A gene fusion test looks for abnormal joins between two genes or between different parts of the same gene. These rearrangements can create a hybrid protein, place a growth-promoting gene under the control of an active regulatory region, or disrupt normal gene function. In cancer, a fusion may help establish the diagnosis, define a tumor subtype, estimate prognosis, or identify a targeted treatment. The test is not a single laboratory method: fusion detection may use RNA or DNA next-generation sequencing, fluorescence in situ hybridization, reverse-transcription PCR, immunohistochemistry, or a combination. The best method depends on the suspected fusion, tumor type, specimen, and clinical question. Results also require context. A rearrangement seen at the DNA level is not automatically an expressed, functional fusion, while a negative RNA result may be limited by degraded RNA or incomplete assay coverage. Understanding what was tested and how it was tested is essential before using the report to guide care.
- Gene fusions are structural changes that join gene segments and may create an oncogenic driver.
- A positive result can support diagnosis, prognosis, or eligibility for a matched targeted therapy.
- RNA-based testing directly evaluates expressed fusion transcripts and is often preferred for broad fusion detection.
- DNA sequencing, FISH, RT-PCR, and immunohistochemistry provide complementary information and have different blind spots.
- A negative result is meaningful only when specimen quality, tumor content, assay coverage, and quality controls are adequate.
- Novel or unusual fusions may need orthogonal confirmation and expert molecular-pathology interpretation.
Table of Contents
- What gene fusions are and how they drive cancer
- When gene fusion testing is ordered
- Testing methods and what each one detects
- Specimens, tumor content, and quality requirements
- How to interpret a positive gene fusion result
- Negative, uncertain, and discordant results
- Important clinical examples
- Questions and next steps after testing
What Gene Fusions Are and How They Drive Cancer
A gene fusion forms when a structural alteration brings together DNA segments that are normally separate. The segments may come from different chromosomes, distant regions of the same chromosome, or nearby regions rearranged by deletion, inversion, duplication, or translocation. The resulting DNA change may be transcribed into a fusion RNA and translated into a fusion protein. Cancer reports often describe the partners with a double colon, such as EML4::ALK, although older reports may use a hyphen.
Not every rearrangement produces a biologically important fusion. For a fusion to act as a cancer driver, it commonly preserves an active protein domain, creates an in-frame transcript, increases expression of a growth-promoting gene, or removes a regulatory region that normally restrains signaling. Many actionable fusions retain the kinase domain of a receptor or signaling protein. The partner gene may contribute a dimerization domain that keeps the kinase switched on, allowing continuous growth signaling without the usual external stimulus.
The distinction among DNA rearrangement, RNA fusion, and protein expression matters. DNA testing identifies the genomic event. RNA testing shows that an abnormal transcript is expressed and clarifies the joined exons. Immunohistochemistry may show abnormal protein expression but usually does not identify the precise partner or breakpoint. A clinically convincing result integrates these levels with the tumor’s morphology, immunophenotype, and other molecular findings [1].
Gene fusions are often mutually exclusive with other dominant oncogenic drivers in the same tumor, but this is not absolute. Coexisting alterations may occur, especially after treatment as resistance evolves. A detected fusion therefore should not be interpreted in isolation from the complete molecular profile and clinical history.
When Gene Fusion Testing Is Ordered
Fusion testing is ordered when the result could clarify what a tumor is, how it may behave, or how it may respond to treatment. In some cancers, a characteristic fusion is part of the diagnostic definition. In others, the main purpose is to find an actionable driver. The same assay can therefore serve different goals depending on the disease.
A pathologist may request testing when microscopic features suggest a fusion-defined tumor. This is common in many sarcomas, pediatric tumors, salivary-gland neoplasms, thyroid cancers, central nervous system tumors, and selected leukemias and lymphomas. Demonstrating the expected fusion can distinguish look-alike tumors that require different management. Conversely, finding an unexpected fusion on broad sequencing may lead to re-review of the pathology and a revised diagnosis.
