When should molecular testing, including Immunohistochemistry (IHC), Fluorescence In Situ Hybridization (FISH), and cytogenetics, be used in oncology to inform treatment decisions?

Molecular Testing in Oncology: When and What to Use

Molecular testing should be performed at diagnosis on all patients with advanced adenocarcinoma (lung, ovarian) and metastatic colorectal cancer using adequate tissue specimens, with multiplexed next-generation sequencing (NGS) panels preferred over single-gene tests to identify actionable mutations that guide targeted therapy selection and improve survival outcomes. 1

When to Use Molecular Testing

Lung Cancer

• Perform molecular testing at diagnosis for all patients with advanced lung adenocarcinoma (stage IV or incurable disease), regardless of clinical characteristics 1
• Testing should be done on either primary or metastatic lesions to guide initial therapy selection 1
• Consider testing in non-adenocarcinoma histologies when clinical features suggest higher probability of oncogenic drivers (age <50 years, light/no tobacco exposure) 1
• Repeat testing for EGFR T790M mutation in patients with EGFR-mutant adenocarcinoma who progress on first-generation EGFR TKIs 1

Ovarian Cancer

• BRCA mutation testing (germline and/or somatic) is mandatory at diagnosis for all patients with high-grade non-mucinous tubo-ovarian carcinoma, regardless of stage 1
• Genomic instability testing is recommended in BRCA wild-type high-grade tumors to identify homologous recombination deficiency for PARP inhibitor eligibility 1

Colorectal Cancer

• Extended RAS mutational testing is mandatory before initiating anti-EGFR therapy, including KRAS and NRAS codons 12,13 (exon 2); 59,61 (exon 3); and 117,146 (exon 4) 1
• BRAF p.V600 testing should be performed for prognostic stratification and in deficient MMR tumors with MLH1 loss to evaluate Lynch syndrome risk 1
• Mismatch repair (MMR) status testing should be ordered for Lynch syndrome identification and prognostic stratification 1

Tests Included in Molecular Testing Panels

Core Genes for Lung Adenocarcinoma

Mandatory testing includes: 1

• EGFR mutations (exons 18-21)
• ALK rearrangements
• ROS1 rearrangements
• BRAF V600E mutations

Appropriate for multiplex panels (not stand-alone): 1

• RET rearrangements
• ERBB2 (HER2) mutations
• KRAS mutations
• MET alterations
• NTRK fusions (in select tumor types) 1

Ovarian Cancer Testing

• BRCA1/2 mutations (germline and somatic) 1
• Homologous recombination deficiency (HRD) testing in BRCA wild-type tumors 1
• Non-BRCA homologous recombination gene mutations (research setting only) 1

Colorectal Cancer Testing

• Extended RAS panel (KRAS/NRAS exons 2,3,4) 1
• BRAF p.V600 mutation 1
• MMR status (IHC or microsatellite instability testing) 1

When to Use Specific Testing Methodologies

Immunohistochemistry (IHC)

Appropriate uses: 1

• ALK testing: IHC is an equivalent alternative to FISH for detecting ALK rearrangements 1
• ROS1 screening: IHC may be used as initial screening, but positive results must be confirmed by molecular or cytogenetic methods 1
• MMR protein expression in colorectal cancer 1
• NTRK screening in tumor types with high fusion prevalence, with confirmatory testing for positives 1

Inappropriate uses: 1

• Do NOT use EGFR expression by IHC to select patients for EGFR-targeted TKI therapy 1

Fluorescence In Situ Hybridization (FISH)

Appropriate uses: 1

• Detecting gene rearrangements (ALK, ROS1, RET) when IHC is unavailable 1
• Copy number analysis and structural alterations 1
• Confirmatory testing for NTRK rearrangements detected on DNA-based NGS 1

Inappropriate uses: 1

• Do NOT use EGFR copy number analysis (FISH or CISH) to select patients for EGFR-targeted TKI therapy 1

Cytogenetics/Molecular Methods

Next-Generation Sequencing (NGS): 1

• Preferred methodology: Multiplexed genetic sequencing panels are preferred over multiple single-gene tests to identify treatment options beyond core biomarkers 1
• Detects multiple alteration types but individual assays vary in what they can identify 1
• Most efficient approach for comprehensive molecular profiling 1

PCR-based methods: 1

• Real-time PCR for highly targeted specific mutations 1
• Sensitivity of 1-5% is considered acceptable 1

Sanger sequencing: 1

• Requires ≥25-30% tumor content after enrichment 1
• Not appropriate for detecting subclonal events or resistance mutations 1

Specimen Requirements and Common Pitfalls

Adequate Tissue Procurement

• Preferred specimens: Surgical specimens or image-guided biopsies of treatment-naive tumor 1
• Minimum tumor cellularity: Preferably 30%, but assays should detect alterations with as little as 20% tumor cells 1
• Cell blocks from effusions are acceptable for molecular analysis 1
• FFPE tissue is standard, but cytology smear preparations are also suitable 1

Critical Pitfalls to Avoid

• Insufficient tissue from minimally invasive procedures: Bronchoscopists and interventional radiologists must procure sufficient tissue for all necessary testing 1
• Tissue prioritization: Deploy "up-front" slide sectioning protocols to maximize tissue for molecular and diagnostic testing 1
• Testing wrong specimen: Metastatic/recurrent tumor tissue is preferred for predictive biomarker testing in colorectal cancer; use primary tumor only if metastatic tissue unavailable 1
• Incomplete RAS testing: Testing only KRAS exon 2 is insufficient; extended RAS testing is mandatory 1
• Using IHC for EGFR selection: This is explicitly contraindicated 1

Quality Assurance

• Validate all testing methods according to accepted clinical molecular diagnostics standards 1
• Confirm unexpected or equivocal results using alternative methods or samples 1
• Report actionable findings clearly: Specify whether NTRK alterations are fusions (actionable) versus amplifications/mutations (not actionable) 1

Testing Algorithm Summary

1. At diagnosis: Obtain adequate tissue (≥20-30% tumor cellularity) 1
2. First-line approach: Multiplexed NGS panel covering all relevant biomarkers for the tumor type 1
3. Alternative/confirmatory: Use IHC for ALK (equivalent to FISH), ROS1 screening (requires confirmation), or MMR proteins 1
4. FISH reserved for: Confirming rearrangements when needed or when NGS unavailable 1
5. Reflex testing: Automatically confirm positive screening tests (e.g., ROS1 IHC) with molecular/cytogenetic methods 1