Technical Validity of a Customized Assay of Sensitivity to Endocrine Therapy Using Sections from Fixed Breast Cancer Tissue.
Aurora Kinase A
/ genetics
Breast Neoplasms
/ drug therapy
Cohort Studies
Estrogen Receptor alpha
/ genetics
Estrogens
/ therapeutic use
Gene Expression Profiling
/ statistics & numerical data
Humans
Oligonucleotide Array Sequence Analysis
/ statistics & numerical data
Paraffin Embedding
RNA, Messenger
/ analysis
Receptor, ErbB-2
/ genetics
Receptors, Progesterone
/ genetics
Reproducibility of Results
Tissue Fixation
Journal
Clinical chemistry
ISSN: 1530-8561
Titre abrégé: Clin Chem
Pays: England
ID NLM: 9421549
Informations de publication
Date de publication:
01 07 2020
01 07 2020
Historique:
received:
01
01
2020
accepted:
20
04
2020
pubmed:
3
7
2020
medline:
30
3
2021
entrez:
3
7
2020
Statut:
ppublish
Résumé
We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II-III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin's concordance correlation coefficient (CCC) was used to measure concordance between measurements. SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1-9% coefficient of variation over 20 weekly tests, linear range 3.0-11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.
Sections du résumé
BACKGROUND
We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II-III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance.
METHODS
We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin's concordance correlation coefficient (CCC) was used to measure concordance between measurements.
RESULTS
SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1-9% coefficient of variation over 20 weekly tests, linear range 3.0-11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts).
CONCLUSIONS
Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.
Identifiants
pubmed: 32613237
pii: 5866248
doi: 10.1093/clinchem/hvaa105
doi:
Substances chimiques
ESR1 protein, human
0
Estrogen Receptor alpha
0
Estrogens
0
RNA, Messenger
0
Receptors, Progesterone
0
ERBB2 protein, human
EC 2.7.10.1
Receptor, ErbB-2
EC 2.7.10.1
AURKA protein, human
EC 2.7.11.1
Aurora Kinase A
EC 2.7.11.1
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
934-945Informations de copyright
© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.