Gene Rearrangement Studies (ALK, ROS1)

Gene rearrangement studies find structural changes in which a gene is broken and joined to another gene, creating a fusion that drives tumour growth. ALK and ROS1 fusions are the best known in lung adenocarcinoma, and their carriers respond very well to tyrosine kinase inhibitors that act on the fusion protein.

The fusion can be found at the DNA level (fluorescence in situ hybridisation with break apart probes, DNA sequencing), at the RNA level (RT-PCR, RNA-based sequencing), or at the protein level (immunohistochemistry, which finds over expression of the fusion protein). Each method has strengths and weaknesses.

Rapid results are needed in advanced disease, because the matched drug is started as soon as the result is available. Immunohistochemistry takes 1 to 2 days, fluorescence in situ hybridisation 2 to 5 days, and sequencing 7 to 14 days.

ALK rearrangements are found in about 3 to 5% of lung adenocarcinomas, ROS1 in 1 to 2%, RET in 1 to 2%, and NTRK in under 1%. They are enriched in younger patients and in never or light smokers, and in tumours with solid or signet ring and mucinous patterns. They also occur in other tumours: ALK in anaplastic large cell lymphoma, inflammatory myofibroblastic tumour and neuroblastoma (mutations and amplification), and ROS1 in glioma and cholangiocarcinoma.

Indications.

• Advanced non-squamous non-small cell lung cancer at diagnosis, and squamous cancer in never smokers or small biopsies.
• Metastatic lung cancer that has progressed on treatment, to look for acquired resistance changes.
• Anaplastic large cell lymphoma, inflammatory myofibroblastic tumour and other tumours with suspected ALK or ROS1 involvement.
• Tumour agnostic testing for NTRK fusions in rare cancers and unknown primaries.

Not indicated for early cancers where results do not change management, though testing is being used more often in resected disease for adjuvant ALK treatment.

Specimen requirements.

• Formalin fixed, paraffin embedded tumour with at least 20% tumour cells for sequencing, and enough tumour nuclei for fluorescence in situ hybridisation (50 to 100 cells are scored).
• Cytology cell blocks, with the same fixation rules.
• Fresh or frozen tissue improves RNA quality for fusion panels.
• Plasma cell free DNA when tissue is not available, with lower sensitivity for fusions.
• Fixation for 6 to 72 hours, and avoidance of acid decalcification.

Normal appearances.

Break apart fluorescence in situ hybridisation shows fused red and green signals (or yellow) in normal nuclei. Immunohistochemistry shows no ALK staining in normal lung tumours. A fusion negative sequencing result means no fusion was detected in the regions covered.

Interpretation.

• Fluorescence in situ hybridisation: a case is positive when 15% or more of tumour nuclei show split signals (separated red and green) or isolated single signals.
• ALK immunohistochemistry: strong, diffuse cytoplasmic staining in tumour cells is positive, and it is accepted as a basis for treatment when a validated antibody and platform have been used.
• ROS1 immunohistochemistry: a screening test with lower specificity, and positive results are confirmed by fluorescence in situ hybridisation or sequencing.
• RNA-based sequencing: identifies the exact partner gene and breakpoint (for example, EML4-ALK, CD74-ROS1), and finds RET, NTRK, MET exon 14 skipping and other events.
• ALK-positive tumours: respond to ALK inhibitors, with high response rates and long disease control, including in brain metastases for newer drugs.
• ROS1-positive tumours: respond to ROS1 inhibitors.
• Resistance: secondary mutations in the kinase domain at progression (for example, ALK G1202R) determine the choice of the next drug.
• Rearrangements are almost always mutually exclusive with EGFR and KRAS mutations, so a second driver in the same tumour questions the result.

Critical values and findings needing immediate action.

• An ALK, ROS1, RET or NTRK fusion in advanced cancer: urgent referral for the matched inhibitor, which is usually preferred to chemotherapy or immunotherapy as first treatment.
• A positive immunohistochemistry screen for ROS1: confirmatory testing without delay.
• Symptomatic brain metastases in a fusion positive patient: urgent specialist care, with a drug that crosses the blood brain barrier.
• Failed sample: repeat on another block, a new biopsy, or plasma testing.

Limitations and artefacts.

• Break apart fluorescence in situ hybridisation can miss subtle rearrangements (inversions with small separation), and it does not show the partner gene.
• Immunohistochemistry can give false positives (ROS1 staining in reactive cells) and false negatives (low expression, poor fixation).
• DNA-based sequencing can miss fusions when the breakpoint lies in a region that is not covered, so RNA-based testing is more sensitive for several targets.
• RNA in formalin fixed tissue degrades with time and poor fixation, and a failed result is not a negative result.
• Low tumour content and necrosis cause false negative results.
• Plasma tests miss fusions in many cases.
• Heterogeneity and changes under treatment require re-testing at progression.
• Complete testing of several targets needs enough tissue, so a stepwise approach that saves material is used.

Special groups.

• Young never smokers with lung adenocarcinoma: the highest rate of fusions, and broad testing is done in all.
• Patients with small biopsies: sequencing panels that cover many targets in a single run save tissue.
• Patients with lymphoma or inflammatory myofibroblastic tumour: ALK immunohistochemistry is part of the diagnostic workup.
• Children with solid tumours: NTRK and ALK fusions are looked for when tumours are unusual.
• Regions: ALK immunohistochemistry is the most accessible test, and confirmation by fluorescence in situ hybridisation or sequencing is done in reference laboratories. ALK and ROS1 inhibitors are costly, so the result may not lead to treatment, and access programmes and generic drugs change this.

Follow up tests.

• EGFR, KRAS, BRAF, MET, RET and HER2 testing, and PD-L1 immunohistochemistry.
• RNA-based fusion panel or next generation sequencing if the first test is negative or equivocal.
• Brain MRI and staging imaging.
• Repeat biopsy or plasma test at progression, for resistance mutations.
• Baseline liver function, and monitoring on treatment.
• Multidisciplinary meeting.