How is Fluorescence In Situ Hybridization (FISH) used in targeted chemotherapy?

FISH in Targeted Chemotherapy

FISH is the current gold standard technique for identifying specific chromosomal abnormalities and gene rearrangements that directly guide selection of targeted therapies in cancer patients, particularly for detecting actionable alterations like ALK rearrangements in lung cancer, HER2 amplification in breast cancer, and high-risk cytogenetics in multiple myeloma. 1

Primary Diagnostic Role in Treatment Selection

FISH serves as a companion diagnostic test that determines patient eligibility for specific targeted therapies by detecting genomic aberrations that predict treatment response 1:

• In lung cancer: Break-apart FISH is FDA-approved for detecting ALK rearrangements (≥15% positive cells) to identify patients who benefit from ALK kinase inhibitors like crizotinib 2, 3
• In breast cancer: HER2 FISH testing (HER2:CEP17 ratio >2.2) predicts response to trastuzumab and other anti-HER2 therapies 1
• In multiple myeloma: FISH identifies high-risk cytogenetic abnormalities including t(4;14), t(14;16), and del(17p13) that guide treatment intensification decisions 1
• In sarcomas: FISH detects NTRK gene fusions that indicate eligibility for TRK inhibitors like larotrectinib and entrectinib 1

Technical Advantages for Targeted Therapy Selection

FISH overcomes critical limitations of conventional karyotyping that make it superior for treatment decisions 1:

• Performed on interphase cells, eliminating the need for dividing cells and enabling analysis when metaphases are difficult to obtain 1
• Detects submicroscopic abnormalities (3-5 Mb resolution) invisible on standard karyotypes 1
• Identifies cryptic translocations with variable breakpoints (e.g., IGH rearrangements) where molecular methods fail due to breakpoint heterogeneity 1
• Enumerates the percentage of cells harboring specific abnormalities, providing quantitative data for treatment monitoring 1, 4

Specific Applications by Cancer Type

Multiple Myeloma

For routine diagnosis in newly diagnosed myeloma, testing should minimally include t(4;14) and del(17p13) by FISH, as these define high-risk disease requiring more aggressive therapy 1. A comprehensive panel may include gain(1q), del(1p), t(14;16), and markers for aneuploidy 1.

Non-Small Cell Lung Cancer

ALK FISH should be performed using break-apart probes with a positivity threshold of ≥15% rearranged cells 1, 3. ROS1 FISH requires break-apart probe design showing signals split by at least 1 probe diameter in ≥15% of tumor cells 1. Importantly, patients with 5' deletion patterns on ALK FISH may have lower response rates to crizotinib compared to those with split signals, and corroborative testing with IHC or NGS is beneficial 2.

Breast Cancer

HER2 FISH is used when IHC results are equivocal (2+) or to resolve discordant cases 1. Approximately 3% of cases show IHC/FISH discordance, and available data suggest benefit from anti-HER2 therapy is limited to patients with gene amplification 1.

TRK Fusion Sarcomas

FISH uses break-apart probes flanking NTRK1, NTRK2, and NTRK3 genes to detect rearrangements without identifying fusion partners 1. However, FISH has a reported false-negative rate up to 30% in pediatric sarcomas and cannot distinguish in-frame from out-of-frame fusions 1.

Critical Limitations Affecting Treatment Decisions

Several technical constraints must be recognized when using FISH for therapy selection 1:

• Does not detect point mutations: TP53 is deleted in only 7% of myeloma by FISH but mutated at much higher frequency by sequencing 1
• Targeted analysis only: FISH requires knowing which abnormality to test for, missing unexpected driver alterations 1
• Cannot distinguish structural from numerical changes: Positive results may reflect duplications rather than therapeutically relevant translocations 5
• Variable sensitivity by probe design: Break-apart probes may miss complex rearrangements or small deletions 1, 3

Monitoring Minimal Residual Disease

FISH enables quantitative assessment of treatment response and residual disease 4:

• In CML patients achieving complete cytogenetic remission (100% Ph-negative by karyotype), FISH detected 0-2.5% BCR-ABL fusion-positive cells in first remission 4
• Post-transplant CML patients with 2-6% BCR-ABL positive cells by FISH had higher relapse rates within 3-24 months compared to those with 0-1.5% 4
• For follow-up studies, a minimum of 200 interphase nuclei must be analyzed (versus 100 for diagnostic samples) 1

Integration with Other Testing Modalities

FISH should not be used as a standalone test but integrated into a comprehensive molecular testing strategy 1:

• IHC can serve as a screening test (e.g., for ROS1, ALK) with FISH confirmation of positive results 1
• Next-generation sequencing detects FISH-negative cases with atypical breakpoints and identifies concurrent mutations 1, 2
• In cases with discordant FISH and IHC results, NGS may reveal alternative driver mutations (EGFR, KRAS) indicating false-positive FISH 2

Quality Requirements for Clinical Use

Validation standards are critical for treatment-guiding FISH assays 1, 5:

• Thresholds must be established using diverse sample preparations reflecting typical diagnostic specimens 1
• Minimum 100 interphase nuclei for diagnostic samples, 200 for follow-up 1
• Concurrent normal controls required for probes with limited clinical experience 5
• Borderline results require extreme caution and clinical context, as cut-off values represent estimates 5