What additional molecular tests should be ordered for a patient with metastatic rectal cancer, beyond the standard tests?

Additional Molecular Testing for Metastatic Rectal Cancer

Beyond the standard RAS, BRAF, and MMR/MSI testing, you should order HER2 amplification testing (by IHC or FISH) in RAS wild-type patients, NTRK fusion testing when feasible, and DPD deficiency testing before initiating fluoropyrimidine-based chemotherapy. 1

Mandatory Additional Tests

HER2 Amplification Testing

• HER2 amplification by IHC or FISH is recommended in all RAS wild-type patients to identify those eligible for HER2-directed therapy in second-line and beyond 1
• While HER2 blockade is only recommended after first-line progression, testing should be done upfront with initial molecular tests to avoid treatment delays when patients progress 1, 2
• This test has Level III, Grade B evidence according to ESMO 2023 guidelines 1

NTRK Fusion Testing

• NTRK fusion testing is recommended when feasible, despite being extremely uncommon (<0.5% incidence in metastatic colorectal cancer) 1
• Screening can be performed by IHC followed by confirmation with next-generation sequencing 1
• NTRK-rearranged tumors are frequently MSI-high and located in the right colon 1
• If an NTRK fusion is detected, treatment with larotrectinib or entrectinib is recommended (Level III, Grade A) 1, 3
• NTRK testing influences treatment decisions only after progression on at least two lines of treatment 1

DPD Deficiency Testing

• Testing for DPD deficiency must be conducted before initiating fluoropyrimidine-based chemotherapy (5-FU or capecitabine), which is used in most metastatic rectal cancer patients 1
• This has Level III, Grade A evidence according to ESMO 2023 guidelines 1
• DPD deficiency affects 3-5% of the population (partial deficiency) and 0.5% have complete deficiency 4
• Treatment-related deaths occur in 2.3% of patients with known DPYD variants versus only 0.1% in those without variants 4

Tests NOT Recommended Outside Clinical Trials

Currently Not Recommended

• PIK3CA mutations, ALK and ROS1 gene fusions, and HER2 activating mutations are not recommended outside clinical trials (Level IV, Grade D) 1
• While PIK3CA mutations are present in 10-20% of colorectal cancers and may contribute to cetuximab resistance, they do not currently guide treatment decisions in routine practice 1, 5, 6, 7, 8
• PTEN loss by IHC is not recommended (Level V, Grade D) 1
• EGFR protein expression, EGFR amplification, HER3 and MET receptor overexpression are not recommended 1

Practical Implementation Algorithm

At Time of Metastatic Diagnosis

1. Order comprehensive molecular panel including:

   • MMR protein IHC or MSI testing (mandatory) 1
   • KRAS/NRAS exons 2,3, and 4 (mandatory) 1
   • BRAF V600E mutation (mandatory) 1
   • HER2 amplification by IHC or FISH (recommended in RAS wild-type) 1
   • NTRK fusion screening by IHC when feasible 1
   • DPD deficiency testing before fluoropyrimidine initiation 1

2. Ensure adequate tissue preparation:

   • Fixation with 10% neutral buffered formalin for 6-48 hours 1
   • Macro-dissection to maximize tumor cell content (>20%) 1

3. Turnaround time should be 10 working days from specimen receipt to final report for >90% of specimens 1

Common Pitfalls to Avoid

• Do not delay HER2 testing until first-line progression, as this creates time pressure when patients are clinically deteriorating 2
• Do not start fluoropyrimidine therapy without DPD testing, as this can cause severe, potentially fatal toxicity 1, 4
• Ensure testing is performed in CLIA-certified laboratories that participate in external quality assessment schemes 1
• Do not assume all molecular testing can wait—simultaneous testing prevents treatment delays and allows comprehensive treatment planning across all lines of therapy 2