet not been possible to isolate or visualize viral particles from GRBV-infected plants, thereby hampering the development of a serological assay that would facilitate GRBV detection in grapevine. In this work, mass spectrometry approaches were applied in order to quantify GRBV in infected plants and identify potential biomarkers for viral infection. We present for the first time the physical detection on the protein level of the two GRBV genes V1 (coat protein) and V2 in grapevine tissue lysates. The GRBV coat protein load in petioles was determined to be in the range of 100-900 million copies per milligram wet weight by using three heavy isotope labeled reference peptides as internal standards. In leaves on the other hand, the V1 copy number per unit wet tissue weight appeared to be about six times lower than in petioles, and about 300 times lower in terms of protein concentration in the extractable protein mass, albeit these estimations could only be made with one reference peptide detectable in leaf extracts. Moreover, we found in leaf and petiole extracts of GRBV-infected plants a consistent upregulation of several enzymes involved in flavonoid biosynthesis by label-free shotgun proteomics, indicating the activation of a defense mechanism against GRBV, a plant response already described for Grapevine leafroll-associated virus infection on the transcriptome level. Finally and importantly, we identified some other microorganisms belonging to the grapevine leaf microbiota, two bacterial species (Novosphingobium sp. Rr 2-17 and Methylobacterium) and one virus, Grapevine rupestris stem pitting-associated virus.

Dataset Information

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

Publications

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets