Comprehensive detection of fusions and mutations in oncogenes^4,9

NGS is a sensitive and specific way to detect fusions and mutations and can also identify other biomarkers in one comprehensive molecular test.
RNA-NGS can also detect fusion partners and the position of gene rearrangements.
Liquid biopsy is emerging as a valuable option to test patients but should always be confirmed with a tissue test if negative

Reliability varies depending on the assay used¹⁷

Different  NGS panels target different regions of genome and depth of coverage varies between assays.  Requirements for tumour content can also vary between assays.¹⁷

Can detect novel fusion partners and evaluates multiple actionable targets¹⁸

Depending on the assay used, NGS can simultaneously evaluate multiple and novel gene fusion partners and mutations while requiring limited tissue. RNA sequencing is focused on coding sequences rather than introns, and is suited for gene fusion detection.^5,18

May not identify all gene fusions and targetable mutations¹⁸

DNA-NGS is limited for fusion detection by intron size. RNA-NGS is suited for gene fusion detection but may be limited by RNA quality.^5,18
Depending on assay, not all genes or alterations might be covered, and certain assays can only detect known fusions due to their design.

When ordering an NGS test, be certain it detects the most common gene fusions and mutations in actionable genes¹⁹

Ensure that driver mutations in EGFR, KRAS and fusions in NTRK, ROS1, ALK and RET are included to help identify the best treatment options for patients.

May detect abnormal expression of proteins encoded by fusion genes indirectly but sometimes requires confirmation by other tests^2,9,10

May be used as a screening tool or diagnostic depending on the biomarker.  Screening assays require confirmation by other methodologies.

Depending on the biomarker, it can detect aberrant protein expression with high sensitivity and specificity^2,5,13

IHC antibodies have been reported to have 95-100% sensitivity and range from 70-100% specificity depending on the threshold used to define positivity.

Low cost and readily available^2,9,18

Lower cost than NGS with a turnaround time of 1-2 days¹⁸.

Cannot reliably differentiate between normal protein expression and proteins resulting from gene rearrangements^9,18

IHC can detect both wild-type and fusion proteins leading to possible false positives.
IHC cannot identify fusion partners and there is also a risk of false negative for fusions in some biomarkers. Therefore, orthogonal techniques are required to confirm the presence of gene rearrangements.

Can detect most common fusions, depending on probes used

Different probes are required for each NTRK gene.²¹ For ROS1 fusion detection by FISH relies on dual color ‘break-apart’ probes which label the fusion breakpoints. Rearrangements are determined by looking at the pattern of fluorochrome labeling^9,21

False positives or negatives might occur^9,18

Reliability depends on the probes used. There is a risk of false-positive results due to complex chromosomal translocations and detection of non-functional fusion proteins.^9,18 Variant or complex rearrangements may be missed.^1,2,9,18 False-negative results may be above 30% in some cases.¹⁸

Readily available, break-apart FISH is a common method for detecting gene fusions^10,21

Allows visualisation of the target within the cell and enables several targets to be detected in one sample using multiple fluorophores.^10,18 The use of break-apart probes allows fusions with unknown partners to be detected.¹⁸

Conventional FISH may require confirmatory testing depending on the biomarker^9,18

Conventional FISH may require multiple tests to detect gene fusions and does not detect novel fusions as compared to NGS.
FISH is primarily DNA based and will not provide information about the expression of the fusion protein and cannot detect mutations. FISH requires technical expertise to interpret and score.

Requires multiple primer sets to detect gene fusions^18,22

Different gene alterations can be detected by RT-PCR with multiple primer sets required for each fusion variant.^18,21,22

Reliable for known gene fusions and mutations, some biomarkers require confirmatory testing

Each variant requires a specific primer set. Therefore, PCR may miss unknown or untested variants. Low specificity for some biomarkers in certain indications will require confirmation using FISH or NGS^11,22.

Low cost per assay with high sensitivity and specificity¹⁸

RT-PCR can also provide information on the expression level of oncogene being tested.

Can only detect known target sequences¹⁸

Target sequences must be known and cannot readily detect novel fusion partners.
A comprehensive multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay might be challenging because of the potentially large number of possible 5’ fusion partners.^10,18