In advanced solid tumors, fusion testing is frequently part of comprehensive biomarker profiling. Lung adenocarcinoma is a prominent example because fusions involving ALK, ROS1, RET, or NTRK1/2/3 can be matched to approved targeted drugs in appropriate settings. Fusion testing can also identify actionable changes involving FGFR2, FGFR3, BRAF, NRG1, and other genes, depending on tumor type and current treatment standards. Because indications change, treatment decisions should use current disease-specific guidelines rather than a generic list of “targetable” genes.
The laboratory request should make the clinical question clear. A narrow test is efficient when one specific fusion is strongly suspected, such as BCR::ABL1 in chronic myeloid leukemia or PML::RARA in suspected acute promyelocytic leukemia. A broad RNA panel is more appropriate when many possible partners or genes could explain the tumor. Testing only a common partner may miss rare but clinically equivalent fusions.
Fusion testing may also be considered after DNA profiling finds no established driver, particularly in tumors enriched for rearrangements or in never-smokers with lung adenocarcinoma. Concurrent DNA and RNA analysis can increase detection of actionable structural variants compared with DNA testing alone [3]. The choice should account for available tissue, turnaround time, cost, and the consequences of a false-negative result.
Testing Methods and What Each One Detects
“Gene fusion test” describes a clinical objective, not one universal technique. The report should state the analyte, platform, targeted genes or regions, and major limitations. Each method answers a slightly different question.
| Method | What it directly detects | Main strengths | Important limitations |
|---|---|---|---|
| RNA next-generation sequencing | Expressed fusion transcripts and exon junctions | Broad partner detection; confirms expression; often efficient because introns are removed | RNA is fragile; expression may be low; panel design and bioinformatics can miss events |
| DNA next-generation sequencing | Genomic breakpoints and structural variants | Can combine fusions with mutations, copy number, and other biomarkers | Large or repetitive introns may be poorly covered; a DNA rearrangement may not yield a functional transcript |
| FISH | Separation, gain, loss, or juxtaposition of fluorescent probe signals | Works on small tissue areas; useful for known loci; independent of exact transcript | Often does not identify the partner or transcript; interpretation thresholds and atypical patterns matter |
| RT-PCR | A specific fusion transcript | Highly sensitive, rapid, and useful for confirmation or monitoring | Requires known partners and breakpoints; a narrow assay misses unexpected variants |
| Immunohistochemistry | Abnormal protein expression or localization | Fast, tissue-sparing, and widely available for some targets | It is an indirect surrogate; sensitivity and specificity vary by protein and tumor type |
RNA-based next-generation sequencing is frequently favored for broad fusion discovery because it reads the transcript after introns have been removed. This can avoid the challenge of sequencing very large intronic breakpoint regions. Hybrid-capture, anchored multiplex PCR, amplicon, and whole-transcriptome approaches differ in partner independence and coverage. An anchored method may identify an unknown partner when one side of the fusion is targeted. A fixed amplicon design may require both partners or exact exon combinations to be represented, which can miss uncommon junctions [2].
DNA-based NGS remains valuable. It can detect rearrangements even when RNA is unavailable and can place fusion analysis within a broader tumor profile. However, a panel may not cover all relevant introns, and repetitive sequences can prevent confident mapping. A DNA structural variant labeled as a potential fusion may need RNA confirmation to show that it creates an expressed in-frame transcript.
FISH testing commonly uses break-apart probes that flank a gene. Separated signals indicate a rearrangement involving that locus but do not necessarily reveal the fusion partner. Dual-fusion probes target two known partners and can provide more specific evidence. Complex or borderline signal patterns may require correlation with sequencing or another orthogonal method.
RT-PCR is highly effective when the expected transcript is known. It is widely used in hematologic malignancies for rapid diagnosis and quantitative monitoring. It is less suitable as a stand-alone discovery test because uncommon partners or breakpoints can escape the primer design. Immunohistochemistry can serve as a screen or companion method, as with ALK or pan-TRK staining, but a positive stain may need molecular confirmation, especially when morphology and prevalence make a true fusion unlikely.
