Quantitative real-time polymerase chain reaction is an alternative method for the detection of HER-2 amplification in formalin-fixed paraffin-embedded breast cancer samples.
Author(s): Pu T, Guo P, Qiu Y, Chen S, Yang L, Sun L, Ye F, Bu H
Publication: Int J Clin Exp Pathol, 2015, Vol. 8, Page 10565-74
PubMed ID: 26617766 PubMed Review Paper? No
Purpose of Paper
This paper compared fluorescent in situ hybridization (FISH), quantitative PCR (qPCR), and immunohistochemistry for the quantification of human epidermal growth factor receptor 2 (HER-2) in formalin-fixed paraffin-embedded (FFPE) breast tumor specimens. The potential impact of cold ischemia time (0-12 h) on HER-2 quantification by the three methods was also investigated using a subset of breast tumor specimens.
Conclusion of Paper
HER-2 amplification results in the 71 breast tumor specimens were nearly identical when evaluated by qPCR and FISH detection methods, but immunohistochemistry yielded a greater number of ambiguous results. When breast tumor specimens were subjected to the 0 to 12 h cold ischemia timecourse, qPCR comparative cycle threshold (Ct) values for HER-2 exhibited nonsignificant fluctuations over the cold ischemia times investigated. Conversely, FISH signal intensity degraded substantially for HER-2 (but not CEP17) beginning after 2 hours.
Studies
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Study Purpose
This study compared FISH, qPCR, and immunohistochemistry methods of HER-2 quantification in 71 surgically resected FFPE breast tumor specimens that contained 70% or more tumor cell content. Potential effects of cold ischemia time on HER-2 quantification was investigated in a subset of breast tumor specimens collected from five patients following a room temperature delay to fixation of 0, 1, 2, 3, 4, 12 h. All specimens were fixed in 10% neutral buffered formalin for 24 hours. FFPE sections (4 µm thick) were used for FISH, immunohistochemistry, and after DNA extraction with a phenol-chloroform based method, qPCR. HER-2 copy number variation was quantified by qPCR using the delta delta comparative threshold cycle (Ct) method and three housekeeping genes (actin, transferrin receptor protein 1 (TFRC), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), while FISH was determined by HER-2/CEP17 ratio.
Summary of Findings:
HER-2 amplification results in the 71 FFPE breast tumor specimens were nearly identical when evaluated by qPCR and FISH detection methods, but immunohistochemistry yielded a greater number of ambiguous results. HER-2 quantification by qPCR yielded 26 positive and 45 negative specimens, of which 24 and 44, respectively, were confirmed by FISH. However of the 26 specimens that were positive by qPCR, only 19 were positive by immunohistochemistry while the remaining 7 specimens generated ambiguous results. Similarly, of the 45 specimens that were HER-2 negative by qPCR only 25 were negative by immunohistochemistry, with the remaining 20 generating ambiguous results. When breast tumor specimens subjected to the 0 to 12 h cold ischemia timecourse were compared, comparative HER-2 mean Ct values exhibited nonsignificant fluctuations over the cold ischemia times investigated. Conversely, FISH signal intensity degraded substantially for HER-2 (but not CEP17) beginning after 2 hours. HER-2 FISH signal was considered to be degraded when one of following was present, an indistinct cellular outline, ≥25% cells unscorable due to weak signal, poor nuclear resolution, or interfering background signal.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Protein Immunohistochemistry DNA FISH Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 h
1 h
2 h
3 h
4 h
12 h
Real-time qPCR Specific Technology platform FISH
Immunohistochemistry
Immunohistochemistry Specific Targeted peptide/protein HER-2
FISH Specific Targeted nucleic acid HER-2/CEP17
Real-time qPCR Specific Targeted nucleic acid Actin
TFRC
GAPDH
HER-2