What are the key molecular biomarkers used to diagnose, prognosticate, and guide targeted therapy in non‑small cell lung cancer?

Key Molecular Biomarkers for Lung Cancer

For non-small cell lung cancer (NSCLC), the essential biomarkers are EGFR mutations, ALK rearrangements, ROS1 rearrangements, and PD-L1 expression, with testing mandatory before initiating treatment to guide targeted therapy selection. 1

Primary Actionable Biomarkers

Genomic Alterations for Targeted Therapy

EGFR (Epidermal Growth Factor Receptor):

• Activating mutations detected in approximately 25% of lung adenocarcinomas 1
• Guides use of tyrosine kinase inhibitors (erlotinib, gefitinib, afatinib, osimertinib) 2
• Testing is mandatory prior to treatment initiation 3, 2
• T790M resistance mutation should be identified when resistance develops, as osimertinib is available for this specific alteration 3

ALK (Anaplastic Lymphoma Kinase):

• EML4-ALK gene rearrangements found in adenocarcinomas 1
• Predicts response to crizotinib, ceritinib, alectinib, and brigatinib 2
• Testing required before administering ALK inhibitors 3

ROS1 Rearrangements:

• Identified in NSCLC subtypes 1
• Predicts response to crizotinib 2
• Should be tested at minimum alongside EGFR and ALK 3

Immunotherapy Biomarker

PD-L1 (Programmed Death-Ligand 1):

• Overexpressed on tumor cells in various lung cancers 1, 4
• Testing recommended for all patients with newly diagnosed advanced NSCLC 4, 5
• Measured by immunohistochemistry assays evaluating membranous staining of tumor cells 4
• Different FDA-approved assays exist: PD-L1 IHC 22C3 for pembrolizumab, PD-L1 IHC 28-8 for nivolumab, and Ventana SP142 for atezolizumab 1
• Scoring thresholds: ≥50% tumor proportion score for first-line pembrolizumab monotherapy, ≥1% for second-line 4, 2

Additional Actionable Biomarkers

Other genomic alterations with available targeted therapies include: 3, 6

• RET rearrangements - actionable with specific inhibitors
• BRAF V600E mutations - targetable alterations
• MET exon 14 alterations and MET amplification 1
• KRAS mutations - identified in adenocarcinomas 1
• NTRK1-3 fusions - emerging therapeutic targets 6
• ERBB2/HER2, NRG1 - additional biomarkers under investigation 6

Histologic Subtype-Specific Patterns

Critical distinction: Adenocarcinomas and squamous cell carcinomas have different molecular profiles 1

Adenocarcinoma (40-50% of NSCLC):

• EGFR mutations and ALK rearrangements in ~25% of cases 1
• Additional alterations: KRAS, LKB1/STK11, NF1, CDKN2A, SMARCA4, KEAP1, MET amplification, ROS1 and RET rearrangements 1

Squamous Cell Carcinoma (20-30% of NSCLC):

• Rarely harbor EGFR mutations or ALK rearrangements 1
• Instead show alterations in RTKs, DDR2, FGFRs, and inactivating mutations in CDKN2A, PTEN, KEAP1, MLL2, HLA-A, NFE2L2, NOTCH1, and RB1 1

Diagnostic Approach for Subclassification

Immunohistochemical markers for NSCLC subtyping:

• Triple marker panel (TTF-1, Napsin A, P40) demonstrates 86% sensitivity and 100% specificity for adenocarcinoma, and 100% sensitivity and 97.1% specificity for squamous cell carcinoma 1
• This approach is cost-effective, improves turnaround time, and preserves tissue for molecular testing 1

Testing Strategy

Next-generation sequencing (NGS) is preferred over sequential single-gene testing: 7

• Enables simultaneous identification of point mutations, insertions, deletions, copy number alterations, fusion genes, and microsatellite instability 7
• Prevents tissue exhaustion from multiple sequential tests 7
• Current guidelines recommend testing at least seven biomarkers upfront at NSCLC diagnosis 7

If adequate testing was not performed initially, molecular testing should be completed before subsequent lines of therapy 3

For patients unable to provide tumor tissue, EGFR mutational analysis may be performed on plasma 2

Critical Pitfalls

PD-L1 testing limitations:

• Not a perfect biomarker - less than half of biomarker-selected patients benefit from treatment 4
• Some responses occur in "biomarker-negative" cohorts 4
• Different antibodies and scoring systems create interpretation challenges 1
• Non-specific staining of alveolar macrophages and bronchial basal cells can confound results 1

Tissue adequacy concerns:

• Neoplastic cell content below threshold renders biomarker-negative results inconclusive 1
• Low tumor fraction in liquid biopsies increases risk of false-negative and false-positive findings 1