Why FISH is Preferred Over PCR for HER2 Amplification Detection in NSCLC
FISH is preferred over PCR for detecting HER2 amplification in NSCLC because it directly visualizes gene copy number at the single-cell level in intact tissue, avoiding the critical limitation of PCR where tumor and non-tumor cells are mixed during RNA/DNA extraction, which dilutes tumor cell influence and leads to false-negative results. 1
Technical Superiority of FISH for Gene Amplification
Direct Visualization vs. Dilution Effect
FISH detects gene amplification by directly visualizing DNA in individual tumor cells within tissue sections, maintaining spatial context and avoiding the mixing of tumor and non-tumor cells that occurs during nucleic acid extraction for PCR. 1
PCR-based methods (both RT-PCR and DNA PCR) require homogenization of tissue samples, which mixes tumor cells with stromal cells, inflammatory cells, and normal tissue, diluting the tumor cell contribution to the final result and increasing false-negative rates. 1
This dilution effect is particularly problematic in NSCLC specimens, which often have lower tumor cellularity compared to surgical resections and may contain significant amounts of normal lung parenchyma, inflammatory infiltrate, and necrotic tissue. 1
Accuracy and Reproducibility
When measured against external "gold standard" molecular characterization, FISH is more accurate, reproducible, and robust than other methods for detecting gene amplification. 1
FISH has demonstrated high specificity (approaching 100%) for detecting gene amplifications in lung cancer, with the ability to distinguish true amplification from polysomy or other chromosomal abnormalities. 1
The concordance between FISH and IHC for gene rearrangements (such as ALK) ranges from 75-100% depending on the antibody and protocol used, with FISH serving as the confirmatory gold standard. 1, 2
Limitations of PCR for Amplification Detection
RNA-Based RT-PCR Issues
RT-PCR for HER2 shows variable concordance with FISH, with some studies meeting the 95% ASCO/CAP concordance threshold while others show significantly lower concordance. 1
Concern has been raised regarding RT-PCR's sensitivity, with reports of high rates of equivocal findings and false-negative results that could lead to patient mismanagement. 1
While RT-PCR appears very specific, the mixing of tumor and non-tumor cells during RNA extraction can dilute the influence of tumor cells, leading to false-negative findings even when gene amplification is present. 1
DNA PCR Limitations
DNA PCR-based assays (including quantitative and digital PCR) are designed to detect specific mutations at known hotspots but are not optimally suited for detecting gene amplifications. 1
These assays require predetermined primers for specific alterations and lack the capability to visualize the actual chromosomal context of amplification, which can include complex patterns like clustered amplification or polysomy. 1
DNA PCR methods cannot distinguish between true gene amplification and increased chromosome copy number (polysomy), a distinction that is clinically relevant and easily made with FISH. 1
Clinical Context for NSCLC
Established Standard for Gene Rearrangements
The current standard method for detecting gene rearrangements in NSCLC (such as ALK and ROS1) is FISH, with a commercially available probe set that has been validated and FDA-approved for clinical use. 1
FISH has been the established method for detecting ALK rearrangements since the initial clinical trials of crizotinib, providing the evidence base for FDA approval and clinical decision-making. 1
European and US guidelines explicitly recognize FISH as an acceptable validated test for gene rearrangement detection in lung cancer, with other methods requiring validation against FISH as the reference standard. 1
Tissue Requirements and Practical Considerations
FISH can be performed on formalin-fixed paraffin-embedded (FFPE) tissue, the standard specimen type in clinical practice, and is relatively less dependent on tumor purity compared to PCR-based methods. 1
The assay works reliably even in samples with lower tumor cellularity (as low as 20% in some protocols), whereas PCR methods require higher tumor content to avoid false negatives from dilution. 1, 3
FISH has a relatively short turnaround time (1-3 days) and is widely available in clinical laboratories, making it practical for routine clinical use. 1
When PCR May Be Appropriate
Mutation Detection vs. Amplification
PCR-based methods (particularly Sanger sequencing and NGS) are the appropriate methods for detecting EGFR mutations, KRAS mutations, and other point mutations in NSCLC, where the goal is to identify specific nucleotide changes rather than gene copy number. 1
For detecting gene fusions where the fusion partner is known, RT-PCR can be used with specific primers, but this approach has sensitivity of only approximately 90% depending on coverage of fusion partners. 1
NGS as Comprehensive Alternative
Next-generation sequencing (NGS) can detect both mutations and some gene rearrangements simultaneously, offering comprehensive molecular profiling, though it may miss some rearrangements with atypical breakpoints. 1, 3
RNA-based NGS panels are increasingly used for fusion detection and can identify novel fusion partners, but FISH remains valuable for confirmation and when RNA quality is insufficient. 1, 4
Common Pitfalls to Avoid
Do not rely solely on IHC for HER2 assessment in NSCLC without confirmatory FISH or NGS, as IHC has lower specificity in lung cancer compared to breast cancer. 5, 6
Recognize that HER2 overexpression by IHC does not always correlate with gene amplification by FISH in NSCLC—studies show only 2-4% of NSCLC cases demonstrate true gene amplification despite 20-23% showing some degree of IHC positivity. 5, 6
Ensure adequate tissue sampling with at least 20-30% tumor cellularity for any molecular testing, as lower cellularity increases the risk of false negatives regardless of method. 3
When using PCR methods, be aware that negative results in samples with low tumor purity may represent false negatives due to dilution effects rather than true absence of the alteration. 1