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Multi-gene mutation and fusion detection (NSCLC)

Pan Lung Cancer real-time PCR assay (AmoyDx®).

The AmoyDx®Pan Lung Cancer real-time PCR assay (AmoyDx) in conjunction with the Quantstudio5 System provides the option of using FFPE tissue for fast and accurate mutation and fusion detection.
The Pan Lung Cancer PCR assay is a real time PCR assay for qualitative detection of 167 hotspot alterations in 11 target genes, being EGFR, ALK, ROS1, KRAS, BRAF, HER2, RET, MET, NTRK1, NTRK2 and NTRK3, using a DNA input of 70ng and an RNA input of 120ng.
In formalin-fixed, paraffin-embedded tumor samples, the presence of mutations is detected with a mutation-specific sensitivity of 1~5% gene mutation in 7.5-15ng FFPE tissue DNA. For RNA, the kit allows detection of fusions from 4ng RNA input from FFPE tissue.

The Pan Lung Cancer PCR assay consists of two major components:

  • 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.

Clinical Implication

Lung cancer is one of the most common malignant tumors, 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 non-small cell lung cancer (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 [1], 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 [2-3]. A large number 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) [4-5]. The presence of the ALK, ROS1, and MET alterations are correlated with the efficacy of ALK/MET inhibitor therapy [6-7], patients with RET fusion could benefit from MET/RET/VEGFR inhibitor [8], BRAF mutated patients will benefit from BRAF inhibitor treatment [9], and KRAS and HER2 mutations are always associated with prognosis of some targeted drugs [10-12]. It is indicated in NCCN Guideline 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 [13].

Specimen Requirements

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


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.


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.

Special Requirements


Turn-Around Time

Five to 7 business days for slides and paraffin blocks, respectively.


  1. Liu SY, Gou LY et al. The Unique Characteristics of MET Exon 14 Mutation in Chinese Patients with NSCLC. J Thorac Oncol 2016; 11(9): 1503-10.
  2. Farago AF, Taylor MS et al. Clinicopathologic Features of Non-Small-Cell Lung Cancer Harboring an NTRK Gene Fusion. JCO Precis Oncol 2018. doi: 10.1200/PO.18.00037.
  3. Lovly C, Horn L, Pao W. 2015. Molecular Profiling of Lung Cancer. My Cancer Genome; 2016
  4. Maemondo M, Inoue A, Kobayashi K et al. Gefitinib or Chemotherapy for Non–Small-Cell Lung Cancer with Mutated EGFR. N Engl J Med 2010; 362: 2380-2388.
  5. Yang JC, Wu YL, Schuler M et al. Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials. Lancet Oncol 2015; 16: 141-151.
  6. Shaw AT, Kim DW, Nakagawa K et al. Crizotinib versus Chemotherapy in Advanced ALK-Positive Lung Cancer. N Engl J Med 2013; 368: 2385-2394.
  7. Shaw AT, Ou SH, Bang YJ et al. Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J Med 2014; 371: 1963-1971.
  8. Drilon A, Wang L, Hasanovic A et al. Response to Cabozantinib in patients with RET fusion-positive lung adenocarcinomas. Cancer Discov 2013; 3: 630-635.
  9. Planchard D, Mazieres J, Riely GJ, et al. Interim results of phase II study BRF113928 of dabrafenib in BRAF V600E mutation-positive non-small cell lung cancer (NSCLC) patients. ASCO Annual Meeting Proceedings. 2013, 31: 8009.
  10. Janne PA, Shaw AT, Pereira JR et al. Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer: a randomised, multicentre, placebo-controlled, phase 2 study. Lancet Oncol 2013; 14: 38-47.
  11. Ohashi K, Sequist LV, Arcila ME et al. Characteristics of Lung Cancers Harboring NRAS Mutations. Clin Cancer Res 2013; 19: 2584-2591.
  12. Mazieres J, Peters S, Lepage B et al. Lung cancer that harbors an HER2 mutation: epidemiologic characteristics and therapeutic perspectives. J Clin Oncol 2013; 31: 1997-2003.
  13. NCCN Clinical Practice Guidelines in Oncology: Non-Small Cell Lung Cancer. Version 1. 2019.
  14. ADx-LG01; AmoyDx® Pan Lung Cancer PCR Panel, instructions for use, version P1.0, 2019.

Updated on March 9, 2021