Specimens, Tumor Content, and Quality Requirements
Most solid-tumor fusion tests use formalin-fixed, paraffin-embedded tissue from a biopsy or resection. Cytology cell blocks, fresh or frozen tissue, bone marrow, peripheral blood, and body-fluid specimens may also be accepted. The laboratory’s validated specimen types should guide collection. A sample suitable for routine microscopy is not automatically suitable for RNA sequencing.
Before testing, a pathologist typically marks the tumor area and estimates tumor cellularity. Macrodissection may enrich tumor cells and reduce dilution by normal tissue. For RNA assays, however, tumor percentage is not the only determinant of sensitivity. Fusion transcript abundance varies, so a lower-cellularity sample may still yield a strong signal, while a high-cellularity sample with severely degraded RNA may fail.
RNA is particularly vulnerable to preanalytic damage. Delayed fixation, overfixation, decalcification, heat, moisture, and prolonged storage can fragment RNA. Acid decalcification can be especially problematic for bone specimens. Laboratories assess RNA quantity and quality using assay-specific measures such as amplifiable fragment size, housekeeping-gene performance, mapped reads, unique reads, or internal expression controls. A report that says “no fusion detected” should be distinguished from one that says the assay failed or was noninformative.
Liquid biopsy can detect some fusion-associated rearrangements in circulating tumor DNA, but sensitivity depends on tumor shedding and the DNA assay’s breakpoint coverage. A negative plasma result does not exclude a fusion in tissue. When tissue is available and the clinical suspicion is substantial, a negative blood test may warrant tissue-based RNA or combined DNA/RNA testing.
The report’s technical section should be reviewed for:
- specimen source and collection date;
- estimated tumor content and whether enrichment was performed;
- RNA or DNA quality-control status;
- genes, exons, or introns covered;
- minimum supporting-read criteria and other calling thresholds;
- known limitations for rare partners, low expression, or complex rearrangements;
- whether a negative result is considered technically valid.
A failed test is not a biologically negative test. Options may include testing another block, obtaining a new biopsy, using a less RNA-dependent method, or applying a targeted assay that requires less material. The best rescue strategy depends on urgency and the suspected alteration.
How to Interpret a Positive Gene Fusion Result
A positive report should identify both genes when possible, the direction of the fusion, the joined exons or genomic breakpoints, the method, and the laboratory’s clinical classification. The interpretation should address whether the fusion is known, likely functional, diagnostically characteristic, prognostic, or therapeutically actionable.
The order of the gene names is meaningful. In an RNA fusion, the 5′ partner contributes the beginning of the transcript and the 3′ partner contributes the remainder. For many kinase fusions, the 3′ gene retains the kinase domain. Reports may also describe the transcript as in-frame or out-of-frame. An in-frame fusion preserves the reading frame and is more likely to produce a stable hybrid protein, although biology cannot be determined from frame alone.
A common, well-characterized fusion in the expected tumor context usually has strong significance. For example, an established kinase fusion in a compatible cancer may support use of a targeted inhibitor. A characteristic fusion in a morphologically compatible sarcoma may confirm the diagnosis. A fusion in a hematologic malignancy may define a disease category and provide a quantitative marker for monitoring.
Novel fusions require more caution. A new partner involving a known oncogene may still be oncogenic if the transcript is expressed, in-frame, and retains the critical functional domain. Evidence may include recurrence in databases or literature, compatibility with known fusion architecture, adequate supporting reads, absence from normal controls, and orthogonal confirmation. Conversely, a fusion between neighboring genes may represent read-through transcription rather than a genomic driver. Low-support calls can arise from template switching, misalignment, index hopping, or other technical artifacts.
