Detecting ROS1+ NSCLC

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines^®) recommend testing for ROS1+ disease in all patients with nonsquamous metastatic NSCLC¹

ROS1+ NSCLC can be detected with various testing methods⁷

Immunohistochemistry (IHC)

• Uses specific monoclonal antibodies to detect overexpression of the ROS1 fusion protein⁷
• Requires dedicated tissue and limits multiplexing^8,9
• Low specificity; molecular confirmation is necessary¹

Fluorescence in situ hybridization (FISH)

• Uses “break-apart” probes to show ROS1 gene rearrangement⁷
• Requires dedicated tissue^9,10
• May underdetect the FIG-ROS1 variant¹

Next-generation sequencing (NGS)

• Multigene parallel sequencing assays^7,9
• Capable of detecting ROS1 fusions¹
• Provides information about multiple biomarkers, including rare and common mutations, and can typically use less tissue^9,10
• Turnaround time for results may be longer than with RT-PCR, IHC, or FISH¹¹

Reverse transcriptase-polymerase chain reaction (RT-PCR)

• Reverse transcription from extracted RNA¹²
• Fast and sensitive detection of specific fusion variants¹²
• May not detect fusions in novel partners¹

NCCN=National Comprehensive Cancer Network; NSCLC=non-small cell lung cancer; ROS1=ROS proto-oncogene 1.

Detecting ALK+ NSCLC

ALK gene rearrangements are present in approximately 5% of patients with NSCLC¹³

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines^®) and CAP clinical guidelines recommend that patients with metastatic NSCLC be routinely tested for ALK+ disease.^1,4

ALK may be detected by using a variety of methods^14-16

Fluorescence in situ hybridization (FISH)

• Uses fluorescently labeled DNA probes that bind to and localize specific regions in the tumor nuclei to detect ALK rearrangements and amplification¹⁴
  

Immunohistochemistry (IHC)

• Uses specific monoclonal antibodies to detect overexpression of the ALK fusion protein^15,17

Next-generation sequencing (NGS)

• NGS allows massive parallel sequencing for the identification of potentially targetable genetic alterations in tumors¹⁸
• Other testing methods are under investigation, including reverse transcriptase-polymerase chain reaction (RT-PCR)^19,20

Reverse transcriptase-polymerase chain reaction (RT-PCR)

• RT-PCR is a technique that utilizes RNA extraction to detect ALK gene fusions, such as EML4-ALK^19,20

Currently, FISH, IHC, and NGS are the only testing methods FDA approved for clinical application in detecting ALK+ NSCLC^14-16

ALK=anaplastic lymphoma kinase; CAP=College of American Pathologists; DNA=deoxyribonucleic acid; EML4=echinoderm microtubule-associated protein-like 4; NCCN=National Comprehensive Cancer Network; NSCLC=non-small cell lung cancer; RNA=ribonucleic acid.

Detecting EGFR mutations in NSCLC

EGFR mutations play an important role in carcinogenesis²¹

• The aberrant signaling influences several key aspects, including cell proliferation, apoptosis, migration, survival, and more complex processes, such as angiogenesis²¹
• Different EGFR mutations have different signaling properties, but most mutations affect the adenosine triphosphate (ATP) binding cleft, where targeting TKIs compete for binding²¹

Ideal testing methods should cover a broad spectrum of EGFR mutations⁴

The 2017 CAP/IASLC/AMP guidelines suggest the following:

• Detect all individual mutations that have been reported in at least 1% of EGFR-mutated lung adenocarcinomas⁴
• Detect mutations in specimens with at least 20% cancer cells^4,5
  
• Use EGFR T790M mutational testing in patients harboring sensitizing EGFR mutations after treatment with EGFR-targeted therapies^4,5
• Total EGFR expression by IHC is not sufficient to select patients for EGFR-targeted therapies^4,5

Note: These may not be all known mutations of EGFR.
Bp=base pair; IHC=immunohistochemistry.