Understanding Next-Generation Sequencing (NGS) in Cancer

If your pathology report includes a section on molecular testing, there is a good chance the testing was performed using next-generation sequencing (NGS). NGS is a powerful laboratory method that reads the genetic code of a cancer and can check many genes at once for changes that affect diagnosis, treatment, and prognosis. Reports that come back from NGS can look intimidating: long lists of gene names, unfamiliar abbreviations, and terms like “variant of uncertain significance.” This article explains, in plain language, what NGS is, what it looks for, how the results are reported, and what those results mean for your care. It is a general guide; the articles on individual biomarkers in this section cover what specific results mean for specific cancers.

What next-generation sequencing is

Next-generation sequencing (NGS) is a method for reading the order of the chemical letters that make up DNA, the molecule that carries the instructions for how cells grow and behave. DNA is written in just four letters, and the exact order of those letters spells out the instructions in each gene. Cancer develops when changes in this code cause cells to grow out of control, so reading the code can reveal what is driving a particular cancer.

What makes NGS “next-generation” is its scale. Older methods could read only one gene at a time, which was slow and used up precious tissue. NGS breaks DNA into many small fragments, reads millions of them at once, and uses a computer to reassemble them and compare them against a reference sequence. This lets the laboratory check dozens or even hundreds of genes from a single small sample in one test. Some NGS tests also read RNA, a closely related molecule that is especially useful for detecting a particular type of change called a gene fusion, described later in this article.

What NGS looks for in a cancer

Next-generation sequencing reads the genetic code of a cancer to identify the changes driving it. These changes, which a pathology report may group under the heading molecular testing or biomarker testing, come in several types, and NGS can detect all of them from the same sample.

Mutations — Small changes in the sequence of a single gene, where one or a few letters are altered, added, or removed. A mutation can switch a growth gene permanently on or disable a protective one. Examples include EGFR mutations in lung cancer and KRAS mutations in pancreatic and colorectal cancer.
Gene fusions — Large changes in which two separate genes become joined together, creating an abnormal combined gene that drives cancer growth. Fusions are often best detected using the RNA-based part of an NGS test. Examples include ALK and NTRK fusions.
Copy number changes (amplifications) — Changes in the number of copies of a gene. Normally, a cell has two copies of each gene; in some cancers, a gene is amplified to many extra copies, producing too much of its protein. HER2 amplification is a well-known example.
Broad genomic measures — Some results are not about a single gene but summarize the whole tumor. Tumor mutational burden (TMB) counts the total number of mutations across the cancer, and microsatellite instability (MSI) reflects a breakdown in the cell’s DNA repair system. Both can help predict response to immunotherapy.

Not every test looks for every type of change. The laboratory selects a panel of genes appropriate to the cancer being tested, which is why an NGS report for lung cancer looks different from one for colon cancer.

DNA panels and RNA panels

Next-generation sequencing can look for two different types of genetic material in a tissue sample, DNA and RNA, and the two are suited to finding different kinds of changes. Understanding the difference explains why some reports mention both.

DNA-based sequencing is the foundation of most NGS testing and excels at detecting mutations and copy-number changes. RNA-based sequencing is particularly good at detecting gene fusions because the junction between two genes is often easier to detect at the RNA level than in DNA. For this reason, comprehensive NGS panels frequently test both DNA and RNA, especially in cancers such as lung cancer where fusions are an important treatment target. If a cancer is strongly suspected to carry a fusion but DNA testing does not detect one, an RNA-based test may be added to ensure a treatable fusion is not missed.

How NGS results are reported

An NGS report summarizes the genetic changes found in the cancer, and learning to recognize a few standard elements makes the report much easier to read. The report typically lists each altered gene, the specific change found, and a comment on its meaning.

The gene and the specific change — The report names the gene (for example, EGFR) and the exact alteration within it (for example, an exon 19 deletion or the L858R mutation). The specific change matters, because different changes in the same gene can have different treatment implications.
Variant classification — Each change is classified by how confident the laboratory is that it is meaningful. A change that is clearly known to cause or contribute to cancer is called pathogenic, meaning it is harmful rather than harmless. A change is considered actionable when it can be acted upon, usually because it matches an approved targeted therapy, makes the cancer eligible for immunotherapy, or opens a clinical trial. A pathogenic change is not always actionable, because not every harmful change has a matching treatment. A change whose meaning is not yet understood is called a variant of uncertain significance (described below).
Variant allele frequency (VAF) — Some reports give a percentage showing how many of the cancer cells carry the change. This can help distinguish a main driver of the cancer from a change present in only a small portion of the tumor.
An interpretation or comment — Many reports include a plain-language note explaining whether a change points to an approved targeted therapy, a clinical trial, or a known effect on prognosis. This is often the most useful part of the report for a patient.
No significant changes found — Sometimes the report states that the panel did not detect any meaningful change, often worded as “no reportable alterations” or “no actionable variants identified.” This is a genuine result, not a failed test. It means the genes on the panel were read but none carried a change that affects treatment or prognosis in a way the laboratory could report.

A result showing no significant changes can feel anticlimactic, but it is still useful information. It tells the care team that the cancer is unlikely to respond to the targeted drugs associated with the genes on the panel, which helps focus attention on other approaches, such as chemotherapy or immunotherapy. Depending on the situation, the team may also consider a broader panel or testing of a different sample if a treatable change is still suspected.

What a variant of uncertain significance (VUS) means

One term that frequently appears in a next-generation sequencing report and often causes concern is variant of uncertain significance, usually abbreviated as VUS. A VUS is a change found in the genetic code whose effect is not yet known. Scientists have not gathered enough evidence to say whether it contributes to cancer or is simply a harmless variation, of which everyone carries many.