The word “actionable” is context dependent. It can mean that an approved therapy is indicated for that tumor type, that a tumor-agnostic approval exists, that evidence supports off-label use, or that a clinical trial is available. These levels are not equivalent. Treatment relevance also depends on disease stage, prior therapy, co-alterations, drug access, and current regulatory and guideline status. The molecular result should be interpreted through a multidisciplinary oncology process when possible.
| Report element | Practical meaning |
|---|---|
| Fusion partners | Identifies the genes involved; one partner may be unknown in a break-apart or single-ended assay |
| Exon junction or breakpoint | Helps determine reading frame, retained domains, and assay confidence |
| Supporting evidence | Read counts, unique molecules, signal percentage, or other method-specific metrics |
| Classification tier | Summarizes diagnostic, prognostic, or therapeutic evidence under the laboratory’s framework |
| Therapy statement | Must be matched to tumor type, stage, guideline, and approval context |
| Confirmation note | Indicates whether another method is recommended because the event is novel or atypical |
Negative, Uncertain, and Discordant Results
A negative fusion result means that the assay did not detect a reportable fusion within its validated scope. It does not mean that the tumor has no rearrangement. Interpretation starts with whether the test passed quality controls and whether the relevant genes and breakpoint regions were covered.
For an RNA assay, a strong negative result generally requires adequate RNA quality, sufficient usable reads, and successful internal controls. If RNA is degraded or control genes perform poorly, the laboratory may issue an indeterminate or failed result. Some assays can still detect a highly expressed fusion in suboptimal material, but absence of a signal in that setting is less reassuring.
For DNA panels, a negative result may reflect incomplete intronic coverage. Important breakpoints can lie within long, repetitive introns that are difficult to capture and map. If the tumor type or clinical features strongly suggest a fusion, RNA sequencing, FISH, or targeted RT-PCR may be appropriate even after negative DNA profiling.
An uncertain result can include a novel fusion with limited functional evidence, an out-of-frame event, a low-read call near the detection threshold, or a rearrangement whose partner is unknown. Such findings should not automatically be used for targeted therapy. The laboratory may recommend confirmation, pathology correlation, or review in a molecular tumor board.
Discordant results are possible because methods measure different analytes. Examples include:
- DNA NGS detects a rearrangement, but RNA NGS does not detect an expressed transcript.
- Break-apart FISH is positive, but sequencing identifies no canonical fusion.
- Immunohistochemistry is positive, but molecular testing is negative.
- RNA NGS identifies a fusion that is not captured by the DNA panel.
Discordance is not resolved by assuming that one platform is always correct. The team should examine specimen differences, tumor content, assay design, fusion architecture, expression level, and quality metrics. A rearrangement may be present in a subclone, the RNA may be degraded, or the FISH pattern may result from a complex copy-number change rather than a functional fusion. Retesting the same material with an orthogonal method or testing a different specimen can clarify the finding.
Important Clinical Examples
Fusion significance varies across diseases. The following examples illustrate why method and context matter rather than provide a complete treatment list.
Lung cancer: Fusions involving ALK, ROS1, RET, and NTRK can drive subsets of non–small cell lung cancers. RNA testing can improve detection when DNA sequencing misses intronic breakpoints or cannot establish an expressed transcript. A confirmed driver may direct matched therapy according to current lung-cancer guidelines. Dedicated pages on ALK testing and ROS1 fusion testing address target-specific interpretation.
NTRK fusions: NTRK1, NTRK2, and NTRK3 fusions occur at high frequency in a small group of rare tumors and at low frequency across many common cancers. Because tumor-agnostic TRK inhibitors exist for eligible patients, accurate detection is important. Pan-TRK immunohistochemistry may be useful as a screen in some settings, but RNA NGS can identify the partner and transcript. Assay design matters because uncommon partners can be missed by fixed-primer approaches [2].
Thyroid and biliary cancers: RET and NTRK fusions may occur in thyroid cancers, while FGFR2 fusions are important in a subset of intrahepatic cholangiocarcinomas. The report should distinguish a true fusion from other rearrangements and confirm that the relevant functional domain is retained.
