Clinical Implications

Lung cancer is one of the most common malignant tumor, and 80~85% of lung cancers are non-small cell lung cancer (NSCLC). Lung cancer is a leading cause of cancer death worldwide. Patients with NSCLC often present with advanced disease and treatment benefit with standard chemotherapy is modest.

There are many driver mutations in NSCLC. The frequency of mutations in NSCLC for EGFR , HER2 , KRAS and BRAF genes are respectively 10-35%, 2-4%, 6 -25% and 1-4%. Approximately 1-3% of lung adenocarcinoma patients harbor MET exon 14 skipping mutations, and about 3-7%, 2%, 1%, 0.12%, 0.02%, 0.08% of NSCLC patients have gene fusions in ALK , ROS1 , RET , NTRK1 , NTRK2 and NTRK3 genes. A large amount of clinical studies showed that gene alteration status is important efficacy predictor for targeted therapy. For instance, NSCLC patients with sensitizing EGFR mutations significantly benefit from EGFR tyrosine kinase inhibitors (TKIs). The presence of the ALK, ROS1 and MET alterations are correlated with the efficacy of ALK/MET inhibitor therapy, patients with RET fusion could benefit from MET/RET/VEGFR inhibitor, BRAF mutated patients will benefit from BRAF inhibitor treatment, and KRAS and HER2 mutations are always associated with prognosis of some targeted drugs. It is indicated in the NCCN Guidelines for NSCLC that gene mutation testing is required before targeted therapy, and it is strongly recommended to conduct multi target test to determine the optimal precision oncology treatment.

Methodology

The Pan Lung Cancer PCR assay (AmoyDx®) consists of two major components, the RNA gene fusion detection and the DNA gene mutation detection.

The RNA gene fusion detection includes two processes:

  1. Reverse Transcription : extracted RNA from FFPE or fresh tumor tissue is employed in this step, reverse transcription of target RNA enables complementary DNA (cDNA) synthesis with the action of reverse transcriptase and specific primers.
  2. PCR Amplification: the specific primers are designed for amplification of cDNA, and ALK, ROS1, RET, MET, NTRK1 NTRK2 and NTRK3 variant amplicon is detected by fluorescent probes.

The DNA gene mutation detection system uses ADx-ARMS technology, which comprises specific primers and fluorescent probes to detect gene mutations. During the amplification, the target mutant DNA is matched with the bases at 3’ end of the primer, and amplified efficiently, then the mutant amplicon is detected by fluorescent-labeled probes. While the wild-type DNA cannot be matched with specific primers, no amplification occurs.

Specimen Requirements

Formalin-fixed, paraffin-embedded NSCLC tissue specimens with a fixation time of 648 hours and ≤ 2 years of age.

Fresh tissue

Volume

One representative paraffin block is preferred. Alternatively, 3-7 unstained tissue sections of 5-10µm are requested.

Storage and Shipment Instructions

Maintain and ship FFPE specimens at ambient temperature.

Maintain and ship fresh specimens in cold formalin at 4°C.

Limitations

Insufficient tumor content may not allow the detection of mutations and fusions (< 30%); tumor content is evaluated by a certified pathologist prior to analysis and macrodissection is performed. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results. The kit can only detect 167 specific hotspot variants. Samples with negative result (No mutation/fusion detected) may harbor mutations or fusions not detected by this assay. Detection of a mutation or fusion is dependent on the number of copies present in the specimen and may be affected by sample integrity, amount of isolated DNA/RNA, and the presence of interfering substances. The presence of PCR inhibitors may cause false negative or invalid results.

Turn-Around Time

Five to 7 business days for slides and paraffin blocks, respectivel