A VUS is neither a positive nor a negative result. It should not be used to make treatment decisions, nor should it be treated as proof of a problem. As laboratories collect more data over time, a variant of uncertain significance may later be reclassified as either meaningful or harmless. Because the meaning of a VUS can change, it is reasonable to ask the care team whether a particular VUS is being monitored for reclassification. The presence of a VUS on a report is common and, on its own, is not a cause for alarm.

Tissue testing and liquid biopsy

Next-generation sequencing requires a sample of the cancer’s genetic material, which can come from two sources. Most often, NGS is performed on tumor tissue obtained during a biopsy or surgery, the same tissue the pathologist examines under the microscope. This is the standard approach and provides a rich sample for testing.

NGS can also be performed on a blood sample, an approach called a liquid biopsy. Cancers shed small fragments of their DNA into the bloodstream, and a liquid biopsy detects and sequences this circulating tumor DNA. A liquid biopsy can be useful when a tissue sample is difficult or risky to obtain, when there is not enough tissue left for testing, or when doctors want to monitor a cancer over time without repeated procedures. Tissue and blood testing are often complementary, and the care team chooses the approach, or combination, that best fits the situation.

Testing the tumor versus testing inherited DNA

An important point about next-generation sequencing is what it does, and does not, tell you about inherited risk. Most NGS performed on a cancer is tumor-only testing, meaning it reads the DNA of the cancer cells to guide treatment. It is designed to find the changes driving that specific cancer, not to determine whether a change was inherited.

This matters because some genes, such as BRCA1 and BRCA2, can be altered either only within the tumor (a somatic change, not inherited) or in every cell of the body from birth (a germline change, inherited and relevant to family members). A tumor-only NGS test that finds a BRCA mutation cannot, by itself, tell which type it is. When a result suggests a possible inherited cause, a separate germline test, performed on blood or saliva and often arranged through a genetic counselor, is used to find out more. Some laboratories perform paired testing, sequencing both the tumor and normal DNA together to separate inherited changes from those that arose in the cancer. The difference between somatic and germline testing is explained in more detail in the article on genetic testing in cancer.

Comprehensive panels versus single-gene testing

When molecular testing is needed, it can be done one gene at a time or many genes at once, and next-generation sequencing makes the second approach practical. Testing genes one at a time can work when only a single result is needed, but it is slow and consumes tissue with each test. A comprehensive NGS panel reads many relevant genes from a single sample simultaneously.

For cancers in which multiple genes could each point to a different treatment, such as lung cancer, a comprehensive panel is generally preferred. It conserves the limited tissue from a biopsy, provides a fuller picture in one step, and can reveal an unexpected but treatable change that single-gene testing might not have detected. The care team decides how broad a panel is appropriate based on the cancer type, the stage, and what treatment decisions depend on the result.

How NGS results guide care

The purpose of next-generation sequencing is to inform decisions, not to make them. The genetic changes NGS finds in a cancer are used by the treatment team, alongside the diagnosis, the stage, and the patient’s overall health, to consider which treatments may be appropriate. NGS guides care in a few main ways.

Matching the cancer to a targeted therapy — Many genetic changes have a matching drug designed to block their effect. Finding such a change can indicate eligibility for that targeted therapy. Some of these drugs are approved based on the genetic change regardless of where the cancer started, an approach called tumor-agnostic treatment.
Identifying immunotherapy candidates — Broad measures, such as high tumor mutational burden or microsatellite instability, can indicate that a cancer may respond to immunotherapy.
Opening clinical trial options — A genetic change with no approved drug for that cancer may still match an investigational therapy clinical trial, which the oncology team can discuss.
Providing prognostic information — Some changes indicate how a cancer is likely to behave, which can inform how intensively it is treated.

Because the meaning of any single result depends heavily on the cancer type, the articles on individual biomarkers in this section explain what specific NGS findings mean in specific cancers.

Practical questions patients often have about NGS

Do I need an extra procedure to have NGS done?

Usually not. Next-generation sequencing is most often performed on tissue that was already collected during the biopsy or surgery used to diagnose the cancer, so no additional procedure is typically needed. If there is insufficient tissue, or if the existing sample is unsuitable, the care team may recommend a fresh tissue sample or a blood-based liquid biopsy.

How long do NGS results take?

Comprehensive next-generation sequencing usually takes longer than a single-gene test, often one to three weeks, because it reads many genes and the data require careful analysis. This wait can feel long when treatment decisions are pending, and it is reasonable to ask the care team when results are expected and whether any treatment can begin in the meantime.

Will NGS find something in every cancer?

Not always. Some cancers have a clear, treatable genetic change; others have changes with no current treatment or no significant change detected by the panel. A result showing no actionable change is still informative, because it helps the care team focus on other treatment approaches such as chemotherapy or immunotherapy.

Does an NGS result tell me about my family’s risk?

Usually not on its own. Most NGS is tumor-only testing aimed at guiding treatment, and it is not designed to determine inherited risk. If a result raises the possibility of an inherited change, a separate germline test, often arranged through a genetic counselor, is used to assess its implications for family members.

Questions to ask your doctor

Was next-generation sequencing performed on my cancer, and which genes were included in the panel?
Was the testing done on tumor tissue, on a blood sample (liquid biopsy), or both?
Did the test read DNA, RNA, or both, and could a gene fusion have been missed?
Were any changes found that point to an approved targeted therapy or a clinical trial?
If a variant of uncertain significance was reported, is it being monitored for reclassification?
Does my result have any implications for inherited risk, and should I have germline testing?
How long will the results take, and can any treatment begin while we wait?
If no actionable change was found, what treatment options does that leave?
Is there enough tissue for testing, or will I need another sample?
How will my NGS results be used together with the rest of my pathology report to plan treatment?