Sarcomas: Many sarcomas are defined by characteristic fusions, including EWSR1-, SS18-, CIC-, or NAB2::STAT6-related entities. FISH may show that a locus is rearranged, but broad RNA sequencing can identify the partner and help distinguish tumors that share a rearranged gene. Morphology remains essential because the same gene can partner with different genes in biologically distinct neoplasms.
Leukemias: BCR::ABL1 defines chronic myeloid leukemia and occurs in a subset of acute lymphoblastic leukemias. RT-PCR can establish the transcript type and later quantify molecular response. PML::RARA is the defining fusion of acute promyelocytic leukemia, a medical emergency in which rapid testing has immediate treatment implications. See the dedicated BCR::ABL1 test and PML::RARA test guides for disease-specific details.
Brain and pediatric tumors: Fusion detection can refine classification in gliomas, infant-type hemispheric gliomas, pediatric spindle-cell tumors, and other rare cancers. Because the relevant genes and partners are diverse, broad RNA-based approaches may be more informative than a sequence of single-gene tests.
The same fusion name does not guarantee identical behavior across all cancers. Drug sensitivity and prognostic meaning can differ by tissue context, fusion partner, breakpoint, co-alterations, and prior treatment. Clinical interpretation must therefore be disease specific.
Questions and Next Steps After Testing
The most useful first question is: what exactly did the assay test? Ask whether it analyzed RNA, DNA, protein, or chromosome signals; whether it was targeted or broad; and whether the suspected gene and relevant partners were included. A concise result line without the technical scope can be misleading.
For a positive result, useful questions include:
- Is the fusion established as a driver in this tumor type?
- Was an expressed transcript demonstrated, or only a DNA rearrangement?
- Is the transcript in-frame and does it retain the critical functional domain?
- Does the finding confirm or change the pathologic diagnosis?
- Is there an approved therapy for this disease and setting, a tumor-agnostic indication, or only investigational evidence?
- Is orthogonal confirmation recommended before treatment?
- Should the result be reviewed by a molecular tumor board?
For a negative result, ask whether the sample passed all quality checks and whether the laboratory considers the result technically informative. Determine whether RNA testing was performed if only DNA was analyzed, especially when the cancer is known to harbor actionable fusions. A repeat biopsy is not always necessary; another block, cytology specimen, blood or marrow sample, or a targeted FISH/RT-PCR assay may provide an alternative.
A pathologist or molecular laboratory specialist can explain whether the finding fits the tumor. The treating oncologist can interpret therapeutic evidence and current guideline status. A genetic counselor may be helpful when the broader history suggests inherited cancer risk, although most tumor fusions are acquired and are not themselves hereditary findings.
Keep the complete report, not only the result summary. Future clinicians may need the assay name, specimen identifier, fusion partners, exon junction, quality metrics, and date because knowledge and treatment indications evolve. Reanalysis may become useful if a fusion was previously classified as uncertain or if a new therapy targets the detected alteration.
Gene fusion testing is most powerful when viewed as part of integrated diagnosis. The molecular event, tumor morphology, immunophenotype, clinical presentation, and other genomic alterations should tell a coherent story. When they do not, discrepancy review is a strength of good care, not a reason to force the result into a predetermined conclusion.
References
- Oncogenic gene fusions in cancer: from biology to therapy. 2025. Review article.
- Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing. 2023. Consensus recommendations.
- Actionable Structural Variant Detection via RNA-NGS and DNA-NGS in Patients With Advanced Non–Small Cell Lung Cancer. 2024. Cohort study.
- Challenges and prospects in utilizing technologies for gene fusion analysis in cancer diagnostics. 2024. Review article.
- Recommendations for the use of next-generation sequencing (NGS) for patients with advanced cancer in 2024. 2024. Professional recommendation.
Disclaimer
This article provides general education about gene fusion testing and cannot interpret an individual pathology or molecular report. Assay capabilities and treatment indications vary, and oncology decisions should be made with qualified clinicians using the complete clinical record and current guidelines. An urgent suspected diagnosis, such as acute promyelocytic leukemia, requires immediate specialist management rather than reliance on general information.
