What is the role of Immunohistochemistry (IHC) testing for ROS1 in diagnosing non-small cell lung cancer (NSCLC)?

ROS1 IHC Testing in Non-Small Cell Lung Cancer

ROS1 immunohistochemistry should be used only as a screening tool in advanced non-squamous NSCLC, with all positive results requiring mandatory confirmation by FISH, RT-PCR, or NGS before initiating targeted therapy. 1

Testing Algorithm for ROS1 Status

When to Test

• Systematic ROS1 testing is mandatory for all patients with advanced non-squamous NSCLC, regardless of clinical characteristics 1
• Testing should be performed on adenocarcinomas and NSCLC-not otherwise specified (NSCLC-NOS) 1
• Do not test patients with confident squamous cell carcinoma diagnosis, except in young patients (<50 years), never/former light smokers (<15 pack-years), or long-time ex-smokers (quit >15 years ago) 1

Recommended Testing Approach

Primary Testing Methods:

• FISH remains the gold standard for detecting ROS1 rearrangements and can be used as a standalone confirmatory test 1
• Validated RT-PCR may be used as an alternative primary method 1
• Next-generation sequencing (NGS) is preferable when available, particularly RNA-based NGS for fusion detection 1

IHC as Screening Only:

• IHC may be used as a screening approach only, not as a standalone diagnostic test 1, 2
• All positive IHC results must be confirmed by FISH, RT-PCR, or NGS before treatment decisions 1, 2
• There is no FDA-approved IHC assay for ROS1, unlike ALK testing 1

Critical Limitations of ROS1 IHC

Specificity Issues

The major limitation of ROS1 IHC is moderate to low specificity, leading to false-positive results that require confirmatory testing:

• ROS1 IHC shows high sensitivity (up to 100%) but specificity ranges from 87-99% depending on the scoring threshold used 3, 4
• In real-world practice, ROS1 IHC positivity rate (4.4%) significantly exceeds the true ROS1 rearrangement rate (1.8-1.9%) detected by FISH or NGS 5
• False positives occur with both commonly used antibody clones (D4D6 and SP384), though SP384 demonstrates higher sensitivity 4

Antibody Clone Differences

• SP384 clone shows 100% sensitivity with ≥2+ staining intensity cutoff and generally produces stronger, easier-to-interpret staining 4
• D4D6 clone shows only 42.86% sensitivity with ≥2+ cutoff but 100% sensitivity with ≥1+ cutoff 4
• High concordance with FISH occurs when IHC shows diffuse (≥60% tumor cells) 2-3+ cytoplasmic staining 6

Practical Workflow Considerations

Turnaround Time and Cost

• IHC turnaround time: 0-8 days at approximately £51 cost 7
• FISH turnaround time: 9-42 days at approximately £159 cost 7
• Combined IHC screening followed by FISH confirmation: average 6 days 5
• NGS as reflex testing: average 3 days, potentially faster than IHC-FISH algorithm 5

Emerging Evidence Against IHC Screening

Recent real-world data suggests abandoning IHC screening entirely in favor of upfront NGS testing:

• NGS detects ROS1 rearrangements in 1.9% of cases (matching true prevalence) versus 4.4% IHC positivity rate 5
• NGS provides faster results (3 days) compared to IHC-FISH sequential testing (6 days) 5
• NGS simultaneously detects multiple actionable alterations, avoiding sequential testing delays 1
• RNA-based NGS is preferred for identifying the expanding range of fusion genes 1

Common Pitfalls to Avoid

Critical Error: Using IHC alone for treatment decisions

• Never initiate ROS1-targeted therapy based solely on positive IHC without molecular confirmation 1, 2
• Unlike ALK testing where validated IHC can be used standalone, ROS1 IHC lacks this validation 1, 2

Interpretation Challenges:

• Variable staining intensity makes standardization difficult across laboratories 3, 5
• Non-specific staining can occur in necrotic areas and extracellular matrix 3
• Heterogeneous staining patterns complicate scoring 3, 4

Resource Allocation:

• In settings with limited tissue, prioritize molecular testing (FISH/NGS) over IHC to avoid tissue exhaustion on a screening test requiring confirmation 1
• If multiplex NGS platforms are available, use them as first-line testing rather than sequential single-gene approaches 1

Optimal Testing Strategy

For laboratories with NGS access:

• Proceed directly to comprehensive NGS panel testing without IHC screening 1, 5
• This approach provides faster results, higher specificity, and simultaneous detection of multiple actionable alterations 5

For laboratories without NGS access:

• Use ROS1 IHC as initial screen with validated protocols and appropriate antibody clones 3, 7
• Confirm all positive IHC results with FISH or RT-PCR before treatment 1
• Consider using H-score readout with adjusted thresholds to balance sensitivity and specificity 3